| ObjectiveMalignant glioma is the most common primary tumor in the central nervous system(CNS), which has become an important brain disease endangering human health. Chemotherapy, as one of the important means of clinical comprehensive treatment methods, is essential for the management of advanced tumor and prevention of its recurrence. However, chemotherapy has not been able to achieve satisfactory result to those solid tumors, which take account for90%of malignant tumors. Chemotherapeutic drugs have poor selectivity between tumor cells and normal cells, and have toxic and side effects. Therefore, it remains an important issure in tumor research to look for efficient drugs with lower toxicity.Nodosin, a pentacyclic6,7-seco-ent-kaurane diterpenoid with an enmein skeleton extracted from the genus Isodon (Labiatae), has the functions of antimicrobial, anti-inflammatory and so on, as well as broad source. In this study, the malignant glioma cells C6, U87and transplantation tumors model with C6cells injection were used as the research objects, to determine the antitumor effect of nodosin in glioma and its mechanism. And the results would suppor to the clinical research of the resist of glioma with powerful experimental and theoretical basis, and expand horizon for the development of the new glioma drugs.MethodsMorphologic observation of nodosin on C6cells was determined by phase contrast microscopy;cell viability was tested by MTT assay; the cytotoxicity of nodosin was evaluated by lactate dehydrogenase(LDH) leakage method; cell proliferation was detected by Brdll assay; and Hoechst33342staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis was used to check the apoptotic cells.The western bloting method was used to detect the cyclin proteins involved in cell cycle of cyclin B1and cdc2and the cell apoptosis related proteins of Bcl-2, Bcl-xL,Bax, Bak and the cell signal pathway protein levels about glioma in glioblastoma C6and U87cells. The activity of caspase-3/7, caspase-8and caspase-9were evaluated by the determination kits, and the cell mitochondrial membrane potential was tested by JC-1staining. All these methods were used to study the mechanism of nodosin in the glioma cell proliferation and apoptosis. In the meanwhile, using the GSK33inhibitor LiCl combined with nodosin to study the effect of nodosin on glioma cell apoptosis.The effects of nodosin on the migration and invasion ability of C6and U87glioma cells were assayed by wound healing method, cell migration transwell and invasion transwell experiments. To study the mechanism of nodosin on migration and invasion in glioma cells, we observed the change of cytoskeleton by nodosin treatment using confocal laser scanning microscope and detected the protein MMP-9using the western blotting method.To establish the in situ in vivo animal model, we innoculated the C6glia cells into Balb/c nude mice, and then determine the effects of nodosin on tumor growth and tumor weight. And the HE staining was used to observe tumor histologic changes in the mice.ResultsMTT assay results showed that nodosin inhibited the rat C6glioma cells and the human U87glioma cells proliferation in time-and dose-dependent manners, and the IC50value of nodosin were about25.8μM and11.05P-M after24h treatment in the C6cells and U87cells, respectively. While the inhibitional effects of nodosin on the normal glia cells HEB and primary rat glia cells is small, with the IC50value were32.06μM and46.69μM, respectively. LDH test found that there is no effect of nodosin on the cell cytotoxicity of C6and U87cells. These results suggested that nodosin has the anti-tumor effect with strong cell selectivity, and its antitumor mechanism may be related to cell cycle arrest, apoptosis induction and invasion and metastasis suppression.BrdU assay and flow cytometry showed that the nodosin have good cell proliferation inhibition effects in C6and U87glioma cells. Cell cycles were significantly arrested the cell cycle at G2/M phase, which inhibited the cell cycle to the next cycle. Western blot experiments results have shown that after24h treatment of nodosin(5,10,15μM) in the C6and U87cells, the cell cycle associated protein levels of cyclinBl and cdc2were decreased.Cell morphological observasion, hoechst33342staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis powerfully testified that nodosin could induce the apoptosis of glioma cells. After24h of nodosin treatment, glioma cells had morphological changes, which show up in the cell number and density decreasing, with the number of death cells increasing as the dose increased in the supernatants, chromatin edge set, the nucleus fragmentation and apoptotic body formation. Caspase activity experiments found that nodosin increases the activity of caspase-3/7, caspase-8and caspase-9in C6and U87glioma cells in a certain dose-response relationship,which reminded us that nodosin induced apoptosis in the glioma cells, through both the death receptor pathway and the mitochondria! pathway. According to the results of western blot method, nodosin can downregulat the Bcl-2, Bcl-X1. protein expression levels, and upregulate Bak protein expression level without influencing Bax. At the same time, we found the nodosin can suppress P38MAPK signaling pathway, Akt signaling pathway and PKC signaling pathway. And using the GSK30inhibitor LiCl combined with nodosin on the glioma cells, we found that LiCl could block cell apoptosis induced by nodosin.Wound healing method, cell migration and cell invasion assays by transwell furtherly indicated that nodosin can inhibit migration and invasion of glioma cells without cytotoxicity against the cancer cells. Cofocal results indicated that nodosin can inhibit microtubules gathered; according to western blot analysis,5,10and15μM of nodosin can decrease MMP-9protein expression level in a dose-dependent manner. These results suggested that the mechanism of nodosin on the glioma cells migration and invasion maybe correlated with cytoskeleton reorganization and the MMP-9protein expression level.In the in vivo experiments, there were no tumor growth and mice weight significantly change between the control group and nodosin group in the third day after innoculated C6glia cells into Balb/c mice. Day4to12days after inoculation, compared with the control group, the nude mice tumor weight and tumor volume growth rate of the nodosin group were significantly less(P<0.05). HE staining showed that the C6glioma cells of control group were of high density and very few of necrosis, while in the nodosin group the density of cells are lower than that in the control group, with the cell numbers reduced, the size of cell becomes smaller, apoptosis of nuclear pyknosis morphological,even part of the region of the liquefaction.Conelusion(1) It is the first time for the study of nodosin on the glioma cell model, and we found that nodosin has obvious selectively inhibitory effect on the cell growth.(2)Nodosin inhibits glioma cells proliferation, which associated with cell cycle blocking in G2/M phase and downrugulating cell cycle associated protein,such as cyclinBl and cdc2protein levels.(3)Nodosin has the effect of inducing glioma cell apoptosis by increasing the activity of Caspase-3/7, Caspase-8, Caspase-9, decreasing mitochondrial membrane potential, downrugulating the Bel-2and Bcl-XL protein expression levels, and upregulating Bak protein expression level. More over, nodosin suppress the signaling pathways proteins of glioma, such as the P38MAPK signaling pathway, Akt signaling pathway and PKC signaling pathway. And the GSK3βis necessary for the glioma cell apoptosis induced by nodosin.(4)Nodosin inhibits migration and invasion metastasis of glioma cells associated with promoting cell microtubule depolymerization and decreasing the protein levels of MMP-9.(5)We found that nodosin has the fuction of resistance malignant glioma in vivo experiments for the first time, which inhibits tumor growth and reduces the tumor weight in nude mice. |
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