CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF...

UNIVERSIDADE DE LISBOAFACULDADE DE CIÊNCIAS

DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA

CYTOTOXIC ACTIVITY AND MECHANISM OF ACTIONOF ORGANOMETALLIC COMPLEXES

Daniel Mosebo Fernandes Bandarra

Mestrado em Bioquímica

Área de especialização em Bioquímica Médica

2010

UNIVERSIDADE DE LISBOAFACULDADE DE CIÊNCIAS

DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA

CYTOTOXIC ACTIVITY AND MECHANISM OF ACTIONOF ORGANOMETALLIC COMPLEXES

Daniel Mosebo Fernandes Bandarra

Dissertação orientada por:Doutora Margarida MeirelesDoutora Mª José Calhorda

Mestrado em Bioquímica

Área de especialização em Bioquímica Médica

2010

Este trabalho foi realizado na Faculdade de Ciências da Universidade de Lisboa

Parte dos resultados incluídos no presente trabalho fazem parte do artigo científico já publicado [73].No cumprimento do disposto no nº2 do artigo 8º do Decreto-lei 388/07, declaro que participei naconcepção e na execução do trabalho que esteve na base do artigo, bem como na interpretação eredacção dos resultados.

Dedico este trabalho a todos os que procuram fazer a diferença neste Mundo, colaborando com assuas ideias e experiências de modo a contribuir para a sua evolução.

Jeg dedikerer dette arbejde til alle dem, der søger at gøre en forskel i denne verden, og somsamarbejder med deres ideer og erfaringer til at bidrage til dens udvikling.

I dedicate this work to all those who try to make a difference in this world, collaborating with theirideas and experiences in order to contribute to its evolution.

How wonderful that we have met with a paradox, now we have some hope of making progress

Niels Bohr

CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | xi

RESUMO

O cancro é uma das doenças com maior mortalidade a nível mundial e, de acordo com a Organização

Mundial de Saúde, prevê-se um aumento do número de mortes, podendo chegar a 12 milhões em

2030. Apesar desta tendência, muitos estudos têm sido feitos para a contrariar, nomeadamente ao

nível da quimioterapia. Actualmente a quimioterapia e a radioterapia constituem as opções mais

recorrentes para o tratamento de cancro, sendo exemplos de tratamentos que exploram a

sensibilidade das células tumorais a danos no DNA.

O uso de metais na medicina remota à Antiga China com o uso de ouro. Outros metais como a

cisplatina têm sido usados desde a segunda metade do século XX. De facto a cisplatina e os seus

análogos são os fármacos actualmente mais usados no tratamento de tumores sólidos, devido à sua

elevada eficácia. No entanto, apresentam diversas limitações, como o desenvolvimento de resistência

do organismo ou a sua elevada toxicidade. Por essa razão, novos complexos com outros centros

metálicos, como o ruténio, o vanádio ou o molibdénio, têm sido testados.

Vários estudos de agentes antitumorais com compostos com molibdénio têm sido feitos, tendo já

sido comprovadas as suas propriedades citotóxicas. No entanto o seu mecanismo de acção encontra-

se por se esclarecer.

O presente trabalho teve como objectivo o estudo das propriedades antitumorais de dois complexos

organometálicos com molibdénio, [Mo(3-C3H5)Br(CO)2(1,10-fenantrolina)], B1, e [Mo(3-

C3H5)CF3SO3(CO)2(2,2’-bipiridil)], T2 (Figura 1), e o estudo do mecanismo de acção citotóxica

destes complexos em linhas tumorais.

A actividade citotóxica de B1 e T2 foi estudada em três linhas tumorais: células HeLa (cancro do

colo do útero), células MCF-7 (cancro da mama) e RPE (epitélio pigmentado da retina imortalizada

pela expressão da proteína telomerase humana), através do ensaio do MTT (brometo de 3-(4,5-

dimetiltiazon-2-il)-2,5-difeniltetrazólio), um ensaio metabólico universalmente usado. Para o

complexo B1 foram obtidos valores de IC50 (inibição da viabilidade celular em 50%) entre 1 e 9 M

e para T2 entre 13 e 46 M para as três linhas celulares estudadas (Tabela 1). Estes valores

encontram-se na gama de valores considerados muito bons ao nível da citotoxicidade em linhas

tumorais, em partícular B1, que apresenta valores de IC50 semelhantes aos da cisplatina.

Figura 1 – Estrutura esquemática de B1 e T2.


RESUMO






























RESUMO





























xii | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES

CompostosIC50 (M)

HeLa MCF-7 RPE

T2 23,7 ± 0,01 44,5 ± 0,7 13,1 ± 2,4

B1 5,1 ± 1,0 8,9 ± 0,5 0,7 ± 0,1

Para compreender o mecanismo da acção citotóxica destes complexos em linhas tumorais, começou-

se por estudar a interacção dos complexos com a membrana plasmática através da determinação do

coeficiente de partição octanol/água dos complexos em solução. Ambos os complexos apresentam

um comportamento hidrofóbico em solução, sendo B1 mais hidrofóbico que T2. De forma a

perceber melhor a interacção composto-membrana plasmática, procedeu-se à quantificação de

molibdénio em células tumurais HeLa após a incubação destas com os compostos, durante 48 horas e

com diversas concentrações. Para além disso, nas mesmas condições, procedeu-se à quantificação de

molibdénio em fracções citosólicas e nucleares. Relativamente às células controlo (sem composto),

observou-se um aumento dos níveis de molibdénio quer no citosol, quer no núcleo, sendo este

aumento dependente da concentração de composto. Estes resultados parecem indicar que B1 e T2

possuem propriedades que lhes permitem entrar na célula e fundamentalmente no núcleo.

Face aos resultados obtidos, colocou-se a hipótese de que os complexos pudessem exercer o seu

efeito citotóxico através da interacção com o DNA, inibindo assim o crescimento das células

tumorais.

Procedeu-se a uma série de ensaios visando a detecção da interacção entre os compostos e o DNA.

Começou-se por estudar a interacção dos complexos com DNA plasmídico, através da realização de

electroforeses, em gel de agarose, de soluções de DNA plasmídico após incubação com os

compostos. Observou-se que ambos os complexos levam a alterações na mobilidade do DNA

palsmídico, sendo este efeito mais evidente para B1. Estes resultados sugerem que há interacções dos

compostos com o DNA.

Efectuaram-se também estudos de titulação de calf thymus DNA (ctDNA) por espectrofotometria

UV-Vis e por dicroísmo circular. Nos ensaios por espectrofotometria UV-Vis mediram-se variações

de absorvência após a adição de quantidades crescentes de ctDNA, fixando-se a concentração de

composto, com os respectivos controlos. Nos ensaios de dicroísmo circular fixou-se a concentração

de DNA e adicionaram-se quantidades crescentes de composto. Os resultados obtidos sugerem que a

interacção entre os compostos e o DNA seja maioritariamente por intercalação, não podendo no

entanto ser excluídos outros tipos de interacções. A partir da titulação determinaram-se as constantes

Quadro 1 – Estudos in vitro da actividade citotóxica dos complexos demolibdénio T2 e B1 em células Hela, MCF-7 [74] e RPE (os valorescorrespondem à média ± desvio padrão de três replicados)

CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | xiii

de ligação entre ctDNA e os complexos, tendo-se obtido valores de 2,08 (±0,98) × 105 e 3,68 (±2,01)

× 105 M-1 para B1 e T2, respectivamente. Os valores das constantes de ligação DNA-composto

encontram-se na mesma gama de valores obtidos para o brometo de etídeo, um intercalador clássico,

confirmando assim a natureza intercalativa do tipo de interacção entre os complexos e o DNA.

A microscopia de força atómica (AFM) de soluções de DNA plasmídico com e sem B1 permitiu

estudar possíveis transições estruturais do DNA face a interacções do complexo. Obtiveram-se

imagens topográficas onde se observaram alterações a nível da estrutura terciária do DNA

plasmídico aquando da presença de B1. Estes resultados são comparativos com os obtidos

anteriormente, onde é demonstrado a interacção dos complexos com o DNA. Infelizmente, devido a

limitações experimentais, não foi possível efectuar os estudos de AFM para o complexo T2.

Em suma, a realização deste trabalho permitiu, a partir dos estudos de actividade citotóxica,

identificar dois compostos como potenciais agentes antitumorais e esclarecer um dos possíveis

mecanismos responsáveis pela sua acção citotóxica em células tumorais. Tendo em conta os

resultados obtidos, conclui-se que ambos os compostos apresentam propriedades que lhes permitam

entrar na célula e chegar ao núcleo. Neste compartimento, os complexos levam à inibição do

crescimento celular através da sua interacção, maioritariamente por intercalação, com o DNA,

levando, possivelmente, a alterações ao nível da sua conformação e ultimamente à morte celular.

O presente trabalho mostra a potencialidade do uso de B1 e T2 na quimioterapia, podendo trazer

novas perspectivas nesta área, de modo a ultrapassar as limitações existentes actualmente, uma vez

que se trata de compostos metálicos com diferentes propriedades químicas relativamente aos

actualmente usados. A utilização destes ligandos e a possibilidade de combinar outros novos,

constituem uma mais valia no tratamento do cancro.

É de referir ainda a oportunidade que este trabalho proporcionou a publicação de um artigo científico

com o título: “Mo(II) complexes: A new family of cytotoxic agents?”, na revista Jounal of Inorganic

Biochemistry [73] (doi:10.1016/j.jinorgbio.2010.07.006).

http://dx.doi.org/10.1016/j.jinorgbio.2010.07.006

xiv | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES

Abstract

Antitumor properties of two molybdenum complexes, [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)]

(B1) and [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) have been tested in vitro against human

cervical cancer cell line (HeLa), human breast cancer cell line (MCF-7), and human telomerase

reverse transcriptase – retinal epithelial cells (RPE) using a metabolic activity test, MTT (3-(4,5-

dimethylthiazon-2-yl)-2,5-diphenyltetrazolium bromide), with IC50 values ranged from 1 to 9 M,

and from 13 to 46 M, approximately, for B1 and T2, respectively. In order to understand the

mechanism of action against cancer cell lines several studies have been made. Cellular uptake of

molybdenum and octanol/water partition assays revealed that both B1 and T2 exhibit a selective

uptake by cells with the ability to reach the nucleus, and a hydrophobic behavior in solution, B1

being more hydrophobic than T2. The interaction of the complexes with DNA was also studied.

