Studies On The Relationship Between Oridonin-induced Autophagy And Apoptosis In Human Cancer Cells

The dissertation investigated the molecular mechanisms of the relationship between oridonin-induced apoptosis and autophagy in different human cancer cell lines, including human cervical carcinoma cell HeLa, human breast cancer cell MCF-7 and human epidermal carcinoma cell A431 in vitro.As the results shown, HeLa, MCF-7 and A431 cells were all induced apoptosis by oridonin in a dose- and time-dependent manner. In the apoptotic process, typical apoptotic features, including apoptotic bodies, membrane blebbing as well as nuclear condensation, were induced by oridonin. The fluorescence density of Hoechst 33258 was enhanced while that of Rhodamine 123 declined. DNA was single-strandedly damaged, which led DNA ladder appearance in agarose gel electrophoresis. Companied with DNA damage, cell cycle arrest was induced in oridonin-challenged cells. Finally, cancerous cells underwent programmed cell death. According to various concentrations of oridonin, different degrees of autophagy emerged at the same time, which was characterized by increase in MDC recruitment. Oridonin augmented apoptosis and autophagy potently, however, autophagy exerted contrary influences on apoptosis signaling pathway in different cell lines.After monodansylcadervarine, (MDC, the specific dye for autophagosomes) labeling, oridonin-treated HeLa cells recruited more MDC-positive particles, and had more significantly fluorescence density, and simultaneous expressions of autophagy-related proteins, microtubule-associated protein 1 light chain 3 (MAP-LC3) and coiled-coil, myosin-like Bcl-2 interacting protein (Beclin 1), were increased by oridonin. In the case of oridonin-application, the autophagic ratio was decreased according to the increased doses of 3-methyladenine (3-MA, the specific inhibitor of autophagy) treatment, companied with downregulation of the protein expressions of MAP-LC3 and Beclin 1. Furthermore, when small G protein Ras inhibitor was applied, the autophagic levels were augmented; whereas p38 kinase and Jun N-terminal kinase (JNK) inhibitors decreased autophagic ratio significantly, indicating that Ras negatively, while p38 and JNK mitogen-activated protein kinase (MAPKs) positively regulated this oridonin-induced autophagic process. Protein kinase raf-1 and extracellular signal-regulated protein kinase (ERK1/2) had not obvious correlation to these signaling pathways. Oligonucleosomal fragmentation of DNA as well as increased activities of Bcl-2-associated X protein Bax proteins were induced by oridonin, but the expression of phosphorylated B-cell leukemia/lymphoma 2 (Bcl-2) protein was reduced. When the inhibitor of phosphatidylinositol 3-kinase (PI3K), wortmannin, was applied prior to oridonin addition, the autophagic level was significantly decreased, while the apoptotic level was increased, indicating that PI3K is a key regulator of both autophagy and apoptosis. Akt, down-stream factor of PI3K, was activated in autophagic process but suppressed in apoptosis study. In addition, when autophagy was blocked by 3-MA, the expression of Sir2-related Protein Type 1 protein (SIRT-1) was decreased, indicating SIRT-1 contributed to autophagy. Taken together, oridonin simultaneously induced HeLa cell both apoptosis and autophagy in HeLa cells, and inhibition of autophagy contributes to upregulation of apoptosis.Oridonin inhibited MCF-7 cell growth. Oridonin downregulated the phosphorylation of ERK, whereas those of JNK and P38 kinase were upregulated. Cell cycle altered through up-regulation of p53 and p21 protein expressions. Pan-caspase inhibitor Z-VAD-fmk and Calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and downregulation of mitochondrial membrane potential (△ψ_mit) were detected in oridonin-treated MCF-7 cell apoptosis, which involved in a post-mitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and heat shock protein Hsp 90 proteins levels and increased Bax and p21 proteins levels were positively correlated with elevated expression of phosphorylated p53. Moreover, PARP was partially cleaved by Calpain rather than capase-3. Therefore, DNA damage provoked alternations in mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-treated MCF-7 cells. Meanwhile oridonin also induced autophagy in MCF-7 cells in vitro. In the condition of oridonin treatment, 3-MA significantly reduced the autophagic level, and the apoptotic cell ratio was also declined. Furthermore, combined treatment with oridonin and 3-MA upregulated ERK phosphorylation and downregulated JNK and P38 kinase phosphorylation compared with oridonin alone treatment groups, indicating that autophagy facilitated apoptosis in oridonin-induced MCF-7 cells. In addition, 3-MA application downregulated DNA ladder and Bax expression but upregulated Bcl-2 expression, compared with oridonin alone treatment. Thus, oridonin simultaneously induced MCF-7 cells both apoptosis and autophagy, and in this settings, inhibition of autophagy induced lowered apoptotic level, therefore, autophagy participated in upregulation of apoptosis.Oridonin suppressed cell proliferation in epidermal growth factor receptor (EGFR) overexpressing A431 cells in vitro, and promoted both apoptosis and autophagy simultaneously. 3-MA enhanced apoptotic ratio and significantly decreased the total PTK activity and EGFR protein expression, as well as enhanced p53 activity. Meanwhile, when 3-MA, inhibitor of autophagy, was applied, autophagic ratio was downregulated, and both expressions of apoptosis-related proteins procaspase-3 and procaspase-9 were also negatively regulated. Therefore it might be concluded that autophagy antagonized apoptosis mediated by p53 and caspases modulation in oridonin-challenged A 431 cells.Taken together, oridonin promoted both apoptosis and autophagy efficiently in human tumor cells. Oridonin-induced autophagy was a double-edged sword displaying two-way regulation which contributed or suppressed apoptosis through mitochondrial and cell cycle arrest signaling pathways, leading to cell survival or cell death according to different cell types.