Sensitization of cancer cells to inhibitors ofheat shock protein 90 (Hsp90) andproteasome by abamectin

Mahesh Balasaheb Tambe

Degree project in applied biotechnology, Master of Science (2 years), 2010Examensarbete i tillämpad bioteknik 45 hp till masterexamen, 2010Biology Education Centre, Uppsala University, and Department of Biochemistry, Boston UniversitySchool of Medicine, USASupervisors: Prof. Dr. Michael Sherman and Dr.Vladimir Gabai

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Summary

Heat shock proteins of Hsp90 family and Hsp70 family function as molecular chaperones for stabilizing and folding of the unfolded or misfolded proteins. Misfolded proteins are highly undesirable for a cell since they can activate apoptosis or senescence. However, in the presence of molecular chaperones these abnormal proteins are stabilized and refolded and this can prevent activation of signal transduction cascades leading to apoptosis or senescence.

A cancer cell is a transformed form of a normal cell that accumulates mutations due to higher degree of genetic instability and gains a survival advantage over surrounding normal cells; this uncontrolled proliferation leads to tumor development. The process may take a long time and many mutated proteins called oncogenic proteins are responsible for transformation of a normal cell to a cancer cell. These oncogenic proteins in the cell are stabilized by Hsp90 and Hsp70 family molecular chaperones, which otherwise would be degraded by the ubiquitin proteasome degradation pathway. Due to high dependence of transformed cells on chaperones and proteasome activity, new classes of anticancer drugs, Hsp90 inhibitors and proteasome inhibitors, have emerged recently.

Hsp90 inhibitor drugs inhibit the chaperone activity of Hsp90 preventing the stabilization of its client proteins (e.g., proteins involved in cell proliferation, survival, etc.) which leads to their degradation via the proteasome, whereas proteasome inhibitors prevent degradation of misfolded or unfolded proteins leading to the formation of insoluble aggregates which are highly toxic to the cell. Unfortunately both of these classes of drugs are reported to induce other heat shock proteins (e.g., Hsp72 and Hsp27), which function as antiapoptic proteins thus reducing their anticancer activity.

Previous research in our lab has reported that Hsp72 knockout cells show a high degree of sensitivity to Hsp90 inhibitors and proteasome inhibitors. Since there are no available drugs for Hsp72 inhibition, we have previously performed high-throughput cell based screening for refolding of luciferase after heat shock which should pick up inhibitors of heat shock response and chaperone (e.g., Hsp72) activity. This screening gave us 19 FDA approved drugs out of totally screened 20,000 compounds, which may be potential inhibitors of heat shock response and chaperone activity. I was responsible to test these compounds on cancer cells in vitro using a Her2-transformed human breast epithelial cell line MCF10A. These 19 compounds were tested for their toxicity as single agents and in combination with Hsp90 inhibitor 17AAG (17-(Allylamino)-17-Demethoxygeldanamycin) and a proteasome inhibitor MG-132, and I identified a compound abamectin which showed no toxicity by itself but markedly sensitized cells to 17AAG and MG-132. I hypothesized that the observed toxicity could be due to inhibitory effects of abamectin on Hsp72 expression, but by further analysis it became clear that the effect was not related to inhibition of Hsp72 expression, but rather to chaperone activity of the heat shock proteins. We plan to check the effect of abamectin specifically in respect to Hsp72 by using functional assays to study Hsp72 ATPase activity and substrate binding to Hsp72 chaperone. We will also check the combination of abamectin with 17AAG and proteasome inhibitor (Velcade) on antitumor activity in NeuT allograft tumor in mice.

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1. Abbreviations

CT Cholera Toxin

CMV Cytomegalo Virus

DMEM Dulbecco’s Modified Eagles Medium

EGF Epidermal Growth Factor

FBS Fetal Bovine serum

GFP Green Fluorescent Protein

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

HS Horse Serum

Hsp Heat Shock Protein

HSR Heat Shock Response

HSE Heat Shock Element

LB Luria Bertani medium

ml Milliliter

mM Millimolar

NaCl Sodium chloride

PBS Phosphate Buffer Saline

TEMED Tetramethylethylenediamine

xg Times gravity

µl Micro liter

17AAG 17-(Allylamino)-17-demethoxygeldanamycin

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2. Introduction

Heat shock proteins are expressed constitutively or in response to the stress in a cell and are found abundantly in cytosol, mitochondria, chloroplast and ER (endoplasmic reticulum) where they play a role in protein folding and stabilization of unfolded proteins which otherwise will aggregate or be degraded by ubiquitin-proteasome pathway. Depending on their molecular weight they are classified as HSP100, HSP90, HSP70, HSP60 and the small Hsps, and they are found to be highly conserved between prokaryotes and eukaryotes (Nollen and Morimoto, 2002;Mosser and Morimoto,2004;Sreedhar, et al., 2004). Classically, chaperones were considered as molecules that assist in protein folding and prevent protein aggregation but the observation that these chaperones interact with several oncogenic proteins like kinases, growth factor receptors, and transcription factors that promote cancer progression raised interest to exploit heat shock proteins in cancer treatment (Workman, et al., 2007).

Cancer is often caused by genetic instability, where a normal cell gathers mutations due to DNA damaging agents. Normally DNA damage response is meant to protect cell from such kind of insults by repairing the damage and by arresting cell proliferation, but in most of cancer cells there are mutations in DNA damage response pathways, which ultimately results in proliferation of cell with the acquired mutations (Giménez, 2010). The mutations cause defects in tumor suppressor genes and oncogenes, of which oncogenes are dominant in nature. When cancer is detected clinically it may contain around 10,000 mutations in its genome and owing to this high genetic diversity in cancer cells it is very hard to treat cancer by concentrating on a single molecular node; therefore, it has been suggested that an efficient strategy to treat cancer would be to target different signaling pathways simultaneously. The involvement of Hsp90 chaperone in interaction with different proteins from various signaling pathways has led to a notion to target Hsp90 function for cancer treatment. Accordingly, blocking the interaction between Hsp90 chaperone and its client proteins is considered to challenge cancer cell and may cause their death (Giménez, 2010;Workman, 2003).

2.1. Heat shock transcription factor 1 (HSF-1)

Heat shock factor 1 is responsible to the transcription of Hsp genes during stress conditions. HSF-1 is under normal conditions present in complex with Hsp90and immunophilins such as Cyp40, FKBP1, FKBP2 and p23. Upon stress signals this complex dissociates, HSF-1 is released and forms trimers; this activated form of HSF-1 enters the nucleus and binds to the HSE elements on promoters for Hsp genes, resulting in elevated levels of heat shock proteins 27, 60, 70, 90 and 110 (Voellmy, 1994; Wu, 1995). HSF-1 functions by recruitment of chromatin remodeling enzymes and histone deacetylase to the Hsp loci with open configuration and a stalled RNA Polymerase II. HSF-1 has multiple phosporylation sites some of which cause activation and some result in inhibition of HSF-1 transcription activity; among kinases involved in phosporylation of HSF-1 are Akt, GSK3, PKC, ERK etc. (Chu, et al., 1998; Knauf, et al., 1996).

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2.2. Heat shock proteins: role in cancer

Several studies have shown that various etiological agents like viruses, radiation, carcinogenic agents, hypermethylation, and oncoproteins which may be the cause of cancer are associated with the alteration of the expression or function of heat shock proteins. Change in the expression or function of heat shock proteins leads to several molecular changes directly related to carcinogenesis by facilitating transformation of a normal cell and giving a cancer cell survival advantage and uncontrolled proliferation ability.

