Discovering and exploiting bacterial proteins as...
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UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO DE BIOLOGIA ANIMAL Discovering and exploiting bacterial proteins as anticancer agents Gonçalo Emanuel Fialho Mourata da Silva DISSERTAÇÃO Mestrado em Biologia Humana e Ambiente 2013
Transcript of Discovering and exploiting bacterial proteins as...
UNIVERSIDADE DE LISBOA
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA ANIMAL
Discovering and exploiting bacterial
proteins as anticancer agents
Gonçalo Emanuel Fialho Mourata da Silva
DISSERTAÇÃO
Mestrado em Biologia Humana e Ambiente
2013
UNIVERSIDADE DE LISBOA
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA ANIMAL
Discovering and exploiting bacterial
proteins as anticancer agents
Gonçalo Emanuel Fialho Mourata da Silva
DISSERTAÇÃO
Mestrado em Biologia Humana e Ambiente
Dissertação orientada por Professor Doutor Arsénio Fialho e
Professora Doutora Ana Crespo
2013
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"To myself I am only a child playing on the beach, while vast oceans of truth lie
undiscovered before me."
Isaac Newton
"I am not apt to follow blindly the lead of other men."
Charles Darwin
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ACKNOWLEDGMENTS
I would like to express my sincere gratitude to my supervisor Professor
Arsénio Fialho (Instituto Superior Técnico), for having granted me this
opportunity to work with him and for all the help and support that he gave me,
every time I needed, throughout my thesis.
I am also grateful to Professor Ana Crespo, at the Departamento de Biologia
Animal (Faculdade de Ciências, Universidade de Lisboa), for having gently
accepted to be my supervisor and for being always available whenever I needed.
I would also like to thank Nuno Bernardes, for all the knowledge transmitted
and for teaching me everything I have learned inside a laboratory this year.
In addition, I am thankful to the people in the Biological Sciences Research
Group (Instituto Superior Técnico), especially to Dalila Mil-Homens and Sofia
Abreu which helped me in many occasions.
I sincerely want to address a special thanks to my closest friends Alexsandro
Costa, João Guerreiro, Duarte Silva, João Serafim, Ana Catarina, Andreia
Ferreira, for always been there, for giving me a kind word, for listening, for
making me laugh, and specially for believing in me and my capabilities. I would
like to extend this acknowledge also with my friends from my master degree
course, especially to Sofia Alves.
I especially want to thank Tânia, for being my fountain of joy and happiness,
and for having the amazing ability of making me smile even in the toughest times.
I would also like to thank my family, especially my parents and my brother, for
all their support, patience and strength they gave me through this entire year. I
could not have finished my master degree without them.
I dedicate the achievement of my master degree to my grandparents José and
Mariana, for being the reason why I came to science and whose memories remain
strictly in my heart and still give me the strength to overcome my fears and
difficulties.
V
ABSTRACT
Azurin is a low molecular weight protein, produced by Pseudomonas
aeruginosa and possesses several antitumor properties, like causing P-cadherin
levels to decrease in invasive breast cancer cells. In this work, we studied the
effect of lysosome and proteasome inhibitors on P-cadherin level, using a breast
cancer cell line, expressing high P-cadherin level (MCF-7/AZ.Pcad), previously
treated with azurin. Additionally, we evaluated how a cholesterol-depleting agent
(MβCD) affects P-cadherin level. The effects of both inhibitors on P-cadherin
were observed by western blot and confirmed that azurin mediates P-cadherin
degradation through lysosome and proteasome proteolytic pathways. We also
described, for the first time, that MβCD causes P-cadherin level to decrease.
Together, these findings have increased our understanding of how the bacterial
protein azurin is acting as anti-cancer agent.
In this work we have also studied the in vitro cytotoxicity of two other bacterial
proteins (MPT 63 and Ndk) against human breast and lung cancer cells. MPT 63
is an antigen secreted by Mycobacterium tuberculosis that induces immunogenic
responses in animal models and its cytotoxicity against several tumor cell lines
was recently described in a patent. Nucleoside diphosphate kinase (Ndk) is a
ubiquitous enzyme which maintains the nucleotide pools within the cells, and can
be secreted by P. aeruginosa. A human Ndk, termed Nm23-H1, also showed an
anti-metastatic role in different cancer models. In order to test possible antitumor
properties of these proteins, MTT cell viability assays were performed in breast
and lung cancer models (MCF-7/AZ.Mock and A549) using increasing azurin,
MPT 63 and Ndk concentrations, and different exposure times. In addition,
matrigel invasion assay was performed in A549 invasive cells treated with Ndk.
Both azurin, MPT 63 and Ndk evidenced cytotoxicity against both cancer models
in a time and dose dependent manner. Ndk revealed cytotoxic activity and
selectivity against tumor cells similar to azurin. We observed a small decrease in
cell invasion using this protein. In summary, we promoted a screening of new
bacterial proteins that demonstrated antitumor potential, especially Ndk.
Keywords: Azurin, MPT 63, Ndk, Cancer, P-cadherin.
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RESUMO
Novas terapias anti-tumorais emergentes baseiam-se em abordagens pouco
convencionais, como a utilização de microorganismos, nomeadamente bactérias
vivas ou produtos purificados a partir das mesmas, como proteínas. A azurina é
uma proteína de baixo peso molecular, produzida por Pseudomonas aeruginosa e
possui diversas propriedades anti-tumorais, entre as quais a indução de apoptose
em células tumorais pela estabilização da proteína supressora de tumores p53.
Mais recentemente, um novo tipo de acção anti-tumoral foi descoberta, tendo sida
descrita a sua capacidade de diminuir os níveis de P-caderina em células tumorais
invasivas de cancro da mama, sem afectar, no entanto, os níveis de E-caderina. O
mecanismo, pelo qual a azurina causa o decréscimo de P-caderina nas células
tumorais não é ainda totalmente conhecido, mas esta parece actuar a nível pós-
transcripcional dado que não se verificam diferenças na expressão de P-caderina
em células tratadas com azurina. Dados relativos a ensaios com análise a
microarrays revelaram que a transcrição de genes associados ao lisossoma e
processos de transporte mediado por vesículas se encontrava mais activa. No
presente trabalho pretendeu-se esclarecer se a diminuição dos níveis de P-
caderina, mediada pela acção da azurina, se deve à sua degradação pelos sistemas
proteolíticos a nível celular, como o lisossoma e proteossoma. Nesse sentido
utilizaram-se células de uma linha celular de cancro da mama, que expressa níveis
elevados de P-caderina (MCF-7/AZ.Pcad), e que foram previamente tratadas com
azurina antes de serem administrados inibidores de lisossoma (cloreto de amónio)
e de proteossoma (MG-132). Do mesmo modo foram também avaliados os efeitos
de um agente sequestrador de colesterol (MβCD) e inibidor de entrada da azurina
nas células, ao nível da P-caderina nesta linha celular tumoral. Os efeitos de
ambos os inibidores, ao nível da degradação da P-caderina, foram observados por
western blot e confirmaram que a azurina medeia a degradação da P-caderina por
sistemas proteolíticos como o lisossoma e o proteossoma. Descrevemos
igualmente, pela primeira vez, que a MβCD provoca a diminuição dos níveis de
P-caderina sem afectar os níveis de E-caderina. Conjuntamente, estes resultados
permitiram aumentar o nosso conhecimento acerca do modo como a azurina actua
como agente anticancerígeno neste caso específico.
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Neste trabalho pretendemos também estudar a citotoxicidade in vitro de duas
outras proteínas bacterianas (MPT 63 e Ndk) em células tumorais humanas de
cancro da mama e de pulmão. A MPT 63 é uma proteína antigénica secretada por
Mycobacterium tuberculosis e capaz de induzir respostas imunogénicas em
diversos modelos animais. Esta proteína apresenta uma estrutura semelhante a
imunoglobulinas, uma característica que é partilhada com a azurina.
Recentemente foi descrita como possuindo elevada actividade citotóxica contra
várias linhas celulares tumorais, assim como um péptido derivado desta proteína
(MB30), tendo esta propriedade de ambas as molécula sido registada numa
patente. A nucleosídeo difosfato cinase (Ndk) é uma enzima ubíqua em diversos
organismos e que tem como função manter as reservas de nucleótidos das células.
Esta proteína pode igualmente ser secretada por várias bactérias como P.
aeruginosa. As Ndks humanas estão agrupadas numa família de proteínas
denominada de Nm23, tendo sidas até hoje descritas dez tipos. A primeira destas
proteínas a ser descrita, denominada Nm23-H1, demonstrou possuir
adicionalmente uma importante acção anti-metastática em diferentes modelos de
cancro. Tendo em conta o vasto leque de acção anti-tumoral da azurina, procurou-
se seleccionar duas proteínas bacterianas (MPT 63 e Ndk) com propriedades
interessantes de serem exploradas, no sentido de testar uma possível actividade
citotóxica das mesmas em células cancerígenas. Para esse efeito foram realizados
ensaios de viabilidade celular (ou ensaios de MTT) em modelos tumorais de
cancro da mama e do pulmão (MCF-7/AZ.Mock e A549), usando concentrações
crescentes de azurina, MPT 63 e Ndk, bem como diferentes tempos de exposição,
com o intuito de entender como estes parâmetros podem afectar o nível de
citotoxicidade destas proteínas. Adicionalmente foi testada a actividade anti-
metastática da Ndk, realizando um ensaio de invasão em matrigel, usando uma
linha celular altamente invasiva de cancro de pulmão, A549. A azurina, assim
como a MPT 63 e a Ndk, evidenciaram citotoxicidade contra ambos os modelos
tumorais testados, de um modo dependente do tempo e concentrações
administradas. A Ndk revelou níveis de actividade citotóxica e selectividade de
acção, relativamente a células tumorais, semelhantes à azurina. Observámos ainda
um pequeno decréscimo da invasão celular das células tumorais de pulmão A549,
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quando esta proteína foi administrada. Em suma, promovemos um rastreio de
novas proteínas bacterianas que demonstraram potencial anti-tumoral,
especialmente a Ndk. O conhecimento acerca destas propriedades necessita de ser
expandido e aprofundado para que, no futuro, se possa avaliar a sua utilização
como agentes anti-cancerígenos úteis, tal como a azurina.
Palavras-chave: Azurina, MPT 63, Ndk, Cancro, P-caderina.
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TABLE OF CONTENTS
ACKNOWLEDGMENTS .................................................................................... III
ABSTRACT ............................................................................................................ V
RESUMO ............................................................................................................. VII
TABLE OF CONTENTS ...................................................................................... XI
LIST OF ABBREVIATIONS ............................................................................. XIII
INDEX OF FIGURES ......................................................................................... XV
INDEX OF TABLES ......................................................................................... XIX
1. INTRODUCTION ........................................................................................... 1
1.1 Cancer ............................................................................................................ 1
1.2 Microorganisms and their products as anti-cancer agents ...................... 2
1.3 Azurin ............................................................................................................ 4
1.4 Cadherins .................................................................................................... 10
1.5 Azurin and P-cadherin interactions ......................................................... 13
1.6 Inhibitors ..................................................................................................... 14
1.7 MPT 63........................................................................................................ 16
1.8 Nucleoside diphosphate kinase ................................................................ 19
2. OBJECTIVES ................................................................................................ 25
3. MATERIALS AND METHODS................................................................... 27
3.1 Bacterial proteins superexpression .......................................................... 27
3.1.1 Bacterial strains and plasmids ......................................................... 27
3.1.2 Inoculum ......................................................................................... 27
3.1.3 Cell sonication ................................................................................. 28
3.1.4 Azurin purification .......................................................................... 28
3.1.5 MPT 63 and Ndk purification ......................................................... 30
3.2 Cell culture and human cell lines ............................................................. 31
3.3 Inhibitors treatment ................................................................................... 32
3.3.1 Lysosome and proteasome inhibitors .............................................. 32
3.3.2 Azurin internalization inhibitor ....................................................... 32
3.4 Protein lysates ............................................................................................ 33
3.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) 33
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3.6 Western blotting ......................................................................................... 34
3.7 MTT cell viability assay ............................................................................ 35
3.8 Matrigel invasion assay ............................................................................. 36
3.9 Bioinformatics ............................................................................................ 36
3.9.1 Sequence analysis and secondary structures ................................... 36
3.9.2 Phylogenetic and structural alignment analysis............................... 37
4. RESULTS....................................................................................................... 39
4.1 Proteolytic pathways inhibitors effect on P-cadherin level .................. 40
4.2 Azurin internalization ................................................................................ 42
4.3 Inhibition of azurin’s effect on E and P-cadherins level using MβCD
44
4.4 Bacterial proteins to treat cancer .............................................................. 45
4.4.1 Purified proteins .............................................................................. 45
4.5 MTT cell viability assays .......................................................................... 46
4.5.1 Azurin .............................................................................................. 47
4.5.2 MPT 63 ............................................................................................ 48
4.5.3 Ndk .................................................................................................. 49
4.6 Matrigel invasion assay ............................................................................. 51
4.7 Bioinformatic analysis on human Nm23 and bacterial Ndks ............... 51
5. DISCUSSION ................................................................................................ 65
6. CONCLUSION .............................................................................................. 73
7. REFERENCES ............................................................................................... 77
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LIST OF ABBREVIATIONS
BCG - Bacille Calmette-Guérin
BSA - Bovine serum albumin
DMBA - Dimethyl-benz-anthracene
DMSO - Dimethyl sulfoxide
E1 - Ubiquitin-activating enzyme
E2 - Ubiquitin-conjugating enzyme
E3 - Ubiquitin-ligase enzyme
ELISA - Enzyme-linked immunosorbent assay
Eph - Ephrin receptor
FBS - Fetal bovine serum
HBS - Hepes buffered saline
HLA-DR - Human Leucocyte Antigen
HUVEC - Human umbilical vein endothelial cells
INF-γ - Gamma interferon
IPTG - Isopropyl-β-D-Thiogalactopyranoside
KSR - Kinase suppressor of ras
Laz - Lipid-modified azurin
LB - Luria Broth
LPS - Lipopolysaccharide
MEM - Minimum Essential Medium
MMP - Matrix metalloproteases
MTT - 3-(4,5 dimethylthiazol-2-yl-2,5 tetrazolium bromide)
MβCD - Methyl-β-cyclodextrin
NaCl - Sodium chloride
NCBI - National Center for Biotechnology Information
Ndk - Nucleoside diphosphate kinase
NDPs - Nucleoside diphosphates
NH4Cl – Ammonium chloride
NSCLC - Non-small cell lung cancer
NTPs - Nucleoside triphosphates
OD640 nm - Optical density
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PBMNCs - Peripheral blood mononuclear cells
PBS - Phosphate buffered saline
PDB - Protein Data Bank
PTD - Protein transduction domain
PVP - Polyvinylpyrrolidone
SCLC - Small cell lung cancer
SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis
sP-cad - Soluble P-cadherin fragment
STRAP - Serine-threonine kinase receptor-associated protein
SURE - Stop Unwanted Rearrangement Events
Th1 - T helper 1
UPS - Ubiquitin-proteasome system
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INDEX OF FIGURES
Figure 1 – Current diverse strategies to use bacteria and bacterial products in
cancer treatment (Bernardes et al, 2013) [11]. ........................................................ 3
Figure 2 – The three-dimensional structure of azurin from P. aeruginosa
(Bonander et al, 1997) [18]. .................................................................................... 4
Figure 3 – Azurin as a promiscuous protein, which possess both anticancer,
antiparasite and antiviral activities (Fialho et al, 2007) [37]. ............................... 10
Figure 4 – Schematic representation of the classical cadherin-catenin complex
and the structural components of cadherins (adapted from Albergaria et al, 2011)
[40]. ....................................................................................................................... 11
Figure 5– Structure of M. tuberculosis MPT 63 protein (adapted from Goulding
et al, 2002) [58] ..................................................................................................... 17
Figure 6 – Three-dimensional structures of human Ndk-A. (A) Diagram of Ndk-
A monomer. (B) Diagram of the homo-hexamer (Han et al, 2010) [79]. ............. 22
Figure 7 – (A): Western Blot analysis of the effect of lysossome inhibitor,
ammonium chloride (NH4Cl), on E and P-cadherins of MCF-7/AZ.Pcad cells,
previously treated with azurin at 0, 50 and 100 μM. Azurin and the inhibitor were
administrated at 0 and 32 hours, respectively, in a 48 hours assay. (B): Charts
illustrated represent average percentage values of protein level for E and P-
cadherins which signal was normalized with actin levels. .................................... 41
Figure 8 – (A): Western Blot analysis of the effect of proteasome inhibitor, MG-
132, on E and P-cadherins of MCF-7/AZ.Pcad cells, previously treated with
azurin at 0, 50 and 100 μM. Azurin and the inhibitor were administrated at 0 and
32 hours, respectively, in a 48 hours assay. (B): Charts illustrated represent
average percentage values of protein level for E and P-cadherins which signal was
normalized with actin levels.................................................................................. 42
Figure 9 - Effects of MβCD on azurin’s internalization on MCF-7/AZ.Pcad cells
after 8 hours of exposure to this inhibitor and 24 hours with fresh medium
(visualized by western blot). PBS was used as double negative control. DMSO
was used as negative control. Azurin was tested at two different concentrations
(50 and 100 μM). ................................................................................................... 43
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Figure 10 – Western blot showing the effect of azurin’s internalization inhibitor
MβCD on E- and P-cadherin levels in MCF-7/AZ.Pcad cells after 8 hours of
exposure and 24 hours with fresh medium. PBS was used as double negative
control. DMSO was used as negative control. A decrease in P-cadherin level,
between control samples and samples treated with MβCD and azurin, is shown in
the figure. ............................................................................................................... 45
Figure 11 - 15% SDS-PAGE of MPT 63 and Ndk purified from E. coli SURE
strain. Proteins appear as a single band at approximately 15 kDa, according to
their expected molecular weight. ........................................................................... 46
Figure 12 – Cytotoxicity (%) caused by azurin on MCF-7/AZ.Mock during 48 h
(one dose) and 72 h (three doses), and A549 48 h (one and two doses). Both cell
lines were tested with 5 different azurin concentrations (0, 10, 25, 50, and 100
μM). Significant values, p-value<0.05 with the Student t-test, are shown as
asterisks (*). ........................................................................................................... 48
Figure 13 – Cytotoxicity (%) caused by MPT 63 on MCF-7/AZ.Mock during 48
h (one dose) and 72 h (three doses), and A549 during 48 h (two doses). Both cell
lines were tested with 5 different MPT 63 concentrations (0, 10, 25, 50, and 100
μM). ....................................................................................................................... 49
Figure 14 - Cytotoxicity (%) caused by Ndk on MCF-7/AZ.Mock during 48 h
(two doses) and 72 h (three doses), A549 48 h (two doses) and 16HBE14o- during
48 h (two doses). Both cell lines were tested with 5 different Ndk concentrations
(0, 10, 25, 50, and 100 μM). Significant values, p-value<0.05 with the Student t-
test, are shown as asterisks (*). ............................................................................. 50
Figure 15 – Cell invasion (%) in a Matrigel invasion assay performed on A549
highly invasive tumor cells, which were treated with Ndk at 0, 50 and 100 μM,
during 24 hours. ..................................................................................................... 51
Figure 16 – ClustalW multiple alignment of human and bacterial Ndks. The
numbers above the sequences indicate the position of amino acid residues.
Secondary structure of E. coli Ndk is represented according to PDB code 2HUR.
Secondary elements are indicate as: single purple curves (turns), yellow arrows
(β-sheets), red lines (bends), blue curved lines (alpha helices), orange curved lines
(3/10-helices), and black lines (no secondary structure assigned). NCBI accession
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number for each protein is given below. Human (Homo sapiens) Nm23 proteins:
Nm23-H1 (CAG46912), Nm23-H2 (NP_001018147), Nm23-H3 (EAW85629),
Nm23-H4 (NP_005000), Nm23-H5 (NP_003542), Nm23-H6 (NP_005784),
Nm23-H7 (Q9Y5B8), Nm23-H7B (NP_932076), Nm23-H8 (AAF20909), Nm23-
H9 (NP_835231), Nm23-H10 (NP_008846). Bacterial Ndks: P. aeruginosa
(EPR01938), M. tuberculosis (EQM19968), M. bovis (AGE68451), E. coli
(ERF95659), M. xanthus (P15266), V. cholerae (WP_001162853). .................... 58
Figure 17 – Sequence alignment and secondary structure comparison between
human Nm23-H1 and P. aeruginosa Ndk. Symbols in Clustal Consensus
sequence indicate standard ClustalW nomenclature: (*) identity, (:) high
conservation and (.) conservation. Secondary structure of Nm23-H1 was
represented according to PDB code 4ENO. Secondary structure of P. aeruginosa
Ndk was predicted using PDB code 3VGU as template. This template was aligned
between its 2 to 141 amino acid residues and share a 79% sequence identity with
P. aeruginosa Ndk. Secondary elements are indicate as: single purple curves
(turns), yellow arrows (β-sheets), red lines (bends), blue curved lines (alpha
helices), orange curved lines (3/10-helices), and black lines (no secondary
structure assigned). NCBI accession number for each protein is given below.
Human (Homo sapiens) Nm23-H1 (CAG46912) and P. aeruginosa Ndk
(EPR01938). .......................................................................................................... 61
Figure 18 – Phylogenetic tree representation of human Nm23 protein family as
well as some bacterial Ndks. Splitstree software was used to construct the tree
based on ClustalW multiple alignment generated. All sequences and its NCBI
accession numbers used were the same as those displayed in Figure 16. Protein
three-dimensional structures represented were retrieved from PDB database:
Nm23-H1 (4ENO), Nm23-H2 (3BBF), Nm23-H3 (1ZS6), Nm23-H4 (1EHW),
Nm23-H10 (2BX6), and Ndks from M. tuberculosis (1K44), E. coli (2HUR) and
M. xanthus (2NCK). Quaternary structures are represented as homohexamers
(Nm23-H1, Nm23-H2, Nm23-H3 and Nm23-H4 and Ndk from M. tuberculosis)
and as homotetramers (Ndk from E. coli and M. xanthus). Tertiary structure is
represented as a monomer (Nm23-H10). Ndk from P. aeruginosa is marked with
an asterisk (*) since it is a predicted three-dimensional model created using Swiss-
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model software tools. This representation was modeled using 2 to 141 amino acid
residues of the Ndk sequence (79% identity) from Halomonas sp. 593 (3VGU) as
template. This template forms a homodimer in its quaternary structure, as well as
the model created. .................................................................................................. 62
Figure 19 – Structural alignment between human Nm23-H1 (4ENO) [blue] and P.
aeruginosa Ndk predicted model (green) was performed using PyMOL software.
Three dimensional structures are displayed at different degrees: 0º (A), 90º (B),
180º (C) and 270º (D). ........................................................................................... 63
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INDEX OF TABLES
Table 1 - Phosphate buffer 8x composition .......................................................... 29
Table 2 - START and elution buffer compositions .............................................. 29
Table 3 - ÄKTA elution program for protein sample desalting ........................... 29
Table 4 - PBS composition ................................................................................... 30
Table 5 - Resolving Gel 8% and 15% composition for one gel ........................... 34
Table 6 – Percent identity matrix of human Nm23 proteins and different bacterial
Ndks. ..................................................................................................................... 59
1
1. INTRODUCTION
1.1 Cancer
Currently cancer represents one of the greatest burdens of our society and will
remain a serious and challenging major public health problem in the future years.
Each year about 12,7 million people are diagnosed with cancer, and
approximately 7,6 million die from it, clearly demonstrating the magnitude of this
disease in human population [1].
