Effects Of Silibinin On Mitomycin C And CH11 Induced Apoptosis In Human Melanoma A375-S2 Cells

Resistance to apoptosis and abnormal increase of autophagy are two essential causes of chemotherapeutic failure. Therefore, to better understand the mechanisms of apoptotic resistance and autophagy induction seems to be critical to devolping good anti-tumor drugs.Silibinin is a classical hepato-protective drug. Due to its anti-tumor efficacies in many solid tumors such as breast cancer, bladder cancer, prostate cancer and lung cancer, it is now become a hotspot in cancer research. However, Li et al found that silibinin possessed bi-directional effects on human malignant melanoma A375-S2 cell survival:On one hand, silibinin suppressed ultraviolet C (UVC)-induced apoptosis efficiently; on the other hand, silibinin synergized Fas agonistic antibody CH11-induced cell apoptosis. But the mechanisms of silibinin's bi-directional effects were not clearly understood. Based on these researchers' studies, we investigated the mechanism of silibinin's bi-directional effects on A375-S2 cells from apoptosis, autophagy and reactive oxygen species (ROS) three aspects.Our study found that silibinin also opposed chemotherapeutic drug mitomycin C-induced apoptosis:Mitomycin C treatment up-regulated the protein levels of p53, induced the collapse of mitochondrial membrane potential (MMP), facilitated the translocation of Bax and cytochrome c, and finally activated caspase 9-mediated apoptosis. Silibinin pre-treatment reversed the down-regulation of p53, up-regulated the expression of p53 deacetylase SIRT-1. Besides, silibinin also escalated the expression of anti-apoptotic Bcl-2 protein, and maintained the stabilization of MMP.Silibinin is capable of suppressing the expression of p53 in a time-dependent manner, and inducing autophagy which is accompanied with the marked Beclinl up-regulation and conversion of LC 3Ⅰto LC 3Ⅱ. The autophagy inducting effect is enhanced by p53 inhibitor pifithrin-a, but attenuated by MG132 which induced the accumulation of p53 in cytoplasm. Meanwhile silibinin was also found to induce the escalation and activation of NF-κB and this process was enhanced by pifithrin-a. Furthermore, NF-κB inhibitor PDTC suppressed autophagy efficiently. Abrogation of autophagy with 3-MA partially reverses the suppressive effect of silibinin on p53 expression.Mitochondrion is an important origin of ROS generation, and the dysfunction of mitochondrial electron transport chain complexes are the main cause of ROS generation. Small amount or mildly formed ROS generation can function as second messenger in regulating cell signaling such as growth and proliferation. But large amount of or server formed ROS will lead to the damage of organelles and proteins, as well as the over-oxidation of lipid. Our study discovered that silibinin time and dose-dependently induced ROS generation, and this process was suppressed by inhibition of complex I, II and III transferring electrons to complex IV but augmented by inhibition of complex IV activity. Further study showed that silibinin dys-regulated complex IV activity. Cytochrome c plays a key role in transferring electron from complex III to IV. Herein cytochrome c was also found to be down-regulated by silibinin in the current study. Superoxide dismutase (SOD) scavenged ROS efficiently, abolished the over-activation of IGF-1R as well as its down-streamed PI3K-Akt and PLC-y pathways, suggesting that silibinin induced the activation of IGF-1R-PI3K-Akt and IGF-1R-PLC y-PKC pathways in a O2·--dependent manner. Pharmacological inhibition of the over-activation of IGF-1R-PI3K-Akt and IGF-1R-PLCγ-PKC pathways led to obvious apoptosis, suggesting that activation of these pathways promoted cell survival. Besides, it was also found that Bcl-2 up-regulation and p53 down-regulation caused by silibinin were also reversed by SOD and by PI3K inhibitor wortmanin.CH11 is Fas agonistic antibody and it mediates extrinsic apoptosis through activating death receptor Fas. In this study, we found that co-treatment of the cells with silibinin and CH11 at very low concentrations induced marked apoptosis and autophagy. Caspase 8 inhibitor and pan-caspase inhibitor suppressed apoptosis but did not affect autophagy in this case. Autophagy inhibitor 3-MA efficiently suppressed autophagy and apoptosis, while autophagy stimulator rapamycin induced autophagy and facilitated apoptosis. Silibinin and CH11 co-treatment up-regulated the protein levels of Fas and FADD potently. Fas antagonistic antibody UB-2 inhibits apoptosis remarkably without any influence on autophagy. Moreover, silibinin and CH11 co-treatment suppressed the activation of IGF-1R as well as PI3K and Akt. Apoptosis and autophagy were attenuated by IGF-1 but enhanced by IGF-1R inhibitor AG1024. Further study demonstrated that the protein levels of Fas and FADD were decreased by IGF-1 stimulation, but increased by IGF-1 R inhibitor AG1024 and PI3K inhibitor wortmannin. Meanwhile the conversion of LC 3 I to LC 3 II was also enhanced by IGF-1, but attenuated by AG1024 and wortmannin. However, the expression of Beclin-1 was not influenced by IGF-1, AG1024 and wortmannin.In conclusion, silibinin suppressed mitomycin C-induced mitochondrial apoptosis via suppressing p53 and up-regulating Bcl-2. Suppression of p53 also induced autophagy via activating NF-κB, and autophagy protected the cells from mitomycin C induced apoptosis. Slibinin also induces the generation of O2·- via dysfunction of complex IV activity and down-regulating cytochrome c expression. IGF-1R-PI3K-Akt and IGF-1R-PLC y-PKC signaling pathways were activated in a O2·--dependent manner and facilitated cell survival in silibinin treated A375-S2 cells. And the activation of IGF-1R-PI3K was also involved in p53 suppression and Bcl-2 up-regulation. In facilitating cell death aspect, silibinin and CH11 co-treatment induced apoptosis and autophagy in A375-S2 cells, and autophagy facilitated apoptosis. Silibinin and CH11 co-treatment suppressed IGF-1R-PI3K-Akt pathway and thereby increasing the proten levels of Fas and FADD expression, finally inducing caspase 8-dependent apoptosis. Furthermore, the suppression of IGF-1R-PI3K-Akt pathway by silibinin and CH11 co-treatment also induced Beclin 1-independent autophagy and therefore further augmented apoptosis.