In general Hsps are found to be over-expressed in most of the cancer types, with differential expression pattern at different stages of a tumor. This can be explained as Hsps like Hsp105 were found to be expressed in highly aggressive tumors and at later stages of tumor invasion, while Hsp27, Hsp60, Hsp72 and other members of Hsp family were upregulated in early stages of cancer progression.

2.2.1. Hsp90 and cancer

Hsp90 family consist of 5 members which include Hsp90α and Hsp90β in cytoplasm, Grp94 in Endoplasmic reticulum, TRAP1 and Hsp90N in mitochondria (Felts, et al., 2000). Hsp90 forms a complex chaperone machine in combination with Hsp70, Hsp40, HOP, AHA1, p23, Cdc27, HARC and Immunophilins. These co-chaperones interact with the Hsp90 dimer at different stages in the chaperone cycle, responsible for stabilization of the various proteins in a normal cell. Along with its role in stabilizing the normal proteins in a cell, it also leads to the stabilization and proper folding of the mutated proteins in a cancer cell. The Hsp90 chaperone machine interacts with various proteins (called ‘client proteins’) involved in cell proliferation, metastasis, angiogenesis, and cell survival, whose stabilization gives cancer cells growth advantage as compared to the normal cells.

The list of Hsp90 oncogenic client proteins is increasing and includes proteins like Her-2 (ERBB2), BCR-ABL, C-RAF, BRAF, CDK4 (Cyclin dependent kinase), PLK-1 (polo like kinase-1), MET or HGFR, mutant p53, steroid receptors like androgen and estrogen receptors, survivin and telomerase, hTERT, VEGFR (vascular endothelial growth factor receptor), FLT3 and hypoxia inducible factor (HIF)-1α which are the members of six ‘acquired characteristic hallmarks of a cancer cell’ as hypothesized by Hanahan and Weinberg (Hanahan and Weinberg, 2000) and shown in Figure 1 below.

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Figure 1 Heat shock protein 90 involved in the interaction with its client proteins which are the members of 6 characteristic hallmarks of a cancer cell

2.2.2. Hsp70 and cancer

A cancer cell is subjected to various extracellular stresses like hypoxia, nutrient deprivation and intracellular stresses due to mutated proteins, unfolded proteins and deregulated signaling pathways. This condition favors the activation of HSF-1 transcription factor for the expression of heat shock proteins, where Hsps act as anti-apoptic proteins to protect cancer cells from stress- induced death (de Billy, et al., 2009; Whitesell and Lindquist, 2009). Due to its chaperone and antiapoptic activity Hsp70 family proteins are considered as a potentially drug target along with Hsp90, and the observation that Hsp90 inhibitors induce Hsp72 has surged the demand to develop drugs inhibiting Hsp72 function.

The Hsp70 family of heat shock proteins expresses 8 different members with different expression levels at different cellular localization. Hsc70 is expressed constitutively in cytoplasm which is associated with most of the housekeeping functions of Hsp70 family of proteins under normal conditions. The expression of Hsp72 (an inducible form of Hsp70) is at higher levels under stress conditions due to the transcription of HSPA1A/HSPA1B gene mediated by heat shock transcription factor 1 (HSF-1) (de Billy, et al., 2009; Whitesell and Lindquist, 2009).

Hsp90

Evading Apoptosis

(IGF-1R, Akt)

Self-sufficiency in

growth signals

(Her-2, KIT, MET,

other RTKs)

Insensitivity to anti-

growth signals

(CDK4, CDK6,

Cyclin D)

Tissue invasion and

metastasis

(MMP2, Urokinase)

Limitless replicative

potential

(Telomerase)

Sustained

Angiogenesis

(HIF, MET, Src,

VEGF, other RTKs)

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Structural similarities are seen in all the members of Hsp70 family: a N-terminal nucleotide binding domain (NBD) is responsible for ATPase activity, a substrate binding domain (SBD) - for binding the proteins and a poorly conserved C-terminal domain with its EEVD motif interact with the co-chaperones containing tertratricopeptide repeat (TPR motif) (Daugaard, et al., 2007). The research leading towards designing potential inhibitor molecules of Hsp72 function is gaining momentum and some drugs have been reported but their molecular target and specificity still remain a question mark. The designed small molecule inhibitors are targeted towards ATP binding site, or disrupting the binding of co-chaperones of Hsp70, or disrupting the peptide binding by Hsp70. Since the ATP binding site is conserved in all the members of Hsp70 family, designing a specific molecule inhibitor for Hsp72 based on disruption of ATP binding is unlikely to be the selective for Hsp72 alone. Furthermore, designing drugs for ATP binding site have some limitations due to highly flexible nature of NBD (Zhang and Zuiderweg, 2004), presence of hydrophilic residues at ATP binding site (Halgren, 2009), and having more binding affinity towards ADP (Borges and Ramos, 2006); the accessibility is further limited as ATP binding site is seen much enclosed even in its most open conformation (Powers, et al.).

Figure 2 below shows the different point of interactions of Hsp70 with the proteins involved in apoptotic pathways, thereby inhibiting apoptosis.

Figure 2

Anti-apoptic role of heat shock protein 70 family proteins (Powers, et al., 2009) Hsp70 family of proteins interacts with various proteins involved in both caspase dependent (Bid, caspase 9, BCl-2, caspase 3) and caspase independent (JNK, AIF, lysosome, p53) apoptic pathway. The death receptors spanning the plasma membrane trigger the apoptic pathway after binding of ligands extracellularly, which results in cascade of intracellular reactions resulting in apoptotic death of the cell. But when Hsp72 is present in a cell at high levels it binds to various factors in this pathway to block apoptosis, giving cell survival advantage.

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2.2.3. Hsp27 and cancer

Heat shock protein 27 belongs to small heat shock protein family, characterized by its large oligomers in the cell and functioning as ATP-independent chaperones. Large oligomers of Hsp27 participate in protein folding where they have a role to store the unfolded proteins which are then folded by high molecular weight ATP-dependent chaperones or degraded by proteasome. Hsp27 has several protective roles in a cell one of which includes preventing cell damage caused by oxidative stress due to reactive oxygen species and oxidants (Arrigo, A.P. 2007). Such Hsp27 activity is mediated by glutathione which co-relates with increased levels of glucose-6-dehydrogenase, glutathione reductase and glutathione transferase, key enzymes in ROS-glutathione pathway (Preville, et al., 1999; Arrigo, A.P. 2007). Several anti-cancer drugs and X-ray irradiation are designed to act as modulators of redox state homeostasis in a cell but the ability of Hsp27 to maintain redox state homeostasis results in resistance to these class of drugs and X-rays irradiation.

Hsp27 is also a key molecule in inhibition of apoptosis during stress conditions caused by several pro-apoptic agents. Several points where Hsp27 act to inhibit the apoptic pathway are inhibition of Bid translocation to mitochondria which inhibits release of cytochrome c by mitochondria, interaction with cytochrome c and apoptosome formation, and also interaction with Signal Transducers and Activator of Transcription (STAT-3), a transcriptional activator of Bcl-xL or surviving, which are inhibitors of apoptosis (Arrigo, 2007). Due to its role as anti-apoptic protein, high expression of Hsp27 in a cell confers resistance to several anti-cancer drugs, so Hsp27 is also considered as a potential target for anti-cancer therapy in combination with apoptosis-inducing drugs.

