Celastrus Orbiculatus Extracts Inhibit Gastric Carcinoma Metastasis Via Targeting Tumor Suppressor Gene Maspin

Gastric cancer (GC) is malignant and highly hazardous to human health. It’s the second leading cause for cancer-related death. The development and prognosis of GC is due to the activation of oncogenes, inactivation of tumor suppressor genes, regulation of complex signaling pathways, etc. The tumor suppressor gene maspin, plays an important role in tumor cell invation and metastasis. The metastasis of tumor cells is more likely to happen when maspin gene is lost. The urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) can secrete a variety of hydrolytic enzymes, which would degrade extracellular matrix and basement membrane. They are also closely related to the tumor metastasis and prognosis. The signaling pathways, phosphatidyl-inositol 3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPK), are close with tumorigenesis and development. They also play a intermediary and signal amplifying role during tumor cell invation. It has been reported that MASPIN protein can reduce the expression of uPA and MMPs through PI3K/Akt and MAPK signaling pathways. Therefore, to enhance the expression of maspin gene in tumor cells can be used as a potential means for the treatment of gastric cancer.Celastrus orbiculatus Thunb. (Nansheteng, celastrus), is widely distributed in China, such as Northeast, East China, Northwest and Hunan province. It is known as a anti-inflammatory and anti-rheumatic drug. We found that Celastrus orbiculatus extracts (COE) could anti-tumor in vivo and vitro, including inhibition of tumor cell proliferation, invasion, metastasis and promotion of apoptosis of tumor cells.In the present study, we overexpressed maspin gene in human MGC-803 gastric cancer cells (MGC-803/maspin++). And studied the mechanism of COE inhibit the metastasis by targeting maspin. As the results show, within limits, COE could increase the expression of MASPIN in a concentration-dependent manner, inhibit metastasis and promote apoptosis of cancer cells compared with controls. Further molecular mechanisms investigations come into contact with PI3K/AKT/mTOR and MAPK signaling pathways.The findings will be described in three parts as follows.Part ⅠOverexpression maspin in human gastric cancer MGC-803Objective:To generate overexpression maspin in human gastric cancer MGC-803 cellsMethods:The human gatric cancer MGC-803 cells were infected with lentiviral, to integrate maspin gene which contains GFP into cellular DNA in vitro. Three days later, the fluorescence intensity was observed by inverted fluorescent microscope. The expression of MASPIN protein was detected by western blot assay.Results:72 hour later, strong fluorescence was observed under inverted fluorescent microscope in gastric carcinoma MGC-803 cells after infection. Western blot showed that the expression of MASPIN protein was higher than negative and wild type control.Conclusions:The construction of human gastric cancer MGC-803/maspin++ is successful.Part ⅡCelastrus Orbiculatus Extracts inhibit metastasis in MGC-803/maspin++ cellsObjective:To investigate the effect of Celastrus Orbiculatus extract (COE) to proliferation, apoptosis, invasion and migration in MGC-803/maspin++ cells.Methods:The logarithmic growth phase human gastric carcinoma MGC-803/maspin++ cells were divided into COE groups (20,40,80,160,320 μg/mL, respectively), wild type, negative and positive control (5-Fu 32 μg/mL) groups. Through MTT assay, wether the ability to proliferate of human gastric carcinoma MGC-803/maspin++ cells change before and after treating with those groups was measured. The cellular morphology was observed under inverted microscope when treated with COE. According to the TUNEL assay, the phenomenon of MGC-803/maspin++ cell apoptosis was detected. The invasion ability of cells was assayed by transwell invasion assay. The human gastric cancer MGC-803/maspin++ cell migration was observed by cell scratch and transwell migration assay.Results:The proliferation, invasion and migration of human gastric carcinoma MGC-803/maspin++ cells was inhibited and the apoptosis was promoted compared with wild type control. What’s more, COE further inhanced the effects in a concentration-dependent manner. (P<0.05 or P<0.01).Conclusions:maspin gene could anti-tumor in vitro and effectively inhibit the proliferation, invasion and migration, promote the apoptosis of human gastric cancer MGC-803 cells, the influence was strengthened cooperate with COE.Part ⅢMechanisms of COE inhibit the metastasis in MGC-803/maspin++ cellsObjective:To investigate the mechanisms of the Celastrus Orbiculatus extract (COE) inhibit the metastasis in MGC-803/maspin++ cells.Methods:The human gastric carcinoma MGC-803/maspin++ cells were classified into groups as COE (20,40,80,160,320 μg/mL, respectively),-wild type, negative and positive control groups. The time is 24h. The protein expressions were detected by western blot.Results:COE could increase the MASPIN protein expression in a concentration-dependent manner. COE treatment concentration-dependently reduced the expression of MMP-2、MMP-9、 uPA、Bcl-2、Akt、p-Akt、mTOR、p-mTOR and p38MAPK of protein in human gastric cancer MGC-803/maspin++ cells, whereas there was no significant effect on Bax and Erkl/2. Expression trend of p-Erkl/2 and p-p38MAPK was rised in general.Conclusions:The mechanisms of the anti-metastasis and pro-apoptosis activity of COE might be due to the PI3K/Akt/mTOR、MAPK signaling pathways and Bcl-2 family respectively.