During the last decades lung cancer has been the most common cancer in the
world, as well as the most common cancer in men [1], [2]. This type of cancer
alone is accountable for 1,4 million deaths each year (18,2% of overall cancers),
being unveiled 1,6 million new cases annually [1]. Among women, lung cancer is
the fourth most frequent, as well as the second most common cause of death from
cancer [1]. Regarding its histopathologic classification, lung cancer can be divided
in 2 major types: Small cell lung cancer (SCLC) and Non-small cell lung cancer
(NSCLC), which comprises 3 subtypes (adenocarcinoma, squamous cell
carcinoma and large cell carcinoma) and accounts for more than 85% of all lung
cancers [3], [4]. NSCLC is typically chemo-resistant and treated primarily by
surgery at early stages, while SCLC progresses more rapidly and metastasizes
earlier than NSCLC, being usually treated by chemotherapy and radiotherapy [3],
[4].
Breast cancer is the second most common cancer worldwide, with 1,4 million
cases being diagnosed annually, and the first cause of cancer-related death among
women (458,000 deaths/year) [1]. This type of cancer changes the size and/or
shape of the breast and can be classified into 2 histopathological categories: ductal
and lobular carcinomas. Each one of these carcinomas can be designated as in situ
or invasive, according to whether the tumor is confined to the glandular area of
the organ or whether it has invaded the stroma [5]. Ductal carcinoma represents
80% of breast cancer cases and it arises from epithelial lining the mammary ducts,
whereas lobular carcinoma is a less common form of breast cancer, that is
originated in the milk-producing lobules of the breast [5], [6].
2
Current cancer treatments rely on surgery, chemo and radiotherapy, or even
hormone therapy, in the case of breast cancer [5]. However these therapies can
reveal serious and systemic side-effects in patient’s health due to its high toxicity
and lack of cancerous tissue specificity [7]. Additionally not every patient
responds efficiently to chemotherapy or other treatments, since cancer cells can
undergo micro-evolution and rapidly render cancer cells resistant to drug therapy
[8], [9]. Another relevant issue is that not always the primary tumor is responsible
for the death of cancer patients, but rather the metastases of cancerous cells to
secondary sites, as brain, bones or lungs for instance [6], [10]. Therefore today we
face new challenges regarding cancer treatment and cancer patients, especially
those which do not respond to conventional therapies, demand for new and more
efficient and selective drugs or therapies to fight this disease.
1.2 Microorganisms and their products as anti-cancer agents
One of the new paths considered in the search for new anticancer therapies
resides on an unconventional approach using microorganisms, namely live
bacteria or their purified products (Figure 1). Although it can be considered, at
first sight, as a revolutionary method, the use of live bacteria and their products
has been investigated and initially proposed a long time ago. In fact, in late
nineteenth century, William Coley, a surgeon in the Memorial Hospital in New
York, used a mixture of extracts of killed bacteria for the first time against
different types of cancer. Surprisingly he observed anti-tumor activity and
complete remission of tumor in some cases, although some patients have
developed systemic infections and died [11], [12]. More recently this line of work
has regained a new insight and several patents, regarding the use of bacteria or its
derived products, have been issued [12].
Since Coley’s experiments until now several live, attenuated or engineered
bacteria, namely Mycobaterium, Clostridium, Salmonella or Listeria, have shown
the ability to act as anticancer agents [13]. The main disadvantage of this method
is clearly the risk of originating undesired infections on patients, caused by live
bacteria themselves, making it a serious risk if applied on humans. Therefore,
3
attenuated or genetic engineered bacteria have been explored in order to minimize
that problem.
However not only bacteria may have applications in cancer therapies, since
bacterial purified products such as protein, enzymes, immunotoxins, antibiotics or
other secondary metabolites have been extensively studied concerning this matter
[11]. With these approaches one could overcome the limitation of using living
bacteria, eliminating the risk of infection. Moreover, some of these products have
proven to cause significant and promising results, such as tumor regression
through growth inhibition, cell cycle arrest or even apoptosis induction [12]. The
use of some bacterial products (as proteins for instance) which are able to target
and lead to the death of tumor cells specifically, would probably overcome the
flaws unspecific cancer treatments.
Figure 1– Current diverse strategies to use bacteria and bacterial products in cancer treatment
(Bernardes et al, 2013) [11].
4
1.3 Azurin
Azurin is a low molecular weight (128 aa-14 kDa), water-soluble, type I
copper-containing protein that belongs to the cupredoxin family [13], [14], [15].
This periplasmic redox protein is secreted by Pseudomonas aeruginosa and act as
electron donor to nitrite reductase during denitrification in this pathogenic
bacterium [14], [16].
Azurin possesses a characteristic single domain structure, which consists of a
rigid β-sandwich core (an immunoglobulin fold), formed by eight antiparallel
strands (Greek key β-barrel structure) stabilized by a disulfide bridge and its
tridimensional structure is presented in Figure 2 [13], [17]. In addition, it has a
neutral hydrophobic patch surrounding the copper site [17]. Azurin displays
structural similarity with variable domains of various immunoglobulins, thereby
demonstrating its single antibody-like structure [13].
However a new and interesting role regarding azurin was revealed in 2000,
when Zaborina et al. reported azurin cytotoxic and apoptosis-inducing activities
towards murine macrophage cell line J774 [19]. Since J774 is a transformed cell
line, derived from reticulum cell sarcoma, it was relevant to verify if azurin could
Figure 2 – The three-dimensional structure of azurin from P. aeruginosa (Bonander et al, 1997)
[18].
5
cause cytotoxic effects in human tumor cell lines. In fact, later it was shown that
azurin can also trigger apoptosis and lead to significant cytotoxicity in different
human tumor cell lines as breast cancer (MCF-7), melanoma (UISO-Mel-2) and
osteosarcoma (U2OS) cells [20], [21], [22]. Moreover, in the referred cell types
the elevation of p53 intracellular levels led to enhanced pro-apoptotic Bax
formation and a rearrangement in its distribution from the cytosol to the
mitochondria, due to azurin treatment, which it is known to occur along with the
release of cytochrome c from the mitochondria to the cytosol [21]. In the case of
MCF-7 cells it was also shown that not only enhanced Bax formation occurs but
also anti-apoptotic Bcl2 levels decrease with the time of azurin treatment [21].
Interestingly azurin exhibits preferentially selectivity against tumor cell lines,
showing much less cytotoxic and apoptotic effects towards normal cell lines [21].
A domain with only 28 amino acids, designated Azu 50-77 or p28, was
identified as the preferential entry domain of azurin and can act as a potential
protein transduction domain (PTD) in cancer cells [23]. Although azurin entry
mode remains unclear, recently some studies regarding p28 showed that this
peptide requires caveolae-mediated endocytosis in order to enter cells, since
microtubule and caveolae-disrupting agents, that inhibit caveosome formation and
transport, were able to inhibit considerably the entry of p28 in different cancer cell
lines [15], [24]. Caveolae are a 50- to 100-nm subset of lipid raft invaginations of
the plasma membrane, which contains caveolin-specific proteins, as caveolin-1,
that act as regulators of signal transduction [24]. Another evidence that supports
the importance of caveolae in p28 internalization is that p28 co-localizes with
caveolin-1 [24], [25]. Moreover cancer cells showed to be more sensitive to the
effects of these inhibitors comparing to normal cells, which suggests that a higher
number of membrane receptors or structures present in cancer cells, such as
caveolae, can help to explain p28 preferential entry in this type of cells [15], [24].
On the other hand several inhibitors of energy-dependent transport mechanisms,
as Na+K
+ ATPase pump, had no inhibitory effect on p28 penetration, suggesting
that non-endocytic pathways may also be involved in the internalization of this
peptide [15], [24].
6
After entering cancer cells, azurin can be found in the cytosol and nuclear
fractions [20]. Azurin has shown the ability to bind and to form a complex with
the tumor suppressor protein p53, thereby stabilizing it and favoring its
intracellular level to raise [20]. This protein is a central and major player in a
complex network responsible for regulating processes like cell growth, genomic
stability and cell death [26]. These different processes can be regulated by the
same protein as it is a transcription factor that acts as a sequence-specific
transcription regulator for many pro-apoptotic genes, namely bax and p21, which
encode protein Bax, involved in apoptosis, and protein 21, involved in the
inhibition of cell cycle progression as well as growth arrest [26]. Although p53-
mediated apoptosis is not yet a fully understood phenomenon, it is clear that p53
has a fundamental role on azurin triggering-apoptosis. As a matter of fact
evidence shows that apoptosis rate and Bax levels are almost inexistent in p53
null cell lines or p53 nonfunctional cell lines, thus supporting a mandatory
relationship between p53 positive cells and azurin apoptotic induction [21].
Therefore azurin can cause an increase in p53 intracellular level which, ultimately,
lead to apoptosis induction in cancer cells, via caspase-mediated mitochondrial
pathways [20]. Another interesting fact is that azurin was shown to be localized in
cytosol and in mitochondria but not in the nucleus of p53 null cell lines, thereby
demonstrating that azurin nuclear transport is p53-dependent [20], [21]. Further
studies on this matter revealed that azurin binds preferentially to the N-terminal
and central domain of p53, but very weakly to the C-terminal domain of this
protein [21]. It was suggested that azurin forms a stable complex by binding the
N-terminal domain of p53 in a 4:1 stoichiometry, and that it can also bind the
DNA-binding domain of this tumor suppressor protein [15]. MDM2, a repressor
oncoprotein which inhibits p53 transcriptional activity and favors its degradation,
also binds p53 in the N-terminal region [27]. However it seems that azurin does
not overlap the MDM2 binding site, although it may sterically shield p53 from
interacting with MDM2 or other ubiquitin ligases [27]. Therefore the increase in
p53 in response to azurin results essentially from a reduction in proteasome
degradation of the tumor suppressor protein, and not from p53 enhanced
transcription [15].
7
More recently, azurin in vitro anti-tumor effects were extended to liquid
cancers as well. Azurin demonstrated selective entry and cytotoxic effects against
HL60, an acute myeloid leukemia cell line, and K562, a chronic myeloid leukemia
cell line [28]. It also showed significant effect on arresting K562 cells at G2/M
checkpoint, which often leads to induction of apoptosis.
Additionally to its entry specificity in cancer cells, azurin-derived peptide p28
has been shown to be capable of interfering with angiogenesis by inhibiting the
formation of capillary tube formation of human umbilical vein endothelial cells
(HUVEC), in a dose-related manner [25]. Although p28 could alter p53
intracellular levels in these cells, inhibition of angiogenesis was not suggested to
be accomplished by a p53-mediated inhibition of cell cycle. In this study azurin
also revealed the same anti-angiogenic activity but not the same efficacy of
inhibition as p28.
Azurin, as well as p28, seems additionally capable of interfering in oncogenic
transformation, since it was shown to inhibit the development of precancerous
lesions in a mouse mammary gland organ culture model, previously exposed to a
carcinogen Dimethyl-benz-anthracene (DMBA) [16].
Considering azurin’s remarkable p53-mediated apoptosis and cytotoxic effects
against several tumor cell lines as in vitro models, it was of interest to verify
whether or not similar results could be observed within in vivo models. On this
subject azurin has shown to be effective on tumor growth inhibition in nude
(athymic) mice with xenotransplanted UISO-Mel-2 and MCF-7 cells [20], [21]. In
both studies tumor regression was justified by an increase of apoptosis in tumor
cells. In another study, using a Dalton’s lymphoma bearing ascites mice model,
Ramachandran et al. also showed azurin’s ability to induce apoptosis in tumor
cells, therefore leading to tumor regression. In this study Bax and caspase-3 levels
in tumor cells, treated with azurin, were shown to be increased, whereas Bcl-2
levels were diminished [29]. These results clearly support in vitro evidence,
already described, regarding p53-mediated apoptosis by azurin, which alters the
balance between pro and anti-apoptotic protein levels in tumor cells in favor of the
first. Another important factor, verified in these three in vivo models, was the lack
8
of azurin’s toxic effects on the animals [20], [21], [29]. In both cases no body
weight changes or any histologic evidence of toxicity were observed.
Since p28 was considered accountable for a significant amount of the overall
tumoricidal activity of azurin, this peptide was tested originally in vivo towards
MCF-7 xenografs in athymic mice, resulting in tumor growth inhibition [15]. A
more recent and broader study evaluated p28 activity against HCT-116 (colon
cancer), UISO-Mel-23 (melanoma) and MDA-MB-231 (breast cancer) xenografs
in athymic mice [30]. In this study tumor cell proliferation decreased in a dose-
related manner in all three xenografs, whereas tumor regression was observed in a
dose-related way in UISO-Mel-23 and MDA-MB-231 xenografs [30]. This
azurin-derived peptide was shown to be non-immunogenic and non-toxic in mice
and non-human primates, which indicates that it can become a promising drug to
be used in a near future [30].
Taking into account all these promising findings about p28, it was proposed its
entry into a phase I clinical trial in order to access its potential role as an anti-
tumor drug in humans. Intravenous p28 was administrated three times a week, for
4 weeks, in 15 adult patients with p53-positive advanced solid tumors (7
melanoma, 4 colon cancer, 1 sarcoma, 1 gastrointestinal stromal cell tumor, 1
prostate cancer and 1 pancreatic cancer) [31]. Five escalating doses of p28 were
used although no significant adverse effects, toxicity, nor any immune response
were observed in any patient. This phase I clinical trial resulted in one patient with
a complete tumor regression, three patients with partial regression, and seven
patients with stable disease [31].
Cupredoxins, like azurin, exhibit topological similarity to a eucaryotic family
of ligands named ephrins, having a common type of Greek key β-barrel [32].
Ephrins are endogenous ligands that bind with Ephrin receptors (Eph), which
constitute the largest family of receptor protein tyrosine kinases [33]. Binding and
heterodimerization between an ephrin and its receptor leads to the trans-
autophosphorylation of the tyrosine kinase domains of the Eph receptor, leading
to several signaling transduction cascades involved in developmental processes
that require organized patterning and movement of cells, as in the remodeling of
blood vessels [32], [33]. However Eph receptors as well as ephrins have shown
9
also to be linked to pathological processes, such as tumor progression,
angiogenesis, migration and invasion [32]. Both types of proteins were shown to
be up-regulated in several different tumors, like EphB2 in breast carcinoma or
lung cancer, for instance [32]. Azurin can bind the EphB2 receptor tyrosine kinase
with a higher affinity than its endogenous ligand, ephrinB2, competing for this
receptor and diminishing tyrosine phosphorylation, thereby interfering with cell
signaling and cancer growth [32].
Despite being involved in the denitrification in P.aeruginosa, an obligatory
role of azurin in this process was excluded, thereby the biological role of this
protein still requires clarification [23]. It has been suggested, however, that
azurin’s physiological function could be involved in bacterial virulence,
characteristic of pathogenic bacteria like P.aeruginosa [23]. Experiments
involving this bacterium and cancer cells have demonstrated that azurin secretion
occurs mainly in the presence of cancer cells in the medium, whereas in the
absence of cancer cells very little secretion of this protein was verified [34]. These
findings point out the possible existence of a sensing mechanism in bacteria that
could lead to azurin secretion in the presence of cancer cells, which they could
sense as a threat or competitor to their own growth [35].
Interestingly it was shown that azurin can inhibit not only growth in cancer
cells, but also in different pathogens as virus (AIDS virus HIV-1), parasites
(Plasmodium falciparum) and protozoans (Toxoplasma gondii) [14], [36]. It
appears that azurin’s ability for binding some pathogen surface proteins interferes
in the entry and inhibit the growth of different pathogens and cancer cells [14],
[36]. This promiscuity in binding different proteins, as seen in Figure 3, may be
attributable to its structural similarity with the variable folds of immunoglobulins,
which could represent a “progenitor” immune response used by prokaryotes, as
suggested by some authors [14].
Azurin’s promiscuity and broader anti-tumor action represent two major
aspects of this bacterial protein. So far it has been shown capable of 3 different
modes of anti-tumor action: induction of apoptosis through p53 stabilization;
inhibition of angiogenesis; and binding ephrin receptor kinases. Acting on
different pathways in cancer progression, azurin differentiates itself from any
10
other current anti-tumor drug available, which could show to be more effective
against cancer cells.
Figure 3 - Azurin as a promiscuous protein, which possess both anticancer, antiparasite and
antiviral activities (Fialho et al, 2007) [37].
1.4 Cadherins
Classical cadherins constitute a family of transmembrane glycoproteins that
mediate calcium-dependent cell-cell adhesion and present themselves as the major
components of cell-cell adhesive junctions [38], [39]. These particular family
includes four different types of cadherins, designated accordingly to their tissue
distribution: CDH1/E-cadherin (epithelial), CDH2/N-cadherin (neuronal),
CDH3/P-cadherin (placental) and CDH4/R-cadherin (retinal) [40]. E- and P-
cadherin can be divided in three major structural domains: 1) an extracellular
domain, which is responsible for cadherins adhesion properties since it’s where
Ca2+
ions will bind to stabilize cadherins conformation; 2) a single membrane-
spanning segment, accountable for protein anchorage to the cellular membrane; 3)
a highly conserved cytoplasmic domain that bind directly to α, β and γ-catenins
and p120-catenin, forming a complex that acts as a bridge between cadherins and
the actin cytoskeleton [38], [39]. This binding is supported by α-catenin and
11
provides the molecular basis for stable cell-cell interactions and its represented in
Figure 4 [40].
The epithelial-calcium dependent cell-cell adhesion is accomplished by the
establishment of hemophilic interactions between proteins, as two cadherin
molecules of adjacent cells to form a homodimer [39]. The cadherin/catenin
complex stability, as well as the signaling pathways controlled by this structure, is
therefore essential for maintaining some cell properties, like cell-cell adhesion and
homeostatic tissue architecture [39]. By regulating these properties the
cadherin/catenin complex has a major role on processes like cell growth,
differentiation, motility and survival [40]. In fact there is wide evidence that
alterations in the adhesion properties between adjacent cells provide them with an
invasive and migratory phenotype. Several data was reported regarding changes in
normal E- and P-cadherin function or expression, which has been associated with
all steps involved in tumor progression [39].
E-cadherin is predominantly expressed in all epithelial tissues, playing a major
role in the formation of epithelia, and being responsible for the maintenance of
cell shape and polarity [40]. E-cadherin gene, CDH1, acts as a tumor suppressor
Figure 4 – Schematic representation of the classical cadherin-catenin complex and the structural
components of cadherins (adapted from Albergaria et al, 2011) [40].
12
gene, regulating the invasion and metastasis of tumor cells. In fact loss of
expression or abnormal function of E-cadherin, by mutations or loss of
heterozygosity, can result in increased ability of tumor cells to invade and to
create metastasis in neighbouring tissues, namely in breast cancer [39], [41].
Another relevant factor that inhibits E-cadherin expression is hypermethylation of
the promotor region of CDH1, which has been implicated at the same time with
the induction of migration in breast cancer cell lines [41]. Reduced expression of
E-cadherin was associated with tumor progression in several types of cancer,
including breast and stomach carcinomas [38], [39].
P-cadherin is a 118 kDa cadherin, that is expressed in ectodermal tissues,
namely in the basal layers of stratified epithelia (as skin, uterine cervix, prostate,
lung) and in myoepithelial cells of the breast [38], [40], [42]. This cadherin has
been associated in growth and differentiation processes, as those during
embryogenesis, for instance, and low levels of this protein were detected in
normal tissues [40]. Unlike CDH1, which is undoubtedly recognized as a tumor
suppressor gene, CDH3 role in cancer is contradictory and less characterized [40].
Distinct P-cadherin behavior was verified in different types of cancer, since it
appears to act as an invasion suppressor in colorectal cancer and melanoma
models whereas it resembles an oncogene in other models, as breast cancer, by
inducing tumor cell motility and invasiveness when overexpressed [43]. Increased
P-cadherin expression was also correlated with cell dedifferentiation and
increased cell proliferation [39]. The opposite effects of E- and P-cadherin in
breast cancer (as well as other models) are moreover unexpected since these two
molecules share more than 67% of homology [40].
Before acquiring an invading behavior, tumor cells often suffer a process
termed epithelial-to-mesenchymal transition, on which epithelial cadherins suffers
downregulation whereas mesenchymal cadherins are expressed de novo [44]. This
cadherin switch results in inhibition of cell-cell contacts and elicits active signals
which prompt cell migration and invasion. In breast cancer, many of the highly
aggressive tumors do not show, however, this cadherin switch. By contrast these
tumors show P-cadherin overexpression while maintaining the normal E-cadherin
expression [44]. P-cadherin is preferentially overexpressed in basal like
13
carcinomas and it was verified that 20% to 40% of invasive breast carcinomas, as
well as 25% of ductal carcinomas in situ showed overexpression of this cadherin
[42], [43]. Another relevant observation is that P-cadherin expression has been
reported as a marker of poor prognosis and reduced patient survival in high
histological grade tumors, with decreased cell polarity [42]. Expression of this
cadherin can also have an important role in the prognosis of invasive breast
carcinomas that maintains normal E-cadherin expression [39]. An in vitro study
supports these findings as it revealed that P-cadherin overexpression, in wild-type
E-cadherin breast cancer cell lines, as MCF-7/AZ, can induce increased cell
invasion, motility and migration [44]. Moreover it was shown that P-cadherin can
only induce invasion in breast cancer cell lines which already express and
endogenous cadherin like E-cadherin, by disrupting the interaction between E-
cadherin and two catenins (p120 and β-catenin) [40].
Additionally it was found that the presence of P-cadherin, in these E-cadherin
positive cancer cells, can provoke the secretion of pro-invasive factors, as matrix
metalloproteases (MMPs), MMP-1 and MMP-2, which in turn lead to P-cadherin
ectodomain cleavage [44]. The 80 kDa soluble P-cadherin fragment (sP-cad)
formed in that cleavage has pro-invasive activity, being responsible for in vitro
invasion of these cancer cells [42], [44]. This protein was found also in body
fluids, as milk from the lactating breast, although its biological role in that context
is still unknown [42]. Although the mechanism by which P-cadherin
overexpression induces the secretion of metalloproteases is unknown, it is clear
that these proteases are responsible for extracellular matrix degradation, for
instance, which increases cell invasion induction [44].
1.5 Azurin and P-cadherin interactions
Considering azurin’s broader anti-tumor properties and knowing that it exhibits
a high affinity level with P-cadherin, it was of interest to evaluate this protein’s
activity in an invasive breast cancer cell lines (MCF-7/AZ.Pcad) that express
endogenous levels of E-cadherin and higher levels of P-cadherin [45]. After
azurin administration with sub-lethal dosages (50-100 µM) to these cells, it was
verified, by western blotting, a specific decrease in P-cadherin total protein level
14
[45]. P-cadherin distribution across the plasma membrane was analyzed through
immunofluorescence, and it was shown that it suffered a decrease, as well, in cells
treated with azurin [45]. On the contrary, evidence showed that E-cadherin level
and its membrane distribution were not affected by azurin’s treatment.
Additionally, it was found that a reduction in P-cadherin’s level (caused by
azurin) was correlated with a less invasive behavior of breast cancer cells in a
Matrigel system, as well as with a lower activity of metalloproteases MMP-2 and
9 in cell conditioned medium. Another extreme relevant finding was that P-
cadherin soluble fragment (sP-cad) level, which has pro-invasive activity, was
found to be decreased when the conditioned medium, on which cells treated with
azurin where cultured, was analyzed. Complementary assays regarding CDH1 and
CDH3 genes showed that P-cadherin decreased levels were not a consequence of
changes in gene expression [45]. Therefore it was suggested that post-
transcriptional regulation processes, mediated by azurin, should be involved in P-
cadherin diminished levels. Microarray analysis on mRNA profiles of MCF-
7/AZ.Pcad cells treated with azurin showed that genes were associated with
membrane organization, vesicle-mediated and endosome transport, and lysosome
were up-regulated (Bernardes et al., 2013, submitted for publication).