Besides regulating apoptosis Hsp27 along with Hsp90 are reported as key regulators of metastasis. Metastasis is a major challenge in cancer treatment, which is attributed to more than 90% of deaths due to cancer (Germanov, et al., 2006); this process involves dissemination of tumor cells from their primary site to distant organs in the body that serves as new site for tumor growth. Here the phosphorylated form of Hsp27 and extracellularly expressed Hsp90α isoform are found to be associated with activation of MMP-2 (Matrix metalloproteinase) which digests several components of extracellular matrix, facilitating the cell invasion and metastasis (Xu, et al., 2006). Hsp27 and Hsp70 are also reported to play a role in DNA damage response according to the study by Nadin, et al., (2007) where they reported the role of elevated levels of Hsp27 and Hsp70 after the heat shock rendering the cell less susceptible to the DNA damaging agent Doxorubicin. They also reported the co-localization of mismatch repair proteins hMLH1 and hMSH2 with Hsp27 and Hsp70 after the heat shock (Nadin, et al., 2007).

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2.3. Hsp90 inhibitors as anticancer drugs

Various Hsp90 inhibitor molecules have been designed to inhibit the function of Hsp90 complex chaperone machine. These inhibitor molecules are designed to interact with the ATP binding site which involves competitive binding between drugs and ATP, or some drugs binding to the C-terminus binding site disrupting the interaction between Hsp90 and co-chaperones (Holmes, et al., 2007). The Hsp90 inhibitor drugs belong to various classes based on chemical compositions which are described below in Table 1.

Table1. List of Hsp90 inhibitor drugs (Whitesell and Mclellan, 2007)

Class Example Source Binding site Development status

Macrolide Radicicol Natural Product

N-terminal ATP Binding pocket

N.A (Toxic in vivo) (Powers and Workman, 2006)

Radicicol 6 oxime Natural Product

N-terminal ATP Binding pocket

Preclinical development dropped

Benzoquinone ansamycin

Geldenamycin Natural Product

N-terminal ATP Binding pocket

N.A. (instable, hepatotoxicity) (Supko, et al., 1995)

Herbimycin A Natural Product

N-terminal ATP Binding pocket

N.A

Macbecin I/II Natural Product

N-terminal ATP Binding pocket

N.A

17-AAG (Tanespimycin)

Semi-synthetic Geldenamycin

N-terminal ATP Binding pocket

In clinical Trial

17 DMAG (Alvespimycin) (more water soluble than 17AAG, orally bioavailable) (Pacey, et al., 2006)

Semi-synthetic Geldenamycin

N-terminal ATP Binding pocket

In clinical Trial

CNF-1010 (www.biogenidec.com)

Semi-synthetic GA analog

N-terminal ATP Binding pocket

In clinical Trial

KOS-953 (www.kosan.com)

Semi-synthetic GA analog

N-terminal ATP Binding pocket

In clinical Trial

10

IPI-504 (soluble derivative of 17AAG) (Sydor, et al., 2006)

Chemical reduction product of 17AAG

N-terminal ATP Binding pocket

In clinical Trial

Purine like CNF2024 (Chiosis, et al., 2001)

Synthetic N-terminal ATP Binding pocket

In clinical Trial

Pyrazolelisoxazole resorcinol

CCT-018159 (good solubility, relatively independent of DT-dipharose and P-glycoprotein) (Sharp, et al., 2007)

Synthetic N-terminal ATP Binding pocket

Late pre-clinical

Peptidomimetic Shepherdin (Pacey, et al., 2006)

Synthetic N-terminal ATP Binding pocket

Preclinical development

Coumarin Novobiocin (Marcu, et al., 2000)

Natural product

C-terminal cryptic ATP binding pocket (inhibit binding of Hsp90 and co-chaperones Hsc70 and P23) (Marcu and Neckers, 2003)

Lead development

DNA cross linker Cisplatin Synthetic N.A Histone deacetylase inhibitor

Depsipeptide FK228 (Hsp90 is acetylated which inhibits ATP binding) (Kristeleit, et al., 2004)

Natural product

In clinical trial

Vorinostat Synthetic

2.3.1. Specificity for Hsp90 inhibitors for normal and cancer cell

The drugs mentioned above in the Table 1 vary greatly in their therapeutic index and their toxic effects. Being toxic, many of them are discarded from the clinical trials but some of them like 17AAG have proved highly selective to their action on cancer cells and is currently in Phase III clinical trials. But in general it has been observed that Hsp90 inhibitor molecules are highly selective to cancer cell comparing to normal cell, which is further explained by several researchers. In normal cell Hsp90 is present in a free state but during stress signals or chaperone activity Hsp90 forms a part of Hsp90 chaperone complex. During stress signals or in case of accumulation of unfolded proteins Hsp90 is recruited to the chaperone complex engaged in

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protein folding activity, and this is the case for cancer cells as well (Kamal, et al., 2003). Reports suggest that during chaperone function, Hsp90 has more affinity towards ATP; this state of Hsp90 is referred to as ‘active state’ and, interestingly, in a cancer cell Hsp90 is found in active state with more affinity towards ATP (Kamal, et al., 2003). Thus the inhibitor molecules which compete for ATP to bind in the ATPase binding site of Hsp90 are more attracted towards active Hsp90 in cancer cell. This high affinity binding results in the accumulation of the inhibitor molecules in a cancer cell whereas in a normal cell lack of such interactions between Hsp90 and inhibitor molecules results in rapid clearance of these molecules. Since a cancer cell is highly addicted to Hsp90 chaperone machine for its survival, this suggests inability of cancer cell to survive in the absence of Hsp90 chaperone function. The data from clinical trials suggest that 17AAG sensitizing activity is limited due to its ability to induce heat shock proteins 72 and 27 (anti-apoptotic proteins), and also to its poor water solubility and its efflux by p-glycoprotein, which has raised question on its use as single agent anti-cancer drug (Kamal, et al., 2003) (Ramanathan, et al., 2005). However, isoforms of 17AAG called 17-DMAG has been developed with more solubility and less degree of metabolism in the body as compared to 17AAG, which could be used as inhibitor of Hsp90 for future experiments.

2.4. Proteasome inhibitors as anticancer drugs

The proteasome is responsible to maintain protein homeostasis in a cell by degradation of polyubiquitin labeled proteins. In recent years the proteasome has become an exciting avenue for cancer researchers and several small molecule inhibitors of proteasome are developed to be used as anticancer drugs. Inhibiting proteasome activity results in accumulation of highly unstable, mutated and unfolded proteins which forms highly toxic insoluble protein complexes in a cell. Some of the molecular events after inhibiting proteasome are p53 accumulation, which induces cell cycle regulators like p21 causing cell cycle arrest (Lashinger, et al., 2005; Nickeleit, et al., 2008), accumulation of pro-apoptic proteins, e.g. Bax, Bik, Bim and NOXA, causing apoptosis (Lomonosova, et al., 2009; Cao, et al., 2008), down regulation of Inhibitors of apoptosis (IAP’s), e.g. survivin (Uddin, et al., 2008), activation of Bone Morphogenetic pathway (BMP), a tumor suppressor pathway, and upregulation of Death receptor 5 (DR5) which leads to TRAIL-induced apoptosis (He, et al., 2004; Wu, et al., 2010). Several small molecule proteasome inhibitors like Bortezomib (Velcade), CEP-18770 (reversible inhibitor), carfilzomib and NPI-0052 (irreversible inhibitor) have shown promising results in preclinical study and are at different stages of clinical trials; whereas Velcade has shown good results in clinical trials and is currently approved for treatment of patients with multiple myeloma and previously treated mantle cell lymphoma. (Chauhan, et al., 2008; Cusack, et al., 2006; Kupperman, et al., 2010 ; Richardson, et al., 2005). The use of proteasome inhibitors are associated with certain drawbacks like causing peripheral neuropathy, myelosuppression, induction of macroautophagy, activation of constitutive NF-kB signaling (cell type dependent) (Dolcet, et al., 2006). In pancreatic cancer cells Bortezomib induce pro-survival signaling pathways like EGFR, Akt, ERK and JNK. It also activates STAT3 (a key growth promoting protein), and, importantly, increased expression of Hsp72, Hsp27 and other chaperones. The ability of proteasome inhibitors to induce heat shock proteins has rendered

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them inefficient to treat cancer when used in vivo. As discussed above, the role of Hsp70 and Hsp27 family of proteins in preventing apoptosis may lead to the major cause of failure of proteasome inhibitors in some clinical trials. The specificity of the proteasome inhibitors for normal cells and tumor cells needs to be further analyzed by thorough investigation, and it is also necessary to find a proper combinatory chemotherapy treatment where proteasome inhibitors could be used along with inhibitors of chaperones.