According to gel electrophoresis studies, both complexes seem to interact with plasmid DNA. The

binding constants of B1 and T2 with calf thymus DNA (ctDNA), as determined by absorption

titration, are 2.08 (±0.98) × 105 and 3.68 (±2.01) × 105 M-1, respectively. These results together with

data obtained from circular dichroism suggest that the complexes interact with DNA, mainly by

intercalation, changing its conformation and possibly inducing cell death. Preliminary studies of

structural transitions in the tertiary structure of plasmid DNA using atomic force microscopy showed

that B1 seems to induce structural changes on the plasmid, plectonemic supercoiling being the

predominant form adopted by the plasmid. These results show that in future both complexes may

provide a valuable tool in cancer chemotherapy.

Keywords:

Molybdenum, antitumor activity, interaction with DNA, chemotherapy

CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 1

Index

1. Index of figures ............................................................................................................................3

2. Index of tables ..............................................................................................................................5

3. Abbreviations list.........................................................................................................................6

4. Introduction .................................................................................................................................7

4.1. The current state of cancer research ......................................................................................7

4.2. The hallmarks of chemotherapy ............................................................................................7

4.3. The limit to chemotherapy.....................................................................................................9

4.4. Medicinal inorganic chemistry ............................................................................................11

4.5. Mode of action of metal anticancer compounds..................................................................12

4.6. Experimental strategies .......................................................................................................13

4.7. Electronic absorption titration .............................................................................................13

4.8. Circular dichroism...............................................................................................................14

4.9. Atomic force microscopy (AFM) ........................................................................................14

5. Aim..............................................................................................................................................17

6. Experimental..............................................................................................................................19

6.1. Instrumentation and materials .............................................................................................19

6.2. Synthesis of molybdenum(II) complexes ............................................................................19

6.2.1. [Mo(3-C3H5)(CF3SO3)(CO)2(2,2’-bpy)] (T2) ............................................................19

6.2.2. [Mo(3-C3H5)(Br)(CO)2(1,10-phenanthroline)] (B1)..................................................20

6.3. Cell cultures.........................................................................................................................20

6.4. Subculture of cells ...............................................................................................................20

6.5. Cell quantification ..............................................................................................................20

6.6. Cryopreservation of cells.....................................................................................................21

6.7. Resuscitation of frozen cells................................................................................................21

6.8. Cytotoxic activity assay in vitro ..........................................................................................21

6.9. Octanol/water partition coefficient ......................................................................................22

6.10. Conductimetry .................................................................................................................22

6.11. Cellular molybdenum uptake...........................................................................................22

6.12. DNA binding studies .......................................................................................................23

6.12.1. Electronic absorption titration .....................................................................................23

6.12.2. Circular dichroism.......................................................................................................23

6.12.3. Gel electrophoresis studies ..........................................................................................23

6.12.4. Atomic force microscopy ............................................................................................24

7. Results and discussion...............................................................................................................25

7.1. Cytotoxic activity assay in vitro ..........................................................................................25

2 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES

7.2. Octanol/water partition coefficient ......................................................................................29

7.3. Conductimetry .....................................................................................................................30

7.4. Cellular molybdenum uptake ..............................................................................................31

7.5. DNA Binding Studies..........................................................................................................32

7.5.1. Electronic absorption titration .....................................................................................32

7.5.2. Circular dichroism.......................................................................................................34

7.5.3. Gel electrophoresis studies ..........................................................................................35

7.5.4. Atomic force microscopy ............................................................................................35

8. Conclusions ................................................................................................................................37

9. Acknowledgements ....................................................................................................................39

10. References ..................................................................................................................................41

11. Annex..........................................................................................................................................47


1. Index of figures

Figure 1 - Cisplatin and its analogues (Adapted from [17]). ............................................................................. 8Figure 2 - Dose-response relationships and proposed resistance mechanisms (Adapted from [24])............... 10Figure 3 – Drawing schemes of the transitions in plasmid DNA tertiary structure in response to an intercalatoragent: (a) predominantly relaxed; (b) toroidally supercoiled; (c) mixed toroidal and plectonemic supercoils; (d)complete plectonemic supercoiling (adapted from [59])............................................................................... 15Figure 4 - Schematic illustration of the overview of the work plan. Mo(II) complexes were tested and acted aspotent cytotoxic drugs, interacting with DNA in vitro. Do they enter the cell and directly damage DNA toinhibit cell growth? ...................................................................................................................................... 18Figure 5 – Haemocytometer (adapted from [62]) ......................................................................................... 21Figure 6 - Schematic structure of B1 and T2.................................................................................................. 25Figure 7 - In vitro cytotoxic assays for T2 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with T2 (right). ............................................................................. 25Figure 8 - In vitro cytotoxic assays for B1 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with B1 (right). ............................................................................. 26Figure 9 - In vitro cytotoxic assays for B1 against RPE cells (left). Dose-response curve obtained by nonlinearregression analysis for RPE cells treated with B1 (right)................................................................................ 26Figure 10 - In vitro cytotoxic assays for T2 against RPE cells (left). Dose-response curve obtained by nonlinearregression analysis for RPE cells treated with T2 (right). ............................................................................... 27Figure 11 - Dose-response curve obtained by nonlinear regression analysis for HeLa cells treated with B1 for1, 2, 24, and 48 hours (left). In vitro cytotoxic activity for B1 against HeLa cells at 1, 2, 24, and 48 hours (right)..................................................................................................................................................................... 28Figure 12 - Dose-response curve obtained by nonlinear regression analysis for HeLa cells treated with T2 for 1,2, 24, and 48 hours (left). In vitro cytotoxic activity for T2 against HeLa cells at 1, 2, 24, and 48 hours (right).29Figure 13 - Mean log octanol/water partition coefficients (Log P) of the molybdenum compounds. .............. 29Figure 14 - Specific conductivity of B1 in 0,5% DMSO in water solution......................................................... 30Figure 15 - Specific conductivity of T2 in water solution................................................................................ 30Figure 16 - Comparison of the intracellular molybdenum concentration in HeLa cells after 48 h of exposure tocompounds T2 and B1. ................................................................................................................................ 31Figure 17 - Molybdenum concentration in cytosolic (a) and nuclear (b) extracts of HeLa cells after 48 h ofexposure to compounds T2 and B1. ............................................................................................................. 32Figure 18 - Left: UV-Vis absorption spectra of T2 (20 M) in Tris buffer in the presence of increasing amountsof ctDNA. [DNA] = 0, 10, 20, 30, 40, 50 M. The arrow indicates the absorbance changes upon increasing DNAconcentration; right: plot of D/ap vs. D for the titration of DNA to complex. Absorbance was monitored at299 nm. ....................................................................................................................................................... 33Figure 19 - Left: UV-Vis absorption spectra of B1 (20 M) in Tris buffer in the presence of increasing amountsof ctDNA. [DNA] = 0, 10, 20, 30, 40, 50 M. The arrow indicates the absorbance changes upon increasing DNAconcentration; right: plot of D/ap vs. D for the titration of DNA to complex. Absorbance was monitored at272 nm. ....................................................................................................................................................... 34Figure 20 – Circular Dichroism of ctDNA incubated with B1 (left) and T2 (right). [Complex] = 0, 10, 25, 50, 75,100, 250 M. The arrow indicates the signal changes upon increasing complex concentration. .................... 35Figure 21 - Electrophoretic mobility pattern of pYES2 plasmid DNA (C) incubated with complex B1 (right) andT2 (left) at 10, 50 and 100 M...................................................................................................................... 35Figure 22 - A Selection of topographic images recorded of plasmid pYES2 (a) and plasmid pYES2 incubatedwith T2 (b) adsorbed to AP-mica. The scale bars correspond to 250 nm........................................................ 36Figure 23 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved inDMSO with no incubation time (0 hours)...................................................................................................... 47Figure 24 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved inDMSO after 2 hours of incubation time at 37 ºC. .......................................................................................... 48


Figure 25 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved inDMSO after 24 hours of incubation time at 37 ºC. ........................................................................................ 49Figure 26 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved inDMSO after 48 hours of incubation time at 37 ºC. ........................................................................................ 50Figure 27 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved inDMSO with no incubation time (0 hours)...................................................................................................... 51Figure 28 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved inDMSO after 2 hours of incubation time at 37 ºC. .......................................................................................... 52Figure 29 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved inDMSO after 24 hours of incubation time at 37 ºC. ........................................................................................ 53Figure 30 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved inDMSO after 48 hours of incubation time at 37 ºC. ........................................................................................ 54Figure 31 - Absorption spectra of B1 ([Mo(3-C3H5)Br(CO)2(1,10-fenantrolina)]). .......................................... 55Figure 32 - Absorption spectra of T2 ([Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipiridil)])............................................. 55


2. Index of tables

Table 1 - Drugs of Choice for Different Types of Cancer (Adapted from [5])..................................................... 9Table 2 - In vitro cytotoxicity assays for molybdenum complexes T2 and B1 against HeLa, MCF-7 [74], and RPEcells (data are mean ±SD of three replicates each)........................................................................................ 27


3. Abbreviations list

AFM – Atomic force microscopy

ALL – Acute lymphoblastic leukaemia

AML – Acute myelogenous leukemia

B1 – [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)]

BC – Before Christ

CD – Circular dichroism

ctDNA – calf thymus DNA

DCR – Dose-response curve

DMSO – Dimethyl sulfoxide

DNA – Desoxyribonucleotic acid

DTT – Dithiothreitol

EDTA – Ethylenediamine tetraacetic acid

FBS – Fetal bovine serum

FDA – Food and drug administration

HeLa – Human cervical cancer cell line

HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IC50 – Inhibitory concentration (dose causing 50% inhibition of cell growth)

