| 【Background】Gastric cancer is the fourth most common type of cancer and the second mostcommon cause of cancer deaths worldwide. Surgical tumor resection remains the primarycurative treatment for GC but the overall5-year survival rate remains poor, rangingbetween20–25%. The addition of combined modality strategies (pre-or postoperativechemo/radiotherapy or perioperative chemotherapy) results in5-year survival rates of only30–35%. Preoperative chemo/radiotherapy produces pathologic complete responses (pCRs)in no more than20–30%of patients, while preoperative chemotherapy alone is only rarelyassociated with pCRs.Worldwide, despite the improvements, estimated cure rates forpatients with advanced stagesremain poor and, in the metastatic setting, chemotherapy isthe mainstay of palliative therapy and results in objective response rates (ORRs) of only20–40%with a median overall survivals (OS) of8–10months。Although novel strategieshave recently been used in the diagnosis and treatment of gastric cancer, the outcome ofgastric cancer patients remains very poor. Improvements in gastric cancer prognosis arelargely dependent on a complete understanding of the molecular biology and the mechanisms of gastric cancer. Over the years, a large number of proto-oncogene andtumor suppressor genes have been identified, but the precise molecular mechanisms ofgastric carcinogenesis remain unclear.Growth arrest-specific-gene1(GAS1), a glycosylphosphatidyinositol-linkedprotein, was originally identified as a protein involved in the contact inhibitionof fibroblasts. It has been directly related to cell cycle arrest in the G0to S phasetransition. In addition, GAS1also suppressed cell growth in different cellsystems. Moreover, GAS1was involved in apoposis in different cell systems incontext-dependent ways. Above all, these studies suggest that GAS1is an negativeregulator of cell growth and apoptosis.Recently, the critical roles of GAS1in tumors have been gradually determined. GAS1has been mapped to a chromosome site that is frequently deleted in cancer cells.Overexpression of GAS1has been found to inhibit tumor growth and progression inglioma cells, and contributes to the prediction of metastasis and recurrence in stage II andIII colorectal cancer. In addition, other studies showed that GAS1expression wasnegatively related to metastasis in melanoma and prostate cancer, suggesting that GAS1might inhibit cancer metastasis. Taken together, all of these findings demonstrate thatGAS1functions as a tumor suppressor gene in various tumors. However, it was reportedthat GAS1knockout mice did not have an increased incidence of cancer, which put therole of GAS1as a tumor suppressor gene into doubt. Therefore, additional efforts areneeded to clarify the functions of GAS1in cancer.We previously showed that GAS1was an epirubicin resistance-related gene in gastriccancer cells with a partially randomized small interfering RNA (siRNA) library GAS1suppression resulted in significant multidrug resistance in gastric cancer cells, andupregulation of GAS1reversed the chemoresistance of gastric cancer. However, the rolesand mechanisms of GAS1in carcinogenesis and the progression of gastric cancer have notbeen well defined.【Aims】1. To evaluate the expression of Gas-1in gastric cancer tissues and its clinicalpathological significance by western bolt and Immunohistochemistry.2. To investigate the effect of Gas-1on gastric cancer cell proliferation and cellapoptosis.3. To explore the possible mechanism by which Gas-1inhibitions gastric cancercell proliferation.【Methods】1. Immunohistochemistry and western bolt were performed to examine theexpression of Gas-1in gastric cancer and the matched adjacent noncancerous tissues.2. western blot analysis was performed to examine protein expression of Gas-1in gastric cancer cell lines (SGC7901、AGS、MKN45、MKN28and GES).3. Liposome2000was used to transfect full length human Gas-1vector andGas-1-siRNA vector into AGS cells and SGC7901cells, respectively. G418was used toselect stable cell clones.4. MTT assay,Plate colony formation assay, Soft agar colony formation assaywere used to analyze the effect of Gas-1on gastric cancer cell proliferation in vitro.5. Subcutaneous tumor formative assay was used to investigate the effect of Gas-1on proliferative ability in vivo.6. FACS and Hochest33258were used to analyze the effect of Gas-1on gastriccancer cell apoptosis.7. Western blot analysis was carried out to detect cell apoptosis relatedandmolecules Bcl-2、Bax and capase-3.