Co-targeting ASCT2 And Glutamine Synthetase Suppresses Growth Of Glutamine-independent Gastric Cancer Cells

Gastric cancer is a tumor of high incidence all over the world.Especially in China,gastric cancer has the second incidence and mortality.Radical surgery remains the main method for gastric cancer.However,many patients in China are diagnosed in the advanced stage.Gastric cancer patients with distant metastasis lymph node are more likely to have tumor recurrence.The number of recurrent Patients is over half,even though they had experienced radical surgery.Nowadays preoperative chemotherapy,neoadjuvant chemotherapy,radiation therapy and molecular targeted therapy are applied in gastric carcinoma treatment.But the overall 5-year survival rate is still too low.So to explore new treatments for gastric cancer is desperately needed.ASCT2(SLC1A5)is a cell surface Na+-dependent neutral amino acid transporter,which can adjust the intake of glutamine.While glutamine are non-essential amino acids in normal cells,but glutamine lack has obviously suppressed effect on cell growth.Glutamine can support increased nutrition metabolic demand for tumor cells because of their rapid proliferation rate in producing ATP,in nucleotide and protien biosynthesis.And elevated intracellular glutamine will activate mTORC1 signal,thus promoting cell growth and reducing cell apoptosis.Decreasing cancer cell glutamine intake is a potential way to treat cancer.The effectiveness of ASCT2 inhibitor has been approved in melanoma cells,prostate cancer,breast cancer and acute myeloid leukemia.In the first part of this study we measured ASCT2 protein expression in 6 gastric cancer cells and normal mucosa tissues by western blot,and then applied ASCT2 inhibitors GPNA and BenSer into cell culture respectively.We found that all gastric cancer cells have higher ASCT2 protein expression compared with normal mucosatissues and that gastric cancer cells had variant sensitivity to ASCT2 inhibitor.Growth of AGS,MGC-803 and MKN-74 gastric cancer cell slowed significantly after ASCT2 inhibitor treatment.However,there was no obvious variation in cell proliferation of gastric cancer cells HGC-27,MKN-45 and NUGC-3.In the second part we tried to explore the reasons of different response to ASCT2 inhibitors in gastric cancer cells on the basis of the first part.Firstly we cultured 6gastric cells in glutamine deficiency.We found the result was consistent with the first part: the growth of AGS,MGC-803,MKN-74 gastric cancer cells stunted in glutamine lack,these cells were called glutamine-dependent cells;HGC-27,MKN-45,NUGC-3 gastric cancer cells continued to grow in glutamine lack,these cells were called glutamine-independent cells.By using western blot experiment,we found glutamine synthetase(GS)protein expression increased for glutamine-independent gastric cancer cells in lack of glutamine,while for glutamine-dependent gastric cancer cells glutamine synthetase protein was rarely expressed,even hardly expressed.So we speculated that glutamine synthetase may be an important factor of glutamine dependence for gastric cancer cells.To verify our speculation,we transfected GS overexpressed plasmid into glutamine-dependent gastric cancer cells by lipofectamine3000 method,and found GS expression enhanced cell tolerance to glutamine deficiency.To further prove the validity of our speculation,we transfected GS knockdown plasmid into glutamine-independent gastric cancer cells by the same method and found GS downregulation significantly suppressed cell growth.Based on these results,we suggested that for glutamine-independent gastric cancer treatment with ASCT2 inhibitor,GS inhibitor should be combined;for Glutamine-dependent gastric cancer,using GPNA or BenSer alone should be enough,and GS inhibitor supplement has no benefit.In the third part,we used immunohistochemistry method to detect ASCT2 protein expression in 193 cases of gastric cancer tissues.We found ASCT2 expression existed in both gastric mucosa and carcinoma tissue,and ASCT2 expression washigher in most cancer tissue than in normal mucosa tissues.In view of our previous experimental results,we also detected the expression of GS in 149 cases of ASCT2 high expressed gastric cancer tissues.We found that GS expression was generally low in gastric cancer tissues,but there were still some cancer tissues with GS expression.We suggested that for treatment with ASCT2 inhibitor in those patients with GS expression,combined use of GS inhibitor should be necessary.