The Research Of Influence And Mechanism Of The Inhibition Of Glycolysis Of Pancreatic Cancer To The Biological Characteristic Of The Adenocarcinoma Cell PANC-1

Partâ… Analysis the activity of the enzyme of glycoysis in PancreaticNormal Tissue,Carcinoma and Adjacent Non-Cancerous TissueObjective To detect the activity of enzyme of glycolysis,concluding the lactatedehydrogenase (LDH) and LDH isoenzyme,and succinate dehydrogenase(SDH),so as toresearch the relation between biological characterictic of the pancreatic cancer andglycolysis.Methods Consecutive 12 cases pancreatic ductal adenocarcinoma and 12 benign lesionssuch an insulinoma from October 2006 to July 2008 were collected,as well as normalpancreatic tissues.The total activity of the LDH was detected by the LDH Testing Kits,andthe iosenzyme pattern of LDH was inspected by the France Sebia hydrasys.The activity ofSDH was detected by the enzyme histochemical staining.Results Compared to the normal tissue,LDH activity of the pancreatic cancer andadjacent non-cancerous tissue was significantly higher (P＜0.05).LDH iosenzyme patternin cancer tissue was also significantly different,i.e.,the percentage of LDH4 and LDH₅increased obviously,and greater than the normal tissue (P＜0.05).SDH actvity of thepancreatic cancer was obviously higher than the adjacent non-cancerous tissur and the normal pancreatic tissues.Conclusion The alteration of SDH,LDH activity and its isoenzyme pattern are possiblyrelated to the pathogenesis of pancreatic cancer.Inhibit the LDH activity may be a newtherapeutic strategy for pancreatic cancer.Partâ…¡Effect of the inhibitor of LDH(oxamate) on growth andapoptosis of human pancreatic cancer cell in vitroObjective To investigate the effect of Lacate Dehydrogenase(LDH) inhibitor-oxamate onthe growth and apoptosis of human pancreatic cancer cell.Methods The inhibitional effect of oxamate was observed at 0 mol/L,0.02 mol/L,0.04mol/L,0.08 mol/L,0.1mol/L on the growth ofpanc-1 cell line in medium containing 10%fetal bovine serum and serum-free medium for 24 h and 48 h by MTT assays.Flowcytometry(FCM),Hochest and PI staining analysis was usedto study the changes of cellapoptosis.Results Oxamate caused dose-dependent inhibition on the growth of human pancreaticcancer cell panc-1.Inhibition was much greater in serum-free medium than in medium with10% fetal bovine serum (p＜0.0 1).Flow cytometry(FCM),Hochest and PI staining analysisshows that the proportion of the apoptosis increased gradually,and it isdose-dependent(p＜0.01).Conclusion LDH inhibitor-oxamate inhibits the growth of human pancreatic cancer cell and induces apoptosis.Oxamate could be a potential targent for anti-pancreatic cancer.Partâ…¢The research of mechasim of pancreatic cancer cell apoptosisintroduced by inhibitor of LDH-oxamate in vitroObjective Investigate and research the possible mechasim for apoptosis of pancreaticcancer cell introduced by LDH-oxamate in vitro.Method The pancreatic cancer cell PANC-1 was treated with different concentration ofoxamate (0mol/L,0.02mol/L,0.04mol/L,0.06mol/L,0.08 mol/L,0.1mol/L) for 48h.Theapoptosis was measured by flow cytometry(Annexin V/PI);then dyed with Fluo-3/AM.Thelight density of cells for different concentration of oxamate under confocal lasermicroscopy were investigated;mitochondrial membrane potential was detected by flowcytometry With Rodamin 123 or Rodamin 123/PI stained.Result With oxamate treated for 48h,the apoptosis of PANC-1 increased obviously.Oxamate can influenced the concentration of Ca²⁺ in PANC-1,and this effect is correspondto the concentration of oxamate.The MMP(0.02mol/L-0.06mol/L)increased,but decreasedsignificantly(0.08mol/L-0.1mol/L).The percentage of Rh123-/PI- cells increased aftertreated with oxamate,that of 0.08mol/L-0.1mol/L increased obviously (p＜0.05),thepercentage of 0.1 mol/L came to (8.94±0.13)%.Conclusion The glycolysis inhibitor-oxamate could induce the pancreatic cancer cellapoptosis effectively by inhibition of glycolysis.Low concentration induce the apoptosismainly by inferring the metabolism,but the 0.08mol/L-0.lmol/L could also influenced theconcentration of Ca,degrade the mitochondrial membrane potential ,lead to the apoptosis. Oxamate can induced the increase of the concentration of Ca²⁺ of PANC-1,and influencedthe following pathophysiologiacn response(mitochondrial membrane potential).Partâ…£Effect of LDH-A shRNA on the Growth,Apoptosis of Humanpancreatic cancer cell PANC-1 and the expression of LDH-AObjective:Most cancer cells exhibit increased glycolysis,Lactate dehydrogenase catalysesthe conversion of pyruvate to lactate,which is essential for the glycolytic pathway.MethodResult Conclusion.The five isoenzymes of LDH are composed of LDH-A(so-called muscletype) and LDH-B(so-called heart type) subunits.This study constructed a short hairpinRNA(shRNA) targeting LDH-A,and analyze its effects on growth,apoptosis of panc-1cells,the expression of LDH-A and the activity of the LDH.Method:Three shRNAs targeting LDH-A were combined to pGCsilencer vector,andtransfected into panc-1 cells.The expression of LDH-A after transfected by the threeshRNAs in pancreatic cancer cell lines panc-1 was detected by Quantitative Real TimePCR.After tranfected by the LDH shRNA-3 with the highest inhibited rate..cell growthwas analyzed by MTT assay,apoptosis was detected by flow cytometry.The LDH-Aexpression was detected by reverse transcription polymerase chain reaction,LDH activitywas observed with enzyme cytochemical method.Results:The 2-△△Ct of LDH-A shRNA-3 was (0.47±0.02),less than the untransfectedpancreatic cancer cell(0.71±0.01),the LDH-A shRNA-3 can inhibit the expression of the LDH-A most obviously.The growth of the pancreatic cancer cell was inhibitted after 12htransfected by LDH-A shRNA-3,all the Absorbance value of transfected cell in24h,36h,48h,72h discreased obviously compared to the normal pancreatic cancer cell(p＜0.01).The apoptosis rates of the transfected cell increased to 61.74%.The inhibition ofLDH-A expression in pane-1 cells transfected by shRNA-3 was significantly and theactivity of LDH was also reduced.Conclusion:LDH-A shRNA can reduce the expression of the LDH-A,inhibit the activityof LDH,inhibit the growth and introduce the apoptosis of panc-1 cells siganificantly.Thetreatment of pancreatic cancer with the LDH-A shRNA could become the a new strategy infuture.