【Results】1. The expression of Gas-1in gastric cancer tissues and gastric cancer cells.GAS1expression was evaluated in the161gastric cancer tissues in two pieces of atissue assay and matched adjacent noncancerous tissues by immunohistochemical staining.It was demonstrated that GAS1was primarily located in the cytoplasm, and onlyoccasionally in membrane, in cancer cells.Further analysis showed that GAS1stainingwas positive in85(52.8%) gastric cancer tissue samples and156(96.3%) of noncanceroustissue samples (P <0.05)The average score in gastric cancer tissues was significantly lower than that in matched noncancerous tissues (8.32±2.43versus1.81±1.32)theexpression of GAS1was higher in well-differentiated tumor tissues than in moderately orpoorly differentiated ones, indicating a correlation between GAS1expression andhistological grade of gastric cancer (P <0.05). With respect to the TNM stage,GAS1expression in stage III and IV patients was much lower than in stage I and II patients (P <0.05).the expression of GAS1was higher in well-differentiated tumor tissues than inmoderately or poorly differentiated ones, indicating a correlation between GAS1expression and histological grade of gastric cancer (P <0.05). With respect to the TNMstage, GAS1expression in stage III and IV patients was much lower than in stage I and IIpatients (P <0.05).However, there was no correlation between GAS1expression and age,gender, or metastasis. At the end of follow-up, the survival times of74patients wereanalyzed with the Kaplan–Meier method, and the results revealed that patients withnegative GAS1expression had shorter survival time than those with positive GAS1expression, demonstrating that reduced or negative GAS1expression was associated withshorter survival time and a poorer prognosis than positive GAS1expression.We examined GAS1expression in fresh tissues by western blot. It was obvious thatGAS1expression was significantly decreased in gastric cancer tissues as compared withmatched noncancerous tissues.We then examined the expression of GAS1in four gastric cancer (SGC7901, MKN45,MKN28, and AGS) cell lines and the immortal gastric epithelial cell line (GES).GAS1expression was lower in all gastric cancer cell lines than GES cells, and GAS1expressionwas relatively higher in SGC7901cells and lower in AGS cells.2. The effect of Gas-1on gastric cancer cell proliferative ability in vitro and in vivo.To evaluate the potential role of GAS1in growth and proliferation of gastric cancercells, SGC7901cells and AGS cells were transfected with GAS1-specific siRNA vectorsand GAS1overexpression vector, respectively.The growth rate of SGC7901-siGAS1-1cells was increased, The ability of plateassays and soft agar assays were increased;There was a significant increased in tumorsize;the proportion of apoptotic was significantly decreased, Hoechst33258staining showed a decreased number of apoptotic cells.GAS1-transfected cells showed asignificantly decreased rate of cell growth;The ability of plate assays and soft agar assayswere decreased;There was a significant decreased in tumor size;the proportion ofapoptotic was significantly increased, Hoechst33258staining showed a increased numberof apoptotic cells.3. Gas-1regulated various cell apoptosis related proteins.We also investigated the effects of GAS1on the expression of apoptosis-relatedmolecules by Western blot.The Bcl-2Bax ratio of SGC7901-siGAS1-1cells wasdecreased，the enzymatic activity of Caspase-3was significantly decreased，the expressionof Bcl-2and Caspase-3in tumor tissues of nude mice were decreased；The Bcl-2Baxratio of GAS1-transfected cells was increased，the enzymatic activity of Caspase-3wassignificantly increased，the expression of Bcl-2and Caspase-3in tumor tissues of nudemice were increased.【Conclusions】1. The expression of Gas-1was significantly lower in gastric cancer tissues,compared with noncancerous tissues. The overexpression of Gas-1was associated with theprognosis of the gastric cancer patients.2. Gas-1could inhibit gastric cancer cell growth and proliferation in vitro and invivo and Gas-1could inhibit cell apoptosis.3. Gas-1inhibit gastic cancer cell growth and proliferation partly by regulating cellapoptosis specific molecules,such as the Bcl-2Bax ratio and the activity of Caspase-3. |
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