ICP-MS – Inductively coupled plasma mass spectrometry

MCF-7 – Human breast cancer cell line

MDR – Multidrug resistance

MOPP – Mustargen-oncovin-procarbazine-prednisone

MTT – 3-(4,5-dimethylthiazon-2-yl)-2,5-diphenyltetrazolium bromide

NCI – American national cancer institute

PBS – Phosphate buffered saline

RNA – Ribonucleic acid

ROS – Reactive oxygen species

RPE – Human telomerase reverse transcriptase – retinal epithelial cells

RPMI – Roswell park memorial institute medium

T2 – [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)]

TAE buffer – Buffer solution containing a mixture of tris base, acetic acid and EDTA

TE buffer – Buffer solution containing tris and EDTA

WHO – World health organization


4. Introduction

4.1. The current state of cancer research

Cancer is one of the major causes of death worldwide [1]. According to World Health Organization

(WHO) cancer is the world’s second biggest killer, despite cancer being one of the research fields

where most money has been invested. As a result, many developments have been achieved regarding

the understanding of the pathogenesis of cancer [2]. Furthermore, properties such as unlimited

proliferation capacity, self-sufficiency in growth signals, insensitivity to growth-inhibitory signals,

evasion of programmed cell death (apoptosis), sustained angiogenesis, tissue invasion and metastasis

are considered to be a set of characteristics acquired during tumor development [3,4]. Even though

remarkable progress is being made in understanding the molecular, biochemical and cellular basis of

cancer, such knowledge has not been sufficient to cure the disease efficiently [5,6]. Improvements in

cancer treatment are however being achieved, namely on chemotherapy, which is the treatment of

choice for patients diagnosed in the late stages of locally and advanced widespread metastatic

cancers [4,5,7].

4.2. The hallmarks of chemotherapy

Chemotherapy emerged in the late 1940s and 1950s with the classical alkylating agents, nitrogen

mustard. First used as sulfur mustard gas during the First World War, it was applied to treat patients

with non-Hodgkin’s lymphoma, whose remission only lasted few weeks [5,8,9]. Cyclophosphamide

and chlorambucil were later developed to treat patients with lymphomas, leukaemias and solid

tumors; however these alkylating agents quickly proved inefficient, as tumors became resistant to

these drugs [8,9,10]. After the Second World War, new approaches arose with the synthesis of

antimetabolites, like methotrexate, a folate analogue which, as single agent, exhibited antitumor

activity against epithelial cancer, along with breast, ovarian, bladder, head and neck cancers. Folate

analogues also became the first drugs to induce successfully remission in patients with acute

lymphoblastic leukaemia (ALL), although the effect did not last long [5,8,10,11]. Methotrexate is

still mainly used on patients with ALL as well as other lymphomas. The application and research

done with these compounds have provided an important model for understanding mechanisms of

resistance for other agents. Further research led to the development of new drugs, such as vincristine,

a Vinca alkaloid, which was found to inhibit cell division [8,12,13]. In the 1960s several experiments

have shown that killing cancer cells might be cell cycle dependent, whereas some DNA synthesis

inhibitors (methotrexate) were more effective against rapidly dividing cells. On the other hand,

alkylating drugs, such as cyclophosphamide, that physically damage DNA, could kill cells in any

stage of the cell cycle. Furthermore, it was shown that cytotoxic activity was dose dependent, and it

was also important to use several therapies together to prevent drug resistance [8]. In the late 1960s

nitrogen mustard, vincristine, procarbazine and prednisone – known as the MOPP regimen – was


successfully used as combination chemotherapy to treat Hodgkin’s and non-Hodgkin’s lymphoma,

proving that drugs were most efficient when used as combination therapy and against tumors of

small volume [8,10,13, 14]. Barnett Rosenberg, a Michigan State University researcher, discovered

in 1964 that cisdiamminedichloroplatinum(II), known as cisplatin, could be used as a successful

agent in the therapy of testicular and ovarian cancer, after having received approval for clinical use

by Food and Drug Administration (FDA) [5,9,15,16] (Figure 1).

Figure 1 - Cisplatin and its analogues (Adapted from [17]).

Despite several successful reported cases using chemotherapy, cancer drug discoveries gained a

reputation of low efficacy and high toxicity risk, leading FDA to approve for marketing only 10%

(29 of 280) of new agents into Phase I (dose-finding) clinical trials in patients in the period of 1975

to 1994 [18]. During those years several new drugs were used in chemotherapy, such as

anthracyclines and epipodophyllotoxins, which are related to the topoisomerase II inhibition, an

enzyme essential for DNA replication, transcription and repair. In the early 1980s chemotherapy

progression decreased, which led the American National Cancer Institute (NCI), division of cancer

treatment, to create an innovative screening system, where panels of 60 human tumor cell lines were

used to test new drugs. This methodology is now widely used by industry, including a rapid

colorimetric cell viability assay, the MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium

bromide) assay, and high-throughput automated screening [8]. Recently, a new era arose with

targeted-therapy, whereas targets such as growths factors, signaling molecules, cell-cycle proteins,

apoptotic signals and molecules related to angiogenesis became a priority in chemotherapy (Table 1)

[4].


Table 1 - Drugs of Choice for Different Types of Cancer (Adapted from [5])

Drug Class Principal mechanism of action Example(s) Types of tumors commonlyused for

Alkylating agents To form covalent bonds with DNAand prevent DNA replication

CyclophosphamideCarmustineCisplatin

Non-Hodgkin’s lymphomaBrain gliobastomaOvarian, head, neck, lung andtesticular

Antimetabolites

Folate antagonist

Pyrimidine analogues

Purine analogues

To block or subvert pathways inDNA synthesisTo inhibit dihydrofolate reductase,preventing generation oftetrahydrofolate, thereby interferingwith thymidylate synthesisTo convert into nucleotides andinhibit thymidylate synthesis6-thiol analogues of the endogenous6-OH purine bases that becomeconverted into nucleotides

Methotrexate

Fluorouracil

MercaptopurineThioguanine

Acute lymphocytic leukemia(ALL)

Colorectal and gastric

Acute myelogenous leukemia(AML)

Antitumor antibiotics To interfere with topoisomerase IIaction, inhibiting DNA and RNAsynthesisTo cause fragmentation of DNAchainsTo intercalate in DNA, interfere withRNA polymerase and inhibittranscriptionTo act as an alkylating agent

Doxorubicin

Bleomycin

Dactinomycin

Mitomycin

Osteogenic sarcoma,Hodgkin’s disease, CML, softtissue sarcomaCervical

Wilms’ tumor

LungPlant alkaloidsVinca alkaloids

Podophyllotoxins

Taxoids

Camptothecins

To inhibit mitosis at metaphase bybinding to tubulinTo inhibit DNA synthesis byinterfering with topoisomerase II, andalso mitochondrial functionTo promote the polymerization oftubulin and inhibit the disassembly ofmicrotubulesTo inhibit topoisomerase I and DNAand RNA synthetases, and alsomicrotubule formation

Vincristine,vinblastineEtoposide

Taxol

Irinotecan, topotecan

Lung, Non-Hodgkin’slymphomaLung, Kaposi’s sarcoma

Ovarian, breast and lung

Refractory colorectal andadvanced ovarian

Nowadays better drugs have been developed and knowledge regarding cancer has grown, so that the

new challenge is to design new strategies where targeted-therapy and cytotoxics can be combined in

the most effective manner [8].

4.3. The limit to chemotherapy

Cytotoxic agents of chemotherapy are conditioned by several factors. Drug metabolizing enzymes

and drug transporters are essential in the understanding of the overall process where antitumor

compounds are involved [19,20]. It is also important to understand the side effects of these agents in

order to make them less toxic to the organism without significant cytotoxic activity loss [21]. Thus it

would be crucial to achieve a balance between drug sensitivity and resistance displayed by target

tumor cells to maximize the efficacy and minimize the toxicity of treatment [21,20]. Although the

use of chemotherapy has improved quality of life and prolonged survival for many cancer patients,


its potential to induce toxicity is also predictable. Several side effects arise, the most common being

gastrointestinal toxicity, alopecia (such as hair loss) and myelosuppression [21]. Such malignancies

are due to the fact that the target of most chemotherapeutic agents consists of rapidly dividing cells,

leading adverse effects into normal cells with a high growth proliferation, such as hair cells. Patients

whose tumor does not respond to the therapeutic agents seem to possess resistance mechanisms,

which are considered to be the major limitation for successful cancer treatment [22,72].

Chemotherapeutic resistance can be classified as ‘acquired’ or ‘intrinsic’. Intrinsic resistance limits

the usefulness of therapeutic agents, while acquired resistance might be related to a resistance

achieved after an initial successful progress, which somehow became powerless [22,23,72]. These

mechanisms of resistance can also be reflected by dose-response relationships: if the dose-response

curve (DCR) has a “shoulder” it will be classified as ‘active’, otherwise ‘passive’ results in a DRC

terminal plateau (saturable passive) or in a decreased DRC slope (non-saturable passive) (Figure 2,

[24]).

Figure 2 - Dose-response relationships and proposed resistance mechanisms (Adapted from [24]).

Examples of active resistance would be efflux pumps, DNA repair systems, anti-apoptotic factors,

etc. Passive resistance would include drug uptake or activating systems, proapoptotic factors or

factors that are part of the apoptotic cascade, cells in a sensitive phase of the cell cycle, etc.

Furthermore, several factors may contribute to resistance: blood flow and drug delivery, extracellular

environment, drug efflux, drug uptake, drug detoxification, drug binding, DNA repair, decreased

DNA mismatch repair, reduced apoptotic response, apoptosis inhibitors, etc [22,24,25]. Although

these mechanisms can act individually, they can also act synergistically, resulting in multidrug


resistance (MDR) [26,72]. To diminish side-effects and reduce propensity to induce drug resistance,

novel antitumor compounds have been designed in order to overpass chemotherapy limitations.

4.4. Medicinal inorganic chemistry

Metals are essential to many life-related processes and their use as therapeutic agents can be traced

back to 2500 BC by the ancient Chinese, who have attributed medicinal proprieties to gold.