2.5. Aim

In recent years Hsp90 and Proteasome inhibitor drugs are gaining importance to be used as anti-cancer drugs and many of them are currently in clinical trials. One molecular event occurring after the use of these drugs is induction of heat shock proteins 72 and 27; both of these Hsp’s are anti-apoptic proteins and prevent the cell death after treatment with Hsp90 and proteasome inhibitors. Therefore, anti-cancer therapy targeting Hsp90 or proteasome needs to be reviewed and a combination therapy along with Hsp72 inhibitors should be utilized.

However, it has been a challenge to design a drug working specifically to inhibit Hsp72 (an inducible form of Hsp70). Our experimental study is meant to find Hsp72 inhibitor drugs from the set of 19 drugs obtained after high throughput cell based screening for the compounds inhibiting luciferase refolding after heat shock. To achieve this we need to standardize the in

vitro assay to test these compounds for their toxic effects as single agent and in combination with Hsp90 and proteasome inhibitors on the transformed cells in vitro. We further plan to reveal the molecular targets of the compounds found effective in sensitizing to Hsp90 and proteasome inhibitors and then test this combination of drugs on tumors grown as allografts in mice.

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3. Results

The compounds which were tested for its toxicity effects on MCF10A cells are in table 2.

Table 2. FDA approved drugs found effective in the screening for inhibiting the luciferase refolding after heat shock

Compound Notations used

throughout the

report

Molecular weight

Cytarabine A 243.2211

Camptothecin B 348.3615

Tyrothricin C 1272.478

Norethindrone acetate D 340.4667

Desipramine hydrochloride E 302.8504

Juglone F 174.1575

Pimozide G 461.5596

Fendiline Hydrochloride H 351.9234

Metergoline I 403.5288

Alexidine hydrochloride J 581.7252

Niclosamide K 327.1257

Aloin L 434.4035

Salinomycin M 773.0014

Bepridil Hydrochloride N 403.0124

Fluphenazine hydrochloride O 510.4532

Abamectin P 873.1006

Perhexiline Maleate Q 393.5716

Triflupromazine hydrochloride R 388.8857

Thiothixene S 443.6345

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3.1. Generation of Her-2/neuT transformed cells

Retroviral vectors for her2/neuT were produced by transfecting 293T cells with plasmids expressing her2/neuT gene together with plasmids containing viral packaging genes. This retrovirus was used for infecting MCF10A cells and 4 days after infection and selection with puromycin I could see the specific change in the morphology of cells: they formed ‘foci’ (Figure 3). In Figure 3A the cells expressing control vector virus were comparable to the normal non-infected cells. In the Figure 3B it was evident that the cells changed their morphology and gained ability to grow in multilayer.

A B

Figure 3

Transformation of MCF10A wt cells by infection of Her-2/neuT virus

A. Wild type MCF10A (mammary epithelial cells) B. MCF10A Her-2/neuT transformed cells forming ‘foci’. The photographs are taken under phase contrast at 10X magnification.

3.2. Screening of compounds using MTS assay

The compounds obtained after primary screening were tested for its toxicity effect on MCF10A wild type and MCF10A neuT cells. After initial failure with the experiments using an XTT assay, a rather simple assay called MTS assay from Promega was used and this worked well in my experiments. Based on the principle of color formation due to the reaction of MTS tetrazolium salt and NADPH/NADH produced by metabolically active cells this assay provided a colorimetric method to determine the number of viable cells in the culture.

The first aim was to find optimum concentrations of 17AAG (Hsp90 inhibitor) and MG-132 (proteasome inhibitor) which were not totally toxic to the cells but kills around 30-40% of the cells. The cells were cultured on 24 well plate at 50% confluency and incubated with the compounds at 200nM, 500nM, 1µM, 2µM, 5µM and 10µM for 15 hours, then after adding 15µl of MTS reagent the plates were incubated for 1 hour at 37oC. Then the plates were scanned at 490nm in a microplate reader. The data in Figure 4 shows the effect of MG-132 at 0.1, 0.2 and 0.5µM, and 17AAG at 0.5µM on MCF10A cells.

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Figure 4

Effect of 17-AAG and MG-132 on cell viability of MCF10A cells, cells seeded in 24 well plate and incubated over night with 0.1µM, 0.2 µM, 0.5 µM MG-132 and 0.5 µM 17AAG, next day the plate was read in 96-well plate reader at 490nm. The cells without addition of any drug were used as control. The graph shows the percent of the cells alive.

As seen in the Figure 4 MG-132 at 0.5µM and 17-AAG at 1µM showed almost the same toxicity for the MCF10A cells, and so I decided to use these concentrations of MG-132 and 17-AAG for further experiments. The same experimental approach was used to test 19 different compounds on cells cultured in 24 well plates. The cells were maintained without any drugs, with the drugs alone (as control) and with these 19 drugs used in combination with 1 µM 17AAG and 0.5 µM MG-132. With a series of experiments at 2, 5 and 10 µM concentration of these 19 compounds I found some of them toxic at 2 µM, some toxic at 5 µM and some at 10 µM when used alone. I continued experiments with the non-toxic compounds and found one compound called abamectin showing the sensitization effect when used at 10 µM in combination with 17AAG (1µM) and MG-132 (0.5µM). Abamectin was not toxic when used alone but showed sensitization of the neuT transformed cells to 17-AAG and at some extent to MG-132. In the graphs below (Figure 5 and Figure 6) the control samples include cells without addition of any drugs, MG-132 control is MG-132 used alone at 0.5µM, 17-AAG control is 17AAG used alone at 1µM and the control for abamectin is abamectin used alone at 2, 5 and 10 µM.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

control 0.1µM MG 0.2µM MG 0.5µM MG 1µM17AAG

% c

ell v

iab

ility

Overnight incubation with compounds

Compounds

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Figure 5

MTS assay for toxicity of compounds on the cells in vitro. The toxicity of the compounds on MCF10A her-2/neuT cells in vitro when used alone at 2µM and in combination with 1µM 17AAG and 0.5µM MG-132. A and B) Her-2 transformed MCF10A cells treated with the different compounds as mentioned in Table2 used as single agent and in combination with 17AAG and MG-132.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

control A B C D E F

control

MG-132

17-AAG

2µM

% c

ell v

iab

ility

Aovernight incubation with compounds

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

control

MG-132

17-AAG

2µM

% c

ell v

iab

ilitY

B

overnight incubation with compounds

Compound

s

Compounds

17

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

control

MG-132

17AAG

C

% c

ell v

iab

ility

10µM

overnight incubation with compounds

0

0.05

0.1

0.15

0.2

0.25

0.3

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0.4

0.45

control A F G I L M O

control

MG

AAG

5µM

% c

ell v

iab

ility

Bovernight incubation with compounds

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

control D E H J N Q R

Control

MG132

17AAG

5µM

A

% c

ell v

iab

ilitY

overnight incubation with compounds

Compounds Compounds

Compounds

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Figure 6

MTS assay for toxicity of compounds on the cells in vitro. The toxicity of the compounds on MCF10A her-2/neuT cells in vitro when used alone at (2, 5 and 10µM) and in combination with 1µM 17AAG and 0.5µM MG-132. A and B) at 5 µM and C and D) at 10 µM, in all the case in combination with 17AAG and MG-132. E) Abamectin used at 2 µM, 5 µM and 10 µM, showing no toxic effects of Abamectin when used alone, but sensitizes the cells for 17AAG and to some extent for MG-132

