The Study Of Mechanisms Of TIGAR In Tumorigenesis And Development Of Gastric Cancer

Background and objectiveGastric cancer is the fourth most common cancer worldwide and the second leading cause of cancer mortality.Current treatment modalities include combinations of surgery,chemotherapy,and radiation therapy.However even with maximal trimodality treatment,prognosis for gastric cancer patients remains poor,with a median survival ranging from 10 to 14 months in advanced cancer.Furthermore,surgery can only remove the tumor physically,which can not eradicate or inhibit the generation of tumor.The introduction of mechanism-based targeted therapies to deal with human cancers has been taken as one of the fruits of three decades of remarkable progress of research into the mechanisms of cancer pathogenesis.Indeed,each targeted agent represents a validation of a particular capability,characterized by strong specialty and impermanent function,followed by unavoidable recurrence.One interpretation of this outcome,supported by growing experimental evidence,is that each of the core hallmark capabilities is regulated by partially multiple signaling pathways.Consequently,a targeted therapeutic agent inhibiting one key pathway in a tumor may not completely shut off a hallmark capability.The second explanation is in response to therapy,cancer cells may also reduce their dependence on a particular hallmark capability,becoming more dependent on another,which will restrict the efficacy of the targeted agents.Such considerations suggest that drug development and the design of treatment protocols will benefit from incorporating the concepts of functionally discrete hallmark capabilities and of the multiple biochemical pathways involved in supporting each of them.So far now,no agent has ever worked permanently,and drug resistance is inevitable,no agent can completely inhibit tumor growth.Thus,searching for more pathways,more targets,developing more agents,selective cotargeting of multiple core and emerging hallmark capabilities and enabling characteristics in mechanism-guided combinations will result in more effective and durable therapies for human cancer.It is becoming clear that certain metabolic alterations are essential for tumor.There is evidence for cross-talk between signaling pathways and metabolic regulation in every multicellular organism studied.Metabolism is involved in essentially everything a cell does.Activation of oncogene and loss or inhibition of tumor suppressor directly affect metabolism and growth of cancer cells.As an tumor suppressor,p53 has gained great interests in its function of metabolic controlling in recent years.p53 may control cell metabolism through regulating several downstream targeted genes,facilitate oxidative phosphorylation through activating SCO2/GLS2/Parkin,prevent uptake of glucose via suppression of GLUT 1/3/4,inhibit glycolysis through expression of TIGAR.The function in regulating metabolism does contribute to the overall tumor suppression function of p53.While p53 may also provide protection for both normal and tumor cells through lowering ROS and maintaining energy homeostasis on metabolic stress,p53 acts as a double-edged sword.TIGAR(TP5 3-induced glycolysis and apoptosis regulator)was discovered through microarray analysis of gene expression following the activation of p53.As targeted gene of p53,TIGAR shows similar duality,suppresses tumor through inhibiting glycolysis,while accelerating proliferation and regeneration of tumor cells by inhibiting ROS accumulation,increasing GSH:GSSG ratio and biosynthesis of nucleotide.Moreover,the regulation of TIGAR on glycolysis and ROS is cell type dependent,which is a manifestation of tumor heterogeneity,metabolic path is different among different tumor and different site in a tumor.It has been researched about TIGAR function in cerebral glioblastoma,hepatic cancer,head and neck neoplasm,osteosarcoma,breast cancer,hematologic disease and colon cancer,and remains blank in gastric cancer.It has been reported about expression of TIGAR in gastric cancer tissue,and only expression status in cancer tissue without normal control and no further function and mechanism research.TIGAR is involved in many other signal pathways other than accept regulation of p53.For example,TIGAR was down regulated in NPC cell line after inhibition of C-Met via decrease of NADPH and increase of cell death.Moreover,expression of TIGAR was demonstrated in HeLa cell line to be controlled by PI3K-AKT-mTORC pathway.AKT is a serine/threonine kinase activated at downstream of integrin,involved in inhibition of cell apoptosis,proliferation,metastasis and several other processes.Somatic mutation of kinase domain of AKT has been commonly documented,including gastric cancer.Hyperactivation of AKT is one of the most common molecular findings in human cancers.In gastric cancer,expression of AKT and phosphorylated AKT(pAKT)are detected in 74%and 78%of tumors respectively.In a report of 50 advanced gastric carcinomas,there was a statistically significant correlation between pAKT expression and depth of tumor,number of involved lymph nodes and poor outcome.Nuclear pAKT expression was also found to inversely correlate with the apoptotic index.AKT not only has great significance in gastric tumor biologically,or is involved in regulation of TIGAR.This study will detect the TIGAR experession in gastric cancer tissues and adjacent noncancerous tissues through immunohistochemical staining and Western blotting approach,and explore the correlation of TIGAR with tumor staging and prognosis by tissue microarray,then construct TIGAR knocked down gastric cancer cell lines for in vitro experiment to explore the effect of TIGAR on gastric cancer cell growth,furthermore,the mechanisms underlying will be analyzed as well.MethodsPart ?:Immunohistochemical staining and Western blotting was employed to detect expression of TIGAR in both gastric cancer and adjacent noncancerous tissues,with the finding that TIGAR was overexpresssed in gastric cancer tissues.Further study was performed by tissue microarray to determine the relation between TIGAR expression with tumor staging and prognosis.Part ?:Gastric cancer cell lines were utilized to conduct in vitro experiment to investigate TIGAR function.TIGAR