Paracelsus (1493-1541), considered to be the ‘true father of modern metallotherapy’, used several

heavy metals to treat patients with different malignancies, such as cancer [9,27,28]. Since then

several reports have described the use of metals in medicine. As already mentioned, the First World

War has changed the era of chemotherapy with the cytotoxic action of nitrogen mustards established

years after, and further investigation led to the development of efficient antitumor drugs, such as

cisplatin and its derivatives [8,9,15]. Nowadays they continue to be the most frequently used

cytostatic drugs worldwide, constituting the main component of combination chemotherapy for the

treatment of solid carcinomas [29]. This knowledge led to the development of other metal

compounds with cytostatic activity, the groups 13 to 15 of the periodic table and certain transition

metals of groups 4 to 11 being very active [9]. Metals can bind to organic fragments affording

organometallic compounds, whose chemistry offers a rich field in medicine with a high potential to

develop effective antitumor agents [30]. Furthermore, the limited selectivity and toxicity of metals

alone can be overcome by the formation of organometallic compounds. Features such as the metal

coordination numbers, the coordination geometries, the redox potential and the thermodynamic and

kinetic properties have to be considered on the design of organometallic agents suitable for

successful interaction with biological molecules [15,29]. This variety of options provides unlimited

possibilities of combination that results in an extensive spectrum of mechanisms open to

organometallic compounds.

Although platinum complexes are widely used for the treatment of cancer, the appearance of drug

resistance among the drug toxicity induced and its side-effects have led to the development of other

non-platinum drugs, metal-based compounds, in order to overcome platinum compounds limitations,

namely ruthenium, vanadium and molybdenum compounds [9,15,29,30].

Molybdenum is an essential trace element commonly used in the cell as cofactor by important

enzymes (e.g. xanthine oxidase/dehydrogenase). In 1979, Köpf and Köpf-Maier reported the

antitumor action of several metal-based complexes with Mo [31]. Since then a number of

molybdenum containing molecules have been described to display cancerostatic activity. These

include Na2MoO4, heteropolyacid Mo salts, polyoxomolybdenum complexes, Mo complexes bound

to small carborane ligands, and chiral octahedral complexes [32,33,34,35]. Portuguese investigators

in 2005 studied several molybdenum(II) compounds, concluding that they were very efficient

cytotoxic agents against six cell lines, and filed a patent [36]. The mechanisms of action of most of

the organometallic complexes of molybdenum are far from being understood. Since cell growth is


slowed down by those compounds, it seems conceivable that the inhibitory activity might in some

way be related to DNA damage. This can be due to a direct action on the DNA molecule (eg.

intercalation in the double helix) or by the oxidative action of oxygen free radicals generated by the

chemical agents (chapter 4.5).

4.5. Mode of action of metal anticancer compounds

Metal compounds have a high propensity to bind and to interact with many important biological

molecules, e.g. DNA, which is considered to be the primary intracellular target of the antitumor

agents [17,27,28,37]. On the other hand, organometallic compounds can act as enzyme inhibitors or

modulators blocking substrate interaction [38]. Other possible mechanism can be the generation of

endogenous reactive oxygen species (ROS), which can directly or indirectly induce oxidative DNA

damage [38,39]. Therefore covalent and noncovalent interactions can be considered in the interaction

of the organometallic compounds with DNA. Thus, covalent interactions lead to complete inhibition

of DNA processes and they usually are irreversible, contrarily to noncovalent bindings which are

reversible [40,41]. One example of a covalent binder is the chemotherapeutic agent cisplatin and its

analogues (Figure 1), which form covalent bonds between platinum and the nitrogen atom of some

of the DNA base pairs [17]. Additionally three major noncovalent interactions can be found: minor

groove binding, major groove binding and intercalation. However, one complex can participate in

more than one mode of interaction, as actinomycin D, a well-known transcription inhibitor [41,42].

These metal compounds-DNA interactions, ultimately, trigger programmed cell death [27,37].

A classification of metal anticancer compounds based on their mode of action has been suggested

[43]. This classification relies on the metal compounds, rather than the nature of the compound

targets (e.g. proteins, DNA, enzymes, transporters, cellular transduction pathways, etc.), since the

knowledge about their interaction is poorly understood and unreliable. Thus five classes were

created:

1) The metal as a functional role

2) The metal as a structural role

3) The metal as a carrier of ligands

4) The metal as a catalyst

5) The metal as a photosensitizer

One of the most successful metals antitumor compounds, cisplatin, with an antitumor activity

associated with interaction with DNA, forming intrastand crosslinks to adjacent guanosine residues,

leading to cell apoptosis [17,28,37], belongs to the first class. The cytotoxic activity of compounds

belonging to this class depends essentially of the thermodynamic and kinetic parameters of the metal

center and the nature of the ligand. The main disadvantage of these compounds is related to the high

toxicity caused by uncontrolled reactivity with molecules other than their targets. The second class


compounds should not bind directly to the biological molecules (e.g. non-covalent interactions with

DNA) and be less toxic than functional compounds [43]. One of the most successful drugs belonging

to this class is the antimalarial ferroquine. Several other platinum compounds can intercalate into

DNA rather than forming covalent adducts as cisplatin does. Compounds of the third class carry a

ligand, which is delivered in vivo. The ligand is protected by the metal until its delivery. The metal as

a catalyst is associated to the generation of ROS that cause cell damage [28,39]. Several metal

compounds need to be primarily induced to be antitumor active (e.g. ruthenium complexes with

polypyridine ligands) [17,43]. Although several classes have been created, all the compounds used in

clinical practice belong to the first class, and no compounds, from any of the others classes, are

expected to enter in clinical use hereafter, even though a lot of research in this field is being made in

order to develop new drugs [43].

4.6. Experimental strategies

Several approaches can be used in order to study possible mechanisms by which antitumor metal

compounds inhibit cell proliferation, particularly by interactions with DNA [44,45]. Gel

electrophoresis studies constitute a rapidly and economical mode to evaluate the effect of metal

complexes on DNA tertiary structure. Absorption titration and circular dichroism are universally

used to examine the binding mode of DNA-complexes [42,46]. The analysis of the transitions in the

tertiary structure of plasmid DNA induced by metals complex constitutes a complementary approach

to the DNA-complex interaction study and it can be achieved by using atomic force microscopy

(AFM) [47].

4.7. Electronic absorption titration

Electronic absorption titration spectroscopy is one of the most useful techniques and widely used in

DNA binding studies [44]. The mode of interaction can be evaluated by the changes on the

absorption spectra. Thus, if the complex intercalates in DNA, a red-shift (bathochromism) along with

a decrease in the intensity of the complex spectral band (hypochromism) can be observed. The

existence of a bathochromism is also indicative of the stabilization of DNA duplex [48,49]. On the

other hand, the increase in the intensity of the complex absorption band (hyperchromism) is related

to the damage of the secondary structure of DNA. Then, hyperchromism and hypochromism are

related to spectral features of the double helix structure of DNA, where hypochromism means that

the DNA-binding mode of complex may be due to electrostatic effect or intercalation and

hyperchromism is related to the loss of secondary structure of DNA.

The complex-DNA binding strength can be evaluated by the intrinsic binding constant, K, which

represents the binding constant per DNA base pair [50]. In order to determine the binding constant,

the neighbor exclusion model was primarily used by Benesi and Hildebrand [76]. It was limited to

molecules that occupy only one binding site, and does not correspond to most biological ligands


which interact with more than one base pair in the DNA [41]. Schemechel and Crothers [77] adapted

the previous model and Wolf et al applied it successfully to determine K according to the following

equation (Eq 1) [48,50,52]:

[DNA]( a f) = [DNA]( b f)+ ( b f) (Eq 1)

where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficients a, f

and b correspond to Absorbance observed/[complex], the extinction coefficient for the free complex

and the extinction coefficient for the free complex in the fully bound form, respectively. K is given

by the ratio of slope to intercept in plots of [DNA]/(a-f) versus [DNA].

4.8. Circular dichroism

Circular dichroism (CD) results from the difference between left- and right-handed polarized

components of the incident light absorbed by chiral molecules. Those components are absorbed

differently by the sample, leading to a difference in the absorption coefficients: = left-right (M-1

cm-1). The signal is measured in units of cm2g-1 although it is widely used as ellipticity () [53]. CD

can be used to follow rapidly and efficiently DNA conformational transitions, which constitute an

important tool to evaluate with high selectively the behavior of complexes that recognize DNA

structures or sequences [49,53]. For these reasons CD represents a further source of useful

information in the study of the interactions of metals complexes with DNA. The most common DNA

conformation observed is the B-form, which is characterized by positive bands at about 260-280 nm

and a negative band at around 245 nm. Since the base pairs of DNA are perpendicular to the double-

helix axis in the B-form, the peak intensities are relatively small, considering the weak chirality of

the molecule [49,54,55]. Nevertheless, CD being an extremely sensitive method, small

conformational changes (e.g. due to the interaction with metal complexes) can be measured

efficiently [53]. The intercalation of the complex in DNA enhances the intensities of both the bands

stabilizing the DNA conformation, as is commonly observed for the classical intercalators [46]. On

the other hand, simple groove binding and electrostatic interactions would lead to no perturbations or

minimal perturbations in both bands of B-DNA, because these binding modes do not influence the

secondary structure of DNA [45,49,55].

4.9. Atomic force microscopy (AFM)

Atomic force microscopy has become a successful technique to investigate DNA structure and

dynamics at very high resolution [56]. AFM is an ultra-high resolution microscopic technique that

does not require complicated sample preparation or any other complex treatment [47]. It can be used

to study DNA tertiary structure as well as the influence of factors that induce conformational

transitions, such as intercalator complexes (e.g. ethidium bromide) [57]. Recent publications have


shown the potential of AFM to investigate the interactions of DNA with anti-tumor agents of metal

complexes (e.g. cisplatin), where conformational structure changes were evaluated [58]. Furthermore

studies carried out in order to study the effect of ethidium bromide, a classical DNA intercalator on

DNA plasmid has led to the definition of some schematic structures (Figure 3) [59]. Four structures

were considered: predominantly relaxed, toroidally supercoiled, mixed toroidal and plectonemic

supercoils, and complete plectonemic supercoiling. Hence, with the interaction of metal complexes

intercalators the DNA plasmid would become increasingly supercoiled.

