Effects Of Anti-energetic Drug Sodium Citrate To Human Gastric Cancer Cell Proliferation And Its Mechanism

Research background and objectiveGastric cancer is a common malignant tumor, it threats to the people health.The treatment of gastric cancer is a worldwide problem, in recent years, itsprognosis has been improving with the application of comprehensive treatment,especially surgical operation, however, the five year survival rate is still low. Inorder to meet the demands for gastric cancer treatment, which is considered tobe a complex disease, it is necessary to find new effective antitumor drugs.Tumor cells are dependent on glycolysis for gaining ATP, glycolytic inhibitioncan induce cell apoptosis, the drug what we call anti-energetic drug targetstumor energetic metabolism, sodium citrate (SCT) is one of the representative.Phosphofructokinase1(PFK-1), hexokinase (HK) and pyruvate kinase (PK)arethe rate limiting enzymes of glycolysis, PFK-1has the strongest control effect. Itis able to inhibit glycolysis by inhibiting the PFK-1activity. Research data wasshown, SCT as a PFK-1inhibitor, can inhibit the enzyme activity by bindingitself with inhibitory site of PFK-1. Moreover, study had confirmed that DNA ispersistently degradated when tumor cell glycolysis was inhibited and ATP was completely depleted, causing intensive permeability of tumor cell mitochondrion,cell apoptosis was induced by releasing cytochrome C (Cyt-C) and activatingcaspase. The mitochondrial pathway is an important pathway of apoptosis signaltransduction, which is influenced by many kinds of apoptosis gene or proteinregulation, such as Bcl-2family, Cyt-C and so on. Through the study of thatgenes or proteins can provide basis for elucidating the mechanism of drug. Ourprevious studies had proved that SCT can inhibit gastric cancer cell proliferationin vitro, but its mechanism and whether that mechanism was correlated withglycolysis remain unclear; in addition, does SCT has inhibitory effect in vivo tohuman gastric cancer implanted tumor in nude mice and its mechanism, has notbeen reported. Through our previous experiments, we think that SCT mayachieve its function by the mechanism of inhibiting PFK-1activity andinhibiting glycolysis, causing changes of apoptosis genes or proteins expression,activating mitochondrial pathway that leads to Cyt-C release increasingly, toplay its role for inducing gastric cancer cell apoptosis and suppressing tumorgrowth of human gastric cancer in nude mice. In order to confirm this conjecture,human gastric cancer cell line SGC-7901and nude mice model of human gastriccancer implanted tumor are applicated by our experiment, studying effects ofSCT to human gastric cancer cell proliferation in vitro and in vivo, and toexplore its mechanism, which will provide experimental basis and new methodfor gastric cancer therapy. Research contentsPART1Experimental study on the SCT to gastric cancer cellproliferation in vitro and its mechanismMethodsCultured human gastric cancer cell line SGC-7901, which was dividedrandomly into5groups: SCT low, medium, high concentration group (5mmol/L,10mmol/L,20mmol/L),5-FU positive control group (0.5mmol/L) and blankcontrol group. Effects of drug to human gastric cancer cell proliferation wasobserved after the treatment of each group. Cell apoptosis and cell cycle changeswere detected by using flow cytometry, PFK-1, HK, PK activity were detectedby using6-glucose phosphate dehydrogenase coupled assay, lactic acid is theend product of glycolysis, and its content was detected by usingspectrophotometry assay, expression of gene mRNA related to apoptosis wasdetected by using RT-PCR.ResultsResults of microscopic observation were shown: cancer cell proliferationwas inhibited significantly by SCT low, medium, high concentration group and5-FU group after24h and48h treatment, the inhibitory effect of SCT based onconcentration and time dependent manner. Typical morphological changes ofapoptosis were found in the cancer cells of different concentration groups ofSCT and5-FU group.Results of flow cytometry were shown: cancer cell apoptosis in differentconcentration