Effect Of Celastrus Orbiculatus Extracts In Suppressing Proliferation And Invasion Of Nasopharyngeal Carcinoma 5-8F Cell Line By Inhibiting EZH2/ROCK1 Pathway

Nasopharyngeal Carcinoma(NPC)is a kind of epithelial cell malignant tumor.China has the highest incidence and mortality rate of NPC in the world.Due to low differentiation and high metastasis of NPC,the major cause for treatment failure is local regional recurrence and distant metastasis.Histologically,the majority of cases of NPC(95%)are undifferentiated or poorly differentiated,and these histological types are able to spread and metastasize easily.Therefore,it is important to investigate the mechanism of migration and invasionin NPC.The enhancer of zeste homolog 2(EZH2)is the core components of Polycomb repressive(Polycomb Repressor complex 2 complexes,PRC2)whose highly conserved SET domain has histone methyl transferase activity.Studies have showed that EZH2 which is not dependent on the histone methyl transferase activity can affect the cytoskeleton aggregation.It suggested that EZH2 plays an important role in the invasion and metastasis of tumor.Studies have found that EZH2 has high expression level in a variety of carcinomas including NPC,prostate cancer and breast cancer.Its expression level was closely related with tumor size,depth of invasion,tumor stage,lymph node metastasis,migration and invasion ability.Those evidences suggesting that EZH2 had high potential to be a novel diagnosis biomarker and target for antitumor therapy in NPC.Rho-associated coiled coil-containing protein kinase(ROCK)is a serine/threonine protein kinase and it is the downstream effector protein of RhoA.In mammals,there are two isoforms of ROCK:ROCK1 and ROCK2.Overexpression of ROCK1 induces reorganization of the cytoskeleton,and promotes the formation of stress fibers and matrix adhesion.As a result,it can regulate cell movement and migration.China’s traditional Chinese herbal medicine Celastrus orbiculatus thumb(Celastraceae,Celastrus)is recorded in the "An Illustrated Book on Plants" in Qing Dynasty.And Celastrus orbiculatus thumb is also known as Celastrus aculeatus,Guo Shanlong,yellow rattan,fragrant dragon grass,and so on.30 kinds of Celastrus are widely distributedin China.Most of them are distributed in mountain,valley,hillside and bushesin wild.Celastrus orbiculatus thumb is mainly used for the treatment of rheumatoid arthritis,diarrhea,infantile convulsion,traumatic injury,swelling and other diseases.Celastrus contains a variety of pharmacological component such as terpenoids,fatty acids and glycosides etc.These compounds have a wide range of pharmacological effects,such as anti-tumor,anti-inflammatory,antioxidant,antimicrobial and anti fibrosis and anti fertility and so on.Its remarkable role in anti-tumor is worthy of special attention.Our previous studies showed that ethyl acetate extract from Celastrus orbiculatus thumb(COE)has significant anti-tumor effect,but its mechanism has not been fully elucidated.At present,the antitumor mechanism of COE including,inhibition of tumor cell proliferation,inhibition of tumor invasion and metastasis,promotion of tumor apoptosis and inhibition of epithelial mesenchymal transformation in digestive system tumors.In this study,we will study the effect of COE in inhibiting EZH2 in nasopharyngeal carcinoma and its molecular mechanism in suppressing proliferation and invasion in nasopharyngeal carcinoma.The research is divided into four parts.Part 1Clinicopathological significance of EZH2 and ROCK1 expression in nasopharyngeal carcinomaObjective:The aim of the present study was to determine the expression of EZH2 and ROCK1 in nasopharyngeal carcinoma(NPC),and to assess the association between the expression of these proteins and the clinicopathological features of NPC.To confirme that EZH2 could regulate the expression of ROCK1 in nasopharyngeal carcinoma cells.Methods:EZH2 and ROCK1 protein expressions were assessed in a tissue microarray of sections prepared from the tumors of NPC patients using immunohistochemistry.Protein and mRNA expression of ROCK1 in shCtrl 5-8F and shEZH2 5-8F cells were analyzed by western blot and real-time PCR,respectively.Results:Expressions of EZH2 and ROCK1 in clinical NPC tissues were evaluated using IHC.