Evaluation of Antioxidant, Anti-inflammatory, and Cytotoxic Activities of Crotalaria pallida Aiton Leaves

Objectives: In this study, the anticancer effect of HMBA was assessed against SK‑MEL‑28 cells. Materials and Methods: DNA damage was assessed using DNA strand break assay (comet assay), apoptosis‑mediated cell death by Annexin‑V, and Terminal deoxynucleotide transferase dUTP Nick End Labeling experiments. Western blot analysis was performed to assess the phosphorylation of ERK, p38, and JNK. Results: Increasing the time of exposure decreases the IC25, IC50, and IC75 value of HMBA. Activity of lactate dehydrogenase in the culture medium of control and HMBA‑exposed cells directly correlate its cytotoxic property. HMBA caused dose‑dependent DNA damage and induced apoptosis in SK‑MEL‑28 cells. AO‑staining showed autophagy in HMBA‑treated cells, whereas Western blot analysis revealed increase in the phosphorylation of ERK, p38, and JNK. Furthermore, our results show that ERK phosphorylation is responsible for the activation of autophagy protein such as LC3 and p62. These observations reveal that the activation of caspase‑3 and commencement of autophagy is mediated through activation of ERK phosphorylation. Conclusion: Therefore, HMBA inhibits propagation of SK‑MEL‑28 cells via stimulation of apoptosis and autophagy. In addition, HMBA promotes apoptosis and autophagy by phosphorylation of vital signaling protein such as ERK, however other vital signaling such as p38 and JNK also phosphorylate. Thus, HMBA may be of therapeutic importance for the treatment of melanoma.