Figure 3 – Drawing schemes of the transitions in plasmid DNA tertiary structure in

response to an intercalator agent: (a) predominantly relaxed; (b) toroidally

supercoiled; (c) mixed toroidal and plectonemic supercoils; (d) complete plectonemic

supercoiling (adapted from [59]).


5. Aims

Several metal-based complexes with molybdenum were reported as having antitumor and

cancerostatic activity [31,36]. The mechanisms of action of most of the organometallic complexes of

molybdenum are far from being understood, although they might in some way be related to DNA

damage [43].

The present study aimed at evaluating the antitumor activity of two molybdenum complexes, B1

([Mo(3-C3H5)Br(CO)2(1,10-phen)]) and T2 ([Mo(3-C3H5)CF3SO3(CO)2(2,2’-bpy)]), and study

their mechanism of action against cancer cell lines.

To evaluate the cytotoxic activity of the complexes a metabolic activity test (MTT) was used.

Octanol/water partition assays and the determination of intracellular molybdenum were carried out in

order to understand how easily the complexes could pass through the membrane and where may start

the process that triggers cell death. Several other studies regarding the interactions between the

complexes and DNA were also performed, namely absorption titration and circular dichroism.

Complementary approaches, such as gel electrophoresis studies and atomic force microscopy, were

used to contribute to the study of the binding mode of DNA-complexes.

Understanding the mechanism of action of B1 and T2 will potentially constitute a valuable tool in

cancer chemotherapy in order to reduce drug resistance of the chemotherapeutic agents used

nowadays and to overcome some chemotherapy limitations. An overview of the work plan is

illustrated in Figure 4.


Figure 4 - Schematic illustration of the overview of the work plan. Mo(II) complexes were tested and acted as potentcytotoxic drugs, interacting with DNA in vitro. Do they enter the cell and directly damage DNA to inhibit cell growth?






6. Experimental

6.1. Instrumentation and materials

Commercially available reagents and all solvents were purchased from standard chemical suppliers.

Octanol was purchased from Riedel-de Haën, Germany. The RPMI 1640 cell culture medium, fetal

bovine serum (FBS) were purchased from LONZA Co. MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-

diphenyl tetrazolium bromide) was purchased from Sigma Chemical Co, USA. Calf thymus DNA

(ctDNA) was purchased from Sigma Chemical Co. Ltd. and a stock solution was prepared by

dilution in a buffer solution (50 mM NaCl/5 mM Tris-HCl, pH 7.1) followed by stirring at 4 ºC for

two days. This solution was stored at 4 ºC. The stock solution of ctDNA gave a ratio of UV

absorbance at 260 and 280 nm (A260/A280) > 1.8, indicating that the DNA was sufficiently free of

protein contamination [60]. The DNA concentration was determined by the UV absorbance at 260

nm after 1:10 dilution using = 6600 M-1 cm-1 [61]. MTT was dissolved (5 mg/ml) in phosphate

buffer saline pH 7.2.

Infrared spectra were measured on a Mattson 7000 FT spectrometer. Samples were run as KBr

pellets. NMR spectra were recorded on a Bruker Avance-400 spectrometer in CDCl3 or deuterated

DMSO. Elemental analyses were carried out at the University of Vigo, Spain. UV-Vis spectra were

recorded on a Shimadzu UV-2450 equipped with a Peltier cell for temperature control.

6.2. Synthesis of molybdenum(II) complexes

6.2.1. [Mo(3-C3H5)(CF3SO3)(CO)2(2,2’-bpy)] (T2)

Thallium triflate (TlCF3SO3) (0.353 g, 1 mmol) was added to a solution of [MoBr(3-

C3H5)(CO)2(2,2’-bipyridyl)] (0.429 g, 1 mmol) in acetonitrile (20 ml), and the mixture was refluxed

for 5 hours. A white solid of TlBr was formed and filtered with celite. The solid was washed 3 times

with acetonitrile. The filtrate was evaporated and the solid residue dissolved in dichloromethane.

Addition of n-hexane resulted in the formation of red crystals after a few days [73].

Yield: 72% (0.359 g)

IR (KBr disc) (cm-1): 3436; 3069; 1947; 1863; 1602; 1573; 1495; 1474; 1441; 1389; 1312; 1302;

1287; 1237; 1219; 1174; 1158; 1127; 1109; 1077; 1034; 930; 795; 764; 734; 657; 650; 630; 577;

570; 516; 504; 439; 418.

1H NMR (400 MHz, DMSO-d6): 1.63 (d, Hanti); 3.76 (d, Hsyn); 4.06 (m, Hmeso); 7.69 (t, H3/H6); 8.08

(t, H2/H7); 8.16 (d, H4/H5); 9.2 (s, H1/H8).


6.2.2. [Mo(3-C3H5)(Br)(CO)2(1,10-phenanthroline)] (B1)

A solution of 1,10-phenantroline (2 mmol, 0.3965 g) was added to a stirring solution of (3-

C3H5)(CO)2(MeCN)2] (0.7100 g, 2 mmol) in dicholoromethane (20 ml) and the mixture was kept

stirring overnight. The red precipitate formed was washed with dichloromethane (10 ml), diethyl

ether (10 ml) and dried under vacuum.

Yield: 87% (0.7875g)

IR (KBr disc) (cm-1): 1926; 1863; 1832; 1626; 1463, 1426;

1H NMR (400 MHz, CDCl3): 1.51 (d, Hanti); 2.86 (m, Hmeso); 3.14 (d, Hsyn); 4.15 (m Hmeso); 7.82 (m,

H7); 7.96 (s, H4/H5); 8.01 (s, H2); 8.5 (m, H3/H6); 9.23 (t, H8); 9.4 (s, H1);

13C NMR (400 MHz, CDCl3): 53 (Csyn); 54 (Canti); 62 (Cmeso); 124 (C7); 127 (C2/C4/C5); 136 (C3/C6); 151 (C8).

6.3. Cell cultures

Different cell lines were used in order to study intracellular processes, as the cytotoxic activity of the

molybdenum complexes synthesized (Chapter 6.2), as well as their mechanism of action. Thus

HeLa (cervical carcinoma), MCF-7 (breast carcinoma) and hTERT-RPE1 (human telomerase reverse

transcriptase – retinal carcinoma) were maintained in RPMI 1640 supplemented with 10% fetal

bovine serum (FBS), 200 U/ml penicillin, 100 µg/ml streptomycin and 0.3 g/ml L-glutamine in a

humidified atmosphere of 95% air /5% CO2 at 37º C. All cells culture procedures were carried out in

a culture cabinet under sterile conditions, as well as all the material used sterilely.

6.4. Subculture of cells

In order to maintain the cells in a healthy and viable state they were subcultured, process also known

as passaging. Thus the adherent cells were harvested enzymatically from culture dishes occupied 80

to 90% of the surface with cells (confluent state). Thus, trypsin was used to detach the monolayer of

adherent cells and, after diluted in phosphate buffered saline (PBS), it was inactivated by the addition

of medium containing serum. The suspension of cells was then distributed to new culture dishes and

incubated in a humidified atmosphere of 95% air /5% CO2 at 37º C.

6.5. Cell quantification

Seed with the appropriate seeding density is crucial to reach the optimum growth. One way to

quantify cells is by using a haemocytometer, which is both simple and cheap. It contains 9 large

squares and inside it has 16 small squares. Each large square measures 1 mm x 1 mm and is 0.1 mm

deep (Figure 5).


Figure 5 – Haemocytometer (adapted from [62])

Furthermore each square has a volume of 0.1 mm3 and from this it is possible to determine the

concentration of cells and the total number of cells per cubic centimeter. Then cells are counted in

each large square and the average of cells is calculated to increase the accuracy. The count can be

converted to the number of cells per mL of suspension according to the following equation (Eq 2).

Cells (cells/mL) = Average of the number of cells counted × 10 (Eq 2)

6.6. Cryopreservation of cells

One way to preserve cells is by freezing stocks in liquid nitrogen. This process, known as

cryopreservation, grants a renewable source of cells for later use. To avoid the formation of ice

crystals inside the cell and changes in pH, DMSO is used to lower the freezing point. In order to

accomplish a successful cell cryopreservation a freezing chamber (‘Mr Frosty’) was used to cool

down slowly from room temperature to -80 ºC at a rate of 1-3 ºC per minute. Cells were then

harvested and resuspended in a solution of 60% medium and 40% of serum, from which was added

DMSO (10%) and stored in vials in a ‘Mr Frosty’ cryo freezing container. After that it was placed in

a -80 ºC freezer at overnight and hereafter transferred to liquid nitrogen.

6.7. Resuscitation of frozen cells

In order to revive frozen stocks from cryogenic vials stored in liquid nitrogen it is necessary to warm

up the vials at 37 ºC for 1-2 min, avoiding that cells warm up to 37 ºC, otherwise they may rapidly

die. The cell suspension is then added to fresh growth medium pre-warmed and placed in a culture

flask, which is incubated in a humidified atmosphere of 95% air /5% CO2 at 37º C.

6.8. Cytotoxic activity assay in vitro

In order to determine the cell viability towards molybdenum complexes, cytotoxic activity assay was

performed by the MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method


previously described with some modifications [63]. Exponentially growing cells were seeded at a

density of approximately 4x105 cells/ml, in a 96-well flat-bottomed microplate, and incubated for 48

h in an atmosphere of 5% CO2/95% air at 37ºC. Then the cells were treated with the complexes and

incubated for 48 h. Compounds were dissolved in the culture medium with 0.5% DMSO and tested

in concentrations ranging from 1 to 1000 M. Control wells contained supplemented media with

0.5% DMSO. After incubation time MTT solution (100 L, 0,5 mg/mL) was added into each well,

and incubated for 2 hours at 37ºC h in an atmosphere of 5% CO2/95% air. After medium removal,

100 L DMSO was added to dissolve the formazan crystals. The optical density was measured at

570 nm using a 96-well multiscanner autoreader. The IC50 values (concentration that caused 50%

growth inhibition) were calculated by non-linear regression analysis. For the kinetic studies, cell

cultures were exposed to different concentrations of compounds for different periods of time (1, 2,

24, and 48 h), after which the medium with the drug was removed and was replaced by fresh

medium. Each experiment included ten replicates for the different concentrations of complexes and

results represent at least 3 independent experiments. UV-Vis and mass spectrometry spectra of

solutions with appropriate concentrations were measured during this period and showed no

decomposition of the complexes being studied (Annex).

