| This dissertation mainly demonstrated that evodiamine induced cell death through different mechanisms in A375-S2, HeLa, and L929 cells.The studies found that evodiamine obviously inhibited proliferation of several lines of tumor cells, including human malignant melanoma A375-S2 (IC50: 14.7±1.9 mol.L-1), human cervical cancer HeLa (IC50: 15.4±0.7mol.L-1), human acute monocytic leukemic THP-1(IC50:18.1±1.1mol.L-1), and human breast adenocarcinoma MCF7 (IC50: 22.7±1.3 umol.L-0), mouse fibrosarcoma L929 (IC50: 20.3±3.5mol.L-1), but no cytotoxic effects were observed on mouse sarcoma 180 cell (S180), mouse uterocervical cancer U14 cell and Buffalo rat liver BRL cell. Moreover, evodiamine had less influence on human peripheral blood mononuclear cells (PBMC) growth, compared to actinomycin D, cisplatin and 5-Fu, suggesting that evodiamine showed selective anti-tumor activity and had less side effects on human normal cells.In evodiamine-treated A375-S2 cells for 12 h, Hoechst staining showed obvious apoptotic bodies. However, at 24 h, DNA electrophoresis appeared smear-like DNA fragmentation, which was a hallmark of necrosis. LDH activity assay demonstrated that before 24 h incubation with evodiamine, apoptosis pathway was mainly responsible for cell death in A375-S2 cells, but necrosis pathway was initiated after 24 h. At the early stage, evodiamine activated caspase-3, 8, and -9, moreover, pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), and caspase-9 inhibitor (z-LEHD-fmk) had apparently inhibitory effects on evodiamine-induced cell death, which disappeared after 24 h. Furthermore, it was revealed that evodiamine up-regulated Bax expression level but down-regulated Bcl-2's with Western blot assay, suggesting that evodiamine-induced apoptosis was mainly mediated by both caspase family and Bcl-2 family. At the early stage, p38 inhibitor (SB203580) partially blocked evodiamine-induced cell death, which was augmented by combination SB203580 and z-VAD-fmk. Oppositely, ERK inhibitor (PD98059) enhanced evodiamine-induced cell death.Evodiamine did not alter p38 expression, but increased the phosphorylation of p38. However, evodiamine down-regulated both ERK expression and the phosphorylation of ERK. These suggested that MAPK family were responsible for evodiamine-induced necrosis at the late stage. Otherwise, evodiamine had no influence on cell cycle in A375-S2 cells, and evodiamine-induced cell death appeared at all of cell cycle phases.In HeLa cells, evodiamine initiated classic apoptotic pathway. Hoechst 33258 staining showed apoptotic bodies, and DNA electrophoresis appeared DNA ladder, which were believed to be the hallmark of classic apoptosis. Pan-caspase inhibitor and caspase-3, caspase-8, and caspase-9 inhibitors blocked evodiamine-induced apoptosis, moreover, caspase-3, 8, and caspase-9 were activated. However, the activation of caspase-8 was delayed, compared to caspase-3 and caspase-9. Bcl-2 expression was down-regulated and Bax expression was up-regulated by evodiamine. LDH activity assay also demonstrated that necrosis pathway did not involve in the evodiamine-induced HeLa cell death. Although INK inhibitor (SP600125) promoted the apoptosis, evodiamine failed to influence the expression of INK and JNK phosphorylation. Moreover, evodiamine induced cell cycle arrest at G2/M phase.It was demonstrated by LDH activity assay that evodiamine induced cell death through apoptosis pathway in L929 cells. Although apoptotic bodies were observed in evodiamine-treated L929 cells stained with Hoechst 33258, DNA fragmentation, a hallmark of apoptosis, was not detected. Unexpectedly, pan-caspase inhibitor (z-VAD-fink) rendered the cells even more sensitive to evodiamine and caspase-3 inhibitor (z-DEVD-fmk) also weakly augmented the cell death. Moreover, evodiamine caused G2/M arrest at the low dose, but GO/G1 arrest at the high dose.In summary, we first demonstrated that evodiamine induced cell death through distinct mechanisms in A375-S2 , HeLa, an...
|
PDF Full Text Request