This work intends to pursue those evidences and to demonstrate that azurin can
display an important role on mediating P-cadherin degradation through proteolytic
systems.
1.6 Inhibitors
In order to maintain regular homeostasis and meet their nutritional and
energetic demands, cells have devolved proteolytic systems which eliminate
instable and incorrect folded proteins. These systems are able to protect cells from
cytotoxic damage, caused by intracellular accumulation of damaged proteins or
organelles, and to recycle amino acids that result from protein degradation,
replenishing the intracellular reserve of these macromolecules, which are essential
in the absence of nutrients [46]. The two main cellular degradation systems in
eukaryotic cells are the autophagy-lysossome system and the ubiquitin-
15
proteasome system (UPS), although the relative contribution of each one of these
pathways may vary greatly between cell types [47], [48].
Lysosomes are single membrane vesicles that contain in their lumen a large
diversity of hydrolytic enzymes, such as proteases, lipases, glycosidases and
nucleotidases [46]. These enzymes reach their optimal enzymatic activity at the
acidic pH verified in the lysosomal lumen, which is maintained by an ATP-
dependent proton pump (V-ATPase) present at the lysosomal membrane [46],
[47]. The lysosomal degradation pathway is preferentially used in proteolysis of
membrane proteins, as receptors or channels, although it can act on cytoplasm
proteins as well [47]. In order to reach the lysosomes, tagged proteins must be
recognized and delivered to this organelle by three different ways:
macroautophagy, microautophagy and chaperone-mediated autophagy [48].
Macroautophagy involves a de novo formed double membrane vesicle or
autophagosome, which is responsible for protein sequestering, and that will fuse
with late endosomes or lysosomes [46]. In microautophagy, it occurs a lysosomal
membrane invagination, resulting in sequestration of regions of the cytosol
directly by the lysosomal membrane into its lumen. Finally, chaperone-mediated
autophagy is a process that requires the recognition of the target protein by a
cytosolic chaperone, and its binding to a lysosomal membrane receptor, which in
turn allows target protein translocation into lysosome’s lumen. There are several
chemical agents which act as lysosome inhibitors, such as ammonium chloride
and chloroquine, two weak bases that accumulate inside the lysosome and
dissipate its low acidic pH, by neutralizing H+
ions [49].
The UPS represents the major pathway accountable for the degradation of
proteins present in the cytosol, nucleus and endoplasmic reticulum [46]. This
proteolytic system is composed by ubiquitin, a small tagging protein that is
covalently linked to proteins, through a three step ATP-consuming reaction, that
involves E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme)
and E3 (ubiquitin-ligase enzyme), selecting the substrate protein for degradation
[48]. These three enzymes are responsible for selecting misfolded proteins to
degradation, as well as catalyzing the binding reaction between ubiquitin
molecules and target proteins [48]. After ubiquitination target proteins are
16
delivered to the proteasome, a multicatalytic enzyme complex composed by the
catalytic 20S core and the 19S regulator [47]. The 20S core complex its divided in
three catalytic subunits, with distinct activities as chymotrypsin-like, trypsin-like
and caspase-like activities [48]. Finally, polyubiquitinated proteins are degraded
to peptides by the proteasome and then free ubiquitin is recycled [48]. Currently
available proteasome inhibitors interfere with chymotryptic-like of the 20S core
complex such as MG132, a reversible but potent peptide aldehyde [48].
Lipid rafts are considered subdomains of cell membrane, and are involved in
processes like cell proliferation, differentiation and apoptosis, which often are
altered during tumor development [50]. These membrane regions are enriched in
cholesterol, or caveolins, for instance. Methyl-β-cyclodextrin (MβCD) is a cyclic
oligosaccharide that binds preferentially to cholesterol from the plasma
membrane, altering lipid composition and causing cholesterol depletion [51], [52].
By disrupting the assembly of lipid rafts and caveolae, this compound can
therefore inhibit caveolae-mediated endocytosis, since caveolin can’t form further
vesicle [53], [54].
Since azurin enter cells through caveolae-mediated endocytosis, we sought to
use MβCD to inhibit azurin entry into breast cancer cells, in order to evaluate its
impact on P-cadherin degradation level.
1.7 MPT 63
MPT 63 is a low molecular weight (16 kDa) protein that comprises 130 amino
acid residues preceded by a secretion signal peptide [55], [56]. Firstly described as
one of the three most abundant extracellular proteins secreted by Mycobacterium
tuberculosis, MPT 63 was later found to be specific of mycobacteria of the M.
tuberculosis complex, as M. africanum, M. bovis and attenuated M. bovis bacille
Calmette-Guérin (BCG) and it is absent in mycobacterial species that do not
belong to this complex, as M. avium [56], [57].
The X-ray crystal structure of MPT 63 (represented in Figure 5) was
determined by Goulding et al as a β-sandwich, consisting of two antiparallel β-
sheets, similar to an immunoglobulin-like fold, with and additional antiparallel β-
sheet [58]. Interestingly this protein exhibits some structural similarity to cell
17
surface-binding proteins such as Homo sapiens β-2 adaptin, bovine arrestin,
eukaryotic fibronectin-binding proteins, major histocompability domains or T-cell
receptors, for instance [58]. This similarity has suggested a possible role of MPT
63 in cell-host interactions to facilitate endocytosis as well as phagocytosis during
bacterial internalization [58]. In fact MPT 63, as well as other proteins secreted by
M. tuberculosis, is considered relevant to the survival of the bacterium within its
host, since it has been shown to be a cell envelope-associated protein that may act
as a virulence factor [58]. Moreover, secretion of proteins by intracellular
pathogens, as M. bacterium, has a central role in determining pathways of antigen
presentation and recognition by effector T-cells involved in protective immunity
[58]. Although it shares some structural similarities with proteins of other
organisms, this protein doesn’t exhibit a very strong homology with any of them,
which leaves its physiological role yet to be unveiled [55].
Figure 5– Structure of M. tuberculosis MPT 63 protein (adapted from Goulding et al, 2002) [58]
As mentioned earlier, MPT 63 is one of the three most abundant extracellular
proteins secreted by M. tuberculosis, and the other two most abundant proteins
were already described as antigens (antigens 85A and 85B), which can indicate
that this protein can also act as an antigen [56]. In fact so far several data have
18
been pointing in that direction, as a study that has shown that guinea pigs,
previously aerosol infected with virulent M. tuberculosis, were able to induce
humoral immune responses, leading to the production of high-level antibodies
against MPT 63 [55]. Moreover, guineas pigs injected with purified MPT 63
revealed cutaneous delayed-type hypersensitivity responses, which indicate that
this protein can induce immunogenic responses, through different pathways [55].
MPT 63 was also shown to be immunogenic in rabbits, since immunization with
the purified protein led to antibodies production against it [59]. Antibodies against
this protein were also identified in serum from tuberculosis patients by enzyme-
linked immunosorbent assay (ELISA) [60]. In a different study, a DNA vaccine
encoding both the MPT 63 and ESAT-6 antigens, has demonstrated to be capable
of inducing a robust protective response in mice, namely through generation of
gamma interferon (INF-γ)-secreting CD4+ T cells [61]. Since MPT 63 has shown,
at some level, structural similarities with major histocompability domains, as well
as T-cell receptors, it is important to mention a study where these two factors were
tested in humans. In this study it was shown that MPT 63 induced a moderate T
helper 1 (Th1) cell reactivity in peripheral blood mononuclear cells (PBMNCs),
obtained from M. bovis BCG vaccinated healthy subjects [62]. Th1 cells reactivity
were evaluated through proliferation and IFN-γ secretion screening tests.
Furthermore the presentation of MPT 63 to Th1 cells were also analyzed, as
PBMNCs from MPT 63-responding donors were typed for Human Leucocyte
Antigen (HLA-DR) molecules revealing heterogenecity between them [62] .
Therefore it seems that MPT 63 exhibits important requirements for a potential
use as a vaccine against tuberculosis, since it could be able to induce a positive
response throughout heterogeneous groups of donors.
Although little is known about the mechanisms behind this protein’s
immunogenic properties it was suggested that these properties could be explained,
at least in part, by the first 30 amino acid sequence in its N-terminal region, which
has shown a high density of T-cell epitopes recognized in immunized guinea pigs
[56].
As mentioned before MPT 63 is not present in mycobacteria which do not
belong to the Mycobacterium complex, and this specificity is also supported by
19
the fact that MPT 63 lacks epitopes that cross-react serologically with M. avium
antigens [55]. Taken altogether, this results regarding the immunogenic properties
of MPT 63, led several authors to propose this protein as a target for vaccine
design and diagnostic tools development in tuberculosis [57], [60].
MPT 63 and azurin share some important features, as they both are low
molecular weight proteins secreted by bacteria. More interestingly, however, is
the fact that both their structures are a β-sandwich, which demonstrates evident
structural similarities with immunoglobulin-like folds. Another interesting
observation is the promiscuity evidenced by these two proteins, which grant them
unique properties, allowing them t be capable of binding to different proteins.
Additionally to MPT 63’s ability to bind T-cell receptors, it also shares some
structural similarity with bovine arrestin, a protein that inhibits receptor activity,
by binding to the cytoplasmic surface, occluding the interaction with G-proteins
[58]. Taking account that azurin can also bind to surface cell receptors, as Eph
receptors in cancer cells, thereby interfering with cancer growth, it is intriguing to
verify if these proteins can provoke similar anti-tumor effects.
Recently a 30 amino acid peptide derived from MPT 63 and termed MB30, has
shown significant cytotoxic activity against several bladder (HTB-9, UM-UC-3),
colon (COLO 205, HCT 116), and cervical (SiHa, CaSki) cancer cell lines [63].
Moreover, MPT 63 and MB30 also showed cytotoxicity in brain (U87), liver
(HepG2) and breast (MDA-MB-231) cancer cell lines, although the MB30 peptide
has demonstrated an even higher cytotoxicity [63].
Since MPT 63, as well as its derived peptide MB30, has shown cytotoxic
effects against different cancer cell lines, our purpose in this work was to unveil a
possible role of MPT 63 as an anticancer agent. Therefore we sought to verify if
MPT 63 could cause possible cytotoxic effects on a breast (MCF-7/AZ.Mock) and
lung (A549) cancer cell lines.
1.8 Nucleoside diphosphate kinase
Nucleoside diphosphate kinase (Ndk) is an enzyme which catalyzes the
reversible transfer of the 5’-terminal phosphate from nucleoside triphosphates
(NTPs) to nucleoside diphosphates (NDPs), thus playing a key role as a
20
housekeeping enzyme, as it maintains the nucleotide pools for the synthesis of
nucleic acids [64]. Ndk is a small protein, with approximately 150 amino acids,
which form an homohexamer in eukaryotes, archae and gram-positive bacteria,
whereas in gram-negative bacteria it forms homotetramers [64], [65]. So far it has
been described in humans and others eukaryotes as Droshophila melanogaster, as
well as in several bacteria such as P. aeruginosa, Escherichia coli, Myxococcus
xanthus, M. tuberculosis or Vibrio cholerae for instance [66], [67]. Interestingly
there were reported two forms of this protein in P. aeruginosa: a 16 kDa
cytoplasmic form (during the early stages of cellular growth), and a 12 kDa
membrane-associated form (at the onset of the stationary phase) which is
originated from a cleavage in the 16 kDa form, by a periplasmic protease termed
elastase [64], [68]. Although the importance of having two forms of this protein is
not clear, it is known that the membrane-associated form, constitutes a complex
with pyruvate kinase, which predominantly synthesizes GTP [69]. In mammalian
cells Ndk can be localized in the cytosol, as membrane-associated or as an
ectoenzyme in the cell surface exposed to the outside medium [70].
Despite the fact that Ndk exhibits a different structure in humans and some
prokaryotes, it was revealed that Ndk form P. aeruginosa shows 40-45% identity
with eukaryotic Ndks, as well as 50-60% identity with other bacterial Ndks,
evidencing that it is a highly conserved enzyme among different species [66] .
Extracellular secretion of Ndk was reported in M. bovis BCG, M. tuberculosis,
P. aeruginosa, Trichenella spiralis and V. cholerae [67]. The physiological
relevance for the secretory nature of Ndk in these bacteria is not clear, although it
has been mentioned to be an important enzyme in host-pathogen interactions [67].
This suggestion is supported from the fact that Ndk from M. tuberculosis was
reported to interfere and block phagosome maturation in murine macrophages
[71]. Moreover, Ndks secreted by M. tuberculosis and V. cholerae were shown to
display cytotoxic effects on mouse macrophage cell lines, which can indicate that
secretable Ndk plays a major role in the modulation of virulence in these
pathogenic species [67], [72].
Additionally to its kinase function, Ndk has been associated in further
biological functions in eukaryotic cells, namely in humans, where it plays a role in
21
normal embryonic development, cell differentiation, tumor progression and cell
migration [73], [74]. In humans, 10 genes have been already identified as part of
the nm23 (non metastatic) gene family, which represents the human Ndk gene
family [75]. Nm23-H1 was the first metastatic gene in humans to be identified in
1988 by Steeg et al. , since the accumulation of correspondent transcripts were
shown higher in tumor cells of low metastatic potential in murine melanoma cell
lines of varying metastatic potentials [76]. The two more expressed nm23 genes in
humans are nm23-H1 and nm23-H2, localized in chromosome 17q21, and encode
the Ndk-A and Ndk-B, respectively [10], [75]. The three-dimensional structure of
human Ndk-A subunit is shown in Figure 6.
Although it is known mainly by its role in tumor progression, Ndk-B has been
also described as a transcription factor for the c-Myc oncogene, and it was
proposed to act as a repair protein in humans, on the basis of sequence homology
with certain glycosylases [73].
Since Steeg et al. initial observations it has been verified, by several authors, a
correlation between reduced Ndk-A expression and high tumor metastatic
potential in different carcinomas: liver, melanoma, colon, breast, ovarian, gastric
and hepatocellular [75]. Transfection experiments with nm23-H1 and nm23-H2
support these findings, since the overexpression of these genes in breast cancer
MDA cell lines resulted in decreased metastatic potential of these cells [77]. The
overexpression of this protein in tumor cells was found to reduce tumor cell
motility and invasion, promote cellular differentiation and inhibits anchorage-
independent growth, as well as adhesion to fibronectin, laminin and vascular
endothelial cells [10]. However, although Ndk-A has shown anti-metastatic
effects on the majority of carcinomas, there are some exceptions. It was reported
that high levels of this protein were detected in aggressive carcinomas derived
from thyroid, pancreatic, squamous cell lung carcinomas, neuroblastoma and
acute myelogenous leukemia [75]. Moreover high tissue levels of Ndk-A were
found in patients with breast carcinoma [78].
22
Figure 6 – Three-dimensional structures of human Ndk-A. (A) Diagram of Ndk-A monomer. (B)
Diagram of the homo-hexamer (Han et al, 2010) [79].
The mechanisms by which human Ndk achieves anti-metastatic effects in
several tumor cell lines is unknown, and contradictory effects of this protein in
other carcinomas constitutes a challenge that requires further investigation on this
subject.
Mutations in nm23 genes are rare in cancer, therefore it has been proposed that
this genes can become deregulated through expression changes at the protein
level, instead through mutation [78].
Recently in vitro studies revealed that extracellular Ndk-B was secreted by
breast cancer, colon and pancreas cell lines, while Ndk-A was secreted by human
leukemia cell lines into the extracellular environment [78]. The same phenomenon
was verified in MDA-MB-435 and MDA-MB-231 metastatic human breast
carcinoma cell lines, which secreted Ndk into their surroundings, while the non-
metastatic breast cancer cell line MCF-12 didn’t exhibit the same effect [78].
Extracellular Ndk-A was also found in blood and its level was shown to be
correlated with poor prognosis in acute myelogenous leukemia, malignant
lymphoma and neuroblastoma patients [74].
Ndk, as well as azurin and MPT 63, are low molecular weight bacterial
proteins, and secreted by some bacteria. Recent evidence has attributed an anti-
metastatic role for human Ndk in several tumor cell lines, which presents this
protein with an interesting property to be explored in cancer research. Contrasting
23
with the human Ndk, bacterial Ndk effects on tumor cells weren’t studied until
now, as far as we know. Therefore, and since bacterial and human Ndk share great
homology we intended to evaluate possible anti-tumor and anti-metastatic effects
caused by a bacterial Ndk in a human breast cancer cell line (MCF-7/AZ.Mock)
and a human lung carcinoma cell line (A549), using a normal lung cell line as
control (16HBE14o-). Moreover it was of interest to verify if a bacterial Ndk
(from P.aeruginosa) could cause cytotoxic effects, as demonstrated in murine
macrophage cell lines by other bacterial Ndks, and if those effects could be higher
in tumor cell lines, as shown by azurin.
25
2. OBJECTIVES
The present work intends to help clarify how does azurin causes a decrease in
P-cadherin level on a breast cancer cell line, with a normal E-cadherin expression
but a high P-cadherin expression level (MCF-7/AZ.Pcad cells). Taking into
account previous experiments, we seek to unveil a strongly suggested link
between azurin and P-cadherin decrease through proteolytic pathways. Our
approach resides in blocking the proteasome and the lysosome systems, using two
inhibitors, MG-132 and ammonium chloride respectively, and analyze by western
blot how that affects P-cadherin level when cells are previously treated with
azurin. Additionally, we want to verify what effect can MβCD originate on E- and
P-cadherin levels, by inhibiting azurin internalization on these cells.
The second main objective in this work was to use two different bacterial
proteins, termed MPT 63 (from Mycobacterium tuberculosis) and Nucleoside
diphosphate kinase (from Pseudomonas aeruginosa), in order to describe and
elucidate their possible anti-tumor and cytotoxic effects on cancer cell lines from
breast and lung carcinomas (MCF-7/AZ.Mock and A549, respectively). Azurin
(from Pseudomonas aeruginosa) was used as positive control in order to compare
their relative effects, since its cytotoxicity toward tumor cells was already
described. We also used a normal cell line, 16HBE14o-, to evaluate if Ndk’s
cytotoxicity is selective towards only tumor cells. Cell viability assay was
performed using the 3 different cell lines and the three bacterial proteins at 5
different concentrations (0, 10, 25, 50 and 100 µM). Also different exposure time
(48, 72 h) and number of protein doses (1 to 3) applied were tested to see how
these parameters affect cell viability.
Additionally, since a human Ndk, termed Nm23-H1, displays anti-metastatic
behavior, we intended to test if Ndk also possesses anti-metastatic potential
against a highly invasive tumor cell line, A549, using a matrigel invasion system.
27
3. MATERIALS AND METHODS
3.1 Bacterial proteins superexpression
3.1.1 Bacterial strains and plasmids
The azurin-encoding gene azu, derived from P. aeruginosa PAO 1 strain, was
amplified and cloned into pWH844 plamid. The same pWH844 plasmid was also
used for cloning the mpt63 and ndk genes, derived from M. bacterium and P.
aeruginosa, respectively. Azurin, MPT 63 and Ndk were expressed in competent
Escherichia coli SURE (Stop Unwanted Rearrangement Events) strain containing
the pWH844 plasmid. E. coli SURE is a protease-deficient strain, which was
chosen to improve the yield of expressed proteins. Each one of the genes that
encode these proteins is regulated by an IPTG (Isopropyl-β-D-
Thiogalactopyranoside) inducible lac promoter. Upstream region of these genes
also contain a nucleotide sequence encoding a six histidine tag, thus allowing
further protein purification through a nickel affinity column.
3.1.2 Inoculum
E. coli SURE cells were pre-inoculated in Luria Broth (LB) medium [10 g/L of
tryptone; 10 g/L of sodium chloride (NaCl); 5 g/L of yeast extract] with ampicillin
(150 µg/mL) and maintained at 37º C, with continuous shaking at 250 rpm
(revolution per minute) overnight. On the next day Super Broth medium (32 g/L
of tryptone; 20 g/L of yeast extract; 5 g/L of NaCl) was inoculated with a volume
of the pre-inoculum equivalent to an OD640 nm (optical density) of 0.1 and
incubated at 37º C with continuous shaking. The antibiotic added to the culture
medium was ampicillin with the same concentration used for the pre-inoculum
(150 µg/mL). When the inoculum OD640 nm value was between 0.6 and 0.8, IPTG
(0.2 mM) was added to the medium in order to induce gene superexpression.
Induced inoculums were incubated for 4-5 hours at 37º C.
After the incubation period the inoculums were centrifuged at 8000 rpm for 10
minutes (Beckman J2-MC centrifuge) with a constant temperature of 4º C. The
retrieved pellet was ressuspended in 15 mL of START buffer (10 mM of
28
imidazole, 0.2 mM of sodium phosphate, 0.5 M of NaCl, pH 7.4) for each 350 mL
of the initial culture medium and stored at -80ºC until its use.
3.1.3 Cell sonication
The cells were sonicated on ice with a Branson 250 sonifier, applying 9 cycles
of 15 pulses each at 50% duty cycle and output control 10. Between each cycle,
cells were left to rest during 5 minutes. The sonicated cells were centrifuged at
17600 g (B. Braun Sigma-Aldrich 2K15) and 4º C, for 5 minutes, after which time
the resulting supernatant was collected and centrifuged for 1 hour at the same
conditions.
3.1.4 Azurin purification
Azurin purification was accomplished using a HisTrapTM
FF 5 mL purification
column (GE Healthcare Life Sciences), previously equilibrated with 25 mL of
START buffer. Cell lysates were then loaded into the column and washed with 25
mL of START buffer before sequential elution with 25 mL of elution buffers. The
elution buffers contain a constant phosphate buffer and an increasing imidazole
concentration (20, 40, 60, 100, 200, 300 and 500 mM) (Table 1, Table 2). The
purified protein was obtained by collecting the 100 and 200 mM imidazole
fractions.
In order to remove excess salt and imidazole from our protein sample, the
collected fractions were desalted through size-exclusion chromatography using
the ÄKTA Prime liquid chromatography system (Amersham Biosciences).
Samples were injected through a HiPrepTM
26/10 Desalting column (GE
Healthcare Life Sciences) with Sephadex G-25, according to the elution program
shown in Table 3. Phosphate buffered saline (PBS) was used as the elution buffer
(Table 4). The eluted fractions were collected according to the chromatogram
generated by PrimeView software, which measures the desalted solution OD280 nm,
thus allowing protein detection.
29
Table 1 - Phosphate buffer 8x composition
Phosphate buffer 8x
Na2HPO4.2H2O (M) 0.08
NaH2PO4.H2O (M) 0.08
NaCl (M) 4
pH 7.4
Table 2 - START and elution buffer compositions
START buffer Elution buffers
Phosphate buffer 8x (mL) 12 3
Imidazole (mM) 10 20; 40; 60; 100; 200; 300; 400; 500
Water until final volume of (mL) 100 24
pH 7.4 7.4
Table 3 - ÄKTA elution program for protein sample desalting
Step Volume (mL) Flow rate (mL/min) Inject valve
Tube wash 50 40 waste
Column wash 5 5 load
Protein injection 15 5 inject
Protein elution 100 5 load
Column wash 30 5 load
After the desalting step the collected fractions were concentrated in a 3 kDa
cut-off column with a cellulose membrane (Ultracel-3K Amicon Ultra centrifugal
filter from Millipore) through successive centrifuging at 5000 rpm and 4º C
(Eppendorf Centrifuge 5804R) until concentrate the sample to an approximated
volume of 2 mL.
