Effect Of Ardipusilloside-â… on Biological Behavior Of Gastric Cancer And Mechanism Study

Gastric cancer is one of the most common carcinomas in the world. In the development and progression of this cancer, various factors are involved, including the expression of tumor-related protein. To date, the etiology and exact mechanism by which gastric cancer occurs are not fully understand. In recent years, although the incidence of gastric cancer has declined worldwide, the incidence is still high in Asian counties including China. Currently, the most common therapies for gastric cancer are surgery, chemotherapy and radiotherapy. Despite the advanced progress in basic and clinical research, making great advance in early diagnosis and treatment of gastric cancer, the 5-year survival rates of gastric cancer patients are still poor in clinical treatment. Due to the unobvious symptoms of patients with early stage gastric cancer, they were diagnosed at late stage and miss the best treatment opportunity. Therefore, looking for therapeutic drug with high sensitivity and specificity, and less side effects is particularly important.Natural medicine, which has the advantages of less side effect, safety, and low price, attracts our attention in antitumor research in recent years. Ardipusilloside, a triterpenoid saponins which is isolated from Ardisia pusilla A. DC(of the family Myrsinaceae), a Chinese medicinal herb also known as Jiu Jie Long, has been found to have anti-tumor, antihypertensive and antiviral activities as well as other biological and pharmacological functions. Previous studies have shown that ardipusilloside- Ⅰ(ADS- Ⅰ) has antitumor activities in many human cancers including lung cancer, hepatocellular carcinoma, human glioblastoma, cervical cancer, and so on. ADS- Ⅰ exerts its antitumor effect through induction of programmed cell death(PCD), induction of autophagy, and inhibition of tumor invasionandmetastasis.In this study, the effect of ADS-Ⅰ on cell proliferation, invasion,metastasis and epithelial-mesenchymal transition(EMT) in gastric cancer cell lines TSGH and N87 was determined. Furthermore, the molecular mechanism by which ADS- Ⅰexerts its antitumor activities in gastric cancer cells was explored. Gastric cancer xenografts in nude mice were established by subcutaneousinjection of gastric cancer N87 cells, and then the effect of ADS- Ⅰ on gastric cancer xenografts and the underlying mechanism were measured. In conclusion, our finding showed that ADS-Ⅰ regulated growth, invasion, metastasis and EMT of gastric cancer through the JAK/STAT3 signaling pathway. The results of this study offered a new way for the treatment of gastric cancer. This research was divided into three parts:Part I: Effect of ADS-Ⅰon proliferation, invasion,metastasis and EMT of gastric cancer cell linesMethods1. After gastric cancer TSGH and N87 cell lines were treated by various concentration of ADS-I(10、20、40μM) for 24, 48, 72, and 96 h, the ability of the cell proliferation in gastric cancer cell lines was measured by MTT assay. Cells in control group were treated by normalsaline.2. After gastric cancer TSGH and N87 cell lines were treated by various concentration of ADS-I(10、20、40μM) for 24 h, the ability of cell invasion was determined by Transwell invasion assay, in which the amount of cells which pass through membrane was used to measure the invasion ability.3. Wound healing cell migration assay was used to determine the ability of cell metastasis in gastric cancer TSGH and N87 cell lines that were treated by various concentration of ADS-I(10 、 20 、 40μM) for 24 h. The cell migration distance is determined by measuring the width of the wound divided by two and by subtracting this value from the initial half-width of the wound. Migration was also quantified by counting cell numbers at the indicated migration distances from the wound edge.4. After gastric cancer TSGH cell line were treated by various concentration of ADS-I(10、20、40μM) for 24 h, the m RNA expression of E-cadherin and N- cadherin were detected by western blot to measure the EMT of gastric cancer