Browsing Theses by Subjects
Now showing items 1-1 of 1
Manipulation of apoptosis in cancer cellsConventional cancer therapies can have severe side effects, so new strategies to limit these needs to be investigated. Several anticancer agents induce the expression of tumour suppressor gene p21 in colorectal cancer cell line HT-29. Interestingly, the stress protein HSPA1A is also often elevated in tumour cells and has an anti - apoptotic activity. The main aim of this study was to examine whether a two - pronged approach, overexpressing p21 (using genetic approach and inhibition of HSPA1A using pifithrin - µ would be effective in inducing apoptosis in tumour cells. Chitosan or BSA based delivery systems were evaluated for cytotoxicity, with the intension of using it for plasmid DNA based cell transfections in this study. The interaction of HSPA1A protein in combination treatments involving UV radiation and hyperthermia at 42℃ were also evaluated to perceive the various roles of HSPA1A in arresting colorectal cancer cells. Colorectal cancer cell lines HT-29 and leukaemia cancer cell lines U937 were used in the study. All experiments were performed with cancer cell lines maintained in culture medium devoid of antibiotics. Cell cytotoxicity were evaluated using MTS and PI assays. The rate of apoptosis was determined using annexin V and PI staining by flow cytometry. Chitosan or BSA based microparticles or microgels were observed for size determination or morphology using scanning electron microscopy. Full length human p21 inserted plasmid DNA was a gift from Mien - Chie Hung, Addgene, USA. HT- 29 cells were subjected to p21 plasmid DNA transfection effects. Cells were treated with pifithrin - µ (15µM) prior to gene transfection to address its combined effect with p21 plasmid DNA transfection. HSPA1A and p21 protein expression studies were analysed using FITC labelled antibodies by flow cytometer. Combination studies with HSPA1A inhibitor pifithrin - µ and UV reflected enhanced cytotoxicity compared with either of the treatments independently. Hyperthermia at 42℃ induced apoptosis by MTS assay, which was confirmed by flow cytometric analysis in both the cell lines tested. Considering the cytotoxicity reflected by the chitosan or BSA delivery systems in drug free states, the p21 plasmid DNA transfection was carried out using lipofectamine 2000. Both overexpression of p21 and inhibition of HSPA1A protein with pifithrin - µ enhanced the rate of apoptosis with statistical significance of (p-<0.0001****) compared to the respected controls. The data in this thesis suggests the inhibition of HSPA1A in combination with increased p21 would be a promising therapeutic strategy for the treatment of colorectal cancers.