Browsing by Author "Naidoo, Vivash"

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    An in-silico analysis of the glycosylation inhibitors Brefeldin A and Tunicamycin C in colorectal cancer; characterization of novel targets
    (University of the Witwatersrand, Johannesburg, 2024) Naidoo, Vivash
    Colorectal cancer (CRC), a prevalent malignancy in South Africa, is significantly influenced by posttranslational modifications such as glycosylation. This study investigates the complex interactions between genes, signalling pathways, and cellular processes involved in CRC progression and glycosylation. The glycosylation inhibitors, Tunicamycin and Brefeldin A, are known to hinder colon cancer cell proliferation, migration, and invasion, making them potential therapeutic agents. We used Swiss Target Prediction Software to identify target proteins for both compounds and revealed that Protein Kinase C Alpha (PRKCA), Peroxisome Proliferator- Activated Receptor Gamma (PPARG), and Mitogen-Activated Protein Kinase 1 (MAP2K1) are specific for Brefeldin A, and TK1 and PRKCA for Tunicamycin, respectively. These proteins were selected based on their potential role in the glycosylation process and their role in CRC-related pathways. Further, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis disclosed significantly enriched pathways, including Epstein-Barr virus infection, cellular senescence, and cancer pathways. The 3D-crystallographic structures of PrKC1 (PDB ID 6ar4), TK1 (PDB ID 1w4r), PrCK1 (PDB ID: 6ar4) and MAPK (PDB ID: 3eqc) were retrieved from RCSB Protein Data Bank. The compounds BSP and EGCG were downloaded from PubChem. All non-relevant co-crystallized molecules, including ions, crystallographic water, and others, were removed. Missing residues in the proteins were filled in using the MODELLER algorithm on the UCSF Chimera Graphic User Interface. Molecular docking of Tunicamycin C and Brefeldin A was performed with UCSF Chimera, and the docked conformations were visualised in Maestro and Chimera. The complexes with the top docking scores were selected and prepared for molecular dynamics simulation studies to offer structural and dynamic perspectives on the inhibitory potential of the compounds against the target proteins. The Origin Lab software tool was used to post- analyze the docking conformations. Molecular Dynamics simulation was conducted using Graphics Processing Units version of the Particle Mesh Ewald Molecular Dynamics engine in the AMBER18 suite. Our investigation into the dynamic events leading to the proximal binding of Tunicamycin at the pockets of TK1 and PrKC1 suggested that the binding of Tunicamycin induced a conformational perturbation of the 3D structures of these proteins, resulting in a structural deviation that inhibited their activity. Tunicamycin's time-based dynamics indicated a stable pattern, leading to optimal interaction and maximal stabilization in the hydrophobic pockets of TK1 and PrKC1. Binding energy calculations showed a high-affinity interaction of Tunicamycin with these proteins. Similarly, the structural investigation revealed that the binding of Brefeldin A to Mitogen- Activated Protein Kinase (MAPK) and Protein Kinase C (PrKC1) inhibited their activity. A detailed analysis of active site residues revealed crucial residues that contributed to the binding stabilization of Brefeldin A. It was noted that the Brefeldin A/MAPK complex produced a binding energy of -22.18±4.50Kcal/mol while the Brefeldin A/PrCK1 complex produced a binding energy of -23.90±5.36Kcal/mol. These findings provide crucial insights into designing novel inhibitors of TK1 and PrKC1, potentially blocking glycosylation progression in cancer treatment. This study underscores the potential for exploiting glycosylation inhibition as a therapeutic strategy against CRC, opening avenues to mitigate cancer progression
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    Effects of a synthetic indoline derivative on apoptosis in MID and late stage colon cancer cell lines
    (2019) Naidoo, Vivash
    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