groups SCT and5-FU group were significantly increased incomparison with blank control group after24h and48h treatment(P＜0.05),apoptosis rate of cancer cell in different SCT concentration group was upregulated with an increase of SCT concentration or medication timeprolonged. The majority cell in different concentration groups of SCT and5-FUgroup were stopped in G2/M phase, and hypodiploid apoptotic peak was found.However, cells in blank control group were neither stopped in G2/M phase norfound hypodiploid apoptotic peak.Results of PFK-1, HK, PK activity and lactic acid content were shown: PFK-1activity in different concentration groups of SCT cells and lactic acid contentwere significantly lower than that of5-FU group and blank control group (P＜0.05) after24h and48h treatment, PFK-1activity of cells in differentconcentration of groups SCT and lactic acid content were upregulated with anincrease of SCT concentration or medication time reduced. To compare5-FUgroup with blank control group, PFK-1, HK, PK activity and lactic acid contenthad no significant difference(P>0.05). To compare different concentrationgroups of SCT with control group respectively, the difference of HK and PKactivity in24h,48h had no statistical significance(P>0.05).Results of RT-PCR test were shown: After24h treatment, Bax and Cyt-CmRNA expression in different concentration groups of SCT were significantlyhigher than those in the blank control group, Bcl-2and Bcl-xL mRNAexpression in different concentration groups of SCT were significantly lowerthan the blank control group (P＜0.05); Bax and Cyt-C mRNA expression indifferent concentration groups of SCT were upregulated with an increase of SCTconcentration while Bcl-2and Bcl-xL mRNA expression were downregulatewith an increase of SCT concentration. Bax expression of5-FU group was lowerthan SCT high concentration group, but higher than SCT medium concentrationgroup (P＜0.05); to compare5-FU group with SCT medium concentration group, the Bcl-2, Cyt-C, Bcl-xL mRNA expression had no significant difference(P>0.05).PART2Experimental study on the SCT to gastric cancer cellproliferation in vivo and its mechanismMethodsHuman human gastric cancer cell line SGC-7901was constructed by usinghuman gastric cancer implanted tumor of nude mice, were divided randomlyinto5groups: SCT low, middle, high dose group,(7.5mg/kg,15mg/kg,30mg/kg),5-FU positive control group (0.35mg/kg), negative control salinegroup(NS), there were12mice in each group. Each group was injectedcontinuously by using medication for two weeks, to observe the effects of SCTto xenograft growth. Apoptosis of cell ultrastructure was detected by electronmicroscopy, cell apoptosis was detected by TUNEL; PFK-1, HK, PK activitywere detected by6-glucose phosphate dehydrogenase coupled assay, lactic acidis the end product of glycolysis, and its content was detected byspectrophotometry assay, immunohistochemical method and western blottingwere used to detect related protein expression of apoptosis. To detect liver andkidney function and blood indicators, liver and kidney tissue of the nude micewere observed by using microscope.ResultsInhibited results of xenograft growth were shown: to compare differentdose groups of SCT and5-FU group with NS group respectively, their differenceof tumor weight, volume, inhibition rate had statistical significance(P＜0.05),meanwhile, to compare SCT high dose group of nude mice with5-FU group,their difference of tumor weight, volume, inhibition rate had no statistical significance (P>0.05).Results of transmission electron microscope were shown: changes of typicalapoptosis shape were found in tumor cells of SCT group and5-FU group byusing microscope: such as cell shrinkage, shrinkage of nuclear shape,condensation and margination of chromatin, endoplasmic reticulum, varyingsizes of vacuoles in the intracytoplasmic, some mitochondria edema, hyperplasiaof vacuolar degeneration, crest was not distinguished, typical apoptosis bodycould be found. However, tumor cells with uniform size, large nuclei andprominent nucleoli, no condensation and margination of chromatin, andapoptotic