6.9. Octanol/water partition coefficient

Water-saturated octanol and octanol-saturated water were prepared by shaking equal volumes of

octanol and water for 5 hours and allowing the mixture to separate into the respective phases for 24

hours. Solutions of molybdenum complexes (20 M) were prepared in water-saturated octanol and

their absorbance was analyzed by UV spectrophotometry. Three and six milliliters of complex

solution were then added to 40 ml of octanol-saturated water. These solutions were shaken

vigorously for 2 hours. The aqueous phase was separated ensuring that there was no contamination

from the octanol phase, and each of these solutions was analyzed by UV spectrophotometry to obtain

the absorbance of the compounds.

6.10. Conductimetry

The specific conductivity of molybdenum complexes solutions was measured at 25ºC using a

Radiometer Copenhagen – Meterlab CMD 230 conductimeter. The relative uncertainty in

determining the specific conductivity of the compounds solution was within 0.5%. The repeatability

of the conductivity measurement, estimated from two successive runs, was about ± 3. The

conductance reading was checked every 20 s until it reached a steady value.

6.11. Cellular molybdenum uptake

The protocol used was as previously described with some modifications [64]. HeLa cells were

seeded in 100 mm dishes at 4x105 cells/ml and incubated at 37 ºC in an atmosphere of 5% CO2/95%


air for 48 h. The culture medium was removed and replaced with medium containing the

molybdenum complex at a concentration of 0, 10, 50 and 100 M for 48 h. Following treatment, the

cell monolayer was scraped off the culture dishes and the molybdenum content was determined by

inductively coupled plasma mass spectrometry (ICP-MS) by standard protocol at the University of

Vigo, Spain.

Molybdenum content of cytosolic and nuclear extracts was also analyzed. Cells were washed twice

with PBS buffer and lysed with cytosolic lysis buffer (HEPES 50 mM, pH 7.2, EDTA 2 mM, NaCl

10 mM, sucrose 250 mM, DTT 2 mM). Cells were then scraped up and centrifuged (3000 g, 4 min,

4ºC) (supernatant - cytosolic fraction). The pellet was washed with cytosolic buffer and

ressuspended with nuclear lysis buffer (HEPES 50 mM, pH 7.2, EDTA 2 mM, NaCl 400 mM,

glycerol 20% (v/v), DTT 2 mM) (pellet - nuclear fraction). The molybdenum content of both

fractions was determined by ICP-MS at the University of Vigo, Spain.

6.12. DNA binding studies

6.12.1.Electronic absorption titration

Calf thymus DNA (ct DNA) solutions of various concentrations (0 – 100 M) were added to 20 M

buffered solutions (5 mM Tris, 50 mM NaCl, pH 7.2) of the metal complexes. Absorption spectra

were recorded after equilibration at 37.0 ºC for 10 min. The intrinsic binding constant, K, was

determined according to Eq 1 (Chapter 4.7).

6.12.2.Circular dichroism

The CD spectra of ctDNA (500 M) in the absence and presence of molybdenum complexes at

various concentrations (0, 10, 25, 50, 75, 100, 250 M) were recorded on a Jasco J810

spectropolarimeter at 37ºC (Julabo F25 temperature control unit). The region of wavelength between

220-360 nm was scanned for each sample using a 1 mm path quartz cell, and the result was displayed

in millidegreed (deg).

6.12.3.Gel electrophoresis studies

E. coli. Bacteria were transformed with pYES2 DNA plasmid (5856 nucleotides, multiple cloning

site, ampicillin resistance gene) by prior treatment with Ca2+ at 4ºC in order to become competent.

DNA was added to the suspension of competent cells and taken up during a brief increase in

temperature (heat shock). After a brief incubation to allow expression of the antibiotic resistance

genes the cells were plated onto medium containing the antibiotic, ampicillin. The plasmid was then

isolated from the bacteria using a kit from GE Healthcare and stored at 4ºC. The pYES2 was

incubated with various concentrations (0, 10, 50 and 100 M) of molybdenum complexes (B1 and

T2) in TE buffer (Tris HCl 10 mM, EDTA 1 mM, pH 8.0). DNA digested with Hind III and plasmid


without complexes were used as marker and control, respectively. Samples were incubated for 10

min. After incubation samples were run on an agarose gel (1% in TEA Buffer [Tris acetate 40 mM,

EDTA 1 mM, pH 8.0]) stained with ethidium bromide, and imaged with a common transilluminator.

6.12.4.Atomic force microscopy

Squares of mica were stuck to steel discs ready for mounting samples onto the AFM instrument. The

mica squares were cleaved with adhesive tape immediately prior to use. A method involving divalent

cations to bridge between the negatively charged mica substrate and DNA backbone was used. The

plasmid, pYES2, was diluted in a solution of Tris-HCl (20 mM, pH 7.5), MgCl2 (5 mM), and B1 (20

M). Solution of plasmid diluted in Tris-HCl and MgCl2 was used as control. The solution was then

spotted directly onto freshly cleaved mica and, following a 2 min incubation period, the mica was

gently rinsed with H2O and blown dry with compressed N2.


Figure 7 - In vitro cytotoxic assays for T2 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with T2 (right).

7. Results and discussion


To evaluate the potential antiproliferative activity of the molybdenum complexes T1 and B2 (Figure

6), human cervical cancer cell line (HeLa), human breast cancer cell line (MCF-7) [74], and human

telomerase reverse transcriptase – retinal epithelial cells (RPE) were incubated for 48 hours with

varying concentrations of compounds and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) assay was used .

The well known MTT assay measures cell viability in terms of metabolic turnover, as indicated by

the oxidation of MTT to purple formazan by mitochondria. The relation between cell viability and

compound concentration obtained for HeLa and RPE cells treated with compound T2 and B1 was

determined (Figure 7 - 10, left).

0

50

100

0 1 10 25 50 100

% C

ell V

iabi

lity

[T2] ( M)

Figure 6 - Schematic structure of B1 and T2.














100 250 500 1000[T2] ( M)

















Figure 8 - In vitro cytotoxic assays for B1 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with B1 (right).

Figure 9 - In vitro cytotoxic assays for B1 against RPE cells (left). Dose-response curve obtained by nonlinear regressionanalysis for RPE cells treated with B1 (right).

From the relation between cell viability and compound concentration, dose-response curves obtained

by non linear regression analysis for HeLa and RPE cell lines treated with the complexes were done

in order to determine the IC50 value (final concentration ≤0.5% DMSO), which corresponds to the

concentration of compound required to inhibit cell proliferation by 50% (Figure 7-10, right).

0

50

100

0 1 10 25 50 100 250 500 1000

% C

ell V

iabi

lity

[B1] ( M)

0

50

100

0 1 2.5 5 10 20 30 50 100

% C

ell V

iabi

lity

[B1] ( M)


Figure 10 - In vitro cytotoxic assays for T2 against RPE cells (left). Dose-response curve obtained by nonlinearregression analysis for RPE cells treated with T2 (right).

The IC50 values of B1 and T2 against HeLa, MCF-7 [74], and RPE were then calculated from the

dose-response curves (Table 2).

Table 2 - In vitro cytotoxicity assays for molybdenum complexes T2 and B1against HeLa, MCF-7 [74], and RPE cells (data are mean ±SD of three replicateseach).

CompoundsIC50 (M)

HeLa MCF-7 RPE

T2 23.7 ± 0.01 44.5 ± 0.7 13.1 ± 2.4

B1 5.1 ± 1.0 8.9 ± 0.5 0.7 ± 0.1

As demonstrated by the IC50 values (Table 2), the molybdenum complexes showed to be very

effective as cytotoxic agents against the in vitro growth of various cancer cell lines. B1 exhibited

activities with IC50 values ranging from 1 to 9 M, approximately, and for T2 ranging from 13 to 46

M, approximately, for the cell lines studied. The complexes exhibited significant potency against

RPE cells, but less toxic toward MCF-7. Thus, it is clear that the RPE cells are the most sensitive,

whereas MCF-7 cells turned out to be the most resistant against the cytotoxic agents. It is also

evident that B1 is more effective than T2. This interesting behavior may be related to the ligand,

1,10-phenanthroline, which already has been shown to be very effective as cytotoxic agent [44].

Furthermore, both the complexes achieved almost total inhibition (<10% cell viability) at the

maximum concentration tested (100 M) for all cell lines. In fact, B1 values are comparable to

0

50

100

0 1 2.5 5 10 20 30 50 100

% C

ell V

iabi

lity

[T2] ( M)


Figure 11 - Dose-response curve obtained by nonlinear regression analysis for HeLa cells treated with B1 for 1, 2, 24,and 48 hours (left). In vitro cytotoxic activity for B1 against HeLa cells at 1, 2, 24, and 48 hours (right).

cisplatin, which has IC50 values below 10 M with a range of cell lines [65,66]. For further

comparison between these two complexes and the two ligands, complementary studies have been

performed.

The effect of the incubation time of cells with the compounds was also investigated. Thus dose-

response curves (Figure 11-12, left) and cytotoxic activity (Figure 11-12, right) for the treatment of

HeLa cells with the compounds for 1, 2, 24, and 48 hours were determined.

