In vitro effects of the tyrosine kinase inhibitor, MAZ-51 in combination with epigallocatechin gallate on tumour survival in melanoma cells

Abstract

The aggressive nature of melanoma continues to present a significant health burden worldwide, compounded by the limited efficacy of traditional treatment options. This study aimed to explore novel treatments for melanoma by investigating the effects of two compounds: the tea-derived polyphenol epigallocatechin gallate (EGCG) and the synthetic compound 3-(4-Dimethylamino-Naphthelen-1-Ylmethylene)-1, 3-Hydroindolo-2-One) (MAZ-51). Various assays were employed to explore the compound's mechanisms of action. The cytotoxicity assay was performed at 24, 48 and 72 h. These results revealed that both EGCG and MAZ-51 significantly induced cell death in B16F10 cells while exhibiting some toxicity to non-cancerous HaCaT and RAW 264.7 cells. However, the 48-hour time showed the most inhibition of B16F10 cells with 50% inhibitory concentration (IC50) values of 107.20 μM and 35.34 μM for EGCG and MAZ-51, respectively. Cell cycle analysis was used to determine the effects of the compounds and indicated that both compounds induced Sub-G1/G1 arrest in B16F10 cells and not the control cell lines, suggesting that the inhibition of cell growth occurred through cell cycle arrest. Polarisation optical differential interference contrast microscopy and light microscopy, using haematoxylin and eosin staining, were employed to detect morphological alterations in cell morphology, thereby indicating the mode of cell death. Thereafter, the ultrastructural changes of the nucleus after exposure to compounds were examined using transmission electron microscopy (TEM). Morphological alterations indicated apoptosis in B16F10 cells, with EGCG inducing cell rounding and autophagy, and MAZ-51 causing apoptotic body formation corroborating the cytotoxicity assay. Non-cancerous cells were seen to be in various stages of cell division, indicative of normal cell growth. Dual staining of Annexin V and propidium iodide (PI) indicated an increase in early apoptosis in B16F10 cells treated with the compounds, while non-cancerous cells showed no significant apoptotic or necrotic phases. Antioxidant assays demonstrated EGCG's potent antioxidant capacity, which showed a dose-dependent protective effect against reactive oxygen species (ROS) production in all cells. Although MAZ-51 also showed protective effects against ROS, EGCG was more effective. Furthermore, the study also evaluated the compounds effect on adhesion proteins and found that EGCG more effectively increased E-Cadherin levels in B16F10 cells, potentially reducing their invasiveness compared to MAZ-51. Both compounds increased cytoplasmic focal adhesion kinase (FAK) expression and decreased paxillin cytoplasmic expression. These results suggested that EGCG and MAZ-51 could potentially reduce the phosphorylation of FAK and increase the expression of E-Cadherin to reduce the adhesive properties of B16F10 cells that promote cancer cell proliferation. Additionally, the compound's inhibitory potential was tested in the presence of vascular endothelial growth factor (VEGF), which typically increases cell proliferation. The results suggested that both compounds effectively reduced B16F10 cell growth in the presence of VEGF while promoting cell growth in non-cancerous cells. In conclusion, this study has established that EGCG and MAZ-51 hold considerable potential as therapeutic agents for melanoma cell treatment, with minimal toxicity to keratinocyte HaCaT and murine macrophage RAW 264.7 cells. It is recommended that future research concentrate on the individual application of these compounds, as their combination did not exhibit a synergistic effect.