3.3. Analysis of HSF-1 activation by luciferase reporter assay

The above results showing sensitizing effects of Abamectin with 17AAG and MG-132 encouraged us to further check whether this compound affects the activation of HSF-1 transcription factor and in turn the transcription of hsp genes mediated by Heat shock transcription factor-1 (HSF1). For this assay I decided to test another compound called Pimozide which also found to be toxic in the MTS assay. I produced cells expressing luciferase protein under the HSE (heat shock element) promoter. In this case HSF-1 binds to HSE in the promoter region for luciferase gene resulting in luciferase expression. The experiment was designed to check if this combination of compounds were affecting HSF-1 activation or transcription of luciferase, which would be visible by less luciferase activity in the assay. The cells were cultured on a white bottom 96 well plate, followed by addition of the compounds (Abamectin and Pimozide) at 10µM, 17AAG at 1µM and MG-132 at 0.5µM) and incubation for 6 hrs.

0

0.1

0.2

0.3

0.4

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Fig7

HSF-1 activation studied by luciferase assay

The luciferase activity after 6 hrs of incubation with the compounds Abamectin and Pimozide alone and in combination with 1µM 17AAG and 0.5µM MG-132. Control is cells without addition of any compound.

From the results it was evident that the Abamectin and Pimozide alone did not affect luciferase activity when used alone but inhibited its activation when in combination with Hsp90 inhibitor and MG-132 where abamectin in combination with 17AAG and MG-132 was a more potent inhibitor of luciferase activity than Pimozide (Figure 7).

We had two explanations for the observed activity of luciferase, one was that the effect seen might be due to inhibition of transcription of Hsp genes by inhibition of HSF-1, and other possibility was that the effect could be the result of loss of luciferase activity which could be due to improper folding of luciferase and nothing to do with HSF-1.

The more specific explanations for the observed results are:

i) In control cells there is no induction of HSF-1 due to lack of any stress signals, and as a result there is no expression of luciferase

ii) After MG-132 treatment the cell senses stress signal due to the accumulation of unfolded or misfolded proteins which leads to the dissociation of binding between HSF-1 and Hsp90 as a result Hsp90 is recruited to complex chaperone machinery for folding the accumulated proteins. So higher luciferase expression is due to HSF-1 activation.

iii) After 17AAG treatment we know that it breaks binding between Hsp90 and HSF-1, so HSF-1 gets activated and genes with HSE in the promoter are transcribed as observed for luciferase here

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iv) After Abamectin treatment there is no effect on HSF-1 activation, so luciferase activity is not observed

v) When abamectin combined with 17AAG, it causes a strong decrease in luciferase activity. We saw earlier that 17AAG leads to increase of luciferase activity but its combination with abamectin completely inhibits the luciferase activity

vi) Here we hypothesize that abamectin could inhibit the chaperone activity of Hsp72 (an inducible form of Hsp70) and it is well supported by various studies which shows that luciferase is stabilized by Hsp70 chaperone, so in absence of Hsp72 luciferase is not folded properly and become inactive. Similar effect can happen with abamectin used with MG-132 when HSF-1 activation results in luciferase transcription but its improper folding due to inhibition of Hsp70 chaperone results in its reduced activity.

A similar scenario can occur with Pimozide but the data with Pimozide in repeated experiments were highly fluctuating and difficult to reproduce, whereas with Abamectin the data was highly stable. Owing to this I discarded Pimozide from the further studies.

3.4. Effect of Abamectin expression levels of Hsp72

Expression levels of Hsp72

To further check the expression levels of heat shock proteins after the treatment of MCF10A neuT cells with abamectin along with 17AAG and MG-132 I did immunoblotting experiments using standard protocol in the lab as described in materials and methods section. The aim was to see whether this selected combination of compounds has effects on the heat shock proteins expression. So I used anti-Hsp72 antibodies to detect the levels of proteins after the treatment of Abamectin alone and in combination with 17AAG (1µM) and MG-132 (0.5µM) on MCF10A her-2/neuT cells.

Abamectin: 2µM 5µM 10µM

C MG AAG Aba Aba Aba Aba Aba Aba Aba Aba Aba +MG +AAG +MG +AAG +MG +AAG

Figure 8

Immunoblotting for expression levels of Hsp72

Treatment of MCF10A neuT/Her-2 cells with Abamectin (2, 5 and 10µM) alone and in combination with 17AAG (1µM) and MG-132 (0.5µM). Control is the cells without any drugs.

Hsp72

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Interestingly Abamectin when used alone and in combination with Hsp90 and proteasome inhibitor did not affect the expression levels of Hsp72 in vitro. So we supposed that the other possibility of abamectin affecting the chaperone activity of heat shock proteins was interesting to check.

3.5. Analysis of firefly luciferase refolding under CMV promoter

To test further the involvement of abamectin in inhibition of chaperone activity under normal conditions in a cell, I decided again to use the luciferase reporter assay to check the luciferase activity, but this time expressed constitutively under CMV promoter (i.e. under normal rather than stress conditions).

This experiment was different from the previous experiment with luciferase assay as this experiment was meant to check for the expression levels of luciferase under normal cell conditions whereas the previous one was meant to check for the expression levels of luciferase mediated by HSF-1 which is seen under stress conditions.

The cells expressing luciferase under CMV promoter were treated with Abamectin 10µM alone and in combination with 17AAG 1µM and MG-132 0.5µM for 3 hrs and 6 hrs.

Figure 9

Luciferase refolding assay for luciferase activity under the control of CMV promoter

The cells expressing luciferase under CMV promoter were treated with Abamectin at 10µM along with combination of 17AAG (1µM) and MG-132(0.5µM). A) The cells incubated for 3 hrs with the compounds and B) The cells were incubated for 6 hrs with the compounds, and the luciferase activity was read.

As seen in the Figure 9, after incubating cells with the abamectin alone and in combination with 17AAG and MG-132 for 3 hrs and 6 hrs, the luciferase activity was clearly seen to be decreased after 3 and 6 hrs in combination with 17AAG and MG-132.

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To make sure that the results obtained from the luciferase reporter assay was not due to the reduced expression levels of luciferase, I immunoblotted the samples as above with anti-firefly luciferase antibody.

3hrs after addition of compounds 6hrs after addition of compounds

C MG AAG Aba Aba Aba C MG AAG Aba Aba Aba +MG +AAG +MG +AAG

Figure 10

Expression of firefly luciferase under CMV promoter

The cells expressing luciferase under CMV promoter were treated with Abamectin at 10µM along with combination of 17AAG (1µM) and MG-132(0.5µM). A) The cells incubated for 3 hrs with the compounds and the luciferase activity read after freezing at -80oC for 3 hrs or more, B) The cells were incubated for 6 hrs with the compounds and the luciferase activity was read after freezing at -80oC.

It was clear that abamectin in combination with 17AAG and MG-132 did not affect the expression of firefly luciferase and the effect seen in luciferase reporter assay should be the effect of improper folding of luciferase enzyme due to reduced chaperone activity of Hsps.

