Nodosin对恶性胶质瘤的作用及其机制研究
摘要
目的：
恶性胶质瘤,是中枢神经系统最常见的原发性肿瘤,已成为危害人类健康的重要脑部疾病。化疗是其临床综合治疗的重要手段之一,是治疗晚期恶性肿瘤以及防止复发必不可少的。然而,对于占恶性肿瘤90%以上的实体瘤,化疗尚未能取得满意的效果。且化疗药物对肿瘤细胞与正常细胞的选择性较差,对人体往往产生一定的毒副作用。因此,寻找高效、特异和低毒的抗肿瘤药物是肿瘤防治研究中的重要课题。
Nodosin是从唇形科香茶菜属植物中提取出来的一种五环二萜类化合物,来源广泛,具有抑菌、抗炎等作用。本课题以恶性胶质瘤细胞株C6、U87和C6移植瘤模型为研究对象,研究nodosin对脑胶质瘤的抑制作用,并探讨其作用机制,为其临床抗胶质瘤的研究提供有力的实验依据和理论基础,为进一步开发新型抗胶质瘤药物扩展新视野。
方法：
采用倒置显微镜对胶质瘤细胞进行形态学观察；采用四甲基偶氮唑蓝(MTT)法测定细胞生存率,乳酸脱氢酶(LDH)法检测细胞毒性,BrdU检测法检测细胞增殖情况；应用流式细胞仪技术研究nodosin对细胞周期的影响；采用Hoechst33342染色法、原位末端标记技术(TUNEL)法观察nodosin对细胞凋亡的影响。
采用western blot方法对细胞周期相关蛋白cyclin B1和cdc2和细胞凋亡相关蛋白bcl-2、Bcl-xL、 Bax、Bak及相关胶质瘤细胞内信号通路的蛋白水平进行检测；以及通过对caspase-3/7、caspase-8、caspase-9活性检测和细胞线粒体膜电位的测定(JC-1染色),来研究nodosin抑制胶质瘤细胞增殖及诱导细胞凋亡的机制。同时,应用GSK3β抑制剂LiCl联合nodosin,研究nodosin对细胞凋亡的影响。
选择划痕实验和Transwell细胞迁移、侵袭实验评价nodosin对胶质瘤细胞的侵袭迁移能力；应用激光共聚焦扫描显微镜对胶质瘤细胞骨架进行观察,并通过western blot方法检测MMP-9的蛋白含量来研究nodosin对胶质瘤细胞的迁移与侵袭的机理。
通过建立大鼠恶性胶质瘤细胞株C6细胞皮下移植瘤模型,探讨在在体条件下nodosin对肿瘤体积、肿瘤重量等的影响,并通过HE染色观察肿瘤病理组织学的改变。
成果：
MTT结果显示nodosin对胶质瘤细胞的生长有明显的抑制作用,且呈现明显的量效、时效关系,处理细胞24h后,对大鼠胶质瘤C6细胞和人胶质瘤U87细胞的IC50分别为：25.81μM、11.05μM;而nodosin在24h时对人正常胶质细胞HEB及大鼠原代胶质细胞的存活率影响不大,其IC50分别为：32.06μM、46.69μM。LDH检测发现,nodosin对C6及U87没有细胞毒作用。这些结果表明,nodosin的抗肿瘤作用具有较强的细胞选择性,其抗肿瘤作用机制可能与细胞周期阻滞、细胞凋亡诱导和侵袭转移抑制有关。
BrdU检测法和流式细胞术检测结果显示,nodosin对C6及U87胶质瘤细胞具有较好的增殖抑制作用,通过使胶质瘤细胞组滞于G2/M期的机制,抑制胶质瘤细胞进入下一增殖周期,从而抑制胶质痈细胞增殖。Western blot法实验显示,5μM、10μM、15μM的nodosin处理C6、U87细胞24h后,cyclinBl和cdc2蛋白表达水平下调。
细胞形态学观察、Hoechst33342染色法染色以及原位末端标记技术(TUNEL)检测结果显示,nodosin能诱导胶质瘤细胞的凋亡,作用胶质瘤细胞24h后,细胞形态发生改变,细胞数量与密度减少,随着剂量增加,培养基中悬浮的死亡细胞增多,出现染色质边集,细胞核碎片化,凋亡小体形成。Caspase活性检测发现,nodosin能够增加C6、U87细胞中caspase-3/7、caspase-8、caspase-9的活性,并成一定的剂量依赖关系,提示着我们,nodosin诱导胶质瘤细胞凋亡的作用,既通过死亡受体通路,亦通过线粒体通路。Western blot法结果显示,nodosin能够下调Bcl-2、Bcl-x1蛋白表达水平,上调Bak蛋白的表达水平,Bax蛋白水平不变；同时,能抑制P38MAPK、 PI3K/Akt和PKC信号通路；应用GSK3β抑制剂LiCl联合nodosin,发现LiCl可阻断nodosin引起的细胞凋亡。
划痕实验和Transwell细胞迁移、侵袭实验结果显示,nodosin能够剂量依赖性地阻止胶质瘤细胞的迁移与侵袭；cofocal结果发现,nodosin可抑制微管聚集；western blot分析结果显示,5、10和15μM的nodosin能以剂量依赖方式减少MMP-9蛋白表达水平。提示,nodosin对胶质瘤细胞的迁移侵袭抑制机制与其促进细胞微管解聚以及下调MMP-9蛋白表达水平相关。
