Shao, Luying; (2024) An in vitro and in silico approach to natural products with cytotoxic and antimigratory properties against melanoma cell lines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Malignant melanomas are of significant concern due to their aggressive metastatic behaviour and their high mortality rate. Many natural products are under study as novel therapeutic molecules to treat cancer; with the potential of offering a safer profile and helping to overcome drug resistance. However, little is known about the phytochemicals’ mechanisms of action. This project aims to identify phytochemicals that have a cytotoxic effect against two human melanoma cell lines (A375 and SK-MEL-28) and to evaluate their potential mechanism of action. Initially, 25 phytochemicals were screened both as single treatments and combination treatments leading to the identification of harmine and berberine chloride as active molecules showing a moderate synergy on SK-MEL-28. Further experiments, including DAPI staining of the nuclei, evaluation of caspase 3/7 activity and mitochondrial membrane potential depolarisation, were performed to evaluate whether the cytotoxic effect on the melanoma cell lines was due to induction of cell death (apoptosis or necrosis) or inhibition of cell growth. The effect of the phytochemicals on migration and invasion activities was also examined. Finally, a bioinformatic approach was taken to computationally model the mechanisms behind melanoma and the effect of harmine and berberine chloride. A list of melanoma genes including the 54 risk loci identified in the latest melanoma-GWAS (Landi et al., 2020), the genes harbouring melanoma-associated germline mutations, and the genes frequently mutated in melanoma with somatic mutations were selected. In total 232 melanoma risk genes were prioritized and used to draw the protein interactome of melanoma. This melanoma risk model allowed for identification of the processes that are more likely associated with the genes involved in melanoma and that are probably dysfunctional during disease onset and progression. RNA from cells treated with harmine and berberine chloride was collected and submitted for RNA sequencing to identify transcripts within the melanoma risk model that are altered in expression upon drug treatment. In conclusion, the study demonstrates that harmine and berberine chloride exhibit an in vitro synergistic cytotoxic effect on human melanoma cell lines. The cytotoxicity of harmine appears to be mediated by the activation of caspase 3/7 but is independent of mitochondrial function. In contrast, berberine chloride and the combination treatment primarily inhibit cell proliferation, leading to reduced cell density, without inducing cell apoptosis, or possibly triggering cell necrosis. Both phytochemicals also display cytotoxic effects by inhibiting cell migration and invasion. In silico, harmine and berberine chloride are able to modify the expression of genes important to sustain the flux of information within the pathways associated with melanoma risk genes.

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