The results shown in Figure 9 and Figure10 demonstrate the involvement of abamectin in inhibition of the chaperone activity in cells. I could see that abamectin alone and 17AAG alone did affect the luciferase activity to some extent, but the effect enhanced significantly when both of them were combined; this meant that abamectin influences chaperone machinery other than Hsp90. Since we know that in mammalian cells the most important chaperone machine second to Hsp90 is Hsp70 family chaperones, we suggest that the effect of abamectin is on Hsp70 family of chaperones, but this needs to be further investigated.

From our knowledge about the Hsp70 family chaperone we could expect the inhibitory effect on ATPase cycle needed for the activity of Hsp70 chaperone machine, or it could be related to inhibition of interaction between Hsp70 and its co-chaperones.

3.6. Expression levels of Hsp90 client proteins

The degradation of Hsp90 client proteins after inhibition of Hsp90 chaperone function is well documented. But based on the results obtained from my experiments we believed that low concentration of 17AAG (1µM), might not lead to the total degradation of Hsp90 client proteins

Β-actin

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23

which would correspond to the low toxicity of 17AAG used at 1µM. We hypothesize that Hsp90 client proteins may not be totally depended on Hsp90 chaperone machinery for its stabilization but another chaperone in a cell would stabilize these proteins in absence of Hsp90 chaperone. We also believed that abamectin would be responsible for the inhibition of chaperone other than Hsp90.

To test this I checked the expression levels of several Hsp90 client proteins C-RAF, GSK3 and Survivin. But it was clear from the results that the Hsp90 client proteins were totally degraded after inhibition of Hsp90 by 17AAG, meaning they are not dependent on any other chaperone machinery for their stabilization. So our hypothesis regarding the involvement of other chaperone machinery for the stabilization of Hsp90 client proteins proved to be wrong but another hypothesis regarding the involvement of Abamectin in inhibition of chaperone other than Hsp90 still stands and needs to be further evaluated in terms of its specific effects on Hsp72.

Abamectin: 2µM 5µM 10µM

C MG AAG Aba Aba Aba Aba Aba Aba Aba Aba Aba

+MG +AAG +MG +AAG +MG +AAG

Figure 11

Expression levels of Hsp90 client proteins

Hsp90 client proteins BRAF, Survivin and GSK3β expression levels after treatment of Abamectin at 2µM, 5µM and 10µM, alone and in combination with 17AAG (1µM) and MG-132 (0.5µM)

Although, in the above experiment the expression levels of GSK3β dropped after treatment of Abamectin at 10µM alone, we were unable to relate this observation to the observed toxic effects of abamectin, therefore this phenomenon needs to be further evaluated.

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4. Discussion

As described in the introduction, research on heat shock proteins has provided a great deal of evidence about their involvement in various types of cancers and the importance of targeting these heat shock proteins for effective anti-cancer treatment. In this respect our group has hypothesized the possibility of using inhibitors of heat shock response in combination with the novel class of anti-cancer drugs, inhibitors of proteasome and Hsp90. This hypothesis was supported by the study of Gabai, et al., (2005), who described the potent effects of Hsp72 inhibition when combined with Hsp90 and proteasome inhibitor by gene silencing approach using retroviral vectors. In searching for inhibitors of heat shock response Zaarur, et al., (2006) developed a high throughput cell-based screen and out of totally screened 20,000 compounds from different chemical libraries, 19 compounds were reported as potent inhibitors of luciferase refolding after heat shock suggesting their role as inhibitors of the heat shock response. These studies were used as a basis for my project where my role was to study toxic effects of these 19 compounds on the transformed mammary epithelial cells in vitro and find out the effective compounds for sensitizing the cancer cells to inhibitors of Hsp90 and proteasome.

Interestingly, I have found a compound called abamectin which being an inhibitor of heat shock response (as found in screening) also sensitized the transformed cells in vitro to Hsp90 inhibitor (17-AAG) and to some extent for proteasome inhibitor (MG-132). The results obtained were highly stable and this is the first study to report the effect of abamectin as inhibitor of heat shock response and possibly its use in anti-cancer treatment with Hsp90 and proteasome inhibitors.

Abamectin is produced by the fungus Streptomyces avermitilis and known for its use as a pesticide and anti-parasitic drug for humans and animals. There have been some studies with abamectin and its effects on cancer cells where it was found that abamectin inhibit the γ-amino butyric acid (GABA) receptors (Kokoz,1999) and the MDR (multi drug resistance) (Didier, 1996) of the tumors. But this study lacks further development for using abamectin in anti-cancer treatment. Owing to very small number of studies using abamectin in cancer research there has been lack of understanding of the molecular targets of the abamectin and also no study reveals its use in anti-cancer treatment using as single agent or as a combination. Here I hypothesize a novel role for abamectin as an Hsp72 inhibitor (inhibitor of its function) and also suggest a novel combination chemotherapy as anti-cancer therapy using abamectin with Hsp90 inhibitor. Hsps are becoming highly popular among researchers which can be co-related with various studies showing the involvement of heat shock proteins in cancer and their induction as side effect of using Hsp90 and proteasome inhibitors. Hsp72 an inducible form of Hsp70 plays a key role in conferring survival advantage to the cancer cells, and several studies related to inhibiting this stress protein opened a new way to combine Hsp72 inhibitors with Hsp90 and proteasome inhibitors for anti-cancer therapy. The field of Hsp70 inhibitors is still in development and still there is lack of a potent Hsp72 inhibitor molecule used in anti-cancer therapy. My study has also supported the fact that cancer cells could be sensitized to the inhibitors of Hsp90 and proteasome when combined with inhibitors of heat shock response.

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Theoretically speaking questions about specificity of targeting heat shock response could be raised which could be quite logical as heat shock proteins are considered to be ‘friends’ for a cell who protect cells in adverse or stress conditions. So targeting these core modulators of protein homeostasis could result in various side effects even for a normal cell. But in contrast to these beliefs heat shock proteins are expressed in different isoforms of which certain are truly considered to be friends for a cell, but some of them turn out to be foe and helps the cancer cells to prevail in the adverse conditions. As stated in the introduction some studies have revealed that use of Hsp90 inhibitors shows high selectivity between normal cells and cancer cells. In case of Hsp70 family, hsc70 is expressed constitutively which is associated with the chaperone function of Hsp70 family and also is a co-chaperone for Hsp90 chaperone machine under normal cell conditions. But in cancer cells or during stress conditions Hsp72 is expressed which is an inducible form of Hsp70 found at very low expression levels during normal cell conditions. As discussed throughout the report Hsp72 is main culprit here whose presence in cancer cells leads to the reduced effects of Hsp90 inhibitor and also other anti-cancer treatments like radiations etc., so designing a highly specific molecule as inhibitor of Hsp72 and its use along with Hsp90 inhibitor could eliminate the risk of being toxic to the normal cells. The field of Hsp90 and Hsp90 inhibitors is much more developed which allows a simple and clear assessment of the Hsp90 inhibitor drugs and its role in a cell. Whereas Hsp70 in anti-cancer treatment is still a new topic but it is gaining importance where much knowledge is obtained on its function in cancer by studying the effects related to its down regulation or over expression. But when it comes to the different molecular inhibitors of Hsp70, there has been a problem to get molecular target details of these inhibitor molecules in relation to Hsp70 and this could be related to the lack of functional assays for studying Hsp70 family of proteins in a cell. There are some compounds inhibiting HSF-1 which are believed to shut down the expression of heat shock proteins during stress, and these compounds have been tested for their effects as anti-cancer drugs. Some of them were found effective to shut down the heat shock proteins expression and have been used in combination with Hsp90 and proteasome inhibitors. However it has been reported that in some cases heat shock proteins are also expressed in the absence of HSF-1 in cancer cells like PC-3 or DU-145 (Zaarur, et al., 2006). Therefore these molecules might have limited use as anti-cancer drugs. It is very early to say anything about wide applications of the combination treatment using abamectin with Hsp90 inhibitor, as it is tested on mammary epithelial cells only and needs to be further evaluated for different tumor cell lines to gain a wide view of this treatment. During the course of this study beyond testing the compounds for their toxic effects on the cells, I was able to generate a hypothesis that abamectin affects chaperone activity of Hsp72 (as it did not affect expression levels) and this hypothesis could be said to still hold true based on our preliminary experiments. To further prove it, using functional assays for studying the ATPase activity of Hsp70 chaperone in presence of abamectin, I did not have enough time to go through the experiments, so we collaborated with another lab where we assigned them the responsibility to carry out these experiments. Due to the time limit it was not possible for me to further carry out mouse experiments where we planned to grow tumors as allografts in mice and study the