The concentrated sample was then applied onto a Detoxi-GelTM
Endotoxin
Removing column (Thermo Scientific) with a gel matrix composed by crosslinked
6% beaded agarose, that has polymixin B immobilized onto it. This antibiotic can
bind bacterial lipopolysaccharide (LPS) therefore assuring that our protein sample
was free of endotoxin contamination. Before applying the sample, the detoxi-gel
column was previously equilibrated with 5 mL of each of these reagents:
30
distillated water, 1% sodium deoxycholate, distillated water and PBS. Protein
sample was then applied and left to rest at 4º C, for 1 hour, after which time it was
collected by gravity flow by eluting it with PBS until reaching a volume of 30
mL. After each detoxing step the columns were regenerated with 5 mL of destilled
water, 1% sodium deoxycholate, distilled water and 20% ethanol. The collected
sample was concentrated until reaching a volume of 2 mL, following the same
specifications as the first concentration step. This final volume obtained was
centrifuged in a 100 kDa cut-off column (Ultracel-100K Amicon Ultra centrifugal
filter from Millipore) in order to guaranty that no contamination with bacterial
cells took place.
Azurin final concentration was calculated after OD280nm reading and using the
Lambert-Beer equation, where azurin’s extinction coefficient ε (280 nm) value
applied was 9.1 x 103
M-1
.cm-1
[80]. Spot test was performed in LB plates at 37º
C overnight to exclude any possible contamination. Azurin was kept at 4º C until
further use.
Table 4 - PBS composition
PBS Concentration (mM)
NaCl 137
KCl 2.7
Na2HPO4.2H2O 4.3
KH2PO4 1.47
3.1.5 MPT 63 and Ndk purification
MPT 63 purification was performed following exactly the same procedures as
in azurin purification, with the exception that the MPT 63 sample was collected at
60 and 100 mM imidazole fractions.
On its turn Ndk samples were collected at 200 and 300 mM imidazole
fractions. Additionally, after being collected from the detoxing column, Ndk
sample was readily passed through a 0.2 µm PuradiscTM
filter (Whatman), which
removes any bacterial cells present from our sample. After this, protein was
concentrated with the 3 kDa cut-off column but did not pass through the 100 kDa
cut-off column.
31
The quantification of MPT 63 and Ndk was done according to the Bradford
method using a bovine serum albumin (BSA) standard solution kit (Thermo
Scientific). Ten microliters of BSA standard solutions and 10 µl of protein sample
diluted in PBS (1:10, 1:50 and 1:100) were loaded, in duplicated, into a 96-well
microplate and 200 µl of diluted Bio-Rad Protein assay dye concentrate (1:5) were
added to each well. The microplate was incubated in the dark, at room
temperature for 15 minutes, and OD was measured at 595 nm using
SPECTROstar Nano (BMG LABTECH) microplate reader. PBS was used as
blank. To convert the obtained mass concentration in molar concentration it were
considered MPT 63 and Ndk molecular weights: 16 kDa and 15 kDa respectively.
3.2 Cell culture and human cell lines
In this study, two human breast cancer cell lines were used: MCF-7/AZ.Mock
and MCF-7/AZ.Pcad [(transduced with empty vector and CHD3/P-cadherin
cDNA, respectively, and kindly provided by Doctor Joana Paredes (IPATIMUP)].
It were also used a human lung cancer cell line A549 and a human bronchial
epithelial cell line 16HBE14o- [kindly provided by Doctor Dieter Gruenert
(University of California, San Francisco, USA].
MCF-7/Az.Mock and MCF-7/Az.Pcad cell lines were routinely maintained in
Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) and A549 cells were
maintained in Ham’s F-12 medium (Gibco). Both cell media were supplemented
with 10% of heat-inactivated fetal bovine serum (FBS, Lonza), 100 IU/ml of
penicillin (Invitrogen) and 100 mg/ml of streptomycin (Invitrogen). In each
passage, culture medium was firstly removed and cells were washed twice with
PBS and trypsinized before being resuspended in fresh medium and transferred to
a new flask with a 1:3 dilution (1 mL of cell suspension to 2 mL of fresh
medium). Cell scrapers were used to remove A549 cells from the bottom of the
flask, instead of using trypsine.
Human bronchial cell line 16HBE14o- was routinely maintained in
fibronectin/vitrogen coated flasks in Minimum Essential Medium with Earle’s salt
(MEM, Gibco) supplemented with 10% FBS, 0.292 g/L of L-Glutamine (Sigma-
Aldrich) and 100 U/mL of Penicillin/Streptomycin (Gibco). On each passage cells
32
were washed with Hepes buffered saline (HBS, Sigma-Aldrich) and detached
using PET solution (Sigma-Aldrich), which contained HBS, 10%
polyvinylpyrrolidone solution (PVP), 0.2% EGTA in HBS and 0.25% trypsin in
0.02% EDTA.
Both cell lines here mentioned were maintained in a humidified incubator at
37ºC, with a 5% carbon dioxide atmosphere (Binder CO2 incubator C150).
3.3 Inhibitors treatment
3.3.1 Lysosome and proteasome inhibitors
In order to test each inhibitor, all cells were previously treated with azurin at
different cell concentrations (0, 50 and 100 µM).
Breast cancer cells were seeded in a 6-well plate with 5x105 cells (MFC-
7/AZ.Pcad) for each well. The following day, all cells were treated with azurin or
PBS (control) and fresh medium was added. After 32 hours cell were treated with
20 µl of the lysosome inhibitor, ammonium chloride (NH4Cl 0.75 M), or 20 µl of
distilled water as control. In the case of the proteasome inhibitor MG-132 (Sigma-
Aldrich), 2.5 µl of this compound or 2.5 µl of ethanol absolute (control) were
added. Both inhibitors were applied during 16 hours, after which time the cells
were lysed.
3.3.2 Azurin internalization inhibitor
In order to test the effects of MβCD on azurin internalization, breast cancer
cells were seeded with the same number of cells used for the experiments with
lysosome and proteasome inhibitors. The following day fresh medium was added
and cells were treated with PBS (control) or azurin (50 and 100 µM) and
simultaneously with 20 µl of MβCD (Sigma-Aldrich) or 20 µl of Dimethyl
sulfoxide (DMSO), used as control. After 8 hours culture medium was exchanged
and, at the end of 24 hours, protein lysates were prepared.
33
3.4 Protein lysates
Culture media were firstly removed from the 6-well plates and washed twice
with 3 mL of PBS. Cell lysis was accomplished by adding 100 µl of catenin lysis
buffer (1% Triton X-100, 1% Nonidet P-40 in deionized PBS) and a 1:7 dilution
of proteases inhibitor cocktail (Roche Diagnostics) to each well. Plates were kept
at 4º C for 10 minutes, after which time cells were scratched from the wells,
ressuspended and transferred to microtubes. These tubes were vortexed 3 times,
for 10 seconds, and centrifuged at 14000 rpm with a constant temperature of 4º C
for 10 minutes (B.Braun Sigma-Aldrich 2K15). If the cell lysates weren’t used
immediately they were stored at -80ºC.
Protein quantification was done according to the Bradford method as explained
earlier for MPT 63 and Ndk quantification. The only difference was that protein
samples were diluted in PBS with a 1:4 dilution for quantification. Protein
samples were prepared to have 20 µg of total protein dissolved in sample buffer
(Laemmli with 5% (v/v) 2-beta-mercaptoethanol and 5% (v/v) bromophenol blue)
and were stored at -20º C if not used immediately.
3.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE)
Polyacrylamide gels were assembled with a gel caster (Amersham Biosciences)
and polymerized between a glass and a porcelain plate separated by two spacers.
Approximately 3 mL of resolving gel were casted and simultaneously overlaid
with isopropanol to create a smooth surface above the gel. After gel
polymerization, the isopropanol was removed and the stacking gel was casted to
the top of the plates, with the comb (10 wells) already inserted. The resolving gel
and stacking gels were left to polymerize for approximately 20 minutes.
In order to separate azurin, MPT 63 and Ndk it was used a resolving gel with a
15% acrylamide concentration, whereas to separate E- and P- cadherins it was
used a resolving gel with a lower acrylamide concentration (8%). Resolving and
stacking gel composition is shown in Table 5.
34
Table 5 - Resolving Gel 8% and 15% composition for one gel
Resolving Gel 8% Resolving Gel 15% Stacking Gel 5%
Distilled water 2.3 mL 850 µl 1.35 mL
Acrylamide-bisacrylamide 30% 1.35 mL 1.9 mL 335 µl
Tris Base 1.25 mL (1.5 M) 950 µl (1.5 M) 250 µl (1M)
10% SDS 50 µl 34 µl 20 µl
APS 50 µl 34 µl 20 µl
TEMED 3 µl 1.5 µl 2 µl
Before electrophoresis, the comb was removed from the top of the gel and the
gel cassette was placed in the electrophoresis apparatus (Amersham Biosciences)
and electrophoresis buffer was added. Protein samples were boiled in dry bath at
100º C, for 5 minutes, and 20 µl of each sample were loaded to the gel wells, as
well as 3 µl of PageRuler Prestained Protein Ladder (Fermentas).
The electrophoresis voltage was set to 150 V (azurin, MPT 63 and Ndk), and
120 V (E- and P-cadherin).
After electrophoresis the gels containing purified MPT 63 and Ndk proteins
were stained with Coomassie Brilliant Blue staining solution for 2 hours and
destained for half an hour with acetic acid destaining solution until the protein
bands were clearly visible against a translucid background. The destaining
solution was removed and the gel was washed with water and finally dried.
3.6 Western blotting
For western blotting purposes protein samples were separated by SDS-PAGE
following the protocol described earlier. After electrophoresis proteins were
transferred into nitrocellulose membranes using Trans-Blot Turbo RTA Transfer
Kit (BioRad). Protein transference was accomplished by using Trans-Blot Turbo
Transfer System (BioRad) at 2.5 V for 7 minutes. Membranes were blocked with
5% (w/v) non-fat dry milk in PBS-Tween-20 (0.5% v/v) for 1 hour and incubated
with primary antibodies overnight at 4° C. On the next day membranes were
washed three times with PBS-Tween 20 for 5 minutes and incubated for 1 hour, at
room temperature, with secondary antibodies conjugated with horseradish
peroxidase enzyme. After washing the membranes for 5 minutes with PBS-Tween
35
20, proteins were detected by adding ECL reagent (Pierce) as substrate and
chemiluminescence was capture by Fusion Solo system (Vilber Fourmat). The
band intensity obtained was measured with ImageJ software, and the results
represent the ration between the signal intensities in azurin treated and non-treated
samples. Moreover, cadherin levels were normalized by the respective actin level
in each sample.
Anti-E-cadherin (HECD 1, Sigma-Aldrich), anti-azurin (AB0048-200 SicGen)
and anti-actin (sc-1616, Santa Cruz Biotechnology) primary antibodies were
diluted 1:1000 in 5% non-fat milk, whereas anti-P-cadherin (clone 56, BD
Transduction Laboratories) antibody was diluted 1:500.
Anti-mouse (sc-2005, Santa Cruz Biotechnology) secondary antibody was used
against cadherins, whereas anti-goat (sc-2354, Santa Cruz Biotechnology)
secondary antibody was used for actin and azurin.
3.7 MTT cell viability assay
MTT [3-(4,5 dimethylthiazol-2-yl-2,5 tetrazolium bromide) ; Sigma-Aldrich]
assay was used to determine cell viability in breast (MCF-7/AZ.Mock) and lung
(A549) cancer cells, as well as a normal lung cell line (16HBE14o-) after
exposing them to azurin, MPT 63 and Ndk. This assay is based on the reduction
of MTT, by the mitochondrial dehydrogenase of intact cells, to a purple formazan
product which can be quantified. Breast cancer cells (MCF-7/AZ.Mock) were
suspended in a 96-well plate at a density of 1 x 104 cells per well, whereas lung
cancer cells (A549) were seeded at a density of 5 x 103 cells per well. A normal
lung cell line, 16HBE14o-, was used as control and seeded at a density of 1 x 104
cells per well. After 24 hours, medium was exchanged and azurin, MPT 63 and
Ndk were administrated. Both proteins were administrated at 10, 25, 50 and 100
uM, and PBS was used as control. After another 24 hours medium was removed
and a new protein dosage was applied. Forty eight hours after the first protein
administration, 20 µl of MTT solution (5 mg/ml, Sigma-Aldrich) were added to
each well and incubated for 4 hours. The supernatant was carefully removed and
150 ul of MTT solvent (3.4 µl of HCl, 10 µl of Nonidet-P40, 10 ml of
isopropanol) was added to each well. The 96-well plate was shacked in an orbital
36
shaker for 15 minutes at 300 rpm, and SPECTROStar Nano microplate reader was
used to measure the absorbance at 590 nm (with a 620 nm reference filter). Each
condition tested was performed in triplicated.
3.8 Matrigel invasion assay
Matrigel invasion assay was performed using BD Biocoat MatrigelTM
Invasion Chambers with an 8 µm pore size PET membrane with a thin
MatrigelTM layer (BD Biosciences), following manufacturer’s instructions.
Chambers were pre-incubated with serum-free medium during 2 hours at 37ºC. In
the chamber’s upper compartment 2.5 x 104 A549 cells were added with complete
medium and Ndk (at 0, 50 and 100 µM), whereas in the lower compartment only
complete medium was added. After 24 hours at 37ºC, non-invasive cells were
cleared and chambers were washed four times with PBS. Cells were fixed in cold
methanol during 10 minutes at 4ºC. Invasive cells attached to the lower surface
were stained with DAPI and counted under the microscope (Zeiss).
3.9 Bioinformatics
3.9.1 Sequence analysis and secondary structures
Primary protein sequences from bacterial Ndks and all human Nm23 family
members were retrieved from the National Center for Biotechnology Information
(NCBI; http://www.ncbi.nlm.nih.gov/pubmed/). Multiple sequence alignment
between human and bacterial Ndks was performed by ClustalW algorithm default
settings (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and edited on Bioedit
software (http://www.mbio.ncsu.edu/). Secondary structures of E. coli Ndk and
human Nm23-H1 were represented according to structural information provided
by their solved structures on Protein Data Bank (PDB, http://www.rcsb.org/).
Swiss-Model default settings were used (http://swissmodel.expasy.org/) in order
to predict P. aeruginosa Ndk secondary structure and a three-dimensional model,
based on the closest template found in the database.
37
3.9.2 Phylogenetic and structural alignment analysis
Multiple sequence alignment, generated by ClustalW algoritm, was used to
construct the phylogenetic tree representation using Splitstree software
(http://www.splitstree.org/) on default settings. All currently protein three-
dimensional structures available on PDB database were retrieved and added to the
phylogenetic tree, as well as the Ndk P. aeruginosa model built. Structural
alignment was performed between Nm23-H1 and Ndk P. aeruginosa model using
PyMOL software (http://www.pymol.org/) default settings.
39
4. RESULTS
Recently, several experiments revealed that azurin can cause a decrease in P-
cadherin levels in MCF-7/AZ.Pcad breast cancer cells, which display invasive
behavior and express high levels of this protein [45]. Interestingly, azurin did not
exhibit a similar behavior on E-cadherin levels, since they remained unchanged.
These results were also corroborated with immunofluorescence analysis regarding
the membrane localization of both cadherins. Since it was found that this decrease
in protein level was not a consequence of changes in gene expression, it was
suggested that post-transcriptional processes should be involved in this process
[45]. Further observations, regarding microarray analysis on mRNA profiles of
MCF-7/AZ.Pcad cells treated with azurin, showed that several genes were up-
regulated in comparison with untreated cells (Bernardes et al., 2013, submitted for
publication). Interestingly, some of these genes were associated with membrane
organization, vesicle-mediated and endosome transport, or even with lysosome.
This work intends to pursue those evidences and to demonstrate that azurin can
display an important role on mediating P-cadherin degradation through proteolytic
systems.
Tacking account that azurin can enter cells preferentially through lipid rafts
and since tumor cells exhibit a higher number of lipid rafts than normal cells, it
has been suggested that P-cadherin can be mainly localized on these regions.
Azurin internalization on tumor cells through lipid rafts involves the formation of
endocytic vesicles, which thereby make us believe that P-cadherin can also suffer
endocytosis along with this internalization process. These vesicles that sequester
P-cadherin can cause its removal from the cell membrane and further degradation
through proteolytic systems, as mentioned before.
In order to support this theory we sought to use MβCD, which has been known
to remove cholesterol from lipid rafts, thereby inhibiting azurin internalization. By
using this inhibitor, we intended to verify what impact occurs on P-cadherin level
in MCF-7/AZ.Pcad cells treated with azurin.
40
4.1 Proteolytic pathways inhibitors effect on P-cadherin level
The mechanism by which azurin cause a decrease on P-cadherin level in breast
cancer cells is not fully understood, although it was excluded an effect on gene
expression. Post-transcriptional processes should probably be relevant in this case
and, in fact, a microarray analysis on MCF-7/AZ.Pcad cells previously treated
with azurin revealed that several genes up-regulated involved in vesicle-mediated,
endosome transport and lysosome. One possible mechanism by which azurin
mediates P-cadherin decrease could then be related to its degradation by
proteolytic systems. We intended to support this theory with our results.
Therefore we selected two major proteolytic systems present in eukaryotic cells
and tested a lysosome inhibitor (ammonium chloride) and a proteasome inhibitor
(MG-132) to inhibit them. We tested the effect of both inhibitors on an invasive
breast cancer cell line, MCF-7/AZ.Pcad. We performed a 48 hours assay, were
azurin was in contact with tumor cells during the first 32 hours, after which time
medium was removed and each inhibitor was added in the last 16 hours. Cell
samples were analyzed by western blot and protein bands were quantified by
ImageJ software. Cadherin levels were normalized by their respective actin levels
and the average results are shown in Figure 7 and Figure 8.
Observing the results obtained from western blot we can see that MCF-
7/AZ.Pcad cells, on which the two inhibitors were not administrated, showed a
decrease tendency in P-cadherin level when azurin’s concentration increases. On
the other hand, in the presence of the inhibitors, P-cadherin level increases at the
same time as azurin concentration administrated increases.
Regarding to E-cadherin level, it suffered a slight increase when cells where
treated with each inhibitor. In samples untreated with MG-132, on the other hand,
we see a decrease in E-cadherin level when azurin concentration increases. In
samples untreated with ammonium chloride we observed that E-cadherin level
remained constant. Although actin level between samples were not constant, the
charts presented in Figure 7 and Figure 8 include average cadherin level
normalized with their respective actin level, and exhibit the same tendency as the
one shown by western blot results.
41
The results obtained clearly show that both the lysossome and the proteosome
are involved in P-cadherin degradation, since when they are inhibited, P-cadherin
does not continue to decrease as azurin concentration increases, but rather the
oposite, it starts to accumulates inside the cells. Thereby it seems that both these
proteolytic systems have a synergetic action on P-cadherin degradation, when
tumor cells are treated with azurin.
Figure 7 – (A): Western Blot analysis of the effect of lysossome inhibitor, ammonium chloride
(NH4Cl), on E and P-cadherins of MCF-7/AZ.Pcad cells, previously treated with azurin at 0, 50
and 100 μM. Azurin and the inhibitor were administrated at 0 and 32 hours, respectively, in a 48
hours assay. (B): Charts illustrated represent average percentage values of protein level for E and
P-cadherins which signal was normalized with actin levels.
42
Figure 8 – (A): Western Blot analysis of the effect of proteasome inhibitor, MG-132, on E and P-
cadherins of MCF-7/AZ.Pcad cells, previously treated with azurin at 0, 50 and 100 μM. Azurin
and the inhibitor were administrated at 0 and 32 hours, respectively, in a 48 hours assay. (B):
Charts illustrated represent average percentage values of protein level for E and P-cadherins which
signal was normalized with actin levels.
4.2 Azurin internalization
Azurin’s ability for entering cancer cell lines, such as those from breast cancer,
has been known for some years now. Previously, it had been demonstrated, by
confocal imaging, that an Alexa-labeled p28-azurin peptide was less efficient
entering MCF-7 cells, upon MβCD treatment [81]. This compound causes
cholesterol depletion, thereby disrupting structures as lipid rafts which are rich in
cholesterol [52]. In this work we tested the effect of MβCD on azurin’s
43
internalization in the human breast cancer cell line MCF-7/AZ.Pcad. Cells were
incubated with azurin and MβCD for 8 hours, and left another 24 hours with fresh
medium. Western blot was performed in order to evaluate the effects of this
inhibitor and two different azurin concentrations were tested (50 and 100 μM).
Cells treated with DMSO were used as negative control since MβCD was
dissolved in this reagent. PBS was used as double negative control.
The results of this experiment are shown in Figure 9, where it can be seen that
anti-azurin antibody was only detected in samples which were previously treated
with azurin at 50 and 100 μM. Anti-azurin antibody signal was stronger in
samples treated with azurin at 100 μM than the ones treated with a 50 μM
concentration. This observation was verifiable in both cells treated with DMSO
and MβCD.
Analyzing azurin corresponding bands it is possible to identify a decrease in
their intensity when comparing samples treated with DMSO and MβCD. This
decrease was verified in both two azurin concentration used, although it was
clearer between DMSO and MβCD samples treated with azurin at 50 μM. Actin
level was constant between all samples thus showing that azurin and MβCD
treatments did not affect this protein.
β-actin
Azurin
Figure 9 - Effects of MβCD on azurin’s internalization on MCF-7/AZ.Pcad cells after 8 hours of
exposure to this inhibitor and 24 hours with fresh medium (visualized by western blot). PBS was
used as double negative control. DMSO was used as negative control. Azurin was tested at two
different concentrations (50 and 100 μM).
44
4.3 Inhibition of azurin’s effect on E and P-cadherins level using MβCD
Recently it has been described that azurin can cause P-cadherin levels to
decrease in MCF-7/AZ-Pcad cell line, without affecting its E-cadherin levels [82].
Additionally, as we mentioned earlier, microarray analysis on the same cell line
revealed that cells treated with azurin show several up-regulated genes involved
with membrane organization and vesicular transport (Bernardes et al., 2013,
submitted for publication). Thereby, it was of interest within this work to evaluate
the effect of MβCD on E- and P-cadherin levels through a possible decrease in
azurin’s internalization on MCF-7/AZ.Pcad cells.
In Figure 10 we can observe a decrease in P-cadherin level in samples treated
simultaneously with MβCD and azurin at 50 and 100 μM, comparing to control. It
seems that even though MβCD causes a decrease in azurin internalization (as
Figure 9 shows), a decrease in P-cadherin level still continues to occur, which can
mean that MβCD contributes to this effect. Regarding to the samples treated with
PBS and DMSO there were no significant differences on P-cadherin level, which
indicates that DMSO does not affect P-cadherin level. E-cadherin level seems to
remain constant in all samples, apparently not being affected with azurin and
MβCD treatments.
These observations represent an important breakthrough in studying the
mechanisms behind azurin and P-cadherin interactions. It appears that, when
MβCD is administrated alone there are no visible effects on P-cadherin level.
However, when azurin is added simultaneously with this inhibitor, it occurs a
decrease in P-cadherin level when both two different protein concentrations are
tested. From previous studies it was known that azurin does not affect E-cadherin
level, which was also seen here when MβCD was tested. Additionally,
microarrays analysis mentioned earlier, showed that MCF-7/AZ.Pcad cells
previously treated with azurin, display several up-regulated genes, including some
involving membrane organization or vesicle-mediated transport. Considering
these evidence altogether we believe that azurin and MβCD have a synergetic
effect on P-cadherin degradation. This inhibitor helps to deplete cholesterol from
lipid rafts at the cell membrane, where azurin enters preferentially, which could
mean that diminishing azurin internalization could cause a smaller decrease on P-
45
cadherin level. However we saw the opposite, which can probably mean that P-
cadherin is highly present in lipid rafts regions. Thereby a depletion of these sites
by MβCD and the formation of transporting vesicles in the cell membrane, caused
by azurin as microarray analysis suggests, can mean that P-cadherin may suffer
endocytosis and vesicles containing this protein should be conducted to
proteolytic systems, as the lysosome. Since E-cadherin was not affect by azurin or
MβCD, we suggest that this protein may be highly expressed in other sites on the
cell membrane but poorly expressed in lipid rafts.
