The Expression And Significance Of MTORC1 Related Proteins In Gastroenteropancreatic Neuroendocrine Neoplasm

Neuroendocrine tumor comes from diffuse neuroendocrine system, which presents the characteristic of heterogeneity. We can suffer from neuroendocrine tumor in our body everywhere, and the rate of Gastroenteropancreatic neuroendocrine neoplasm(GEP-NET) is 65%~75%. According to a epidemiological investigation of many countries and regions, the morbidity of GEP-NET steadily rising, which follows from colorectal cancers and arranged in the second place in all of the digestive tract malignant tumors.The symptoms of GEP-NET are complicated and various, which can develop from benign to malignancy and this process is tardiness. Besides, because of the confusion comparing with endocrine disease and deficiency of diagnosis, misdiagnose is normal. Surgery is the most important way to cure GEP-NET.Other medicines, including cytotoxic drug and somatostation analogue, which are used to alleviate symptoms. Besides, both anti-angiogenesis and antigrowth factor are new treatment of GEPNET.m TOR is a Serine/threonine protein kinase, which regulates cell growth, proliferation, differentiation, apoptosis, autophagy, cell cycle and so on.Everolimus as a specific inhibitor of mammalian target of rapamycin complex 1(m TORC1) can inhibit tumor cell growth and promote apoptosis, which presents prominent clinical effects. However, the response of GEP-NET patients to everolimus is various and its mechanism is unkown.The development of tumor needs sufficient nutrients and energy, Its supply modes mainly include glycolysis.glutamine metabolism and lipid metabolism. An important feature of malignant tumor is mechanism abnormal.During the process of tumor proliferation, the change of energy mechanism has played an important role.On clinic,we found almost 50% pancreatic neuroendocrine tumors are negative expression in PET-CT, and the higher in grade, the higher expression in PET-CT. These show that the metabolism of some GEP-NET is various and glycolysis is not the most important. Recently, some researchers found the activity of m TORC1 is lin ked to nutrients. However, whether m TORC1 is linked to glutamine or not is little known. In our study, we found NETs were high expression of GLS1. Besides, m TORC1 inhibitions made an influence on GLS1 and inhibit cell growth through a change of glutamine m etabolism. In this study, we showed m TORC1 took an important role in energy mechanism, which provide a new way of treatment about NETs.METHODS:In part I,(1)Western Blot test the expression level of metabolism related enzymes in human gastroenteropancreatic neuroendocrine neoplasm cell lines BON- 1;(2)Selected the gastrointestinal pancreatic neuroendocrine tumor clinical tissue specimens.Using the immunohistochemical detection the expression status of metabolism related enzymes including LDHA,PKM2,SLC1A5,GS,GOT2,GLS1,GLS2. Meanwhile, detection the expression status of m TORC1 and m TORC2 pathways related proteins in Raptor, Rictor.In part II,(1)In vitro cultured human gastroenteropancreatic neuroendocrine neoplasm cell lines BON-1,and then deprive of glucose and/or glutamine,CCK-8assay were used in observation of cell proliferation,cell apoptosis and cell cycle distribution was analyzed by flow cytomety.;(2)Using RNA interference impaired GLS1 activity,detection the cell proliferation and clone growth situation;(3)Detection of cell growth after joined the glutamate in the deprived of glutamine medium;(4)Use m TORC1 inhibitors treated BON-1 cells,Western Blot test the expression level of GLS1 and intracellular glutamine level, cell proliferation detected by CCK-8 assay.RESULTS:1.The expression of metabolism related enzymes in human gastroenteropancreatic neuroendocrine neoplasm clinical tissue specimens and cell line(1)Western Blot test the expression level of metabolism related enzymes in human gastroenteropancreatic neuroendocrine neoplasm cell lines BON- 1 found that glutamine metabolic enzymes GLS has high expression;(2)In the gastrointestinal pancreatic neuroendocrine tumor tissue samples, immunohistochemical results show glutamine metabolic enzyme GLS1 has high expression relative to the adjacent mucosa;(3)The expression of GLS1 and ki67 grading showed a negative trend;(4)Relative to the adjacent carcinoma tissue,Raptor expression in tumor tissue is strong positive in 90%(27/30)GEP-NET samples;(5)Rictor in GEP-NET tissue specimens were negative or weakly positive expression, the expression has no obvious difference in e ach classification.2.m TORC1 pathway adjust GLS1 mediated glutamine metabolism(1)In vitro cultured human gastroenteropancreatic neuroendocrine neoplasm cell lines BON-1,and then deprive of glucose and/or glutamine,found that BON-1 cells arrest of growth after deprived of glutamine,apparent blockage of cell growth and increased apoptosis;(2)Using RNA interference impaired GLS1 activity,GLS1 knockdown markedly reduced BON-1 cells growth;(3)Joined the glutamate in the deprived of glutamine medium can reply to cell growth;(4)Use mTORC1 inhibitor treated BON-1 cells,western blot showed GLS1 protein expression level reduced and intracellular glutamine level rised, suppressed the cell growth at the same time.CONCLUSIONS:mTORC1 signaling pathways in gastrointestinal pancreatic neuroendocrine tumor frequently actively.Human gastroenteropancreatic neuroendocrine neoplasm cell lines BON-1,s growth obviously depends on glutamine,m TORC1 specific inhibitor can impair glutamine metabolism by influence the expression of glutamine catabolism key enzyme GLS1,thereby inhibiting tumor cell proliferation.