Studies On The Mechanisms Of Shikonin-induced Tumor Cell Apoptosis

We studied on the molecular mechanisms of tumor cell apoptosis induced by shikonin. Especially, the involvement of caspases and MAPKs in the apoptotic process was analyzed. Caspases activation was required for shikonin-induced apoptosis in HeLa or A375-S2 cells. Specifically, caspase-3 and -8, become cleaved/activated during the process.It was found that shikonin markedly inhibited proliferation of several lines of tumor cells, including human cervical cancer cells, (HeLa, IC50: 18.9+0.7uzM), human malignant melanoma cells (A375-S2, IC50: 7 + 0.4 uM), human breast adenocarcinoma (MCF-7, IC50: 11.5+ 0.7 uM), and mouse fibrosarcoma cells (L929, IC50: 7.2 + 1.5 uM). Moreover, shikonin had less influence on non-tumorigenic human keratinocyte (HaCaT) cell growth, suggesting that shikonin had anti-tumor activity and less side effects on human normal cells.Shikonin induced A375-S2 cells to activate caspase-3, -8, and -9. Caspase substrates such as PARP and ICAD are cleaved in a caspase-specific manner. Shikonin also up-regulated p53 expression level in the apoptotic process of A375-S2 cells. p53 mediated cell cycle arrest at GO/G1 phase in shikonin-treated A375-S2 cells. Decreased Bcl-xL and increased Bax protein levels were positively correlated with elevated expression of p53 protein. Activation of Bax mediated mitochondrial membrane permeabilization and release of cytochrome c, leading to caspase activation. Shikonin up-regulated p-ERK and p53 expression level. This processing was prevented by MEK inhibitor, PD98059, indicating that phosphorylation of ERKactivated p53. Shikonin induced JNK activation and JNK inhibitor blocked shikonin-induced apoptosis. Shikonin did not alter p38 and p-p38 expression, suggesting that this kinase was not involved in shikonin-induced A375-S2 cell apoptosis.In HeLa cells, shikonin initiated classic apoptotic pathway. Pan-caspase inhibitor, casepase-3 and caspase-8 inhibitor blocked shikonin-induced cell apoptosis. LDH activity assay demonstrated that before 24 h incubation with shikonin, apoptotic pathway was mainly responsible for HeLa cell death, but necrotic pathway was initiated after 48 h. Moreover, shikonin induced the expression of ERK and JNK phophorylation. Phosphorylation of ERK resulted in up-regulation of p53 expression, which were blocked by MEK inhibitor PD 98059, suggesting that ERK acts on upstream of p53. JNK inhibitor prevented shikonin-induced HeLa cell apoptosis, demonstrating that JNK plays a crucial role in shikonin-induced cell apoptosis.In summary, we demonstrated that shikonin induced cell apoptosis through distinct mechanisms in A375-S2 cells and HeLa cells.