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effect of abamectin in combination with 17AAG on tumor regression. For this we are planning to collaborate with another lab with high expertise in animal experiments. After we get the data from these experiments of the effect of abamectin on Hsp72 chaperone function and in vivo study in mice the picture will become much clearer for further possibility of this combination treatment to be tested on humans in clinical trials.

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5. Materials and Methods

5.1. Selection of cell line

The MCF10A cell line was selected for the experimental work. It is an immortalized human mammary epithelial cell line considered as a proper model for the study of breast cancer, due to its ability to be easily transformed by Her-2/neuT oncogene and can also grow as xenograft on nude mice. Her-2 is a human epidermal growth factor which encodes for a transmembrane tyrosine kinase receptor which is overexpressed in 20-30% of breast cancer. The extracellular ligands binds to the receptor on cell membrane leading to effects on cell proliferation, survival, adhesion and cell motility which is related to the involvement of various signaling pathways like Ras/mitogen-activated protein kinase pathway, the phosphatidylinositol 3 kinase (PI3K)/Akt pathway, the Janus kinase/signal transducer and activator of transcription pathway (JAK), and the phospholipase C pathway (Ross, et al., 2009).

5.2.In vitro mammalian cell culture

The medium of growth for MCF10A cells used was DMEM/F-12 (50-50) from Cellgro, with L-glutamine, 5% of HS (Horse serum), 500 units Penicillin and Streptomycin, 100µl EGF (20ng/ml), 100µl of cholera toxin (100ng/ml), 500µl human Insulin solution from Sigma (recombinant insulin at 10 mg/ml in 25mM HEPES solution, pH 8.2) and 100 µl Hydrocortisome (2.5mg/ml) were added to the medium. The water jacketed CO2 incubator from Thermoforma was used to grow the cells at 37oC in presence of 5% CO2.

Flat bottom cell culture plates of different well types (6 well, 12 well, 24 well and 96 well plates) purchased from Costar® were used. The tissue culture dishes were purchased from Falcon, and included 35mm, 60mm and 10cm dishes. The 25cm2 and 75cm2 tissue culture flasks used were purchased from Corning®.

The general procedure followed for subculture of cells growing in the tissue culture plates, or dishes or flasks was to aspirate the media and wash the cells with PBS (to remove the traces of media which could interfere with the activity of the trypsin to be used in further steps).Then, depending on the tissue culture vessel used, 0.05% trypsin was added and incubated at 37oC. The incubation time with trypsin depends on the cell line, for MCF10A cells it was 10-15 minutes. As the cells detached from the surface of the vessel, they were collected by adding growth medium and cultured in another tissue culture flask for further experiments.

Freezing and storage of cells

Cells can be stored in liquid nitrogen for further use. For freezing the cells freezing media containing horse serum and 10% DMSO (as a cryo protectant) was prepared. The cells from the tissue culture flask were transferred to a 15ml test tube with growth medium as described earlier, and centrifuged at 1000 xg for 3-4 min. The supernatant was discarded and the cell pellet obtained was suspended in 2ml freezing media. 2 Cryo tubes (2ml) were taken and the cells suspended in freezing medium were transferred 1ml to each tube, these tubes were kept at -80oC for a week and then transferred to liquid N2 for further long term storage.

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Retrieving cells

The cells freezed using above method were thawed for use again. The cells from cryo tube were thawed at room temperature and collected in a 15ml test tube with growth medium, thereafter this tube was centrifuged at 1000 xg for 3-4 min. After discarding the supernatant the cells were mixed properly in tissue culture growth medium and cultured in a suitable tissue culture dish.

5.3. Immunoblotting

Buffers used

i) Lysis buffer: 5ml of 2X lysis buffer(80mM HEPES, 100mM KCl, 10mM EDTA, 10mM EGTA, 100mM B-glycophosphate, 2mM Na3VO4, 100mM NaF, 2% triton X-100, final volume 250ml ddH2O), dissolve a protease inhibitor tablet in 5 ml dd H2O, add 100µl of PMSF (100X)

ii) Stacking gel buffer: 4X: 363ml dd H2O, 125ml 2M tris, 10ml 20%SDS, 2ml TEMED iii) Protein separating gel buffer: 4X: 114ml dH2O, 375ml 2M Tris-HCl, pH 8.8, 10ml 20%

SDS, 1ml TEMED iv) Running buffer:5X: 15.14g Trizma base, 72.1 g Glycine, 5.0g SDS (or 25ml 20%

SDS), 1 L ddH2O, pH 8.3 v) Western Transfer buffer: 5X: 15.14g Trizma Base, 72.1g Glycine, 1L ddH2O, add 200

ml methanol for 1 L every time for 1X transfer buffer vi) Blocking solution (5%) : 0.5grams of milk solution in 10ml of TBS-T (1X) vii) Primary antibody: 1:2000 times dilution in 3%BSA solution with 100µL/10 ml of

sodium azide 100mM viii) Secondary antibody: 1:1000 dilution in 1% milk solution in TBS-T(1X) ix) TBS-T: 10X: Tris Buffer Saline-Tween 20

Preparation of cell lysates

The cells were grown in 6 well cell culture plates. The lysis buffer (LB) was prepared as described above. 100-150 µl LB added to each individual well of 6 well plate and cell scraper used to detach cells from the surface. Cell lysates were collected in 1.5ml ependorf tubes on ice. The collected cell samples were diluted in Protein Biorad assay reagent (5X, Biorad, catalog # 500-0006) and read at 600nm in spectrophotometer for quantification and adjustment of protein concentration for equal loading on to gels.

Gel running procedure

Protein separation gel and stacking gel were prepared as per the manufacturer’s instructions. Depending on the protein to be analyzed the samples loaded were around 10-20µl along with the protein standard ladder (low range). The gel running apparatus was filled with the gel running buffer and after setting the apparatus properly 25mA current was applied till the samples run to

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the bottom of the gel. To transfer the protein samples which were separated based on their molecular weight on the gel, the gel was embedded in a sandwich in the following order with 1 spunch, 2 whatman papers, gel, nitrocellulose membrane, 2 whatman papers and one spunch, and put in transfer apparatus with transfer buffer. The current was adjusted to 90volts for 1 hour. This setting of time and current was optimal for the transfer of proteins from gel to the membrane; the transfer was further checked by Ponceau staining of the membrane. The membrane was incubated in blocking solution (3-5% milk solution in TBS-T) for 30 min and then kept overnight in primary antibody solution on shaker in the cold room. Next day the primary antibody was aspirated and the membrane washed 3 times with TBS-T (1X) for 15 min each followed by incubation of the membrane for 1 hour in secondary antibody solution at room temperature on the shaker. The membrane was developed on a X-ray film by adding 1ml developing reagent A and reagent B each to the membrane for 1 to 11/2 min. This gave the imprint of the proteins on the membrane to the X-ray film.