β-actin
β-actin
E-cadherin
P-cadherin
Figure 10 – Western blot showing the effect of azurin’s internalization inhibitor MβCD on E- and
P-cadherin levels in MCF-7/AZ.Pcad cells after 8 hours of exposure and 24 hours with fresh
medium. PBS was used as double negative control. DMSO was used as negative control. A
decrease in P-cadherin level, between control samples and samples treated with MβCD and azurin,
is shown in the figure.
4.4 Bacterial proteins to treat cancer
4.4.1 Purified proteins
Before applying MPT 63 and Ndk proteins in MTT (cell viability) assays it
was mandatory to establish if these proteins were successfully purified using the
purification methods already described. As we can see in Figure 11, an SDS-
PAGE was performed in order to confirm this, and the single bands visualized on
46
the gel clearly show that both proteins were purified from E. coli SURE with
success.
~15 kDa
MPT63 Ndk
Figure 11 - 15% SDS-PAGE of MPT 63 and Ndk purified from E. coli SURE strain. Proteins
appear as a single band at approximately 15 kDa, according to their expected molecular weight.
4.5 MTT cell viability assays
Azurin has already been described as a protein capable of producing effects on
several tumor cell lines, including some derived from humans, namely through
p53 stabilization and apoptosis induction. In this work we tested azurin, as well as
MPT 63 and Ndk, other two bacterial proteins with different origins and
properties.
Since MPT 63 shares some structural similarities with azurin and some
experiments supported anti-tumor activity of this protein, we sought to verify the
same properties [63]. Human Ndk-A isoform (or Nm23-H1) has been described as
an anti-metastatic protein, and it was of interest to verify if bacterial Ndk from P.
aeruginosa could exhibit similar properties, as well as cytotoxic activity, since
human and bacterial Ndks share a high homology level, as we will show after
bioinformatic analysis (section 4.7).
Both cytotoxic effects of these proteins were tested by MTT cell viability
assays, that were performed in two tumor cell lines from breast (MCF-
7/AZ.Mock) and lung (A549) and one normal lung epithelial cell line
(16HBE14o-).
47
4.5.1 Azurin
In order to verify if azurin display its cytotoxic properties against tumor cell
lines in a dose dependent-manner, MTT assay was performed using increasing
number of doses and exposure time. Azurin was administrated on MCF-
7/AZ.Mock for 48 (one dose) and 72 hours (three doses). A549 tumor cells were
incubated during 48 hours, where it was tested one and two protein doses. Five
different azurin concentrations (0, 10, 25, 50 and 100 µM) were tested and
cytotoxicity percentages are presented for each condition tested.
Regarding MCF-7/AZ.Mock cells our results shows that azurin caused a
general increase in cell cytotoxicity across increasing concentrations of this
protein, in both 48 and 72 hours assay, as it can be seen in
Figure 12. Moreover, we see that this increase in cytotoxicity occurs not only as
azurin concentration increases, but also when we increase the exposure time and
number of doses applied. About 26% of cell population was killed using three
doses of this protein during 72 hours, in MCF-7/AZ.Mock cells.
A549 cells exhibited a similar cytotoxic response across the different azurin
concentrations tested, as the one observed in MCF-7/AZ.Mock cell line, when
azurin was used. Contrarily to what we have seen on MCF-7/AZ.Mock cells,
adding a second dose of azurin to A549 cells does not appear to cause a
significant increase in cytotoxicity, which was only verified when the highest
protein concentration was used.
Overall we can verify that azurin caused a slightly higher cytotoxicity on MCF-
7/AZ.Mock than on A549 tumor cell line. Statistical analysis using T-student test
on these results revealed a significant value when A549 cells were treated with
azurin at the highest concentration, as shown in
Figure 12. Considering a 0.05 significance level tested, we may assume that
such decrease in cell viability was provoked by azurin administration and its
cytotoxic activity.
48
Figure 12 – Cytotoxicity (%) caused by azurin on MCF-7/AZ.Mock during 48 h (one dose) and 72
h (three doses), and A549 48 h (one and two doses). Both cell lines were tested with 5 different
azurin concentrations (0, 10, 25, 50, and 100 μM). Significant values, p-value<0.05 with the
Student t-test, are shown as asterisks (*).
4.5.2 MPT 63
In the same way as azurin, MPT 63 was tested in two different tumor models.
This protein was administrated in MCF-7/AZ.Mock cells during 48 (one dose)
and 72 hours (three doses), and in A549 for 48 hours (two doses). When it was
applied on MCF-7/AZ.Mock breast cancer cells, MPT 63 caused an increase in
cell citotoxicity across all different concentrations tested, as it can be seen in
Figure 13. It can also be observable that MPT 63 cytotoxicity increases, in this
cell line, when a higher number of protein doses and exposure time are applied.
Regarding A549 cells, MPT 63 caused only a maximal 10% decrease on cell
viability, which is about half of the cytotoxic level seen for azurin, within the
same conditions tested.
Tacking an overview of these results we observed that, once again, A549 cell
line appears to be not as sensitive, as MCF-7/AZ.Mock cells, to cytotoxic effects
caused by MPT 63. It also seems that this protein does not exhibit a cytotoxic
activity as higher as azurin demonstrates.
*
49
Figure 13 – Cytotoxicity (%) caused by MPT 63 on MCF-7/AZ.Mock during 48 h (one dose) and
72 h (three doses), and A549 during 48 h (two doses). Both cell lines were tested with 5 different
MPT 63 concentrations (0, 10, 25, 50, and 100 μM).
4.5.3 Ndk
Ndk was also administrated as double dose, for 48 hours, against breast and
lung cancer cell lines (MCF-7/AZ.Mock and A549), in order to verify if it could
require more doses to provoke cytotoxic effects in those cells. Additionally, a lung
epithelial cell line (16HBE14o-) was used to analyze if Ndk could exhibit
selective cytotoxic effects against cancer cells or if it was effective against normal
cell lines as well. A 72 hours assay (three doses) was also performed with MCF-
7/AZ.Mock tumor cell line.
Analyzing the results showed on Figure 14 we can see that, when two doses of
Ndk are administrated for 48 or 72 hours, with two or three doses respectively,
cell cytotoxicity increases in MCF-7/AZ.Mock and A549 cells, across each
increasing protein concentrations tested. The higher cytotoxicity values were
found when the 100 µM concentration was used. In this case, A549 cells suffered
a 27% decrease in cell survival, which was the same value verified in MCF-
7/AZ.Mock cells after three doses were applied during 72 hours. Therefore, it
seems that also Ndk cytotoxic effect on both tumor cell lines follows a dose and
time dependent tendency. More interestingly, Ndk appears to be slightly more
50
effective on A549 lung cancer cell line, which was not verified for azurin or MPT
63.
Another interesting fact retrieved from Figure 14 is that Ndk has shown a
similar cytotoxic effect as potent as azurin, in MCF-7/AZ.Mock cells. A similar
observation was made in A549 tumor cell line as well, although for a two dose
treatment only, as already described.
Concerning to 16HBE14o- cells, it can be seen, in Figure 14, that Ndk
produces cytotoxic effect on this normal cell line, although not as extended as in
the breast and lung cancer cell lines tested. Moreover, in all Ndk concentrations
tested, the lower cytotoxicity values are seen in 16HBE14o- cell line. This can
possibly indicate that Ndk displays selective cytotoxicity towards tumor cell lines,
in the same way as azurin does.
Statistical analysis using T-student test revealed significant values, with a
significance level inferior to 0.05, in A549 cells treated with Ndk. These results
strongly support the fact that Ndk’s cytotoxic effect is responsible for a decrease
in cell viability in this tumor cell line. When we used the same T-student function
on data retrieved from MTT assay using 16HBE14o- cells, we also found that
samples treated with Ndk showed significant values, thereby there is strong
evidence that Ndk clearly has cytotoxic effect on this epithelial lung cell line.
Figure 14 - Cytotoxicity (%) caused by Ndk on MCF-7/AZ.Mock during 48 h (two doses) and 72
h (three doses), A549 48 h (two doses) and 16HBE14o- during 48 h (two doses). Both cell lines
were tested with 5 different Ndk concentrations (0, 10, 25, 50, and 100 μM). Significant values, p-
value<0.05 with the Student t-test, are shown as asterisks (*).
* *
*
* *
51
4.6 Matrigel invasion assay
Since Ndk exhibited significant cytotoxicity towards tumor cell lines, we
intended to test if it could also display anti-metastatic activity, like Nm23-H1,
against A549 which is a highly invasive tumor cell line. Ndk was administrated,
as single dose, to A549 cells in a matrigel invasion system, during 24 hours.
However, only a 6-8% decrease in cell invasion was observed in Figure 15, which
is not significant. Moreover, no differences were seen between the two different
protein concentrations tested.
Figure 15 – Cell invasion (%) in a Matrigel invasion assay performed on A549 highly invasive
tumor cells, which were treated with Ndk at 0, 50 and 100 μM, during 24 hours.
4.7 Bioinformatic analysis on human Nm23 and bacterial Ndks
As mentioned earlier, a human Ndk (Nm23-H1) has been described as an
anti-metastatic protein is several human tumor models, even though exhibiting
opposite results in a minority of cases. In this work we intended to use a bacterial
Ndk, from P. aeruginosa, in order to evaluate if other Ndks manage to inflict the
same cytotoxic and/or anti-metastatic effects on human cancer cells. Since our
MTT cell viability assays also revealed promising results about the cytotoxicity of
this protein, we thought to explore how similar human Nm23 proteins and
bacterial Ndks can be, especially Nm23-H1 (which has anti-metastatic properties)
and P. aeruginosa Ndk, on which we hope to have a similar effect. With that
52
thought in mind we used several bio-informatic tools, thereby taking a different,
but quite relevant approach on this subject.
A multiple sequence alignment was performed using ClustalW algorithm to
align primary protein sequences from all human Nm23 family members, and some
bacterial Ndks, namely from P. aeruginosa. This alignment is shown in Figure 16,
and as it can be seen, there is a clear similarity between the sizes of each bacterial
Ndk sequence. On the other hand, regarding human Nm23 sequences we face a
great disparity, since some of them (as Nm23-H1) have approximately the same
size of bacterial Ndks but others (like Nm23-H8) are larger. The most significant
and interesting fact that is raised from the multiple alignment is that between all
different Ndks it appears to be several blocks composed by segments of
approximately 40/50 amino acid residues exhibiting a high level of similarity
between the sequences. Since these conserved regions within the protein
sequences, could be attributable to important protein secondary structures, the
secondary structure of E. coli Ndk is represented in Figure 16. If we look at this
structure it is observable that typical secondary elements, as β-sheets or α-helices
for instance, clearly overlap with the blocks of amino acids aligned. This
observation could possibly demonstrate the importance of these conserved
structures, throughout evolution, for the activity of this protein. Moreover, this
may help to explain the fact that P. aeruginosa Ndk seems to exhibit similar
cytotoxic effects on tumor cells, as human Nm23-H1.
Analyzing the percent identity matrix, in Table 6, generated by the multiple
alignment results, we can observe that all bacterial Ndks sequences share higher
identity values (44-65%) between themselves. Relatively to the identity shared
between bacterial Ndks and human Ndks, the highest value was reached with M.
tuberculosis and M. bovis sequences against Nm23-H3 protein (51%), although
not far from Nm23-H1 (45%). Regarding exclusively to Nm23-H1, and the P.
aeruginosa Ndk used in our MTT assays, there is an approximately 43% identity
between these two protein primary sequences. In the case of this specie, the
highest identity value was also found against Nm23-H3 (45%).
Through the evaluation of the results in Table 6, it is possible to verify that
Nm23-H10 is by far the Ndk that reveals the lowest identity level across all Ndks
53
analyzed, including between the other members of human the Nm23 protein
family. Regarding human Nm23 proteins, the ones that could be more related to
each other are Nm23-H1, -H2, -H3 and -H4, since they share between 53-88%
identity level, where the highest value is seen between Nm23-H1 and Nm23-H2.
Taking into account the results obtained with multiple sequence alignment, the
E. coli Ndk secondary structure revealed and the overlapping between secondary
elements and conserved amino acid sequences, we sought to compare Nm23-H1
and P. aeruginosa Ndk regarding their secondary structure. In Figure 17 it can be
seen the result of the alignment between these two proteins as well as a
representation of their respective secondary structures. Both primary sequences
are extremely similar in size. The secondary structure of Nm23-H1 was
represented according to a solved three-dimensional structure presented in PDB
database, whereas P. aeruginosa Ndk represented was based on a protein template
(79% identity) and modeling software (Swiss-model). Through comparison
between the two secondary structures we can see that Nm23-H1 structure exhibits
more detailed information on different elements present in that protein, such as the
localization of turns and bends. However the main observation was that we found
an almost perfect overlapping between the location of β-sheets and α-helices on
both proteins. More importantly, it is evident the higher number of identical
amino acid residues between the two sequences, especially within β-sheets and α-
helices. The same observation is verified for conserved and high conserved
residues, which can indicate the importance of those amino acid residues for
maintaining the correct structure and function of these proteins throughout the
different species.
The multiple sequences involving several Ndks were also used to build a
phylogenetic tree using Splistree software. The resultant representation is
exhibited in Figure 18, and additionally to the phylogenetic tree, all the three-
dimensional structures of Nm23 and bacterial Ndks are displayed. As it can be
visualized in this figure, the closest related human Nm23 seems to be Nm23-H1, -
H2, -H3 and -H4. Nm23-H1 and Nm23-H2 are the two closest members of the
Nm23 protein family. Nm23-H4, a mitochondrial Ndk, shows to have the closest
phylogenetic relationship between a human Nm23 protein and a bacterial Ndk (M.
54
tuberculosis and M. bovis). They both diverge from an initial common branch,
which is very close to the one respective to M. tuberculosis and M. bovis Ndks.
These findings were also verified when analyzing the percent identity matrix.
These Ndks seem to be originated from a different branch than the one from all
the other bacterial Ndks analyzed.
A second higher Nm23 branch diverges in a second group of Nm23 proteins,
where Nm23-H8 and Nm23-H9 are closer to each other, as well as Nm23-H7 and
Nm23-H5 between themselves. As mentioned earlier, Nm23-H10 is by far the
most divergent Nm23 protein relatively to the other members of this human Ndk
protein family.
The protein three-dimensional structures represented on Figure 18 indicate all
solved structures available regarding the proteins presented in the phylogenetic
tree. Human Nm23 (-H1, -H2,-H3 and -H4) quaternary structures are defined as
homohexamers, clearly contrasting with the majority of bacterial Ndks present,
which are known to form homotetramers. Nm23-H1 and Nm23-H2 quaternary
structures also reveal to be extremely resemblant. Interestingly, not only M.
tuberculosis Ndk branch is closest to human Nm23 proteins, but it also shares the
same quaternary structure as homohexamers, contrarily to all the others bacterial
Ndks. Concerning P. aeruginosa Ndk, since no solved structure is yet available, a
model was built using Swiss-model software. Surprisingly, the model predicted as
well as the template used (Halomonas sp. 593 Ndk) are displayed as homodimers.
Nm23-H10 protein is represented as a monomer, thereby it only reveals a tertiary
structure, contrarily to both other human and bacterial Ndks and supports the idea
that this protein is evolutionarily distant from these proteins.
55
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
10 20 30 40 50 60 70 80 90
Nm23-H8 MASKKREVQL QTVINNQSLW DEMLQNKGLT VIDVYQAWCG PCRAMQPLFR KLKNELNEDE ILHFAVAEAD NIVTLQPFRD KCEPVFLFSV
Nm23-H9 ---------- ---------- --MLSSKGLT VVDVYQGWCG PCKPVVSLFQ KMRIEVGLD- LLHFALAEAD RLDVLEKYRG KCEPTFLFYA
Nm23-H1 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H2 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H3 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H4 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(M.tuberculosis) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(M.bovis) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(E.coli)* ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(V.cholerae) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(P.aeruginosa) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(M.xanthus) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H7 ---------- ---------- ---------- ---------- ---------- ---------- --MNHSERFV FIAEWYDPNA SLLRRYELLF
Nm23-H7B ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H5 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H6 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H10 ---------- ---------- ---MGCFFSK RRKADKESRP ENEEERPKQY SWDQREKVDP KDYMFSGLKD ETVGRLPGTV AGQQFLIQDC
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
100 110 120 130 140 150 160 170 180
Nm23-H8 NGKIIEKIQG ANAPLVNKKV INLIDEERKI AAGEMARPQY PEIPLVDSDS EVSEESPCES VQELYSIAII KPDAVISK-- ---KVLEIKR
Nm23-H9 ---------- -------IKD EALSDEDECV SHGKNNG--- ------EDED MVSSERTCT- ------LAII KPDAVAHG-- ---KTDEIIM
Nm23-H1 ---------- ---------- ---------- ---------- ---------- MAN------- --CERTFIAI KPDGVQRG-- ---LVGEIIK
Nm23-H2 ---------- ---------- ---------- ---------- ---------- MAN------- --LERTFIAI KPDGVQRG-- ---LVGEIIK
Nm23-H3 ---------- ---------- ---------- ---------- --MICLVLTI FANLFPAACT GAHERTFLAV KPDGVQRR-- ---LVGEIVR
Nm23-H4 ---------- ---------- ------MGGL FWRSALRGLR CGPRAPGPSL LVRHGSGGPS WTRERTLVAV KPDGVQRR-- ---LVGDVIQ
Ndk(M.tuberculosis) ---------- ---------- ---------- ---------- ---------M LGT------- -VTERTLVLI KPDGIERQ-- ---LIGEIIS
Ndk(M.bovis) ---------- ---------- ---------- ---------- ---------M LGT------- -VTERTLVLI KPDGIERQ-- ---LIGEIIS
Ndk(E.coli)* ---------- ---------- ---------- ---------- ---------- MAI------- ---ERTFSII KPNAVAKN-- ---VIGNIFA
Ndk(V.cholerae) ---------- ---------- ---------- ---------- ---------- MAL------- ---ERTFSII KPDAVKRN-- ---LIGEIYH
Ndk(P.aeruginosa) ---------- ---------- ---------- ---------- ---------- MAL------- ---QRTLSII KPDAVSKN-- ---VIGEILT
Ndk(M.xanthus) ---------- ---------- ---------- ---------- ---------- MAI------- ---ERTLSII KPDGLEKG-- ---VIGKIIS
Nm23-H7 YPGDGSVEMH DVKNHRTFLK RTKYDNLHLE DLFIGNKVNV FSRQLVLIDY GDQYTARQLG SRKEKTLALI KPDAISK--- ----AGEIIE
Nm23-H7B --------MH DVKNHRTFLK RTKYDNLHLE DLFIGNKVNV FSRQLVLIDY GDQYTARQLG SRKEKTLALI KPDAISK--- ----AGEIIE
Nm23-H5 ---------- ---------- ---------- ---------- ---------- -MEISMPPPQ IYVEKTLAII KPDIVDK--- ----EEEIQD
Nm23-H6 ---------- ---------- ---------- ---------- ----MTQNLG SEMASILRSP QALQLTLALI KPDAVAHPL- ---ILEAVHQ
Nm23-H10 ENCNIYIFDH SATVTIDDCT NCIIFLGPVK GSVFFRNCRD CKCTLACQQF RVRDCRKLEV FLCCATQPII ESSSNIKFGC FQWYYPELAF
* Ndk (E.coli)
Secondary structure
56
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
190 200 210 220 230 240 250 260 270
Nm23-H8 KITKAGFIIE A--------- ------EHKT VLTEEQVVNF YSRIADQCDF EEFVSFMTSG LSYILVVSQG SKHNPPSEET EPQTDTEPNE
Nm23-H9 KIQEAGFEIL T--------- ------NEER TMTEAEVRLF YQHKAGEEAF EKLVHHMCSG PSHLLILT-- ---------- ----------
Nm23-H1 RFEQKGFRLV G--------- ------LKFM QASEDLLKEH YVDLKDRPFF AGLVKYMHSG PVVAMVWEG- ---------- ----------
Nm23-H2 RFEQKGFRLV A--------- ------MKFL RASEEHLKQH YIDLKDRPFF PGLVKYMNSG PVVAMVWEG- ---------- ----------
Nm23-H3 RFERKGFKLV A--------- ------LKLV QASEELLREH YAELRERPFY GRLVKYMASG PVVAMVWQG- ---------- ----------
Nm23-H4 RFERRGFTLV G--------- ------MKML QAPESVLAEH YQDLRRKPFY PALIRYMSSG PVVAMVWEG- ---------- ----------
Ndk(M.tuberculosis) RIERKGLTIA A--------- ------LQLR TVSAELASQH YAEHEGKPFF GSLLEFITSG PVVAAIVEG- ---------- ----------
Ndk(M.bovis) RIERKGLTIA A--------- ------LQLR TVSAELASQH YAEHEGKPFF GSLLEFITSG PVVAAIVEG- ---------- ----------
Ndk(E.coli)* RFEAAGFKIV G--------- ------TKML HLTVEQARGF YAEHDGKPFF DGLVEFMTSG PIVVSVLEG- ---------- ----------
Ndk(V.cholerae) RIEKAGLQII A--------- ------AKMV RLSEEQASGF YAEHEGKPFF EPLKEFMTSG PIMVQVLEG- ---------- ----------
Ndk(P.aeruginosa) RFEKAGLRVV A--------- ------AKMV QLSEREAGGF YAEHKERPFF KDLVSFMTSG PVVVQVLEG- ---------- ----------
Ndk(M.xanthus) RFEEKGLKPV A--------- ------IRLQ HLSQAQAEGF YAVHKARPFF KDLVQFMISG PVVLMVLEG- ---------- ----------
Nm23-H7 IINKAGFTIT K--------- ------LKMM MLSRKEALDF HVDHQSRPFF NELIQFITTG PIIAMEILR- ---------- ----------
Nm23-H7B IINKAGFTIT K--------- ------LKMM MLSRKEALDF HVDHQSRPFF NELIQFITTG PIIAMEILR- ---------- ----------
Nm23-H5 IILRSGFTIV Q--------- ------RRKL RLSPEQCSNF YVEKYGKMFF PNLTAYMSSG PLVAMILAR- ---------- ----------
Nm23-H6 QILSNKFLIV R--------- ------MREL LWRKEDCQRF YREHEGRFFY QRLVEFMASG PIRAYILAH- ---------- ----------
Nm23-H10 QFKDAGLSIF NNTWSNIHDF TPVSGELNWS LLPEDAVVQD YVPIPTTEEL KAVRVSTEAN RSIVPISRG- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
280 290 300 310 320 330 340 350 360
Nm23-H8 RSEDQPE--- -VEAQVTPGM MKNKQDSLQE YLERQHLAQL CDIEEDAANV AKFMDAFFPD FKKMKSMKLE KTLALLRPNL FHERKDDVLR
Nm23-H9 RTEG------ -FEDVVTT-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H1 ---------- --LNVVKT-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H2 ---------- --LNVVKT-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H3 ---------- --LDVVRT-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H4 ---------- --YNVVRA-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(M.tuberculosis) ---------- --TRAIAA-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(M.bovis) ---------- --TRAIAA-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(E.coli) ---------- --ENAVQR-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(V.cholerae) ---------- --ENAIAR-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(P.aeruginosa) ---------- --EDAIAK-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Ndk(M.xanthus) ---------- --ENAVLA-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H7 ---------- --DDAICE-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H7B ---------- --DDAICE-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H5 ---------- --HKAISY-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H6 ---------- --KDAIQL-- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Nm23-H10 --QRQKSSDE S--------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
* Ndk (E.coli)
Secondary structure
* Ndk (E.coli)
Secondary structure
57
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
370 380 390 400 410 420 430 440 450
Nm23-H8 IIKDEDFKIL EQRQVVLSEK EAQALCKEYE NEDYFNKLIE NMTSGPSLAL VLLRDNGLQY WKQLLGPRTV EEAIEYFPES LCAQFAMDSL
Nm23-H9 ---------- ---------- ---------- ---------- ---------- ---------- WRTVMGPRDP NVARREQPES LRAQYGTE-M
Nm23-H1 ---------- ---------- ---------- ---------- ---------- ---------- GRVMLGETN- -PADSK-PGT IRGDFCIQ-V
Nm23-H2 ---------- ---------- ---------- ---------- ---------- ---------- GRVMLGETN- -PADSK-PGT IRGDFCIQ-V
Nm23-H3 ---------- ---------- ---------- ---------- ---------- ---------- SRALIGATN- -PADAP-PGT IRGDFCIE-V
Nm23-H4 ---------- ---------- ---------- ---------- ---------- ---------- SRAMIGHTD- -SAEAA-PGT IRGDFSVH-I
Ndk(M.tuberculosis) ---------- ---------- ---------- ---------- ---------- ---------- VRQLAGGTD- -PVQAAAPGT IRGDFALE-T
Ndk(M.bovis) ---------- ---------- ---------- ---------- ---------- ---------- VRQLAGGTD- -PVQAAAPGT IRGDFALE-T
Ndk(E.coli)* ---------- ---------- ---------- ---------- ---------- ---------- HRDLLGATN- -PANAL-AGT LRADYADS-L
Ndk(V.cholerae) ---------- ---------- ---------- ---------- ---------- ---------- YRELMGKTN- -PEEAA-CGT LRADYALS-M
Ndk(P.aeruginosa) ---------- ---------- ---------- ---------- ---------- ---------- NRELMGATD- -PKKAD-AGT IRADFAVS-I
Ndk(M.xanthus) ---------- ---------- ---------- ---------- ---------- ---------- NRDIMGATN- -PAQAA-EGT IRKDFATS-I
Nm23-H7 ---------- ---------- ---------- ---------- ---------- ---------- WKRLLGPANS GVARTDASES IRALFGTD-G
Nm23-H7B ---------- ---------- ---------- ---------- ---------- ---------- WKRLLGPANS GVARTDASES IRALFGTD-G
Nm23-H5 ---------- ---------- ---------- ---------- ---------- ---------- WLELLGPNNS LVAKETHPDS LRAIYGTD-D
Nm23-H6 ---------- ---------- ---------- ---------- ---------- ---------- WRTLMGPTRV FRARHVAPDS IRGSFGLT-D
Nm23-H10 ---------- ---------- ---------- ---------- ---------- --------CL VVLFAGDYTI ANARKLIDEM VGKGFFLVQT
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
460 470 480 490 500 510 520 530 540
Nm23-H8 PVNQLYGSDS LETAEREIQH FFPLQ----- ---------- STLGLIKPHA TSEQREQILK IVKEAGFDLT QVKKMFLTPE QIEKIYPKVT
Nm23-H9 PFNAVHGSRD REDADRELAL LFP------- ---------- -SLKFSDKDT EAPQGE---- ---------- ---------- ----------
Nm23-H1 GRNIIHGSDS VESAEKEIGL WFH------- ---------- --PEELVDYT SCAQNWIYE- ---------- ---------- ----------
Nm23-H2 GRNIIHGSDS VKSAEKEISL WFK------- ---------- --PEELVDYK SCAHDWVYE- ---------- ---------- ----------
Nm23-H3 GKNLIHGSDS VESARREIAL WFR------- ---------- --ADELLCWE DSAGHWLYE- ---------- ---------- ----------
Nm23-H4 SRNVIHASDS VEGAQREIQL WFQ------- ---------- --SSELVSWA DGGQHSSIHP A--------- ---------- ----------
Ndk(M.tuberculosis) QFNLVHGSDS AESAQREIAL WFP------- ---------- --GA------ ---------- ---------- ---------- ----------
Ndk(M.bovis) QFNLVHGSDS AESAQREIAL WFP------- ---------- --GA------ ---------- ---------- ---------- ----------
Ndk(E.coli)* TENGTHGSDS VESAAREIAY FFG------- ---------- --EGEVCPRT R--------- ---------- ---------- ----------
Ndk(V.cholerae) RYNSVHGSDS PASAAREIEF FFP------- ---------- --ESEICPRP ---------- ---------- ---------- ----------
Ndk(P.aeruginosa) DENAVHGSDS EASAAREIAY FFA------- ---------- --ATEVCERI R--------- ---------- ---------- ----------
Ndk(M.xanthus) DKNTVHGSDS LENAKIEIAY FFR------- ---------- --ETEIHSYP YQK------- ---------- ---------- ----------
Nm23-H7 IRNAAHGPDS FASAAREMEL FFPSSGGCGP ANTAKFTNCT CCIVKPHAVS EGLLGKILMA IRDAGFEISA MQMFNMDRVN VEEFYEVYKG
Nm23-H7B IRNAAHGPDS FASAAREMEL FFPSSGGCGP ANTAKFTNCT CCIVKPHAVS EGLLGKILMA IRDAGFEISA MQMFNMDRVN VEEFYEVYKG
Nm23-H5 LRNALHGSND FAAAEREIRF MFP------- ---------- EVIVEPIPIG QAAKDYLNLH IMPTLLEG-- ---------- ----------
Nm23-H6 TRNTTHGSDS VVSASREIAA FFP------- ---------- -DFSEQRWYE EEEP------ ---------- ---------- ----------
Nm23-H10 KEVSMKAEDA QRVFREKAPD FLPLLN---- ---------- --KGPVIALE FNGDGAVEVC QLIVN----- ---------- ----------
* Ndk (E.coli)
Secondary structure
* Ndk (E.coli)
Secondary structure
58
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|..
