Research On Oridonin Inhibits Tumor Growth In Glioma Cells And Its Mechanism

Background:Glioma is the most common malignant intracranial tumors. It is characteristics of high malignant degree, pessimistic prognosis, with serious threat to human health The current therapeutic strategies of glioma include traditional treatments such as sugical resectiong, radiotherapy and chemotherapy. Although these treatments were used for more than a century, still fail to find any breakthrough progress. Despite newly developed therapies such as immunotherapy, gene therapy, et al, these treatments mainly target oncogenic signals, and unfortunately, fail to provide enough survival benefit in both human patients and mouse xenograft models. Oridonin is purified from the Chinese herb Rabdosia rubescens and considered to exert extensive anti-cancer effects on human tumorigenesis. Oridonin has been widely studied and has been considered having extensive anti-cancer effects in human cancers, including lung cancer, hepatocellular carcinoma, pancreatic cancer, gallbladder cancer and osteosarcoma et al. However, the anti-cancer effects of Oridonin on glioma still haven’t been studied.Objective:Through cells culture and cell proliferation assess to investigate the role of Oridonin in cell proliferation; Anaylsis the effects of Oridonin on glioma cells’cell cycle by flow cytometry cell cycle analysis and cell cycle-related proteins analysis. Through cell apoptosis and apotosis-reated proteins analysis to assess Oridonin induces cell apotosis and inhibits tumor growth in glioma. Use Xenograft model of glioma process experiment to analysis the effects of Oridonin on tumor growth and glioma cells apotosis and proliferation in vivo.Methods:U87 cells and U251 cells were cultured and treated with various concentrations of Oridonin. Use MTT assay and clony formation assay to analysis the proliferation in glioma and the abilities of glioma cells to form colonies. Through Annexin V-FITC/PI double-staining and flow cytometry to investigate the propotion of suvival, early apotosis and late apotosis glioma cells. For nuclei morphological apotosis examination, Hoechst 33342 staining was sued. PI staining and flow cytometry were employed to analysis the effects of Oridonin on glioma cell cycle. Total proteins in glioma cells, mitochondria and cytoplasm was extracted after Oridonin treatments, quantified by BCA protein quantity kit, then use Western blot analysis the expression level of apotosis-related and cell cycle-related proteins. U87 cells (1×106) were injected intraperitoneally to nude mice for xenograft model. After 28 days of Oridonin administration, all the mice were sacrificed and tumors were dissected, measure and weighted. The dissected tumors were performanced with immunohistochemistry staining, analysis the cleaved-caspase-3 and PCNA expression level.Results:MTT assay found that both U87 cells and U251 cells howed increasing sensitivity to Oridonin. Cell viability was decreased with the raised concentrations and prolonged treatments of Oridonin. Clony formation assay found that colonies were dramatically decreased in response to Oridonin treatments in both U87 cells and U251 cells. Flow cytometry assay found that cell proportion in S phase was significantly increased, while cell proportion in G2/M phase and G1 phase was dramatically decreased. Western blot analysis found that Cyclin A1 was decreased, Cyclin D1 and Cyclin B1 were also decreased after Oridonin treatments. Hoechst 33342 staining found that Oridonin induced fragmented and condensed nucleus in both U87 cells and U251 cells. Flow cytometry found that apoptotic cell proportion in U87 cells raised from 2%in the control to 17% under lOμM/L of Oridonin. In U251 cells, apoptotic cells under 2.5μM/L of Oridonin were nearly the same with control cells, but increased dramatically to 17.5% under 10μM/L of Oridonin. Western Blot analysis found that Oridonin down-regulated the Bcl-2 and NF-κB, whereas up-regulated cleaved-casepase-3 and cleaved-caspase-9. PARP-1 was also increased in this process. Moreover, we further detected that Bax mainly re-localized into mitochondrial and cytochrome c in cystol was elevated in response to Oridonin treatments. Xenograft model expriments found that tumor volume and weight in Oridonin-administrated groups was significantly smaller than the control group. IHC analysis showed that cleaved-caspase-3 was elevated in Oridonin groups, whereas PCNA was decreasedConclusion:Oridonin inhibited cell proliferations in a dose- and time-dependent manner in glioma. Oridonin could exert anti-cancer effects on tumor growth in human glioma by inducing cell cycle arrest and eventual cell apoptosis. The identification of Oridonin as a critical mediator of glioma growth may potentiate Oridonin as a novel therapeutic strategies in glioma treatments.