knocked down and over expression gastric cell lines were constructed with lentivirus transfection approach,cell growth was detected by MTT,the competency of colony formation and growth speed was analyzed by colony formation,cell cycle and apoptosis were detected by FCM,the tumorigenicity potential of TIGAR was detected by in vivo tumor growth analysis?Part ?:The expression of pAKT,Cyclin D1,BCL-2 and NADPH,ROS was measured after TIGAR was knocked down for the purpose of disclosing the mechanisms underlying.Statistical analysisKaplan-Meier survival analysis and log-rank statistics was employed to perform survival univariate analysis,p<0.05 is considered as statistical significance.The expression of TIGAR in cancer tissues and adjacent non tumor tissues were analyzed by Chi-square test,p<0.05 was regarded as statistical significance.Western blotting results were undergone data process through Image Studio Lite,PS,AI,followed by statistically handled and analyzed with prism5,SPSS22.0.ResultsPart ?:Expression of TIGAR in gastric cancer tissues and its relationship with cilinical datus and prognosis.1.TIGAR was overexpressed in human gastric cancer specimen.18 out of 32 cases were shown a higher expression of TGIAR in gastric cancer tissues than adjacent non-tumor tissues detected by Western blotting of frozen gastric cancer specimens.IHC demonstrated that the expression of TIGAR in gastric cancer tissues is higher than that in adjacent non-tumor tissues,which has a significant statistical difference(p<0.01).2.TIGAR expression was significantly correlated with survival rate of gastric cancer patients,which indicated a poor prognosis.Our results indicated that there was significant difference between TIGAR high expression and low expression patients in respect of mean age(p=0.04),N staging(p=0.02),AJCC staging(p=0.015).The univariate and multivariate analysis showed that the survival rate of gastric cancer patients is significantly correlated with age(p=0.034),tumor size(p=0.036),histological grading(p=0.001),T staging(P=0.008),N staging(P=0.000),M staging(P=0.014),AJCC staging(p=0.000)and TIGAR expression(P=0.034).The 5-year survival rate of TIGAR high expression patients was significantly shorter than that of TIGAR low expression patients(p=0.033).Part ?:The exploration of function of TIGAR in gastric cancer cell lines1.TIGAR was highly expressed in human gastric cancer cell lines.2.TIGAR knockdown inhibited proliferation and colony formation of SGC7901 and AGS cells in vitro and in vivo.The growth of SGC7901 and AGS cells was decreased and colony formation demonstrated a deterioration after TIGAR was knocked down in vitro,but cells stably overexpressing TIGAR showed increased growth.Tumorigenicity was inhibited after TIGAR was knocked down in vivo,the growth of tumor was slow from the beginning and from 14th day on,the trialed group maintained a low growth speed while the control group grow rapidly.3.TIGAR protects cancer cells against oxidative stress and sensitizes GC cells to glycolysis inhibitor Our results showed the percentages of apoptotic cells were increased after 50Um H2O2 treatment at the early and the total apoptosis quantiles(P<0.05).Meanwhile,the percentages of apoptotic cells were significantly increased in the early and total apoptosis quantiles in cells with TIGAR knockdown(P<0.001).These data indicated that TIGAR protected cancer cells from oxidative stress-induced cell deaths.Our results showed that the combination of TIGAR knockdown with 2-DG addition significantly increased the cell apoptosis.These data indicated that TIGAR knockdown sensitized gastric cancer cells to the glycolysis inhibitor.4.TIGAR knockdown induce SGC7901 and AGS cells G2/M phase cycle arrest in vitro.Cell cycle distribution curve of SGC7901 measured by FCM indicated that the percentage of cell population in G0/G1,S,G2/M phase is 53.4%,30.21%,16.07%respectively.While after TIGAR was knocked down,the cell distribution of G0/G1,S,G2/M phase was 47.13%,28.55%,23.98%in SGC7901 shRNA TIGAR B6 group and 50.69%,27.09%,22.07%in SGC7901 shRNA TIGAR B5 group.The proportion of G0/G1 phase cells was significantly decreased(p<0.05),while the proportion of G2/M phase cells was significantly increased(p<0.05),but no significant increase of S phase cells was observed,which indicated a cell arrest in G2/M phase.Similar result was observed in AGS cell line.Part ?:The mechanisms of functions of TIGAR in gastric cancer cell line1.TIGAR knockdown induced decreased generation of NADPH,increased ROS production in SGC7901 and AGS cellsOur data showed that TIGAR knockdown significantly reduced the generation of NADPH,leading to reduced NADPH/NADP+ratios,and increased ROS generation.In accordance with our in vitro results,the in vivo data showed that TIGAR knockdown significantly reduced the NADPH/NADP+ratios(p<0.05),which indicated that TIGAR knocked down inhibited the phosphate pentose pathway in cells,and hence induced cell apoptosis and inhibition of proliferation through reduction of NADPH.2.TIGAR knockdown induced cell cycle arrest via reduction of Cyclin D1 expression,induced cell apoptosis through reduction of BCL-2 expression.BCL-2 and Cyclin D1 were significantly down regulated among detected proteins extracted form nude mice tumorigenicity specimen after TIGAR was knocked down.The above results suggested that TIGAR induced cell cycle arrest via reduction of Cyclin D1 expression,induced cell apoptosis through reduction of BCL-2 expression.AKT was clearly increased after addition of H2O2 for 12 hours,though there was no significant difference between TIGAR KD group and the control group,and so was TIGAR upregulated,which indicated that pAKT pathway was activated under oxidative stress and induced TIGAR upregulation,but pAKT was not affected following TIGAR knocked down.Conclusions1.TIGAR was highly expressed in human gastric cancer as an indication of poor prognosis.2.TIGAR was highly expressed in human gastric cancer cell lines.3.TIGAR knockdown induced cell apoptosis through down regulation of BCL-2 expression,reduction of NADPH generation and ROS accumulation.4.TIGAR knockdown induced cell cycle arrest via reduction of Cyclin D1 expression and so inhibited cell proliferation.5.TIGAR Knockdown sensitizes GC cells to glycolysis inhibitor.