550 560 570 580 590 600 610 620
Nm23-H8 GKDFYKDLLE MLSVGPSMVM ILTKWNAVAE WRRLMGPTDP EEAKLLSPDS IRAQFGISKL KNIVHGASNA YEAKEVVNRL FEDPEEN
Nm23-H9 ---------- ---------- ---------- ---------- --------SS TQPRLKITDL D--------- ---------- -------
Nm23-H1 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Nm23-H2 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Nm23-H3 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Nm23-H4 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Ndk(M.tuberculosis) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Ndk(M.bovis) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Ndk(E.coli) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Ndk(V.cholerae) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Ndk(P.aeruginosa) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Ndk(M.xanthus) ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- -------
Nm23-H7 VVTEYHDMVT EMYSGPCVAM EIQQNNATKT FREFCGPADP EIARHLRPGT LRAIFGKTKI QNAVHCTDLP EDGLLEVQYF FKILDN-
Nm23-H7B VVTEYHDMVT EMYSGPCVAM EIQQNNATKT FREFCGPADP EIARHLRPGT LRAIFGKTKI QNAVHCTDLP EDGLLEVQYF FKILDN-
Nm23-H5 ---------- ---------- ---------- LTELCK---- -----QKPAD PLIWLADWLL KNNPNKPKLC HHPIVEEPY- -------
Nm23-H6 ---------- ---------- ---------- ---------- -----QLRCG PVCYSPEGGV HYVAGTGGLG PA-------- -------
Nm23-H10 ---------- ---------- ---------- ---------- -----EIFNG TKMFVSESKE TASGDVDSFY NFADIQMGI- -------
Figure 16 – ClustalW multiple alignment of human and bacterial Ndks. The numbers above the sequences indicate the position of amino acid residues. Secondary
structure of E. coli Ndk is represented according to PDB code 2HUR. Secondary elements are indicate as: single purple curves (turns), yellow arrows (β-sheets), red
lines (bends), blue curved lines (alpha helices), orange curved lines (3/10-helices), and black lines (no secondary structure assigned). NCBI accession number for each
protein is given below. Human (Homo sapiens) Nm23 proteins: Nm23-H1 (CAG46912), Nm23-H2 (NP_001018147), Nm23-H3 (EAW85629), Nm23-H4
(NP_005000), Nm23-H5 (NP_003542), Nm23-H6 (NP_005784), Nm23-H7 (Q9Y5B8), Nm23-H7B (NP_932076), Nm23-H8 (AAF20909), Nm23-H9 (NP_835231),
Nm23-H10 (NP_008846). Bacterial Ndks: P. aeruginosa (EPR01938), M. tuberculosis (EQM19968), M. bovis (AGE68451), E. coli (ERF95659), M. xanthus
(P15266), V. cholerae (WP_001162853).
59
Table 6 – Percent identity matrix of human Nm23 proteins and different bacterial Ndks.
Nm23-H8 Nm23-H9 Nm23-H7 Nm23-H7B Nm23-H5 Nm23-H6 Nm23-H1 Nm23-H2 Nm23-H3
Nm23-H8 100.00 41.83 21.05 23.08 26.42 20.73 24.34 24.34 21.89
Nm23-H9 41.83 100.00 22.58 25.93 32.32 28.99 27.78 25.69 24.68
Nm23-H7 21.05 22.58 100.00 100.00 33.49 27.23 30.67 28.00 23.95
Nm23-H7B 23.08 25.93 100.00 100.00 33.49 27.23 30.67 28.00 23.95
Nm23-H5 26.42 32.32 33.49 33.49 100.00 33.15 29.53 32.21 27.85
Nm23-H6 20.73 28.99 27.23 27.23 33.15 100.00 28.57 31.29 30.86
Nm23-H1 24.34 27.78 30.67 30.67 29.53 28.57 100.00 88.16 67.11
Nm23-H2 24.34 25.69 28.00 28.00 32.21 31.29 88.16 100.00 65.13
Nm23-H3 21.89 24.68 23.95 23.95 27.85 30.86 67.11 65.13 100.00
Nm23-H4 19.25 21.21 20.54 20.54 26.25 27.16 55.26 53.95 52.66
Ndk
M.tuberculosis 25.71 30.60 37.68 37.68 34.56 36.43 45.26 45.26 51.08
Ndk
M.bovis 25.71 30.60 37.68 37.68 34.56 36.43 45.26 45.26 51.08
Ndk
E.coli 32.87 32.85 34.04 34.04 35.71 39.57 42.66 42.66 44.76
Ndk
V.cholerae 33.10 32.85 37.86 37.86 38.85 36.69 39.44 41.55 41.55
Ndk
P.aeruginosa 31.47 32.12 40.43 40.43 32.86 35.97 42.66 41.96 45.45
Ndk
M.xanthus 26.90 30.66 30.77 30.77 30.28 32.37 45.14 45.83 46.53
Nm23-H10 11.59 8.95 9.19 10.12 7.96 9.33 10.53 9.21 8.28
60
Table 6 – Percent identity matrix of human Nm23 proteins and different bacterial Ndks. (continued)
Nm23-H4 Ndk
M.tuberculosis
Ndk
M.bovis
Ndk
E.coli
Ndk
V.cholerae
Ndk
P.aeruginosa
Ndk
M.xanthus Nm23-H10
Nm23-H8 19.25 25.71 25.71 32.87 33.10 31.47 26.90 11.59
Nm23-H9 21.21 30.60 30.60 32.85 32.85 32.12 30.66 8.95
Nm23-H7 20.54 37.68 37.68 34.04 37.86 40.43 30.77 9.19
Nm23-H7B 20.54 37.68 37.68 34.04 37.86 40.43 30.77 10.12
Nm23-H5 26.25 34.56 34.56 35.71 38.85 32.86 30.28 7.96
Nm23-H6 27.16 36.43 36.43 39.57 36.69 35.97 32.37 9.33
Nm23-H1 55.26 45.26 45.26 42.66 39.44 42.66 45.14 10.53
Nm23-H2 53.95 45.26 45.26 42.66 41.55 41.96 45.83 9.21
Nm23-H3 52.66 51.08 51.08 44.76 41.55 45.45 46.53 8.28
Nm23-H4 100.00 45.32 45.32 37.06 34.51 37.76 37.93 9.09
Ndk
M.tuberculosis 45.32 100.00 100.00 44.12 53.68 48.53 50.00 13.57
Ndk
M.bovis 45.32 100.00 100.00 44.12 53.68 48.53 50.00 13.57
Ndk
E.coli 37.06 44.12 44.12 100.00 64.79 62.24 56.64 13.29
Ndk
V.cholerae 34.51 53.68 53.68 64.79 100.00 65.49 52.11 14.08
Ndk
P.aeruginosa 37.76 48.53 48.53 62.24 65.49 100.00 60.84 13.99
Ndk
M.xanthus 37.93 50.00 50.00 56.64 52.11 60.84 100.00 12.41
Nm23-H10 9.09 13.57 13.57 13.29 14.08 13.99 12.41 100.00
61
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
10 20 30 40 50 60 70 80 90
Nm23-H1 MANCERTFIA IKPDGVQRGL VGEIIKRFEQ KGFRLVGLKF MQASEDLLKE HYVDLKDRPF FAGLVKYMHS GPVVAMVWEG LNVVKTGRVM
Clustal Consensus ** :**: ****.*.:.: :***:.***: *:*:*. *: :* ** .*.: *:*** * .**.:* * ****. * ** :.: ..* :
Ndk (P.aeruginosa) MA-LQRTLSI IKPDAVSKNV IGEILTRFEK AGLRVVAAKM VQLSEREAGG FYAEHKERPF FKDLVSFMTS GPVVVQVLEG EDAIAKNREL
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ..
100 110 120 130 140 150
Nm23-H1 LGETNPADSK PGTIRGDFCI QVGRNIIHGS DSVESAEKEI GLWFHPEELV DYTSCAQNWI YE
Clustal Consensus :* *:* .:. .****.**.: .:..* :*** ** ** :** . :* . *: :
Ndk (P.aeruginosa) MGATDPKKAD AGTIRADFAV SIDENAVHGS DSEASAAREI AYFFAATEVC ERIR------ --
Figure 17 – Sequence alignment and secondary structure comparison between human Nm23-H1 and P. aeruginosa Ndk. Symbols in Clustal Consensus sequence
indicate standard ClustalW nomenclature: (*) identity, (:) high conservation and (.) conservation. Secondary structure of Nm23-H1 was represented according to PDB
code 4ENO. Secondary structure of P. aeruginosa Ndk was predicted using PDB code 3VGU as template. This template was aligned between its 2 to 141 amino acid
residues and share a 79% sequence identity with P. aeruginosa Ndk. Secondary elements are indicate as: single purple curves (turns), yellow arrows (β-sheets), red
lines (bends), blue curved lines (alpha helices), orange curved lines (3/10-helices), and black lines (no secondary structure assigned). NCBI accession number for each
protein is given below. Human (Homo sapiens) Nm23-H1 (CAG46912) and P. aeruginosa Ndk (EPR01938).
Prediction of Ndk
(P.aeruginosa)
Secondary structure
Nm23-H1
Secondary structure
Nm23-H1
Secondary structure
Prediction of Ndk
(P.aeruginosa)
Secondary structure
62
Figure 18 – Phylogenetic tree representation of human Nm23 protein family as well as some
bacterial Ndks. Splitstree software was used to construct the tree based on ClustalW multiple
alignment generated. All sequences and its NCBI accession numbers used were the same as those
displayed in Figure 16. Protein three-dimensional structures represented were retrieved from PDB
database: Nm23-H1 (4ENO), Nm23-H2 (3BBF), Nm23-H3 (1ZS6), Nm23-H4 (1EHW), Nm23-
H10 (2BX6), and Ndks from M. tuberculosis (1K44), E. coli (2HUR) and M. xanthus (2NCK).
Quaternary structures are represented as homohexamers (Nm23-H1, Nm23-H2, Nm23-H3 and
Nm23-H4 and Ndk from M. tuberculosis) and as homotetramers (Ndk from E. coli and M.
xanthus). Tertiary structure is represented as a monomer (Nm23-H10). Ndk from P. aeruginosa is
marked with an asterisk (*) since it is a predicted three-dimensional model created using Swiss-
model software tools. This representation was modeled using 2 to 141 amino acid residues of the
Ndk sequence (79% identity) from Halomonas sp. 593 (3VGU) as template. This template forms a
homodimer in its quaternary structure, as well as the model created.
*
63
Structural alignment between human Nm23-H1 and the predicted model,
created for P. aeruginosa Ndk, was performed using PyMOL software tools and is
presented in Figure 19. This kind of analysis clearly supports other data retrieved
from bioinformatics approaches mentioned above, since a remarkable structural
superimposition between the two proteins was successfully accomplished.
Moreover, this superimposition is even more patent between important secondary
elements as β-sheets and α-helices, which indicate that the three-dimension model
created for P. aeruginosa Ndk is extremely resemblant to human Nm23-H1.
Figure 19 – Structural alignment between human Nm23-H1 (4ENO) [blue] and P. aeruginosa
Ndk predicted model (green) was performed using PyMOL software. Three dimensional structures
are displayed at different degrees: 0º (A), 90º (B), 180º (C) and 270º (D).
65
5. DISCUSSION
Recently, azurin was shown to be capable of cause a decrease in P-cadherin
levels, without interfering with E-cadherin levels, in three different breast cancer
cell lines, including MCF-7/AZ.Pcad [45]. In the same study, an evaluation on the
CDH3/P-cadherin gene expression revealed that no evident decrease took place.
Further experiments based on microarray analysis revealed that when MCF-
7/AZ.Pcad cells were treated with azurin, several genes showed to be up-
regulated. Some of these genes are involved in membrane organization, vesicular
transport and lysosome. Considering these results altogether it can mean that other
mechanisms must be responsible for a decrease in P-cadherin levels when azurin
is administrated. Since post-transcriptional mechanisms should be behind these
observations, we intended to verify if proteolytic systems are involved in P-
cadherin decreased levels, as microarray analysis also pointed out. Following that
line of thought we tested a proteasome and a lysosome inhibitor (MG-132 and
ammonium chloride, respectively) on MCF-7/AZ.Pcad cells, in order to study
how those inhibitory effects could affect P-cadherin levels, after the cells have
been treated with azurin.
Regarding to the samples treated with azurin, but which did not receive any
inhibitor, we showed that P-cadherin level diminishes with increasing azurin
concentrations, as Bernardes et al already described [82]. Also these authors
reported that E-cadherin levels are not affected when azurin is administrated and
this was corroborated looking at the samples untreated with ammonium chloride.
Nevertheless, we found a decrease in E-cadherin level in samples untreated with
MG-132. Since MG-132 was dissolved in ethanol, we administrated absolute
ethanol on control (untreated) samples, whereas water was used in the case of
samples untreated with ammonium chloride. Some authors found that ethanol can
increase E-cadherin expression on B16-BL6 melanoma cells, which is a contrary
observation to what our results show [83]. On the other hand, it was described that
ethanol causes a decrease in vascular endothelial (VE)-cadherin levels by
disrupting junctional VE-cadherin and inducing endocytosis of this protein in
human umbilical vein endothelial cells (HUVEC) [84]. Even though VE-cadherin
is a different cadherin, and that HUVEC are not tumor cells, it does not exclude
66
completely the possibility that ethanol can cause a similar decrease in E-cadherin
level in MCF-7/AZ.Pcad cells, since no similar study was ever done in this case.
When we treated MCF-7/AZ.Pcad cells with each inhibitor, we saw that E and
P-cadherin levels increased, especially when azurin at 100 μM, comparing to
control samples. MDM2 is a protein involved in negative regulation of p53
pathway by inhibiting p53 transactivation and promoting p53 degradation via a
ubiquitin-mediated proteolytic process [85]. However it has been also reported
that this MDM2 can mediate E-cadherin degradation by promoting its
ubiquitination and that MG-132 was able to block E-cadherin degradation by this
protein, in breast cancer cells [85]. It seems that those findings clearly support the
fact that we observed an increase on E-cadherin levels when cells were treated
with MG-132. Even though similar findings have not been yet described regarding
P-cadherin, it is likely that the same can happen since both cadherins are very
similar between themselves. The higher cadherin levels were seen at the highest
azurin concentration tested. This could probably be explained by the fact that
azurin can disrupt p53-MDM2 association and, therefore, contribute for a higher
free intercellular MDM2 concentration, which in turn causes E- or P-cadherin
endocytosis and its cytoplasmic accumulation, since proteolytic systems are being
inhibited.
Regarding to ammonium chloride, this inhibitor seemed to exhibit a similar
effect as MG-132 on both cadherin levels. In fact, E-cadherin degradation was
already found to be blocked by ammonium chloride in MCF-7 cells, since
lysosome has been described to be involved in E-cadherin degradation by several
authors [85], [86]. Unfortunately no studies were made about the proteolytic
systems involved in P-cadherin degradation, although once again it is probable to
be similar to E-cadherin. Our results showed thereby that two major proteolytic
systems in eukaryotic cells, as proteasome and lysosome, appear both to be
involved in E- and P-cadherin degradation.
MβCD depletes cholesterol from the cell membranes and blocks azurin
internalization, especially in lipid rafts, where this molecule is extensively present
and where azurin seems to enter preferentially [25], [81]. Therefore, we thought
to study how MβCD affects azurin internalization on MCF-7/AZ.Pcad cells and,
67
more importantly, what kind of effect does it provokes on E and P-cadherin levels.
Our results showed that as we were expecting, when MβCD is applied on MCF-
7/AZ.Pcad it occurs a decrease in azurin internalization (in both concentrations
used). These observations are supported by the mechanism of action of this
inhibitor, and the theory that argues that the formation of caveolae/caveossome
complex in the cell membrane can be an important route of entry for azurin,
especially in tumor cells where these membrane receptors are overexpressed [25],
[81]. The most remarkable observation was, nevertheless, that when MβCD and
azurin were applied simultaneously in these cells, a decrease on P-cadherin level
was verified. On the contrary, E-cadherin level remained constant when both these
molecules were applied together. Since only P-cadherin level decreases in MCF-
7/AZ.Pcad cells when both MβCD and azurin are applied, and as both these
molecules act preferentially on lipid rafts sites, we suggest that P-cadherin should
be highly expressed in lipid rafts, within tumor cells. These would also explain
why some genes, involved in membrane organization and vesicular transport were
seen to be up-regulated when breast cancer cells were treated with azurin. It
appears that azurin can enter tumor cells through lipid rafts and interact directly
with P-cadherin or just cause the formation of vesicles containing this protein and
its endocytosis. Moreover, as our treatment with lysosome and proteasome
inhibitors showed, it is likely that azurin can also be involved in these proteolytic
pathways, and somehow directing vesicles, containing P-cadherin, for endosome,
lysosome or proteasome, thereby causing P-cadherin degradation. As reported by
Bernardes et al, E-cadherin level is not affected by azurin, and here we found that
also MβCD does not interfere with this protein level [45]. In light of these facts,
this can signify that E-cadherins should be present mainly in other sites in the cell
membrane other than lipid rafts, contrarily to P-cadherin.
In this work we used cell viability assay (MTT) to evaluate the impact of
azurin’s cytotoxicity in human breast (MCF-7/AZ.Mock) and lung (A549) cancer
cell lines when tested regarding different exposure time and protein doses. In the
case of both cell lines this protein induced a clear dose and time dependent
reduction of cell viability. These results are supported by other similar MTT
assays, performed with azurin, towards other tumor cell lines from breast cancer
68
and melanoma, on which a decrease in cell viability was observed at 24, 48 or 72
hours [20], [24], [87]. Recently, a cell viability assay showed a maximal 20%
cytotoxicity when azurin at 100 µM was used on MCF-7/AZ.Mock cells after 48
hours (one dose), which was the same value verified when we treated those cells
during the same conditions [45]. Moreover, this was the same cytotoxicity level
observed on A549 after 48 hours, but after two doses have been applied, which
can mean that this lung cancer cell line is slightly more resistant to azurin’s
cytotoxic effects. Regarding the three-dose treatment on MCF-7/AZ.Mock,
during 72h, 74% of cell viability was reached at the highest protein concentration,
which was exactly the same value observed on an identical MTT assay where
UISO-Mel 2 cells were used [24]. Therefore our results confirmed azurin
cytotoxicity towards different tumor cell lines.