These results indicate that the molybdenum complexes inhibit cell proliferation in a duration-

dependent manner. The IC50 value of both complexes decrease as the exposure time increases,

although the difference between 24 h and 48 h does not seem relevant. Indeed, it seems that some

time is needed in order to achieve the maximum cytotoxic effect, which may be related to the

formation of new species from the molybdenum complexes. In fact, observing the MS spectra for

both the complexes (Annex, Figure 23-30), a cationic complex is formed

([(Mo)(C12H8N2)(C3H5)(O)2]+ , for B1 and [(Mo)(C10H8N2)(O)2]+ for T2). For B1 it is formed after

two hours of incubation time and for T2 is formed immediately. Thus, it is reasonable to admit that

this cationic form may be related to the cytotoxic effect of both the complexes, since it appears after

24 hours with a significative relative abundance, as the cytotoxic activity starts to achieve the

maximum effect. This relation can be seen clearly with B1, which seems to inflict more cytotoxic

effect as the cationic form is created. T2 seems to form more of this cationic complex at 24 hours,

the maximum effect; after that, no relative abundance increase is observed. This may be related to

the stabilizing landing observed for the cytotoxic effect after 24 hours (Figure 12). The increase of

the cationic form may be the key to achieve the maximum cytotoxic effect.


Figure 13 - Mean log octanol/water partition coefficients (Log P) ofthe molybdenum compounds.

Figure 12 - Dose-response curve obtained by nonlinear regression analysis for HeLa cells treated with T2 for 1, 2, 24,and 48 hours (left). In vitro cytotoxic activity for T2 against HeLa cells at 1, 2, 24, and 48 hours (right).

7.2. Octanol/water partition coefficient

Partition coefficients, P, determinations were carried out to estimate how easily the compounds T2

and B1 are able to pass through a biological membrane. The P measurements are based on the

difference in solubility that a given compound exhibits in an aqueous versus a hydrophobic medium

[67]. Complex B1 presented a

hydrophobic behavior (log P = 0.760 ±

0.039). For complex T2 it was not

possible to determine a conclusive value

of log P, nevertheless T2 seems to be

hydrophobic; however, less than B1

(Figure 13). In order to study further the

behavior of the complexes in solution,

the determination of the conductance

were carried out (Chapter 4.3).0

1/2

1

T2 B1

LogP


Figure 15 - Specific conductivity of T2 in water solution

7.3. Conductimetry

Conductimetry is the measurement of the conductance of a solution, where conductance is the ability

of a material to conduct electric current. It is expressed in siemens (S), although since conductivity

values are affected by electrode dimensions, it is commonly expressed in specific conductivity units

(S.cm-1).

This assay contributes to the understanding of the

metal complexes capability for ionizing in

solution, and it functions as a complement of the

octanol/water partition coefficient determination,

telling to interpret the results observed. The

conductance was determined by the measurement

of the current through the solution subject to a

potential applied between two plates. The

measurements made for both complexes were

done in different conditions; for B1 was used

water with 0.5% of DMSO (amount necessary to

complete solubilization of the complex), and for T2 was used only water (at low concentrations it

can be dissolved only in water).

The conductance measured for the water solution was significantly lower than the conductance

measured for the T2 solution, which means that T2 leads to the formation of ionic specimens that

makes the T2 solution more conductive than the

water solution (Figure 15). In fact T2 has a weak

bond to triflate ion, which may lead to T2 loss of

triflate when in contact with oxygen (from water)

originating probably the cationic

[(Mo)(C10H8N2)(O)2]+. As a result, the absorbance

of T2 fraction in octanol solution becomes lower

when ionic specimens are created (Figure 13),

which means that the log P determined does not

correspond effectively to a log P, but a log D (of

distribution). Relatively to B1, and opposite to

the observed for T2, the conductance measured

for the water solution with 0.5% of DMSO was significantly higher than the conductance measured

for the B1 solution (Figure 14). This indicates that B1 has a low tendency to form ionic specimens,

as suspected from inert Mo-Br bond. This effect is consistent with the result obtained for B1 partition

coefficient (Figure 13). Thus, the hypothesis that B1 has a higher hydrophobic behavior than T2 can

now be confirmed.

*P<0.001

0

2

4

Water T2

Spec

ific

Con

dutiv

ity(

S.cm

-1)

*P<0.001

0

13

25

Water B1

Spec

ific

Cond

utiv

ity (m

S.cm

-1)

Figure 14 - Specific conductivity of B1 in 0,5% DMSO inwater solution.


Figure 16 - Comparison of the intracellular molybdenum concentrationin HeLa cells after 48 h of exposure to compounds T2 and B1.

7.4. Cellular molybdenum uptake

In order to understand the beginning of the processes that trigger cell death, cellular molybdenum

content was determined after treatment of HeLa cells with T2 and B1 (Figure 16). The correlation of

the Mo cell uptake with the cytotoxicity previously determined (Chapter 7.1) provides useful

information regarding the intracellular cytotoxic potential and mechanism of action of the

complexes. Each compound was tested

at three different concentrations (10,

50 and 100 M), based on the

cytotoxicity data reported previously.

The intracellular molybdenum levels

of the cells treated with B1 revealed a

significant increase and a dose

dependent relation, contrasting to the

cells treated with T2. However, this

single analysis does not seem to be

enough to conclude about the relation

between the intracellular molybdenum

and T2 concentration, so more studies

are needed in order to avoid

significative experimental errors.

To understand better if the complexes can enter the cell and reach the nucleus, nuclear and cytosolic

fractions of cells pre-incubated with compounds for 48 hours were analyzed (Figure 17). Noted that

during the cell fractionation the plasma membrane is released as vesicles [75], and the molybdenum

content from the plasmatic membrane will be measured in the cytosolic fraction, leaving the nuclear

fraction with low plasmatic membrane contamination.

0

25

50

Control 10 50 100

Intr

acel

lula

r M

o×

104

(m

ol/m

illio

n ce

lls)

Concentration of Mo complex ( M)

B1

T2


As observed in Figure 17, the molybdenum levels of the cytosolic and nuclear extracts treated with

compounds B1 and T2 showed a significant increase and also a dose dependent relation. B1 has

clearly higher values than T2 in both the nucleus and cytosol fractions, which is consistent with the

octanol-water partition coefficient determined (chapter 7.2), as B1 having an extended hydrophobic

behavior would explain the ease with which it interacts with the membrane, contrarily to T2. These

results may also explain the cytotoxic activity of both the complexes, as B1 showed a stronger

cytotoxicity effect than T2 (Chapter 7.1, Table 2). The presence of both the complexes in the

nucleus supports the hypothesis that the complexes reach the nucleus, may interact with DNA and

take part in the process that leads to cell death (Chapter 7.5).

7.5. DNA Binding Studies

Extensive investigations have established DNA as a primary intracellular target of antitumor agents

[27]. Metal complexes can bind to DNA via covalent or/and noncovalent interactions. Whereas in

covalent binding a ligand is replaced by a nitrogen in a base of DNA, noncovalent binding is related

to the interactions of the complexes outside the DNA helix, by intercalative mode for example [48].

Absorption titration and circular dichroism were used to test the hypothesis that B1 and T2 inhibit

cancer cell proliferation by interacting with DNA. The binding mode of DNA-complexes was also

examined.

7.5.1. Electronic absorption titration

Electronic absorption spectroscopy is universally employed to study the binding mode of DNA to

complexes [44]. The absorption spectra changed with the addition of the complexes (Figure 18-19).

The addition of ctDNA (0-50 M bp-1) to T2 led to spectral changes with hyperchromism of the 299

0

20

40

Control 10 50 100

Nuc

lear

Mo

×10

10(m

mol

/mill

ion

cells

)


B1T2

b)

0

5

10

Control 10 50 100

Cyt

osol

ic M

o×

1010

(mm

ol/m

illio

n ce

lls)


B1

T2

a)

Figure 17 - Molybdenum concentration in cytosolic (a) and nuclear (b) extracts of HeLa cells after 48 h of exposure tocompounds T2 and B1.

(a) (b)


0

8

15

0 25 50

D/

ap×

1010

(M2

cm)

D ( M)

Figure 18 - Left: UV-Vis absorption spectra of T2 (20 M) in Tris buffer in the presence of increasing amounts of ctDNA.[DNA] = 0, 10, 20, 30, 40, 50 M. The arrow indicates the absorbance changes upon increasing DNA concentration; right: plotof D/ap vs. D for the titration of DNA to complex. Absorbance was monitored at 299 nm.

nm band, although no obvious red shift can be observed. Generally, hyperchromism is related to the

loss of secondary structure of DNA [48,49]. For the absorbance data at 299 nm, a plot of D/ap vs.

D was done (Figure 18, right), in order to determine the binding constant value (Chapter 4.7, K). It

shows a linear relationship with a K value of 2.08 (±0.98) × 105 M-1.

The binding of B1 to ctDNA also led to similar spectral changes (Figure 19), with hyperchromism

of the 272 nm absorption band. The K value for B1 was calculated to be 3.68 (±2.01) × 105 M-1. The

results indicate that the complex T2 binds more strongly than B1. Both K values are similar to those

of a typical classical intercalator, ethidium bromide, with K values 1.23 (±0.07) × 105 M-1 [68].

These results suggest that both complexes can bind to ctDNA, probably by electrostatic interaction

leading to the stabilization of DNA duplex. Nevertheless, both complexes have aromatic ligands with

extended systems, which means that intercalation could be one of the binding patterns, since the

extend systems can intercalate in DNA. Other approaches, such as circular dichroism, need to be

pursued in order to confirm this result (Chapter 7.5.2).

0

1

2

240 295 350

Abs

orba

nce

Wavelength (nm)


7.5.2. Circular dichroism

CD can be used to follow rapidly and efficiently DNA conformational transitions [53]. Generally, in

noncovalent interactions, such as electrostatic interaction, no spectral chances are observed in the

DNA spectra, since these modes of binding do not affect the secondary structure of DNA. On the

other hand, intercalation mode leads to changes in the DNA spectra [55]. DNA show a positive band

at 275 nm and a negative band at 245 nm, which is consistent with the characteristic B conformation

of DNA (Figure 20) [54]. With the addition of B1, the intensity of the positive band increased and

no significant changes was observed for the negative band. T2 also led to changes in the ctDNA

spectra, with the decrease of positive and negative bands. These changes in the CD spectrum of ct

DNA indicate strong interaction between the complexes and DNA mainly by intercalation mode

[55]. This effect can be seen clearly with complex T2. Despite the existence of intercalation

interactions, electrostatics and groove binding may also occur, mainly with B1 where the alterations

in the DNA spectra are not so obvious. Thus, as far as the binding mode of complexes is concerned it

seems to take place mainly via intercalation.