接种胶质瘤C6细胞后第3d,对照组和nodosin组的肿瘤生长没有明显差异。接种后第4d到第12d,对照组的肿瘤生长快速,nodosin组的肿瘤生长缓慢,差异有显著性意义(P<0.05)；裸鼠肿瘤重量、肿瘤体积增长率均明显小于对照组。HE染色显示,对照组C6胶质瘤细胞丰富密集,坏死很少,nodosin组C6胶质瘤细胞生长密度低于对照组,并可见到细胞体积变小,核固缩深染的凋亡形态学改变,部分区域出现液化坏死。
结论：
(1)首次在胶质瘤细胞模型上研究nodosin抗恶性胶质瘤的作用,发现nodosin对胶质瘤细胞的生长具有明显的选择性抑制作用；
(2) Nodosin对胶质瘤细胞具有细胞增殖抑制作用,这种作用通过阻滞细胞周期于G2/M期以及下调cyclinBl和cdc2蛋白水平来实现；
(3) Nodosin具有诱导胶质瘤细胞凋亡的作用,其机制为：nodosin通过增加Caspase-3/7、Caspase-8、Caspase-9活性、降低线粒体膜电位、下调Bcl-2、Bcl-x1蛋白的表达水平以及上调Bak蛋白的表达水平来诱导胶质瘤细胞凋亡；nodosin能抑制P38MAPK、Akt和PKC等相关胶质瘤细胞内信号通路的蛋白水平；GSK3β在nodosin诱导细胞凋亡中是必需的。
(4) Nodosin能通过促进细胞微管解聚以及下调MMP-9蛋白水平来抑制胶质瘤细胞的迁移侵袭。
(5)首次在体内实验中,发现nodosin能抑制裸鼠内胶质瘤的肿瘤生长及减少肿瘤重量,具有体内抗恶性胶质瘤作用。
Objective
Malignant 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.
Methods
Morphologic 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.
Results
MTT 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.
恶性胶质瘤,是中枢神经系统最常见的原发性肿瘤,已成为危害人类健康的重要脑部疾病。化疗是其临床综合治疗的重要手段之一,是治疗晚期恶性肿瘤以及防止复发必不可少的。然而,对于占恶性肿瘤90%以上的实体瘤,化疗尚未能取得满意的效果。且化疗药物对肿瘤细胞与正常细胞的选择性较差,对人体往往产生一定的毒副作用。因此,寻找高效、特异和低毒的抗肿瘤药物是肿瘤防治研究中的重要课题。
Nodosin是从唇形科香茶菜属植物中提取出来的一种五环二萜类化合物,来源广泛,具有抑菌、抗炎等作用。本课题以恶性胶质瘤细胞株C6、U87和C6移植瘤模型为研究对象,研究nodosin对脑胶质瘤的抑制作用,并探讨其作用机制,为其临床抗胶质瘤的研究提供有力的实验依据和理论基础,为进一步开发新型抗胶质瘤药物扩展新视野。
方法：
采用倒置显微镜对胶质瘤细胞进行形态学观察；采用四甲基偶氮唑蓝(MTT)法测定细胞生存率,乳酸脱氢酶(LDH)法检测细胞毒性,BrdU检测法检测细胞增殖情况；应用流式细胞仪技术研究nodosin对细胞周期的影响；采用Hoechst33342染色法、原位末端标记技术(TUNEL)法观察nodosin对细胞凋亡的影响。
采用western blot方法对细胞周期相关蛋白cyclin B1和cdc2和细胞凋亡相关蛋白bcl-2、Bcl-xL、 Bax、Bak及相关胶质瘤细胞内信号通路的蛋白水平进行检测；以及通过对caspase-3/7、caspase-8、caspase-9活性检测和细胞线粒体膜电位的测定(JC-1染色),来研究nodosin抑制胶质瘤细胞增殖及诱导细胞凋亡的机制。同时,应用GSK3β抑制剂LiCl联合nodosin,研究nodosin对细胞凋亡的影响。
选择划痕实验和Transwell细胞迁移、侵袭实验评价nodosin对胶质瘤细胞的侵袭迁移能力；应用激光共聚焦扫描显微镜对胶质瘤细胞骨架进行观察,并通过western blot方法检测MMP-9的蛋白含量来研究nodosin对胶质瘤细胞的迁移与侵袭的机理。
通过建立大鼠恶性胶质瘤细胞株C6细胞皮下移植瘤模型,探讨在在体条件下nodosin对肿瘤体积、肿瘤重量等的影响,并通过HE染色观察肿瘤病理组织学的改变。
成果：