MPT 63 shares an immunoglobulin-like fold as azurin, and recently this protein
and a derived peptide (MB30) were described to display high cytotoxicity in
several tumor cell lines [63]. Taking into account these findings and considering
the fact that this protein’s activity toward cancer cells is far less studied than
azurin, we thought to evaluate its effects on MCF-7/AZ.Mock and A549 cell
lines. Ours results showed that MPT 63 displayed cytotoxicity towards both tumor
cell lines tested, even though, once again, A549 cells seemed to be not as much
sensitive to cytotoxicity as MCF-7/AZ.Mock, since a 10% decrease in cell
viability was the highest value verified in the latter cell line. On the other hand,
MPT 63 produced cytotoxic effects comparable to azurin between 48 and 72
hours (with one and three doses respectively), in MCF-7/AZ.Mock cells.
Therefore, we can affirm that this protein displays anti-tumor activity against
different cancer cell lines, and that its effect can be intensified by increasing
exposure time and protein doses applied. These results are supported by a study
where MPT 63 and a derived peptide, MB30, were able to cause high cytotoxic
levels in a wide variety of different tumor cell lines, including breast cancer cells
[63]. In the same study it were seen slight differences between cell viability
results across different tumor cells, and the same also clearly happened in our
case. Moreover, it was also observed a time dependent effect regarding this
protein anti-tumor activity of this protein. Concerning to MB30 peptide, it showed
69
to be more effective comparing to MPT 63 against all tumor cell lines tested,
thereby it could be relevant to explore this peptide in future experiments, since it
can harbor the protein sequence accountable for this protein anti-tumorigenic
properties, in the same way that p28 has higher anti-tumor activity than azurin
itself [63].
Nucleotide diphosphate kinases are extremely common in all organisms since
they have important functional activities in cells as maintaining the nucleotide
pools for the synthesis of nucleic acids. In humans, however, these proteins
constitute a Nm23 protein family which includes 10 proteins so far, and have
shown to be involved in other biological functions. Nm23-H1 has been known for
its anti-tumor and anti-metastatic properties, for some years now, and a higher
expression was associated with the inhibition of cell invasion in several tumor
models [75]. In this work we intended to verify the same cytotoxic and anti-
metastatic properties shown by Nm23-H1, using P. aeruginosa Ndk.
The results retrieved from cell viability assays indicate that also Ndk caused a
decrease in cell viability of MCF-7/AZ.Mock and A549 cells, treated with this
protein during 48 hours and when two doses were applied during this period of
time. Unlike the other two proteins tested, this one revealed cell viability values
very similar between the two different tumor cell lines within the same assay.
Moreover, in this case when the highest concentration was used, a 27% cell
mortality was seen in A549 cells (48 hours, 2 doses applied), which was the same
value verified for MCF-7/AZ.Mock in a 72 hours assay (with 3 doses applied).
This can probably means that A549 cells are more sensitive to the cytotoxic
effects of this protein than MCF-7/AZ.Mock cells. Regarding to the cell viability
assay using Ndk, a normal epithelial lung cell line 16HBE14o- was also tested to
evaluate if Ndk’s cytotoxicity activity was selective towards cancer cells or not.
After two doses have been applied, during 48 hours, it was visible a much lower
decrease in cell viability, which never reached below 90%. Considering the results
obtained, we can say that Ndk appears to exhibit a selective cytotoxic activity
against distinct tumor cells in a dose and time dependent response, which starts to
be visualized after a two dose treatment, during 48 hours. It seems that Ndk has
70
higher cytotoxic activity than MPT 63 against both cell lines tested, and displays
much similar results comparing to azurin than MPT 63 does.
In some tumor cells, as MCF-7, it was already described that Nm23-H1 and its
binding partner STRAP (serine-threonine kinase receptor-associated protein)
interact with p53 at the DNA binding domain and increases p53 transcription and
at the same time, activate this protein by removing the negative regulator MDM2
from the p53-MDM2 complex [88], [89]. In MTT assay we only tested p53-
positive cells, thereby it cannot be excluded that Ndk could also have increased
p53 expression or even stabilize it, as azurin does, in the same manner that Nm23-
H1.
Another extremely relevant point would be to know if MPT 63 and Ndk can
enter tumor cells or not, which could help us understand what mechanism
underlies their cytotoxic effects. Actually, we tried to answer this question by
treating tumor cells with these proteins and then using an anti-his-probe antibody,
against the histidine-tag present in both purified proteins, to visualize them by
western blot. Unfortunately, our results were inconclusive since this antibody
seems to be unsuitable for this purpose. On future experiments specific anti-MPT
63 and anti-Ndk antibodies should be designed in order to evaluate the
internalization of these two proteins in human tumor cell lines.
Since human Nm23-H1 was described as an anti-metastatic and that P.
aeruginosa Ndk exhibited relevant cytotoxic effects, it was of interest to use
bioinformatics tools to compare the two proteins at a sequence and structure level.
Taking account all the results obtained, we can see that human Nm23 proteins
seem to be arranged in the phylogenetic tree (Figure 18) as two distinct clusters: a
Group I, which includes Nm23-H1, -H2, -H3 and –H4; and a Group II, which
includes Nm23-H5,-H6,-H7,-H7B,-H8 and –H9. Nm23-H10, on the other hand,
appears to have a different ancestor. This phylogenetic analysis on human Nm23
proteins is corroborated by results obtained by Desvignes et al., on which the
same clusters were identified when analyzing different Nm23 proteins from
chordate organisms, including the human proteins [90]. In the same study it was
found that Nm23-H4 revealed a strongly supported divergence from other Nm23
proteins of the Group I, which was also verified in our results. Since this protein is
71
present predominantly in mitochondria, it is very plausible that this may represent
the human Nm23 ancestor and its closest evolutionary proximity to M.
tuberculosis Ndk clearly supports this theory [91]. Moreover, regarding
quaternary structures of Group I cluster, they are displayed as homohexamers, as
well as M. tuberculosis Ndk. Surprisingly, the main bacterial Ndk cluster seen in
the phylogenetic tree includes Ndks as homotetramers, as the one from M.
xanthus, which seems to be closer to the other mitochondrial Nm23 (-H6). This
can possibly indicate that the Group II cluster of human Nm23 proteins could
have diverged from the main bacterial Ndk cluster and that Nm23-H6 could have
been the first protein to appear within this group. Nm23-H10 is the most divergent
human Nm23 protein as the results show. One of the reasons of its divergence
could be the fact that it only possesses a partial Ndk domain in chordate
organisms, and none at all in some cases [90]. In humans the lack of Ndk activity
of this protein is also known [90].
When comparing secondary structures of Nm23-H1 and the model created for
P. aeruginosa Ndk we see that all α-helices and β-sheets present in Nm23-H1
were also proposed to be present in the bacterial Ndk, in the same relative
positions. Even though the model was proposed as a homodimeric protein, it was
built according to its template, thereby no relevant conclusions can be made
concerning to its quaternary structure. Even so, the structural alignment performed
(Figure 19) showed that both protein structural units represented overlap each
other almost perfectly. These results clearly indicate that the model created for P.
aeruginosa Ndk is very robust and that Ndks have maintained higher sequence
and function conservation throughout evolution, as it has been analyzed by some
authors [92]. More importantly this may support the idea that Ndk and Nm23-H1
resemblance at a sequence and structural level, could permit an association of Ndk
to p53, or other important proteins that bind to Nm23-H1.
Although we used P. aeruginosa Ndk in MTT assays, and this protein shares a
very relevant identity percentage with Nm23-H1, M. tuberculosis Ndk revealed a
slightly higher sequence identity. Another important fact is that the three-
dimensional model built for P. aeruginosa Ndk shows a homodimeric protein,
where M. tuberculosis Ndk is a homohexameric protein and therefore have a
72
similar quaternary structure as human Nm23-H1. This can suggest that this
bacterial Ndk may be suitable to achieve higher cytotoxic and anti-metastatic
effects against tumor cells, when compared to P. aeruginosa Ndk.
We attempt to verify the same anti-metastatic potential reported for Nm23-H1
in different tumor cells, using P. aeruginosa Ndk to complement our study upon
this protein. As mentioned before, Nm23-H1 has been successively associated
with a decrease in tumor cells motility and migration, thereby we performed a
matrigel invasion assay on A549, a highly invasive cell line, and administrated
Ndk in hope to verify similar results [93], [94]. Our preliminary results only
showed a minimal decrease in cell invasion when using Ndk, comparing to
control (between 6 and 8%), and no differences between the two concentrations
applied were visible. However, additionally to the fact that this assay requires to
be repeated to acquire statistical relevance, it is important to refer that this was a
24 hours assay, on which only one protein dose was applied. This fact could be
extremely important since we only observed cytotoxic effects on A549 cells with
Ndk, after 48 hours and a two dose treatment. Therefore, perhaps it is necessary to
increase the number of protein doses and the exposure time to Ndk in future
experiments.
Additionally to its nucleotide diphosphate kinase activity, Nm23-H1 was also
reported to exhibit a protein histidine kinase activity that mediates its anti-motility
function, probably via phosphorylation of the kinase suppressor of ras (KSR) and
signaling mechanisms associated to this pathway [95]. Experiments involving
site-directed mutagenesis and protein activity analysis, regarding Nm23-H1
protein, revealed a requirement of K12 and Y52 residues for histidine kinase
activity and H118 for the metastasis suppressor activity of this protein [96]. Both
these amino acid residues are identical between human Nm23-H1 and P.
aeruginosa Ndk sequences, which can mean that this histidine kinase activity may
also be present on Ndk, and thus it may display anti-metastatic activity. Another
relevant question here may be the importance of quaternary structure in Nm23-H1
anti-metastatic function, since Ndk is not a hexameric protein, which indicate that
more experiments must be done regarding its putative anti-metastatic effect.
73
6. CONCLUSION
Cancer remains one of the major human diseases, representing the leading
cause of death in the world and a terrible burden, which is specially accentuated in
developed countries. Newer and alternative anti-cancer therapies are under
development and are inevitable to reach more successful results against cancer
than the conventional radio- and chemotherapies. The use of microorganisms and
its derived products, especially proteins, to treat this disease are being broadly
explored and can create new windows of hope for these patients.
In this work we used azurin, a well described P. aeruginosa protein with anti-
tumor properties, to understand if it could also mediate a decrease in P-cadherin
levels by inducing its degradation within an invasive tumor cell line which
express high P-cadherin levels, like MCF-7/AZ.Pcad. From our results, it is clear
that the two major proteolytic systems in eukaryotic cells, lysosome and
proteasome, are involved in P-cadherin degradation, even though it was not
possible to declare what is the exact contribution of each system for that matter.
We also found that, when MβCD and azurin were administrated simultaneously to
this tumor cell line, a decrease in P-cadherin level was evident, contrarily to E-
cadherin level, which remained unaltered. Thus we suggest that, by depleting
cholesterol and interfering with lipid rafts on cell membrane, MβCD must be
causing P-cadherin to be displaced from these sites where it is supposedly mainly
present. Since azurin internalization is not completely blocked by this compound,
it is likely that this protein enters tumor cells preferentially through lipid rafts, and
mediates P-cadherin endocytosis and latter proteolysis by systems like the
lysosome. Further investigation is required in order to clarify how exactly does
azurin mediate P-cadherin degradation and what pathways can be involved.
Here we also intended to explore a possible anti-tumor role for new bacterial
proteins as MPT 63 (from M. tuberculosis) and Ndk (from P. aeruginosa) using
two tumor cell lines, derived from two of the deadliest cancers, lung and breast
cancer. Both these proteins share some important features as azurin, since they are
low molecular weight and secreted by pathogenic bacteria, probably as a survival
mechanism against other organisms. MPT 63 also shares an immunoglobulin-like
folding with azurin, which may be relevant for the cytotoxic effect verified on
74
tumor cells as our results showed. Both proteins evidenced cytotoxicity against
cancer cells in a time and dose dependent manner.
Nevertheless, Ndk exhibited a far extensive cytotoxicity than MPT 63 against
both MCF-7/AZ.Mock and A549 cells. In fact, this protein revealed a similar
effect on cell viability as azurin. Another interesting observation was that it
appears to display selective cytotoxicity against tumor cell lines and a lower effect
on normal cells, a feature that also azurin presents. This could be a promising
feature since we seek anti-tumor agents that should act mainly on tumor cells and
limit side effects on normal cells.
Although we have shown that MPT 63 and Ndk display cytotoxicity against
cancer cells in vitro, the mechanisms by which that occurs remain a mystery. It is
likely that they increase apoptotic rate by activating p53, Bax and other pro-
apoptotic proteins in some way, as it was already described for azurin. For this
motive it will be crucial to study the effect of these bacterial proteins on several
major pro-apoptotic proteins, which could provide other important clues and tell
us more about their anti-tumor activities, since it cannot be excluded that they
share similar mechanisms as azurin.
In this work, we also tested anti-his-probe antibody towards these purified
proteins in order to evaluate their internalization in tumor cells, but the results
obtained clearly showed that this is not a suitable antibody to test this. It should be
designed specific antibodies against each one of these proteins to assure the real
impact of their internalization in tumor cell lines. This finding will help us to
understand if these proteins require full cell internalization or not to exert their
cytotoxic activity. If internalization reveals to be a truly requirement for these
protein anti-tumor activities we must develop strategies to increase protein
delivery as the use of liposomes, for instance.
Since human Ndk homologue protein Nm23-H1 is known by its anti-metastatic
activity in several tumor models, we sought to test Ndk in A549 invasion assay.
We only observed a 10% approximated decrease in cell invasion using this
protein, which still is a lower value even though we only have made a single
experiment. It is then important to repeat this experiment using increased
exposure time and higher number of protein doses to test Ndk’s anti-metastatic
75
effect, which could extent the relevance of the anti-tumor effects of this bacterial
protein toward human cancers.
Our study on multiple sequence alignment between Ndks with different origins
showed that M. tuberculosis Ndk has a higher identity level to Nm23-H1 than P.
aeruginosa Ndk. Moreover, this was so far the unique bacterial Ndk to display a
hexameric three-dimensional strucuture, which is the same kind of structure seen
in human Nm23-H1. Therefore, we think that it should be important to consider
this protein in future similar experiments since three-dimensional structure may be
relevant for the anti-metastatic activity of Ndk.
In summary, our results showed that some bacterial proteins with anti-tumor
properties as azurin, Ndk and MPT 63, can induce important cytotoxic effects on
tumor cells but, in order to represent a solid alternative to conventional therapies
in a near future, some issues still need to be solved. The scientific knowledge
gathered until now and the tools at our disposal in the present could be the key to
enhance the anti-tumor properties of these proteins in order to make them more
efficient. For instance, the use of genetically modified bacteria derived from
human microbiome, to express these bacterial proteins can represent one way of
delivering these proteins with efficiency and to increase bioavailability in the site
of the tumors. Moreover, this could limit side effects on normal cells and a more
targeted anti-tumor response. The possibility of these proteins to act on different
pathways, like controlling apoptosis and cell invasion processes make them a
unique weapon to be explored in the future years.
77
7. REFERENCES
[1] J. Ferlay, H.-R. Shin, F. Bray, D. Forman, C. Mathers, and D. M. Parkin,
“Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008.,”
International journal of cancer. Journal international du cancer, vol. 127,
pp. 2893–917, Dec. 2010.
[2] R. Meuwissen and A. Berns, “Mouse models for human lung cancer.,”
Genes & development, vol. 19, pp. 643–64, Mar. 2005.
[3] J. Yokota and T. Kohno, “Molecular footprints of human lung cancer
progression.,” Cancer science, vol. 95, pp. 197–204, Mar. 2004.
[4] A. Fucic, M. Gamulin, Z. Ferencic, D. S. Rokotov, J. Katic, A. Bartonova,
I. B. Lovasic, and D. F. Merlo, “Lung cancer and environmental chemical
exposure: a review of our current state of knowledge with reference to the
role of hormones and hormone receptors as an increased risk factor for
developing lung cancer in man.,” Toxicologic pathology, vol. 38, pp. 849–
55, Oct. 2010.
[5] B.-R. Yi, K. J. Choi, S. U. Kim, and K.-C. Choi, “Therapeutic potential of
stem cells expressing suicide genes that selectively target human breast
cancer cells: evidence that they exert tumoricidal effects via tumor tropism
(review).,” International journal of oncology, vol. 41, pp. 798–804, Sep.
2012.
[6] H. Lahlou and W. J. Muller, “Β1-Integrins Signaling and Mammary Tumor
Progression in Transgenic Mouse Models: Implications for Human Breast
Cancer.,” Breast cancer research : BCR, vol. 13, p. 229, Jan. 2011.
[7] K.-L. Dao and R. N. Hanson, “Targeting the estrogen receptor using
steroid-therapeutic drug conjugates (hybrids).,” Bioconjugate chemistry,
vol. 23, pp. 2139–58, Nov. 2012.
[8] S. Douglass, S. Ali, A. P. Meeson, D. Browell, and J. a Kirby, “The role of
FOXP3 in the development and metastatic spread of breast cancer.,”
Cancer metastasis reviews, vol. 31, pp. 843–54, Dec. 2012.
[9] K. Yanagisawa, B. J. Xu, D. P. Carbone, and R. M. Caprioli, “Molecular
fingerprinting in human lung cancer.,” Clinical lung cancer, vol. 5, pp.
113–8, Sep. 2003.
[10] A. W. Wong, Q. X. Paulson, J. Hong, R. E. Stubbins, K. Poh, E. Schrader,
and N. P. Nunez, “Alcohol promotes breast cancer cell invasion by
regulating the Nm23-ITGA5 pathway.,” Journal of experimental & clinical
cancer research : CR, vol. 30, pp. 1–11, Jan. 2011.
78
[11] N. Bernardes, A. M. Chakrabarty, and A. M. Fialho, “Engineering of
bacterial strains and their products for cancer therapy.,” Applied
microbiology and biotechnology, vol. 97, pp. 5189–5199, May 2013.
[12] A. M. Fialho, N. Bernardes, and A. M. Chakrabarty, “Recent Patents on
Live Bacteria and their Products as Potential Anticancer Agents,” Recent
Patents on Anti-Cancer Drug Discovery, vol. 7, pp. 31–55, Jan. 2012.
[13] N. Bernardes, R. Seruca, A. M. Chakrabarty, and A. M. Fialho, “Microbial-
based therapy of cancer: current progress and future prospects.,”
Bioengineered bugs, vol. 1, pp. 178–90, 2010.
[14] A. Naguleswaran, A. M. Fialho, A. Chaudhari, C. S. Hong, A. M.
Chakrabarty, and W. J. Sullivan, “Azurin-like protein blocks invasion of
Toxoplasma gondii through potential interactions with parasite surface
antigen SAG1.,” Antimicrobial agents and chemotherapy, vol. 52, pp. 402–
8, Feb. 2008.
[15] T. Yamada, R. R. Mehta, F. Lekmine, K. Christov, M. L. King, D.
Majumdar, A. Shilkaitis, A. Green, L. Bratescu, C. W. Beattie, and T. K.
Das Gupta, “A peptide fragment of azurin induces a p53-mediated cell
cycle arrest in human breast cancer cells.,” Molecular cancer therapeutics,
vol. 8, pp. 2947–58, Oct. 2009.
[16] R. R. Mehta, M. Hawthorne, X. Peng, A. Shilkaitis, R. G. Mehta, C. W.
Beattie, and T. K. Das Gupta, “A 28-amino-acid peptide fragment of the
cupredoxin azurin prevents carcinogen-induced mouse mammary lesions.,”
Cancer prevention research (Philadelphia, Pa.), vol. 3, pp. 1351–60, Oct.
2010.
[17] A. M. Fialho, P. Salunkhe, S. Manna, S. Mahali, and A. M. Chakrabarty,
“Glioblastoma multiforme: novel therapeutic approaches.,” ISRN
neurology, vol. 2012, pp. 1–10, Jan. 2012.
[18] N. Bonander, T. Vänngård, L. C. Tsai, V. Langer, H. Nar, and L. Sjölin,
“The metal site of Pseudomonas aeruginosa azurin, revealed by a crystal
structure determination of the Co(II) derivative and Co-EPR
spectroscopy.,” Proteins, vol. 27, pp. 385–94, Mar. 1997.
[19] O. Zaborina, N. Dhiman, M. L. Chen, J. Kostal, I. A. Holder, and A. M.
Chakrabarty, “Secreted products of a nonmucoid Pseudomonas aeruginosa
strain induce two modes of macrophage killing : external-ATP- dependent ,
P2Z-receptor-mediated necrosis apoptosis,” Microbiology, vol. 146, pp.
2521–2530, 2000.
[20] T. Yamada, M. Goto, V. Punj, O. Zaborina, M. L. Chen, K. Kimbara, D.
Majumdar, E. Cunningham, T. K. Das Gupta, and A. M. Chakrabarty,
“Bacterial redox protein azurin, tumor suppressor protein p53, and
79
regression of cancer.,” Proceedings of the National Academy of Sciences of
the United States of America, vol. 99, pp. 14098–103, Oct. 2002.
[21] V. Punj, S. Bhattacharyya, D. Saint-Dic, C. Vasu, E. a Cunningham, J.
Graves, T. Yamada, A. I. Constantinou, K. Christov, B. White, G. Li, D.
Majumdar, A. M. Chakrabarty, and T. K. Das Gupta, “Bacterial cupredoxin
azurin as an inducer of apoptosis and regression in human breast cancer.,”
Oncogene, vol. 23, pp. 2367–78, Mar. 2004.
[22] D. Yang, X. Miao, Z. Ye, and J. Feng, “Bacterial redox protein azurin
induce apoptosis in human osteosarcoma U2OS cells,” Pharmacological
Research, vol. 52, pp. 413–421, 2005.
[23] T. Yamada, A. M. Fialho, V. Punj, L. Bratescu, T. K. Das Gupta, and A.
M. Chakrabarty, “Internalization of bacterial redox protein azurin in
mammalian cells: entry domain and specificity.,” Cellular microbiology,
vol. 7, pp. 1418–31, Oct. 2005.
[24] B. N. Taylor, R. R. Mehta, T. Yamada, F. Lekmine, K. Christov, A. M.
Chakrabarty, A. Green, L. Bratescu, A. Shilkaitis, C. W. Beattie, and T. K.
Das Gupta, “Noncationic peptides obtained from azurin preferentially enter
cancer cells.,” Cancer research, vol. 69, pp. 537–46, Jan. 2009.
[25] R. R. Mehta, T. Yamada, B. N. Taylor, K. Christov, M. L. King, D.
Majumdar, F. Lekmine, C. Tiruppathi, A. Shilkaitis, L. Bratescu, A. Green,
C. W. Beattie, and T. K. Das Gupta, “A cell penetrating peptide derived
from azurin inhibits angiogenesis and tumor growth by inhibiting
phosphorylation of VEGFR-2, FAK and Akt.,” Angiogenesis, vol. 14, pp.
355–69, Sep. 2011.
[26] T. Yamada, Y. Hiraoka, M. Ikehata, K. Kimbara, B. S. Avner, T. K. Das
Gupta, and A. M. Chakrabarty, “Apoptosis or growth arrest: Modulation of
tumor suppressor p53’s specificity by bacterial redox protein azurin.,”
Proceedings of the National Academy of Sciences of the United States of
America, vol. 101, pp. 4770–5, Apr. 2004.
[27] E. Gabellieri, M. Bucciantini, M. Stefani, and P. Cioni, “Does azurin bind
to the transactivation domain of p53? A Trp phosphorescence study.,”
Biophysical chemistry, vol. 159, pp. 287–93, Dec. 2011.