χ2 analysis revealed that upregulation of EZH2 was significantly associated with advanced T stage,advanced N stage and TNM stage cancer and increased ROCK1 expression was significantly associated with advanced N stage cancer.Furthermore,EZH2 expression was significantly positively correlated with ROCK1 expression.ROCK1 protein expression level and mRNA expression level were significantly reduced in shEZH2 5-8F cells compared with control group.Conclusion:The results of the present study indicate that high levels of EZH2 and ROCK1 expression may be associated with advanced stages in NPC.EZH2 koncked down can inhibited ROCK1 protein and mRNA expression levels.The proliferation and invasion of NPC may be inhibited by suppressing EZH2/ROCK1 signal pathway.Part 2Effect and mechanism of EZH2 on the proliferation and invasion of nasopharyngeal carcinomaObjective:The present study was undertaken to determine whether EZH2 was involved in inhibiting proliferation and invasion of human nasopharyngeal carcinoma and the underlying mechanism.Methods:In vitro,cell proliferation of 5-8F,shCtrl 5-8F,shEZH2 5-8F cells were analyzed by MTS assay.Invasion and migration of 5-8F,shCtrl 5-8F,shEZH2 5-8F cells were analyzed by transwell assay.Human Oncogenes and Tumor Suppressor Genes RT2 ProfilerTM PCR Array was applied to identify genes that regulated by EZH2 in nasopharyngeal carcinoma cells.In vivo,the growth-inhibiting effect of EZH2 on the nude mice model of NPC was tested.Results:The proliferation rate of shEZH2 5-8F cells was significantly decreased compared with control cells when measured using an MTS assay(P<0.05).The proliferation rate of shCtrl 5-8F cells only slightly changed compared with 5-8F cells(P>0.05).Thus,EZH2 plays essential roles in cell growth.The migration and invasion rates of shEZH2 5-8F cells were significantly decreased compared with control cells when measured using a Transwell assay(P<0.05).These results showed that EZH2 konckdown significantly reduced the number of cells migrated and invaded to the lower chamber.Human Oncogenes and Tumor Suppressor Genes RT2 ProfilerTM PCR Array containing 84 genes was applied to identify oncogenes and tumor suppressor genes that regulated by EZH2 in nasopharyngeal carcinoma cells.The mRNA expression profiles showed that 8 genes up-regulated>3 folds in shEZH2 5-8F cells and 2 gene down-regulated>3 folds in shEZH25-8F cells,comparing with control cells.Compared with the control group,the shEZH2 5-8F group showed a significant inhibition of tumor growth.At the end of the experiment,all the tumors were weighed.It was found that shEZH2 5-8F group significantly decreased the tumor weight compared with the control group(P<0.05).The average tumor weight of shEZH2 5-8F group was 0.179 ± 0.060g,and the average tumor weight of shCtrl group 5-8F was 0.064 ± 0.033g.Conclusion:EZH2 knockdown inhibited growth,migration and invasion in 5-8F NPC cells.EZH2 knockdown significantly reduced tumor weight in nude mice model.These findings revealed the value of EZH2 in treating invasive nasopharyngeal carcinoma.The mechanism may be related to the regulation of several oncogenes and tumor suppressor genes.But the downstream signaling pathways components need to be further investigated.Part 3Effect of Celastrus Orbiculatus extracts in suppressing proliferation and invasion of nasopharyngeal carcinoma 5-8F cell line by inhibiting EZH2/ROCK1 signaling pathway in vitroObjective:The present study was undertaken to determine if COE was involved in inhibiting proliferation and invasion of human nasopharyngeal carcinoma 5-8F cells in vitro