Neochamaejasmine A Promotes Apoptosis and Cell Cycle Arrest in B16F10 Melanoma Cells via JNK and p38 MAPK Signaling Pathway.

Abstract

The incidence of melanoma has been increasing over the last 30 years. The most common treatments, such as surgery, chemotherapy, and radiotherapy, frequently cause serious damage to the body. It is therefore critical to develop a new therapeutic strategy for the treatment of melanoma. This research aims to evaluate the anti-tumor effect of Neochamaejasmine A (NCA) on B16F10 melanoma cells and the underlying molecular mechanisms. The CCK-8 kit was utilized to assay the influence of NCA on the vitality of B16F10 cells. Modifications in B16F10 cells morphology were observed using a phase-contrast microscope. Apoptosis of B16F10 melanoma cells was assessed by Hoechst 33258, Annexin V and propidium iodide staining. Cell cycle was detected using a commercial kit by flow cytometry. The mRNA and protein expression levels associated with apoptosis and cell cycle arrest were detected by RT-PCR and Western blot. The expression level of pathway proteins was assessed using Western blot. It was found that the proliferation of B16F10 cells was inhibited by NCA in concentration- and time-dependent manners. NCA promoted apoptosis by halting the cell cycle at the G2/M phase. After treatment with NCA, cell apoptosis was confirmed by Hoechst 33258 staining. NCA triggered the cell cycle to seize at the G2/M stage by downregulating cyclin B1 and cyclin-dependent kinase 2 (CDC2) expression. Moreover, the mRNA and protein expression of cleaved caspase- 9 and Bcl-2-associated X-protein (Bax) were increased, whereas there was a decline in the expression of B-cell lymphoma 2 (Bcl-2). The p-p38/p38 and phosphorylated c-Jun N-terminal kinase (p-JNK/JNK) ratio were also elevated by NCA. The apoptosis and G2/M cell cycle arrest were inhibited in cells co-treated with the p38 inhibitor SB203580 and JNK inhibitor SP600125. The expression of apoptosis-related proteins Bax was decreased, and Bcl-2 was increased. The findings of this study showed that NCA could induce apoptosis and cell cycle arrest in B16F10 melanoma cells by activating JNK and p38 MAPK signaling pathway.