MTT结果显示nodosin对胶质瘤细胞的生长有明显的抑制作用,且呈现明显的量效、时效关系,处理细胞24h后,对大鼠胶质瘤C6细胞和人胶质瘤U87细胞的IC50分别为：25.81μM、11.05μM;而nodosin在24h时对人正常胶质细胞HEB及大鼠原代胶质细胞的存活率影响不大,其IC50分别为：32.06μM、46.69μM。LDH检测发现,nodosin对C6及U87没有细胞毒作用。这些结果表明,nodosin的抗肿瘤作用具有较强的细胞选择性,其抗肿瘤作用机制可能与细胞周期阻滞、细胞凋亡诱导和侵袭转移抑制有关。
BrdU检测法和流式细胞术检测结果显示,nodosin对C6及U87胶质瘤细胞具有较好的增殖抑制作用,通过使胶质瘤细胞组滞于G2/M期的机制,抑制胶质瘤细胞进入下一增殖周期,从而抑制胶质痈细胞增殖。Western blot法实验显示,5μM、10μM、15μM的nodosin处理C6、U87细胞24h后,cyclinBl和cdc2蛋白表达水平下调。
细胞形态学观察、Hoechst33342染色法染色以及原位末端标记技术(TUNEL)检测结果显示,nodosin能诱导胶质瘤细胞的凋亡,作用胶质瘤细胞24h后,细胞形态发生改变,细胞数量与密度减少,随着剂量增加,培养基中悬浮的死亡细胞增多,出现染色质边集,细胞核碎片化,凋亡小体形成。Caspase活性检测发现,nodosin能够增加C6、U87细胞中caspase-3/7、caspase-8、caspase-9的活性,并成一定的剂量依赖关系,提示着我们,nodosin诱导胶质瘤细胞凋亡的作用,既通过死亡受体通路,亦通过线粒体通路。Western blot法结果显示,nodosin能够下调Bcl-2、Bcl-x1蛋白表达水平,上调Bak蛋白的表达水平,Bax蛋白水平不变；同时,能抑制P38MAPK、 PI3K/Akt和PKC信号通路；应用GSK3β抑制剂LiCl联合nodosin,发现LiCl可阻断nodosin引起的细胞凋亡。
划痕实验和Transwell细胞迁移、侵袭实验结果显示,nodosin能够剂量依赖性地阻止胶质瘤细胞的迁移与侵袭；cofocal结果发现,nodosin可抑制微管聚集；western blot分析结果显示,5、10和15μM的nodosin能以剂量依赖方式减少MMP-9蛋白表达水平。提示,nodosin对胶质瘤细胞的迁移侵袭抑制机制与其促进细胞微管解聚以及下调MMP-9蛋白表达水平相关。
接种胶质瘤C6细胞后第3d,对照组和nodosin组的肿瘤生长没有明显差异。接种后第4d到第12d,对照组的肿瘤生长快速,nodosin组的肿瘤生长缓慢,差异有显著性意义(P<0.05)；裸鼠肿瘤重量、肿瘤体积增长率均明显小于对照组。HE染色显示,对照组C6胶质瘤细胞丰富密集,坏死很少,nodosin组C6胶质瘤细胞生长密度低于对照组,并可见到细胞体积变小,核固缩深染的凋亡形态学改变,部分区域出现液化坏死。
结论：
(1)首次在胶质瘤细胞模型上研究nodosin抗恶性胶质瘤的作用,发现nodosin对胶质瘤细胞的生长具有明显的选择性抑制作用；
(2) Nodosin对胶质瘤细胞具有细胞增殖抑制作用,这种作用通过阻滞细胞周期于G2/M期以及下调cyclinBl和cdc2蛋白水平来实现；
(3) Nodosin具有诱导胶质瘤细胞凋亡的作用,其机制为：nodosin通过增加Caspase-3/7、Caspase-8、Caspase-9活性、降低线粒体膜电位、下调Bcl-2、Bcl-x1蛋白的表达水平以及上调Bak蛋白的表达水平来诱导胶质瘤细胞凋亡；nodosin能抑制P38MAPK、Akt和PKC等相关胶质瘤细胞内信号通路的蛋白水平；GSK3β在nodosin诱导细胞凋亡中是必需的。
(4) Nodosin能通过促进细胞微管解聚以及下调MMP-9蛋白水平来抑制胶质瘤细胞的迁移侵袭。
(5)首次在体内实验中,发现nodosin能抑制裸鼠内胶质瘤的肿瘤生长及减少肿瘤重量,具有体内抗恶性胶质瘤作用。
Objective
Malignant 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.
Methods
Morphologic 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.
Results
MTT 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.
引文
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