by EZH2/ROCK1 signaling pathway.Methods:Cell proliferation and apoptosis of human nasopharyngeal carcinoma 5-8F cells wasanalyzed by MTS assay,flow cytometry,respectively.Invasion and migration of 5-8F cells were analyzed by transwell assay.Protein and mRNA expression of 5-8F cells were analyzed by western blot and real-time PCR,respectively.Results:COE reduced proliferation of 5-8F cells in a concentration-dependent manner.And COE promoted the apoptosis rate of 5-8F cells in a concentration-dependent manner.However,the apoptosis rates of 5-8F cells did not vary significantly in different doses below 100 μg/ml of COE for 24 h.Therefore,concentrations below 10μg/ml of COE were chosen for the following experiments to ensure that the effect of COE on NPC cells was not caused by direct cytotoxicity of COE.COE significantly reduced the number of cells migrated and invaded to the lower chamber in a dose-dependent manner.Treatment with COE(12.5,25 and 50 μg/ml)inhibited 59.3%,80.6%and 94.21%of cell migration and inhibited 61.54%,82.2%and 94.23%of cell invasion in 5-8F cells,respectively.These results showed that COE inhibited migration and invasion of 5-8F cells in a dose-dependent manner.To assess the mechanisms of COE in NPC cells,EZH2 and ROCK1 protein expressions were evaluated.Treatment of cells with COE suppressed EZH2 and ROCK1 protein expressions in a dose-dependent manner.And EZH2 and ROCK1 protein expression levels were significantly reduced following treatment with COE at 50 μg/ml concentration and 2μM 3-Deazaneplanocin A(DZNeP)for 24 h.Quantitative Real-time PCR was used to evaluate mRNA expression levels of EZH2 and ROCK1 in 5-8F cells.Treatment of cells with COE suppressed EZH2 and ROCK1 mRNA expressions in a dose-dependent manner.qRT-PCR showed that EZH2 mRNA expression levels were significantly reduced following treatment with COE at 50 μg/ml concentration for 24 h but not DZNeP.ROCK1 mRNA expression levels were significantly reduced following treatment with various doses of COE and 2μM DZNeP for 24 h.These results suggested that expressions of EZH2 and ROCK1 mRNA were significantly inhibited by COE in a dose-dependent manner in 5-8F cells.Conclusion:COE inhibited the proliferation,migration and invasion by suppressing EZH2/ROCK1 signal pathway in vitro.These data strongly supported COE to be a novel anti-cancer drug candidate,especially for the cancer with abnormal high expression of EZH2.Part 4Effect of Celastrus Orbiculatus extracts in suppressing proliferation of nasopharyngeal carcinoma 5-8F cell line by inhibiting EZH2/ROCK1signaling pathway in vivoObjective:The present study was undertaken to determine if COE was involved in inhibiting proliferation of human nasopharyngeal carcinoma 5-8F cells in vivo by EZH2/ROCK1 signaling pathway.Methods:The effects of COE on the nude mice subcutaneous tumor models of NPC were tested and the mechanisms were explored.The expression of EZH2/ROCK1 signaling pathway was evaluated by using immunohistochemistry.Results:To investigate the effects of COE on tumor growth in vivo,we used 5-8F cells in subcutaneous tumor models.Compared with the control group,the COE treated group showed a significant inhibition of tumor growth.At the end of the experiment,all the tumors were weighed.It was found that COE treated group significantly decreased the tumor weight compared with control groups.In conclusion,results from the current study demonstrated that COE had potent antitumor effects in nasopharyngeal cancer xenograft model.We examined the expression levels of EZH2 and ROCK1 in the tumor tissue in subcutaneous tumor models by immunohistochemisty.The results showed that COE could reduce the expressions of EZH2 and ROCK1 in subcutaneous tumor models.In addition,EZH2 and ROCK1 proteins were inhibited by COE in a dose-dependent manner.Conclusion:COE inhibited the proliferation of subcutaneous tumors by suppressing EZH2/ROCK1 signal pathway in vivo.