[28] J. M. Kwan, A. M. Fialho, M. Kundu, J. Thomas, C. S. Hong, T. K. Das
Gupta, and A. M. Chakrabarty, “Bacterial proteins as potential drugs in the
treatment of leukemia.,” Leukemia research, vol. 33, pp. 1392–9, Oct.
2009.
[29] S. Ramachandran and M. Mandal, “Induction of apoptosis of azurin
synthesized from P. aeruginosa MTCC 2453 against Dalton’s lymphoma
80
ascites model.,” Biomedicine & pharmacotherapy, vol. 65, pp. 461–6, Oct.
2011.
[30] L. Jia, G. S. Gorman, L. U. Coward, P. E. Noker, D. McCormick, T. L.
Horn, J. B. Harder, M. Muzzio, B. Prabhakar, B. Ganesh, T. K. Das Gupta,
and C. W. Beattie, “Preclinical pharmacokinetics, metabolism, and toxicity
of azurin-p28 (NSC745104) a peptide inhibitor of p53 ubiquitination.,”
Cancer chemotherapy and pharmacology, vol. 68, pp. 513–24, Aug. 2011.
[31] M. a Warso, J. M. Richards, D. Mehta, K. Christov, C. Schaeffer, L. Rae
Bressler, T. Yamada, D. Majumdar, S. a Kennedy, C. W. Beattie, and T. K.
Das Gupta, “A first-in-class, first-in-human, phase I trial of p28, a non-
HDM2-mediated peptide inhibitor of p53 ubiquitination in patients with
advanced solid tumours.,” British journal of cancer, vol. 108, pp. 1061–70,
Mar. 2013.
[32] A. Chaudhari, M. Mahfouz, A. M. Fialho, T. Yamada, A. T. Granja, Y.
Zhu, W. Hashimoto, B. Schlarb-Ridley, W. Cho, T. K. Das Gupta, and A.
M. Chakrabarty, “Cupredoxin-cancer interrelationship: azurin binding with
EphB2, interference in EphB2 tyrosine phosphorylation, and inhibition of
cancer growth.,” Biochemistry, vol. 46, pp. 1799–810, Feb. 2007.
[33] C. Hafner, G. Schmitz, S. Meyer, F. Bataille, P. Hau, T. Langmann, W.
Dietmaier, M. Landthaler, and T. Vogt, “Differential gene expression of
Eph receptors and ephrins in benign human tissues and cancers.,” Clinical
chemistry, vol. 50, pp. 490–9, Mar. 2004.
[34] M. Mahfouz, W. Hashimoto, T. K. Das Gupta, and A. M. Chakrabarty,
“Bacterial proteins and CpG-rich extrachromosomal DNA in potential
cancer therapy.,” Plasmid, vol. 57, pp. 4–17, Jan. 2007.
[35] A. M. Fialho and A. M. Chakrabarty, “Recent patents on bacterial proteins
as potential anticancer agents.,” Recent patents on anti-cancer drug
discovery, vol. 2, pp. 224–34, Nov. 2007.
[36] A. Chaudhari, A. M. Fialho, D. Ratner, P. Gupta, C. S. Hong, S. Kahali, T.
Yamada, K. Haldar, S. Murphy, W. Cho, V. S. Chauhan, T. K. Das Gupta,
and A. M. Chakrabarty, “Azurin, Plasmodium falciparum Malaria and
HIV/AIDS,” Cell Cycle, vol. 5, pp. 1642–1648, 2006.
[37] A. M. Fialho, F. J. Stevens, T. K. Das Gupta, and A. M. Chakrabarty,
“Beyond host-pathogen interactions: microbial defense strategy in the host
environment.,” Current opinion in biotechnology, vol. 18, pp. 279–86, Jun.
2007.
[38] J. Paredes, C. Stove, V. Stove, F. Milanezi, V. Van Marck, L. Derycke, M.
Mareel, M. Bracke, and F. Schmitt, “P-cadherin is up-regulated by the
81
antiestrogen ICI 182,780 and promotes invasion of human breast cancer
cells.,” Cancer research, vol. 64, pp. 8309–17, Nov. 2004.
[39] J. Paredes, J. Figueiredo, A. Albergaria, P. Oliveira, J. Carvalho, A. S.
Ribeiro, J. Caldeira, A. M. Costa, J. Simões-Correia, M. J. Oliveira, H.
Pinheiro, S. S. Pinho, R. Mateus, C. a Reis, M. Leite, M. S. Fernandes, F.
Schmitt, F. Carneiro, C. Figueiredo, C. Oliveira, and R. Seruca, “Epithelial
E- and P-cadherins: role and clinical significance in cancer.,” Biochimica et
biophysica acta, vol. 1826, pp. 297–311, Dec. 2012.
[40] A. Albergaria, A. Ribeiro, A. Vieira, and B. Sousa, “P-cadherin role in
normal breast development and cancer,” The international journal of
developmental biology, vol. 55, pp. 811–822, 2011.
[41] R. van Horssen, A. Hollestelle, J. a P. Rens, A. M. M. Eggermont, M.
Schutte, and T. L. M. Ten Hagen, “E-cadherin promotor methylation and
mutation are inversely related to motility capacity of breast cancer cells.,”
Breast cancer research and treatment, vol. 136, pp. 365–77, Nov. 2012.
[42] J. Paredes, A. L. Correia, A. S. Ribeiro, A. Albergaria, F. Milanezi, and F.
C. Schmitt, “The effect of psychosocial factors on breast cancer outcome: a
systematic review.,” Breast cancer research : BCR, vol. 9, pp. 1–12, Jan.
2007.
[43] A. Albergaria, C. Resende, A. R. Nobre, A. S. Ribeiro, B. Sousa, J. C.
Machado, R. Seruca, J. Paredes, and F. Schmitt, “CCAAT/enhancer
binding protein β (C/EBPβ) isoforms as transcriptional regulators of the
pro-invasive CDH3/P-cadherin gene in human breast cancer cells.,” PloS
one, vol. 8, pp. 1–9, Jan. 2013.
[44] A. S. Ribeiro, A. Albergaria, B. Sousa, A. L. Correia, M. Bracke, R.
Seruca, F. C. Schmitt, and J. Paredes, “Extracellular cleavage and shedding
of P-cadherin: a mechanism underlying the invasive behaviour of breast
cancer cells.,” Oncogene, vol. 29, pp. 1–11, Jan. 2009.
[45] N. Bernardes, A. S. Ribeiro, S. Abreu, B. Mota, R. G. Matos, C. M.
Arraiano, R. Seruca, J. Paredes, and A. M. Fialho, “The bacterial protein
azurin impairs invasion and FAK/Src signaling in P-cadherin-
overexpressing breast cancer cell models.,” PloS one, vol. 8, pp. 1-8, Jan.
2013.
[46] E. Wong and A. M. Cuervo, “Integration of clearance mechanisms: the
proteasome and autophagy.,” Cold Spring Harbor perspectives in biology,
vol. 2, pp. 1–19, Dec. 2010.
[47] M. J. Clague and S. Urbé, “Ubiquitin: same molecule, different degradation
pathways.,” Cell, vol. 143, pp. 682–5, Nov. 2010.
82
[48] X. Chen and X.-M. Yin, “Coordination of autophagy and the proteasome in
resolving endoplasmic reticulum stress.,” Veterinary pathology, vol. 48, pp.
245–53, Jan. 2011.
[49] A. Ciechanover, “Intracellular protein degradation: from a vague idea,
through the lysosome and the ubiquitin-proteasome system, and onto
human diseases and drug targeting (Nobel lecture).,” Angewandte Chemie
(International ed. in English), vol. 44, pp. 5944–67, Sep. 2005.
[50] B. Park, Y.-M. Lee, J.-S. Kim, Y. Her, J. H. Kang, S.-H. Oh, and H.-M.
Kim, “Neutral sphingomyelinase 2 modulates cytotoxic effects of
protopanaxadiol on different human cancer cells.,” BMC complementary
and alternative medicine, vol. 13, p. 194, Jan. 2013.
[51] R. Cubí, L. a Matas, M. Pou, J. Aguilera, and C. Gil, “Differential
sensitivity to detergents of actin cytoskeleton from nerve endings.,”
Biochimica et biophysica acta, vol. 1828, pp. 2385–2393, Jun. 2013.
[52] V. S. Silva, L. Oliveira, and P. P. Gonçalves, “Alteration of aluminium
inhibition of synaptosomal (Na(+)/K(+))ATPase by colestipol
administration.,” Journal of inorganic biochemistry, vol. 128, pp. 208-214,
Nov. 2013.
[53] D. Jensen, C. B. Godfrey, C. Niklas, M. Canals, M. Kocan, D. P. Poole, J.
E. Murphy, F. Alemi, G. S. Cottrell, C. Korbmacher, N. a Lambert, N. W.
Bunnett, and C. U. Corvera, “The Bile Acid Receptor TGR5 Does Not
Interact with β-arrestins or Traffic to Endosomes but Transmits Sustained
Signals from Plasma Membrane Rafts.,” The Journal of biological
chemistry, vol. 288, pp. 22942-22960, Jul. 2013.
[54] Z.-J. Huang, S.-T. Kang, J.-H. Leu, and L.-L. Chen, “Endocytic pathway is
indicated for white spot syndrome virus (WSSV) entry in shrimp.,” Fish &
shellfish immunology, vol. 35, pp. 707-715, Jun. 2013.
[55] C. Manca, K. Lyashchenko, H. G. Wiker, D. Usai, R. Colangeli, and M. L.
Gennaro, “Molecular cloning, purification, and serological characterization
of MPT63, a novel antigen secreted by Mycobacterium tuberculosis.,”
Infection and immunity, vol. 65, pp. 16–23, Jan. 1997.
[56] B. Y. Lee and M. a Horwitz, “T-cell epitope mapping of the three most
abundant extracellular proteins of Mycobacterium tuberculosis in outbred
guinea pigs.,” Infection and immunity, vol. 67, pp. 2665–70, May 1999.
[57] T. Redchuk, N. Korotkevich, O. Gorbatiuk, P. Gilchuk, A. Kaberniuk, O.
Oliynyk, D. Kolibo, and S. Komisarenko, “Expression of Mycobacterium
tuberculosis proteins MPT63 and MPT83 as a fusion: purification,
refolding and immunological characterization,” Journal of Applied
Biomedicine, vol. 10, pp. 169–176, Jan. 2012.
83
[58] C. W. Goulding, A. Parseghian, M. R. Sawaya, D. Cascio, M. I. Apostol,
M. L. Gennaro, and D. Eisenberg, “Crystal structure of a major secreted
protein of Mycobacterium tuberculosis — MPT63 at 1 . 5-Å resolution,”
Protein Science, vol. 11, pp. 2887–2893, Dec. 2002.
[59] S. Nagai, H. G. Wiker, M. Harboe, and M. Kinomoto, “Isolation and partial
characterization of major protein antigens in the culture fluid of
Mycobacterium tuberculosis.,” Infection and immunity, vol. 59, pp. 372–
82, Jan. 1991.
[60] B. L. Wang, Y. Xu, Z. M. Li, Y. M. Xu, X. H. Weng, and H. H. Wang,
“Antibody response to four secretory proteins from Mycobacterium
tuberculosis and their complex antigen in TB patients.,” The international
journal of tuberculosis and lung disease : the official journal of the
International Union against Tuberculosis and Lung Disease, vol. 9, pp.
1327–34, Dec. 2005.
[61] H. M. C. Shane, R. Brookes, S. C. Gilbert, A. V. S. Hill, and I. N. I. Mmun,
“Enhanced Immunogenicity of CD4 ϩ T-Cell Responses and Protective
Efficacy of a DNA-Modified Vaccinia Virus Ankara Prime-Boost
Vaccination Regimen for Murine Tuberculosis,” Infection and immunity,
vol. 69, pp. 681–686, Feb. 2001.
[62] A. S. Mustafa, “Th1 cell reactivity and HLA-DR binding prediction for
promiscuous recognition of MPT63 (Rv1926c), a major secreted protein of
Mycobacterium tuberculosis.,” Scandinavian journal of immunology, vol.
69, pp. 213–22, Mar. 2009.
[63] A. Suri, D. Kanojia, P. Salunkhe, S. Mahali, S. K. Manna, A. Surolia, and
A. Chakrabarty, “Anticancer agent.” WIPO Patent Application
WO/2012/042540, Apr. 5, 2012.
[64] G. W. Sundin, S. Shankar, and a M. Chakrabarty, “Mutational analysis of
nucleoside diphosphate kinase from Pseudomonas aeruginosa:
characterization of critical amino acid residues involved in
exopolysaccharide alginate synthesis.,” Journal of bacteriology, vol. 178,
pp. 7120–8, Dec. 1996.
[65] H. Tokunaga, M. Ishibashi, F. Arisaka, S. Arai, R. Kuroki, T. Arakawa, and
M. Tokunaga, “Residue 134 determines the dimer-tetramer assembly of
nucleoside diphosphate kinase from moderately halophilic bacteria.,” FEBS
letters, vol. 582, pp. 1049–54, Apr. 2008.
[66] a M. Chakrabarty, “Nucleoside diphosphate kinase: role in bacterial
growth, virulence, cell signalling and polysaccharide synthesis.,” Molecular
microbiology, vol. 28, pp. 875–82, Jun. 1998.
84
[67] S. Tiwari, K. V. R. Kishan, T. Chakrabarti, and P. K. Chakraborti, “Amino
acid residues involved in autophosphorylation and phosphotransfer
activities are distinct in nucleoside diphosphate kinase from
Mycobacterium tuberculosis.,” The Journal of biological chemistry, vol.
279, pp. 43595–603, Oct. 2004.
[68] S. Kamath, M. L. Chen, and a M. Chakrabarty, “Secretion of nucleoside
diphosphate kinase by mucoid Pseudomonas aeruginosa 8821: involvement
of a carboxy-terminal motif in secretion.,” Journal of bacteriology, vol.
182, pp. 3826–31, Jul. 2000.
[69] B. a Chopade, S. Shankar, G. W. Sundin, S. Mukhopadhyay, and a M.
Chakrabarty, “Characterization of membrane-associated Pseudomonas
aeruginosa Ras-like protein Pra, a GTP-binding protein that forms
complexes with truncated nucleoside diphosphate kinase and pyruvate
kinase to modulate GTP synthesis.,” Journal of bacteriology, vol. 179, pp.
2181–8, Apr. 1997.
[70] O. Zaborina, N. Misra, J. Kostal, S. Kamath, V. Kapatral, M. E. El-Idrissi,
B. S. Prabhakar, and a M. Chakrabarty, “P2Z-Independent and P2Z
receptor-mediated macrophage killing by Pseudomonas aeruginosa isolated
from cystic fibrosis patients.,” Infection and immunity, vol. 67, pp. 5231–
42, Oct. 1999.
[71] J. Sun, X. Wang, A. Lau, T.-Y. A. Liao, C. Bucci, and Z. Hmama,
“Mycobacterial nucleoside diphosphate kinase blocks phagosome
maturation in murine RAW 264.7 macrophages.,” PloS one, vol. 5, pp. 1–
12, Jan. 2010.
[72] H. H. Dar, D. Prasad, G. C. Varshney, and P. K. Chakraborti, “Secretory
nucleoside diphosphate kinases from both intra- and extracellular
pathogenic bacteria are functionally indistinguishable.,” Microbiology, vol.
157, pp. 3024–35, Nov. 2011.
[73] J. H. Miller, P. Funchain, W. Clendenin, T. Huang, A. Nguyen, E. Wolff,
A. Yeung, J. Chiang, L. Garibyan, M. M. Slupska, and H. Yang,
“Escherichia coli strains (ndk) lacking nucleoside diphosphate kinase are
powerful mutators for base substitutions and frameshifts in mismatch-
repair-deficient strains,” Genetics, vol. 162, pp. 5–13, Jun. 2002.
[74] K. T. Wright, R. Seabright, a Logan, a J. Lilly, F. Khanim, C. M. Bunce,
and W. E. B. Johnson, “Extracellular Nm23H1 stimulates neurite
outgrowth from dorsal root ganglia neurons in vitro independently of nerve
growth factor supplementation or its nucleoside diphosphate kinase
activity.,” Biochemical and biophysical research communications, vol. 398,
pp. 79–85, Jul. 2010.
85
[75] I. Andolfo, D. De Martino, L. Liguori, G. Petrosino, G. Troncone, N. Tata,
A. Galasso, C. Roma, F. Chiancone, S. Zarrilli, G. Arrigoni, S. Staibano, C.
Imbimbo, and M. Zollo, “Correlation of NM23-H1 cytoplasmic expression
with metastatic stage in human prostate cancer tissue.,” Naunyn-
Schmiedeberg’s archives of pharmacology, vol. 384, pp. 489–98, Oct.
2011.
[76] P. S. Steeg, L. Kopper, U. P. Thorgeirsson, E. Talmadge, L. A. Liotta, and
M. E. Sobep, “Tumor metastasis, the process by which cells leave a tumor
and colonize distant sites, is the major cause of death,” Journal of the
Nation Cancer Institute, vol. 88, pp. 200–204, Apr. 1988.
[77] A. J. Lilly, F. L. Khanim, R. E. Hayden, and Q. T. Luong, “Nm23-H1
Indirectly Promotes the Survival of Acute Myeloid Leukemia Blast Cells
by Binding to More Mature Components of the Leukemic Clone Nm23-H1
Indirectly Promotes the Survival of Acute Myeloid Leukemia Blast Cells
by Binding to More Mature,” Cancer research, vol. 71, pp. 1177–1186,
Feb. 2011.
[78] I. L. O. Buxton and N. Yokdang, “Extracellular Nm23 signaling in breast
cancer: Incommodus verum,” Cancers (Basel), vol. 3, pp. 1–12, Sep. 2011.
[79] B.-G. Han, K.-S. Min, B.-I. Lee, and S.-H. Lee, “Refined Structure of
Human NM23-H1 from a Hexagonal Crystal,” Bulletin of the Korean
Chemical Society, vol. 31, pp. 1397–1399, May 2010.
[80] I. M. C. van Amsterdam, M. Ubbink, M. van den Bosch, F. Rotsaert, J.
Sanders-Loehr, and G. W. Canters, “A new type 2 copper cysteinate azurin.
Involvement of an engineered exposed cysteine in copper binding through
internal rearrangement.,” The Journal of biological chemistry, vol. 277, pp.
44121–30, Nov. 2002.
[81] B. N. Taylor, R. R. Mehta, T. Yamada, F. Lekmine, K. Christov, A. M.
Chakrabarty, A. Green, L. Bratescu, A. Shilkaitis, C. W. Beattie, and T. K.
Das Gupta, “Noncationic peptides obtained from azurin preferentially enter
cancer cells.,” Cancer research, vol. 69, pp. 537–46, Jan. 2009.
[82] N. Bernardes, A. S. Ribeiro, S. Abreu, B. Mota, R. G. Matos, C. M.
Arraiano, R. Seruca, J. Paredes, and A. M. Fialho, “The Bacterial Protein
Azurin Impairs Invasion and FAK/Src Signaling in P-Cadherin-
Overexpressing Breast Cancer Cell Models.,” PloS one, vol. 8, pp. 1-8, Jul.
2013.
[83] K. Kushiro and N. Núñez, “Ethanol inhibits B16-BL6 Melanoma
Metastasis and Cell phenotypes associated with metastasis,” In Vivo, vol.
26, pp. 47–58, Feb. 2012.
86
[84] M. Xu, G. Chen, W. Fu, M. Liao, J. a Frank, K. a Bower, S. Fang, Z.
Zhang, X. Shi, and J. Luo, “Ethanol disrupts vascular endothelial barrier:
implication in cancer metastasis.,” Toxicological sciences : an official
journal of the Society of Toxicology, vol. 127, pp. 42–53, May 2012.
[85] J.-Y. Yang, C. S. Zong, W. Xia, Y. Wei, M. Ali-Seyed, Z. Li, K. Broglio,
D. a Berry, and M.-C. Hung, “MDM2 promotes cell motility and
invasiveness by regulating E-cadherin degradation.,” Molecular and
cellular biology, vol. 26, pp. 7269–82, Oct. 2006.
[86] N. Tikhmyanova and E. a Golemis, “NEDD9 and BCAR1 negatively
regulate E-cadherin membrane localization, and promote E-cadherin
degradation.,” PloS one, vol. 6, pp.1-10, Jan. 2011.
[87] S. Ramachandran, S. Sarkar, A. Mazumadar, and M. Mandal, “Azurin
Synthesis from Pseudomonas Aeruginosa MTCC 2453, Properties,
Induction of Reactive Oxygen Species, and p53 Stimulated Apoptosis in
Breast Carcinoma Cells,” Journal of Cancer Science & Therapy, vol. 3, pp.
104–111, Mar. 2011.
[88] H. Jung, H.-A. Seong, and H. Ha, “NM23-H1 tumor suppressor and its
interacting partner STRAP activate p53 function.,” The Journal of
biological chemistry, vol. 282, pp. 35293–307, Nov. 2007.
[89] S.-L. Chen, Y.-S. Wu, H.-Y. Shieh, C.-C. Yen, J.-J. Shen, and K.-H. Lin,
“P53 is a regulator of the metastasis suppressor gene Nm23-H1.,”
Molecular carcinogenesis, vol. 36, pp. 204–14, Apr. 2003.
[90] T. Desvignes, P. Pontarotti, C. Fauvel, and J. Bobe, “Nme protein family
evolutionary history, a vertebrate perspective.,” BMC evolutionary biology,
vol. 9, pp. 1–25, Jan. 2009.
[91] J. Yoon, P. Singh, D. Lee, J. Qiu, S. Cai, R. O. Connor, Y. Chen, B. Shen,
and G. P. Pfeifer, “Characterization of the 3’ to 5' exonuclease activity
found in human nucleoside diphosphate kinase 1 (NDK1) and several of its
homologues,” Biochemistry, vol. 44, pp. 15774–15786, 2005.
[92] D. Perina, M. H. Bosnar, R. Bago, A. Mikoč, M. Harcet, M. Deželjin, and
H. Cetković, “Sponge non-metastatic Group I Nme gene/protein - structure
and function is conserved from sponges to humans.,” BMC evolutionary
biology, vol. 11, p. 87, Jan. 2011.
[93] E. Suzuki, T. Ota, K. Tsukuda, A. Okita, K. Matsuoka, M. Murakami, H.
Doihara, and N. Shimizu, “nm23-H1 reduces in vitro cell migration and the
liver metastatic potential of colon cancer cells by regulating myosin light
chain phosphorylation.,” International journal of cancer. Journal
international du cancer, vol. 108, pp. 207–11, Jan. 2004.
87
[94] F. Liu, Y. Zhang, X.-Y. Zhang, and H.-L. Chen, “Transfection of the
nm23-H1 gene into human hepatocarcinoma cell line inhibits the
expression of sialyl Lewis X, alpha1,3 fucosyltransferase VII, and
metastatic potential.,” Journal of cancer research and clinical oncology,
vol. 128, pp. 189–96, Apr. 2002.
[95] M. T. Hartsough, D. K. Morrison, M. Salerno, D. Palmieri, T. Ouatas, M.
Mair, J. Patrick, and P. S. Steeg, “Nm23-H1 Metastasis Suppressor
Phosphorylation of Kinase Suppressor of Ras via a Histidine Protein
Kinase Pathway Nm23-H1 Metastasis Suppressor Phosphorylation of
Kinase Suppressor of Ras via a Histidine,” Journal of Biological
Chemistry, vol. 277, pp. 32389–32399, Jun. 2002.
[96] Q. Zhang, J. R. Mccorkle, M. Novak, and M. Yang, “Metastasis Supressor
Function of NM23-H1 Requires its 3’-5' Exonuclease Activity,”
International journal of cancer, vol. 128, pp. 40–50, Jan. 2012.
