| Gastric cancer is a malignant tumor that is highly harmful to human health,and its mortality rate ranks second in all tumor mortality.China is a high incidence of gastric cancer country.About 17 million people died of gastric cancer each year,accounting for more than 50%of the number of deaths in digestive system tumors.Clinical treatmentsare often surgery and chemotherapy-based chemotherapy.Due to early clinical symptoms are not obvious,the early diagnosis and treatment rate is low.The prognosis is poor,because gastric cancer cells have the ablity of invasion and metastasis.The development of gastric cancer is closely related to the activation of oncogenes and the inactivation of tumor suppressor genes.Maspin,a tumor suppressor gene,plays a key role in tumor cell proliferation,adhesion,migration and apoptosis.The loss of maspin can lead to the decrease of tumor cell apoptosis.Therefore,maspin gene may become a potential target for the treatment of gastric cancer.Celastrus orbiculatus Thunb.,which is widely distributed in China,has been used as a anti-inflammatory and anti-rheumatic drug.We found that Celastrus Orbiculatus Extracts(COE)have significant anti-tumor capacity.It could inhibit the proliferation,migration and invasion of tumor cells and promote apoptosis of tumor cells.In this study,we first constructed a human gastric cancer MGC-803 cell with low maspin expression.We investigated the molecular mechanism of COE inducing the apoptosis of human gastric cancer cells.Compared with the control group,a certain range of COE can increase the expression of maspin in a concentration-dependent manner,and promote the apoptosis of tumor cells.The molecular mechanism may be related to the bcl2 family,casepase protein,PI3K/Akt and MAPK signaling pathways.This study will be described in three parts as follows.Part ⅠMASPIN expression level in human gastric carcinoma and the construction of MGC-803/maspin-cellsObjective:To observe the expression of maspin in human gastric cancer tissue samples and to construct a human gastric cancer MGC-803 cell model with low maspin expression.Methods:The expression of maspin in human gastric cancer tissue was observed by immunohistochemistry.The human gatric cancer MGC-803 cells were infected with transfection reagent,to integrate maspin siRNA into cellular DNA in vitro.24 hours after transfection,expression of maspin mRNA and protein was assessed by qRT-PCR and western blot respectively.Results:The western blot results showed that MASPIN protein was significantly decreased in MGC-803 cells compared with negative control and wild-type cells.QRT-PCR results showed that maspin RNA was significantly reduced in MGC-803 cells compared with negative control and wild-type cells.Conclusions:The construction of human gastric cancer MGC-803/maspin-is successful.Part ⅡEffects of Celastrus Orbiculatus extracts on the proliferation and the apoptosis in MGC-803/mas/pin-cellsObjective:To investigate the effect of COE to apoptosis,proliferation,migration and invasion in MGC-803/maspin-cells.Methods:The human gastric carcinoma MGC-8803/maspin-cells were divided into wild type,negative and positive control(5-Fu 32 μg/mL)groups and COE groups(10,20,40,80,160,320μg/mL).The morphological changes of MGC-803/maspin-cells were observed under microscope.Apoptosis was detected by transmission electron microscopy of MGC-803/maspin-cells.The effects of COE on the proliferation of MGC-803/maspin-cells were observed by MTT assay.TUNEL assay and flow cytometry were used to detect the apoptosis of MGC-803/maspin-cells.The mitochondrial membrane potential change of MGC-803/maspin-cells was detected by JC-1 assay to detect the apoptosis.The invasive ability of cells was detected by transwell invasion assay.The migration of MGC-803/maspin-was detected by transwell migration assay and cell scratch test.Results:After inhibiting the expression of maspin,the apoptosis of MGC-803 cells was decreased,and the invasion and migration ability of MGC-803 cells were enhanced(P<0.05 or P<0.01).The IC50 of COE was 101.711 g/mL.And the expression level of MASPIN was increased in different concentrations of COE.Conclusion:Maspin has anti-tumor effect in vitro,COE can enhance the expression and the function of MASPIN.COE promotes the apoptosis of human gastric cancer cell MGC-803.COE inhibits the proliferation,invasion and migration ability of human gastric cancer MGC-803 cells.Part ⅢMechanisms of COE induce the apoptosis in MGC-803/maspin-cellsObjective:Effects of COE on the expression of apoptosis-related proteins(Bax,Bcl-2 and casepase-3)in human gastric cancer MGC-803/maspin-cells.And to explore the molecular mechanism of COE in promoting the apoptosis of gastric cancer cells.Methods:The human gastric carcinoma MGC-803/maspin-cells were divided into groups as wild type,negative and positive control(5-Fu 32 μg/mL)groups and COE groups(0,20,40,80μg/mL,respectively).After 24h,western blot was used to detect the key proteins of PI3K/Akt/mTOR and MAPK signaling pathways(MAPK,Bax,Bcl-2,casepase-3,PI3K,Akt,mTOR,Erkl/2,p38MAPK and corresponding phosphorylated proteins).Results:COE decreased the expression of Bcl-2 and PI3K,Akt,p-Akt,p-mTOR,Erkl/2 protein in a concentration-dependent manner,but had no significant changes in mTOR and p38MAPK Protein.The expression of Bax,casepase-3,p-Erkl/2,p-p38MAPK protein increased with the increase of COE concentration.Conclusions:The mechanism of COE-induced apoptosis of MGC-803/maspin-may be related to Bcl-2 family,casepase-3 protein and PI3K/Akt/mTOR,MAPK signaling pathway. |
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