Manipulation of apoptosis in cancer cells

Conventional 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

Evaluation of an In-house Indirect ELISA for Differential Detection of IgM and IgG anti-Brucella Antibodies in Human Brucellosis

Brucellosis caused by various species of the genus Brucella is one of the most important zoonotic diseases of global importance with veterinary, public health, and economic concerns. The study aimed to standardize IgM and IgG-based iELISA to detect anti-Brucella antibodies for serodiagnosis of acute and chronic human brucellosis. The test was standardized using 1:320 dilution of smooth lipopolysaccharide (sLPS) antigen from B. abortus S99 strain, 1:80 serum dilution, 1:4000 anti-human IgM and IgG conjugates, respectively for both IgM and IgG iELISA. The cut-off using 50 each brucellosis positive and negative human sera panel samples was set at ≥ 42 for both IgM and IgG iELISA. A total of 700 human sera samples were evaluated (137 veterinary doctors, 157 artificial inseminators, and 406 veterinary assistants). Overall, the study detected 8.3%, 8.1%, 8%, and 6.1% positivity by in-house IgG iELISA, RBPT, IgM iELISA, and SAT tests, respectively. Considering commercial iELISA kit as a gold standard, the sensitivities of IgM and IgG iELISA were 90% and 97.9%, respectively, whereas, specificities were >99%. The study established >98% specificity and >90% sensitivity for differential detection of immunoglobulin classes in the standardized iELISA. The developed assay outperformed the other evaluated tests with a shorter assay time and can be implemented in both endemic and non-endemic regions for surveillance and diagnosis of human brucellosis