The role of Hsp90/Hsp70 organising protein (Hop) in the Proliferation, Survival and Migration of Breast Cancer Cells.
- Authors: Willmer, Tarryn
- Date: 2012
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4130 , http://hdl.handle.net/10962/d1015720
- Description: Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy.
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The synthesis and breast cancer inhibitory activity of cinnamic acid analogues based on the halogenated monoterpene pharmacophore
- Authors: Chiwakata, Maynard Tendai
- Date: 2012
- Subjects: Halocarbons , Cancer -- Treatment , Breast -- Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3866 , http://hdl.handle.net/10962/d1016129
- Description: Breast cancer is one of the leading causes of death, with mortality rate estimates of 465 000 deaths per annum. It is estimated that 1.3 million women are diagnosed with the disease each year especially in the developing countries. Current chemotherapy relies on the use of high doses of non-specific toxic agents that possess adverse side effects and compromise patient’s compliance and adherence to treatment. Paclitaxel, one of the common drugs used in breast cancer chemotherapy results in sensory and motor neuropathy, whilst hormonal therapy e.g. Herceptin causes severe cardiovascular, gastrointestinal and cutaneous side effects. There has been a demand in developing newer cancer agents that demonstrate selective cytoxicity with minimal effect on normal body tissue. Numerous studies have shown that marine organisms produce a wide range of halogenated compounds that possess cytotoxic properties, and hence can be a source of new drug hits or leads for cancer therapy. Halomon, a polyhalogenated monoterpene from Portieria hornemannii, displayed interesting activity against brain, renal and lung cancer tumours with selective/differential cytotoxicity. This inspired us to focus our project on halogenated monoterpenes isolated from the same Rhodophyta class as P. hornemannii but with particular attention to Plocamium species. Several metabolites have been isolated from P. cornutum, P. corallorhiza and P. suhrii that possess interesting cytotoxicities against a breast cancer cell line (MCF7) and an oesophageal cancer line (WHCO1). The aim of the project was therefore centred at isolating target compounds for preliminary structure-activity studies against a breast cancer cell line, and use this information to synthesize a series of analogues that are more stable than the natural products and yet as active using a fragment-based type approach to map out pharmacophoric elements. Five metabolites were isolated from P. cornutum and five from P. corallorhiza. Cell-based assays were conducted using an MTT assay kit against MCF7 and MDA-MB-231 breast cancer cell lines and (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene, isolated from P. cornutum was the most active with IC50 values of 3.0 μM and 6.15 μM respectively. Introduction of a terminal aromatic ring to enhance stability, together with varying substituents (H, CH3, CF3, Br, CN, CHO, CHCl2) on position 7 of the molecule, gave rise to a series of cinnamate ester derivatives inspired by (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene. The analogues were synthesized from their benzaldehyde precursors via Aldol condensation, esterification and Wittig reactions. Their carboxylic acid counterparts were synthesized by hydrolysis of the parent esters in an attempt to promote water solubilities of the analogues. Biological activity assays were then conducted with the cinnamate analogues against the MDA-MB-231 breast cancer cell line using an MTT assay kit. Ester derivatives with -CHO and -CHCl2 functionalities had IC50 values of 43.45 μM and 100.01 μM respectively whilst the other ester derivatives were inactive. It was concluded that either an aldehyde (-CHO) or gem-dichlorides (-CHCl2) is specifically required for cytotoxic activity to be observed. None of the carboxylic acids were active which could have been due to failure of the compounds to enter the breast cancer cells and reach the target site because of their polar nature. Compounds with -CHO and -CHCl2 functionalities were therefore selected for future SARs studies.
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