cells.Results1. Results from MTT assay showed that ADS- Ⅰ treatment significantly inhibited the cell proliferation of gastric cancer TSGH and N87 cell lines.2. Results from Transwell invasion assay showed that various concentration of ADS-Ⅰ(10、20、40μM)significantly inhibited the cell invasion of gastric cancer TSGH and N87 cell lines. Compared with control groups, the amount of cells which pass through membrane was decreased by ADS-Ⅰtreatment.3. Results from wound healing cell migration assay indicated that ADS- Ⅰtreatment significantly inhibited the cell metastasis in gastric cancer TSGH and N87 cell lines.4. Western blot analysis showed that ADS- Ⅰ treatment significantly down-regulated the m RNA expression of N-cadherin and up-regulated the m RNA expression of E-cadherin, which indicated the inhibitory effect of ADS-Ⅰon EMT in gastric cancer TSGH cell line.Part II: Effect of ADS-Ⅰon the JAK/STAT3 signaling pathway in gastric cancer TSGH cell line1. After gastric cancer TSGH cell line were treated by various concentration of ADS-I(10、20、40μM) for 24 h, the protein expression levels of JAK1, JAK1, p-JAK2,JAK2, p-STAT3, and STAT3 were determined by western blot.2. After gastric cancer TSGH cell line were treated by various concentration of ADS-I(10、20、40μM) for 24 h, the m RNA expression levels of Ki67, Bcl-2, MMP-9,Snail, and Twist were determined by quantitative real-time PCR.MethodsResults1. Western blot analysis showed that ADS- Ⅰ treatment inhibited the protein expression levels of JAK1, JAK1, p-JAK2, JAK2, p-STAT3, and STAT3 in gastric cancer TSGH cell line. ADS- Ⅰ inactivated the JAK/STAT3 signaling pathway through translational regulation.2. Compared with control group, the m RNA expression levels of Ki67, Bcl-2,MMP-9, Snail, and Twist were decreased in gastric cancer TSGH cell line.Part Ⅲ: Effect of ADS-Ⅰon gastric cancer xenograftsMethods1. Gastric cancer xenografts in nude mice were established by subcutaneousinjection of gastric cancer N87 cells. The xenografts were divided into two groups after the tumor volume was greater than 100 mm3, the nude mice with gastric cancer xenografts were treated with either normal saline(200μL/day) or ADS- Ⅰ(50mg/kg/day) by intragastricadministration for continuous period of 12 days.2. The longest diameter and the shortest diameter of the xenografts were measured with caliper every three days.Tumor volume was calculated as follows:Tumor volume(mm3) =(the longest diameter)×(the shortest diameter)2/2. Two days after the ADS-Ⅰadministration, all the nude mice were killed by cervical dislocation.The weight of each gastric cancer xenograft was measured by electronic balance.3. Western blot analysis was used to detected the protein expression levels of JAK1, JAK1, p-JAK2, JAK2, p-STAT3, and STAT3 in gastric cancer xenografts to determine effect of ADS-Ⅰon JAK/STAT3 signaling pathway.4. The protein expression levels of Ki67, Bcl-2, MMP-9, Snail, and Twist were measured in gastric cancer xenografts by western blot analysis.Results 1. Compared with control group, The intragastricadministration of ADS-Ⅰat the concentration of 50mg/kg/day significantly suppressed gastric cancer xenografts in nude mice.2. Compared with control group, after intragastricadministration of ADS-Ⅰfor a continuous period of 12 days, the weight of gastric cancer xenografts was significantly decreased(P< 0.01).3. Western blot analysis showed that ADS- Ⅰ treatment inhibited the protein expression levels of JAK1, JAK1, p-JAK2, JAK2, p-STAT3, and STAT3 in gastric cancer xenografts.4. Compared with control group, the protein expression levels of Ki67, Bcl-2,MMP-9, Snail, and Twist were decreased in gastric cancer xenografts.Conclusions1. ADS- Ⅰ can inhibit proliferation, invasion, metastasis, and epithelial-mesenchymal transition of gastric cancer cells.2. ADS- Ⅰ suppresses proliferation, invasion, metastasis, and epithelial-mesenchymal transition of gastric cancer cells via the JAK/STAT3 signaling pathway.3. ADS- Ⅰ can inhibit the growth of gastric cancer xenografts in nude mice through inactivation of the JAK/STAT3 signaling pathway.