Studies On The Anti-tumor Activity And Mechanisms Of UAE And ZCU Separated From Gelsemium Elegans Benth

Graceful jessamine herb belongs to Gelsemium elegans Benth of Loganiaceae, Gelsemium elegans Benth has long been identified in Traditional Chinese Medicine to have broad spectrum of pharmacological activities, i.e. antitumor, analgesia, immunoregulation and acetylcholine-like effects. This dissertation reported that uncarinic acid E (UAE) and 3β-hydroxy-27-p- (Z)-coumaroyloxyursan-12 -en-28-oic acid (ZCU) were obtained from non-alkaloid components of Gelsemium elegans Benth through activity screening, and their molecular mechanisms of tumor cell apoptosis induced by UAE and ZCU in HepG2 and KB cell line were studied in vitro respectively. The results showed that different components of non-alkaloid of Gelsemium elegans Benth possessed effects of resisting tumor and immune function were enhanced on tumor-bearing mice in vivo. The toxicity were corresponded in vitro and in vivo.The studies demonstrate that UAE and ZCU significantly inhibited proliferation of several tumor cell lines, including human melanoma A375-S2, human mammary adenocarcinoma MCF-7 (including drug resistance strain drug resistance cell line), human gastric adenocarcinoma SGC-7901, human oral epithelioma KB and human hepatoma HepG2. Moreover, UAE and ZCU promoted proliferation for bone marrow (BMC) in mice and human peripheral blood mononuclear cells (PBMC), and showed minor cytotoxity against normal spleen cells and induced differentiation of T cells and B cell, suggesting that UAE and ZCU had anti-tumor activity and less side effects or immunologic enhancement on normal cells.In UAE-treated HepG2 cells, the results demonstrate that UAE could provoke HepG2 cells apoptosis demonstrated by transmission electron microscope and DNA fragmentation. UAE induced HepG2 cells to activate caspase-3, -6. Caspase substrate PARP was cleaved in a caspase-specific manner, and caspase-3 were cleaved to actives. LDH activity assay and flow cytometry demonstrated that apoptosis and necrosis were induced by UAE simultaneously. UAE also up-regulated p53 expression level in the apoptotic process of HepG2 cells, p53 mediated cell cycle arrest at G0/G1 phase in UAE-treated HepG2 cells. UAE increased the expression of the apoptosis inducer, Bax, decreased the expression of the anti-apoptotic protein, Bcl-x_L and Bcl-2, promoted the release of cytochrome c, and activated down-stream caspase-3 in mitochondrial apoptotic pathway. The mitochondrial apoptotic pathway positively correlated with elevated expression of p53 protein and ERK. This process was prevented by MEK inhibitor, PD98059, and PI3K inhibitor, wortmannin, indicating that ERK activated p53. PKC reactivator, PMA, reversed cell death induced by UAE.In KB cells, ZCU initiated classic apoptotic pathways. Caspase inhibitors and MAPK inhibitors blocked ZCU-induced cell apoptosis. ZCU induced KB cells apoptosis and necrosis simultaneously. Moreover, ZCU inhibited the expression of ERK and up-regulated the expression of p38. ERK resulted in the up-regulation of p53 expression, which was blocked by MEK inhibitor PD98059, suggesting that ERK acted on the upstream of p53. Accumulation of p53, inhibition of ERK and activation of p38 increased the ratio of Bax/Bcl-x_L or Bax/Bcl-2 protein expression, and promoted the release of cytochrome c into cytosol, resulting in apoptotic cell death, at the same time, PKC plays an important regulatory role in the activation of MAPKs.In summary, the pathways of signal transduction could be that UAE and ZCU inhibited phospholipase C_γ1, and then inhibited PKC, the up regulation of p53 induced by the down regulation of ERK due to PKC or PI3K inhibition. The up regulation of p53 changes the ratios of Bcl-2/Bax or Bcl-xL/Bax, then it induces the release of cytochrome c, followed by the amplification of caspase cascade reaction, finally leads cell to apoptosis.