0

1

2

240 295 350

Abs

orba

nce

Wavelength (nm)

0

15

30

0 25 50D

/ap

×10

10(M

2cm

)D ( M)

Figure 19 - Left: UV-Vis absorption spectra of B1 (20 M) in Tris buffer in the presence of increasing amounts of ctDNA.[DNA] = 0, 10, 20, 30, 40, 50 M. The arrow indicates the absorbance changes upon increasing DNA concentration; right: plotof D/ap vs. D for the titration of DNA to complex. Absorbance was monitored at 272 nm.


Figure 20 – Circular Dichroism of ctDNA incubated with B1 (left) and T2 (right). [Complex] = 0, 10, 25, 50, 75, 100,250 M. The arrow indicates the signal changes upon increasing complex concentration.

Figure 21 - Electrophoretic mobility pattern of pYES2 plasmid DNA (C) incubated with complex B1 (right) and T2 (left)at 10, 50 and 100 M.

7.5.3. Gel electrophoresis studies

Gel electrophoresis studies were performed in order to evaluate the effect of molybdenum complexes

on DNA tertiary structure. Thus, the effect of T1 and B2 on plasmid DNA, pYES2, was studied

(Figure 21).

Plasmid DNA is visualized as supercoiled (high mobility form), linear (intermediate mobility form)

and circular (low mobility form). With the increasing of B1 concentration, a mobility decrease can

be observed. This effect can be observed more clearly with circular and linear forms. The effect of

B1 is similar to the one observed for intercalator agents, such as cisplatin, which means that

interaction of the complex with DNA occurs and probably by intercalation [70]. This pattern, where

plasmid forms retardation are observed, can be visualized for T2, although more slightly and only in

the supercoiled form, the only form visualized.

7.5.4. Atomic force microscopy

AFM can be used to study the influence of factors, such as intercalators, that induce conformational

transitions on the DNA tertiary structure [57]. Thus, preliminary studies of structural transitions in

the tertiary structure of plasmid DNA were performed. AFM images of the free plasmid pYES2 and

-8

-4

0

4

8

230 270x

103

(deg

)

(nm)

T2B1


-5

-3

0

3

5

230 270x

103

(deg

)

(nm)






(Figure 21).












310

T2B1


270 310

(nm)






(Figure 21).












T2B1


pYES2 incubated with T2 were taken (Figure 22). Images were obtained after a very short time of

incubation (2 min) in order to allow the observation of the changes produced on DNA forms. At

longer times it was not possible to observe the images properly, probably due to the excessive

amount of DNA adhered to the mica. These results showed that T2 induce changes in the DNA

structure as a consequence of its interaction with DNA chains. T2 seems to originate mixed toroidal

and plectonemic supercoiling forms (see Figure 3, Chapter 4.9). As observed in previous studies,

intercalation of aromatic rings between base pairs of DNA seems to be the most probable way of

interaction. The image obtained is similar to others obtained for intercalators [70,71]. Unfortunately,

the study of structural transitions in the tertiary structure of plasmid pYES2 using B1 was not

possible owing to experimental limitations.

Figure 22 - A Selection of topographic images recorded of plasmid pYES2 (a) and plasmidpYES2 incubated with T2 (b) adsorbed to AP-mica. The scale bars correspond to 250 nm.


























8. Conclusions

Organometallic compounds offer a rich chemistry field in medicine with a high potential to develop

effective antitumor drugs [30]. This variety of options provides unlimited possibilities of

combination that result in an extensive spectrum of mechanisms open to organometallic compounds.

Two organometallic compounds [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)], B1, and [Mo(3-

C3H5)CF3SO3(CO)2(2,2’-bipyridyl)], T2, have been studied in the search for innovative and valuable

tools in cancer chemotherapy.

Complexes T2 and B1 exhibited a high cytotoxic activity toward several human cancer cell lines:

HeLa, MCF-7, and RPE. MCF-7 showed to be the most resistant cell line, whereas RPE was the

most sensitive against the complexes. Although both the complexes showed very significant IC50

values, B1 showed to be a little more effective than T2, which may be related to the 1,10-

phenanthroline ligand. Furthermore, B1 presents a range of IC50 values comparable to cisplatin,

which makes it a good candidate as a substitute of cisplatin, avoiding some of the cisplatin

limitations, such as acquired resistance. The kinetic studies revealed that a cationic form of

molybdenum compound may be related to the maximum cytotoxic effect. According to the cellular

uptake of molybdenum and octanol/water partition assays, both complexes seem to enter the cells in

a selective and dose dependent manner and reach the nucleus. These results may be related to the

hydrophobic behavior of the complexes in solution, which showed that B1 is more hydrophobic than

T2. B1 might then be able to pass more easily through a biological membrane than T2. As far as

complex-DNA interaction is concerned, both the complexes showed interaction with DNA (DNA

plasmid and ctDNA). B1 and T2 bind to ctDNA with binding constants of 2.08 (±0.98) × 105 and

3.68 (±2.01) × 105 M-1, respectively. The data from absorption, circular dichroism and atomic force

microscopy support a specific binding mode of both the complexes with DNA, via intercalation,

although other modes of binding can also occur. The interaction of the complexes with DNA, mainly

by intercalation, changing its conformation, may be one pathway responsible for cell death.

It has been shown that both complexes are present in the nucleus and that they interact with DNA in

vitro, so it becomes clear that this process may also occur in vivo.

As both the complexes showed promising results, they may be able to become a successful

alternative to the existing platinum chemotherapeutic agents, the only compounds with clinical use in

chemotherapy currently, and they might exhibit unique properties, such as the reactivity of the

ligands or differences in coordination geometry, that can be used in order to overcome chemotherapy

current limitations.


However, compounds with high cytotoxicity against cancer cell lines do not necessarily mean that all

the criteria for anticancer drugs are fulfilled, and despite the fact that these compounds presented a

high cytotoxic potency, further studies involving in vivo experiments need to be done in order to

raise these compounds to a proper therapeutic window.

Future work will address the intracellular localization of molybdenum, for example by the use of

fluorescents ligands, and the study underlying the metabolism responsible for DNA damage repair,

which would be useful to understand the global picture related to the mechanism of action of these

molybdenum complexes. Long-term work would be related to the use of molybdenum compounds in

in vivo studies.


9. Acknowledgements

É com enorme gratidão que venho agradecer à Professora Margarida Meireles e à Professora Maria

José Calhorda, as orientadoras responsáveis pelo meu projecto de mestrado, por me receberem no

laboratório e por me integrarem no grupo de Química Inorgânica e Teórica. Foi para mim uma honra

partilhar ideias e trabalhos com diversas pessoas experientes e de diferentes áreas. Também queria

agradecer a oportunidade de integrar neste projecto onde pude aprender e aquirir imensa prática

laboratorial. O facto de haver sempre disponibilidade de todos os membros do grupo contribuiu para

o sucesso do trabalho, que culminou com a publicação do artigo científico. Manifesto assim a minha

gratidão a todo o grupo e, em particular, às minhas orientadoras.

Deixo um agradecimento aos diversos grupos de investigação que colaboraram neste projecto e que,

com toda a disponibilidade e empenho, tornaram possível a realização das várias experiências:

Ao Dr. Alexandre Quintas e Dr. Luís Oliveira pela colaboração nos ensaios de dicroísmo circular no

Instituto Superior de Ciências da Saúde Egas Moniz;

Ao grupo de enzimologia da FCUL com especial ênfase para a Dra. Marta Silva pela colaboração

nos ensaios com DNA plasmídico;

À Dra. Filomena Martins e à Dra. Cristina Ventura pela colaboração nos ensaios de partição

octanol/água;

À Dra. M. Soledade Santos pela colaboração nos ensaios das condutividades;

À Marta Saraiva e à Dra. Carla Nunes pelo apoio na síntese química dos complexos;

E à Faculdade de Ciências, nomeadamente ao Departamento de Quimíca e Bioquímica, pela

oportunidade da concretização deste projecto e por tornar possível a minha formação quer científica,

quer pessoal, oferecendo todas as condições necessárias.

Um sincero agradecimento ao CQB pela oportunidade que me deram de apresentar um seminário,

juntamente com a minha colega de grupo Marta Saraiva, com o título “Synthesis and cytotoxic

characteristics of a family of molybdenum(II) complexes”.

Queria também agradecer ao meu colega de laboratório Miguel Lopes, cuja presença tornou este

trabalho mais interessante e desafiador. Com ele pude contar sempre em todos os momentos, quer na

realização dos vários ensaios, quer na vida pessoal. Aqui fica um profundo e sincero agradecimento

de uma amizade que já dura 17 anos.

A todas as pessoas com quem fui partilhando experiências ao longo da vida e que me influenciaram

de algum modo, a todos os meus amigos, a todos os meus colegas de licenciatura, com especial

ênfase para Ana Filipa Ribeiro, Armando Cruz, Bruno Moraes, Carlos Neves, Maria João Lima,


Mariana Oliveira, Miguel Lopes, Sara Carvalhal e Soraia Martins, aqui fica um forte abraço.

Obrigado pelas companhias nos almoços, pelas visitas motivadoras ao laboratório, pelo

conhecimentos partilhados ao longo da licenciatura e do mestrado e, acima de tudo, pela diversão

que tão bem sabemos desfrutar! Um especial agradecimento para a Sara, “a rapariga do laboratório

ao lado”, cuja companhia muito me ajudou nesta experiência.

Por fim, deixo um sincero e especial agradecimento aos meus pais, irmãs e madrinha pelo suporte

sólido, com quem posso sempre contar para o que quer que seja, em qualquer altura.


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11.Annex

Figure 23 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO with no incubation time (0 hours).


11.Annex



11.Annex



Figure 24 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 2 hours of incubation time at 37 ºC.






Figure 27 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO with no incubation time (0 hours).






Figure 28 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 2 hours of incubation time at 37 ºC.






Figure 31 - Absorption spectra of B1 ([Mo(3-C3H5)Br(CO)2(1,10-fenantrolina)]).

Figure 32 - Absorption spectra of T2 ([Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipiridil)]).

0.00

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48 h

B1

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T2

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