bodies were not found in NS group.Results of apoptosis by using TUNEL assay were shown: mean indices oftumor cell apoptosis in the different dose groups of SCT and5-FU group werehigher than the NS group (P＜0.05), meanwhile, to compare5-FU group withSCT medium dose group, the difference had no statistical significance(P>0.05).The mean apoptosis index in different dose groups of SCT was raised with anincrease of SCT dose (P＜0.05).Results of PFK-1, HK, PK activity and lactic acid content in xenografts wereshown: PFK-1activity and lactic acid content in different dose groups of SCTwere much lower than than5-FU group and NS group (P＜0.05), PFK-1activityand lactic acid content in different dose groups of SCT were decreased with anincrease of SCT dose, that was dose-effect relationship. To compare PFK-1, HK,PK activity and lactic acid content of5-FU group with NS group, there was nostatistically significant difference (P>0.05). To compare different dose groups ofSCT with control groups respectively, the difference of HK and PK activity hadno statistical significance(P>0.05). Immunohistochemistry results were shown: cleaved caspase-3and cleavedcaspase-9protein expression in different dose groups of SCT and5-FU groupwere higher than NS group (P＜0.05), meanwhile, the cleaved caspase-3proteinexpression of5-FU group was lower than SCT high dose group, but that washigher than medium dose group. Cleaved caspase-9protein expression of5-FUgroup cleaved was higher than SCT high dose group (P＜0.05), cleavedCaspase-3and cleaved caspase-9protein expression in different dose groups ofSCT was upregulated with an increase of SCT dose (P＜0.05).Results of western blotting were shown: Bax and Cyt-C protein expression indifferent dose groups of SCT were significantly higher than NS group, Bcl-2andBcl-xL protein expression were significantly lower than NS group (P>0.05);Bax and Cyt-C protein expression in different dose groups of SCT wereupregulated with an increase of SCT dose, but Bcl-2and Bcl-xL proteinexpression were downregulated with a decrease of SCT dose. To compare Baxand Bcl-xL protein expression of5-FU group with SCT high dose group, thedifference had no statistical significance (P>0.05), to compare Cyt-C and Bcl-2protein expression of5-FU group with SCT medium dose group, the differencehad no statistical significance (P>0.05).Results of liver and kidney function and blood system test in nude mice wereshown: to compare different dose groups of SCT with NS group in nude mice,the difference of glutamic-pyruvic transaminase (GPT), albumin, urea nitrogen,creatinine was not statistically significant(P>0.05); to compare5-FU group withNS group, the levels of GPT, urea nitrogen, creatinine were increased in variousdegrees in nude mice, but albumin was decreased (P＜0.05). To comparedifferent dose groups of SCT with NS group, the difference within levels of white blood cells, platelets, hemoglobin was not statistically significant(P>0.05),however, to compare5-FU group with NS group, the levels of white blood cells,platelets, hemoglobin in nude mice was significant decreased (P＜0.05). Liverand kidney tissue sections of nude mice were showen that necrotic tissues werefound in liver and kidney sections in5-FU group, meanwhile, the SCT groupand NS group had no obvious abnormalities.Research conclusionsAnti energetic drug SCT has strong inhibitory effects to the human gastriccancer cell line SGC-7901proliferation in vitro, it also has strong inhibitoryeffects to the xenograft growth of human gastric cancer of nude mice in vivo.Maybe SCT achieves its function through the mechanism of inhibitiing PFK-1activity, then inhibiting glycolysis, causing Bax protein upregulation, Bcl-2andBcl-xL protein downregulation, activating the mitochondrial pathway andcausing an increase of Cyt-C release, activating caspase-3and caspase-9,thereby to induce gastric cancer cell apoptosis and inhibit growth of gastriccancer. SCT in nude mice shows that its safety is higher. SCT, as a new effectiveantitumor drug is worthy of further study. This study provides experimentalbasis and new method for gastric cancer therapy.