Studies On The Correlative Mechanisms Between Evodiamine And Interleukin-1-induced Human Melanoma A375-S2 Cell Death

This dissertation reports mechanisms of human melanoma A375-S2 cell death induced by interleukin-1β (IL-1β) and evodiamine, respectively. Based on the correlative mechanisms between them, the specialty of A375-S2 cell death was investigated.First, recombinant human IL-1β was purified, then sub-cloning of A375-S2 cells was carried out and we separated the hypersensitive cell line to IL-1β. The studies demonstrated that IL-1β apparently inhibited the proliferation of A375-S2 cells in vitro. Photomicroscopic observation and Hoechst 33258 staining showed typical apoptotic changes in IL-1β-treated cells. LDH-based assay and DNA fragmentation analysis further affirmed that the type of IL-1β-induced cell death was apoptosis. The result of cell cycle analysis showed that IL-1β arrested A375-S2 cells at G₀/G₁ phase. Then, we found that the inhibitors of caspase family and caspase-1, -3, -8, -9, -10 partially inhibited IL-lp-induced apoptosis, and the activities of caspase-1, -3, -8, -9 were augmented after IL-1β treatment, indicating that caspase activation was involved in IL-1β-induced apoptosis. Caspase-1 activation not only participated in caspase cascade; but also cleaved endogenous IL-1β precursor (pro-IL-1β) into bioactive IL-1β, which acted in synergy with exogenous IL-1β. Caspase-3 activation contributed to the degradation of its substrates, PARP (poly-(ADP-ribose) polymerase) and ICAD (inhibitor of caspase dependent DNase). In mitochondrial pathway, IL-ip decreased the expression of the anti-apoptotic proteins, Bcl-2 and Bcl-xL; increased the expression of the apoptosis inducer, Bax, and promoted the release of apoptosis inducing factor (AIF). Simultaneously, IL-ip activated p53 and its downstream p21, and the activation of p53 might be responsible for the cell cycle arrest and up-regulation of Bax expression. Moreover, p38 and JNK were activated, ERK activity was inhibited in this process, suggesting that ERK presented protective effect from cell death, while p38 and JNK promoted cell death induced by IL-1β.