Effects of a synthetic indoline derivative on apoptosis in MID and late stage colon cancer cell lines

Naidoo, Vivash
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Colorectal cancer (CRC) is the fourth most common and the sixth most lethal cancer in South Africa. Although, there have been advances in anticancer drug development, there is nevertheless a need for the development of novel cost-effective drugs for treatment. Several reports have suggested that the indole ring contained in various natural and synthetic compounds, is an important chemical moiety with certain biological activities that confers anticancer properties against several cancers. Thus, the aim of this study was to evaluate the potential apoptotic effects of a synthesized Indoline molecule (N-(2-hydroxy-5-nitrophenyl (4methylphenyl) methyl) (HNPMPI) on HT-29 (mid stage) and DLD-1 (late stage) colon cancer cell lines. In particular, the possible effects of HNPMPI on cell morphology and apoptosis (Annexin V) were assessed. After establishing the apoptotic profile of the drug treated cells, changes in the expression of key apoptotic proteins were evaluated using a focused human apoptosis protein array. Based on the identification of differentially regulated proteins identified from the array study, gene expressions of selected genes were analyzed using RTPCR. Further, the subcellular localization of key proteins was determined using Confocal Microscopy. After treatment with HNPMPI, the Annexin V profiling demonstrated that HNPMPI more effectively induced apoptosis in HT-29 cells as compared to the DLD1 cell line. The protein profiler assay revealed differential expression of the apoptotic proteins and it showed that the extrinsic pathway of apoptosis is triggered in HT 29 cells; and Caspase mediated apoptosis was activated in DLD-1 cells, after the treatment. With regards to gene expression levels, the transcription levels of the pro-apoptotic genes BAD, BAX and p53 decreased in DLD-1 cells with HNPMPI treatment. In comparison in the HT-29 cells, while mRNA levels of BAX and p53 decreased, BAD levels were increased. The transcription of the two anti-apoptotic genes, Bcl-2 and CASP-3, had diverse responses to the drug treatment in the two cell lines CASP-3 transcription levels increased in both cell lines; while Bcl-2 levels were constant in the HT-29 cells, but decreased in the DLD-1 cells. In support of this, immunofluorescence confirmed that the intracellular protein expression of Bcl-2 and P53 reflected the results obtained from the protein array, after drug treatment. The indole derivative HNPMPI showed effective apoptotic activity against both cell lines, representing mid and late stage colon cancer. A balance between an over-expression of proapoptotic genes and under expression of anti-apoptotic genes is responsible for carcinogenesis. Although gene expression of selected genes were altered, the protein expression results obtained here confirm that HNPMPI has promising pro-apoptotic properties acting to induce both the intrinsic and extrinsic pathways of cell death in a stage specific manner in the two colon cancer cell lines
A dissertation submitted to the Faculty of Heath Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science Johannesburg, 2019