5.4.Bacterial culture and Plasmid Isolation

LB medium: 2L: 20g bacto-trypton, 10g Bacto Yeast Extract, 20g NaCl, 2L ddH2O, 960ml 5N NaOH, pH 7.0, transfer to 5 flasks 250ml and 2 bottles, autoclave 25min, store at room temperature

LB agar: above mixture + 30g Agar (Bacto-agar)

The colony of Escherichia coli was streaked on a Petri plate with LB agar medium and kept at 37oC overnight. Amphicillin was used as a selection marker in the culture medium as the E. coli strain was resistant to amphicillin. Next day a single colony was picked from the petri plate and grown in 250ml flask with liquid LB and amphicillin. The culture was incubated overnight at 37oC on a shaker. Next day the plasmid isolation was done using plasmid Midi and Maxi kit from Qiagen. For further details please refer to the HiSpeedR plasmid purification handbook by Qiagen, (www.Qiagen.com/goto/plasmidinfo). At the end of this extraction procedure the plasmid obtained was eluted in TE buffer. To check the plasmid yield a small fraction of the above obtained plasmid solution was diluted with TE buffer (2µl of plasmid obtained from above purification method was added to 50µl of TE buffer in a small cuvette), and then measured for double stranded DNA content using Biophotometer at 260/280 nm.

5.5.Production of retroviruses and lentiviruses

Human 293T cells were used for retrovirus and lentivirus production. Cells were seeded in a 60 mm culture dish and grown in Dulbecco’s modified eagle medium (DMEM) 1x at 37oC at a 50-60% confluency. For retrovirus production the Rv plasmid G (0.4µg) and Rv Plasmid GP (1.2µg) were mixed to our Rv plasmid of interest (1.5µg) in 0.5ml Opti-MEM medium (without serum), which was further mixed with 0.5ml Opti-MEM containing 10µl Lipofectamine 2000 (Invitrogen). After incubation at room temperature for 30 min this mixture was added to the cells seeded in 60mm dish and 1 ml more of Opti-MEM medium was added (final volume 2 ml). This was followed by incubation at 37oC in incubator overnight. Next day after aspirating the medium from the wells regular DMEM medium with 10% FBS was added to the 60mm dishes with the

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cells. 48-72 hrs after the start of transfection the viruses were collected by filtering the medium through 0.45µm filter (Millipore corp.) using a 5ml syringe (from BD, USA)

5.6.Virus mediated transduction of mammalian cells

MCF10A cells were seeded in a 35mm culture dish at 50-60% confluency. When the cells attached to the surface the viruses (retro and lenti viruses) prepared as described above were added to the cells in the culture dish, as for one 35mm culture dish 0.5ml culture medium + 0.5ml virus + 1 µl Polybrene (1000X). The cells were kept overnight at 37oC in CO2 incubator and next day the medium was changed to the regular DMEM/F-12 50-50 medium. The next day GFP expression was checked which was kept as a control to check the efficiency of infection; if GFP was seen expressed under the fluorescence microscope then the cells were seeded in a 65mm culture dish with puromycin (0.75mg/ml) for selection of transformed cells from untransformed cells.

5.7.MTS cell proliferation and toxicity assay

CellTiter 96® AQueous One Solution Cell Proliferation Assay kit from Promega was used to determine the number of viable cells in the culture. This assay is based on the reaction of NADPH or NADH produced by dehydrogenase enzyme with the tetrazolium compound [3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent PES (phenazine ethosulfate). This reaction yields a colored product soluble in tissue culture medium which can be measured in 96 Microplate reader at 490nm.

The cells were seeded in a 24 well plate to 50-60% confluency. After the cells had attached to the surface the compounds (19 compounds described above and 17AAG and MG-132) were added to the medium and incubated overnight at 37oC with 5% CO2 incubator. Next day 15µl of MTS reagent was added to each well in a 24 well plate and incubated at 37oC in CO2 incubator for 1-4 hours depending on the formation of color. Then these plates were read in a 96-Microplate reader at 490nm.

For detailed procedure kindly refer to promega, CellTiter 96® AQueous One Solution Cell Proliferation Assay, Part# TB245.

5.8.Luciferase reporter assay

This assay works on the principle of luciferase catalyzing the luciferin oxidation which forms a bio-luminescent product called oxyluciferin; this provides a non-radioactive and fairly simple way to measure luciferase activity for different assays.

MCF10 A cells (expressing for luciferase under HSE promoter and under CMV promoter) were seeded to 50-60% confluency in white bottom 96 well plates (NUNC, Denmark) and incubated overnight at 37oC. Next day the compounds were added at 10µM concentration alone and in combination with 17AAG (1µM) and MG-132(0.5µM), and incubated at 37oC (time point

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different for different experiments). After aspirating the growth medium 20µl of Luciferase assay buffer (Promega, Part# E152A) was added to each well. The plate was kept in -80oC for at least 3 hrs (as recommended for complete lysis of the cells and to detach them from the surface). The plates and the luciferase assay reagent (Promega) were thawed simultaneously and the plates were read in 96-Microplate Luminometer (GloMax™) using Gene5 software. For detailed procedure in luciferase reporter assay and the use of 96-Microplate Luminometer please refer to technical bulletin for Luciferase reporter assay from promega (http://www.promega.com/tbs/tb281/tb281.pdf)

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6. Acknowledgements

First of all I express my sincere thanks to Prof. Dr. Michael Sherman for providing me with an excellent opportunity to work in his exciting research group and moulding me in a very fine way. I consider myself lucky enough to get a chance to work with Dr. Sherman and his team. To mention specifically, his leadership and the way he got us involved in the discussions regarding the projects was very impressive. The lessons learnt here have helped me to become a better scientist and even a better person than what I was before, and also I shall cherish them for the entire of my life.

His support during the entire process of visa and also after arriving at Boston was really appreciable as he never let me feel alone in this new place. His friendly and kind nature made me feel comfortable right from the first day of my work in his lab.

I would like to thank Dr. Vladimir Gabai for his very kind and efficient supervision. I was impressed with the way he led me to think independently in this project. He was the one with whom I spent most of my time in the lab, where he maintained my interest in the subject by asking me questions which increased my curiosity and understanding about this research topic. He always told me that ‘science is fun’ and one should enjoy it, which completely changed my way to look at the problems (which are always expected in science).

Being very new to all the experimental work I was a bit nervous and somewhat uncomfortable to begin with but I consider myself blessed to have such a wonderful team around me who guided me throughout without any grudge and made me comfortable with the work.

Special thanks to Dr. Nava Zaarur and Dr. Anatoli Meriin who were very kind to talk to me regarding my project and critically commented on my work. It helped me to polish my thoughts and understanding about my work. Also many thanks to the colleagues in the lab Dr. Julia Yaglom, Le Meng, Kim Geunwon and Teresa Mills for helping me in all the possible way they could.

I take this opportunity to thank the administrative officers Vicki Glenn and Jane Woodson from Boston University who helped me with all my paperwork to get legal residence in USA.

I received a very warm support from the professors at Uppsala University which included my course coordinator Prof. Dr. Staffan Svard, the degree project coordinators Dr. Karin Carlson and Dr. Ulf Lagercrantz, and also to specifically mention Eva Damm and Ylva Lutnes were very kind to me and helped me throughout my stay in Sweden.

Very importantly I want to thank my parents, my friend Mr. Ganesh Pathare and my uncle Mr. Mahesh Mhase for their encouragement, love and moral support throughout.

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7. References

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