Effects Of Epigallocatechin Gallate On Glycolysis And Proliferation Of Pancreatic Cancer

Objective:Pancreatic cancer is one of the most common malignant tumor.Due to its insidious onset,the best treatment period has been missed in most cases.Further,it is insensitive to radiotherapy and chemotherapy.Thus,its 5-year survival rate is less than 8%.In China,the morbidity and mortality of pancreatic cancer are also on the rise,and the annual mortality is almost equal to the morbidity.Unlike normal cells,tumor cells tend to rely more on aerobic glycolysis to produce energy and obtain intermediate metabolites,which are then directed to other biosynthetic pathways that promote tumor growth.Glycolysis can alter the intrinsic metabolism of cancer cells and is an important marker of cancer.Hypoxia-inducing factor-1α（HIF-1α）in cancer cells plays a key role in cancer-induced glycolysis.In addition,pancreatic cancer cells activate key glycolytic enzymes such as hexokinase（HK）and phosphofructo kinase（PFK）even when oxygen is sufficient,increasing glycolytic activity,known as the Warburg effect.There are two main reasons for this advantage in promoting tumor cell growth:first,cancer cells can survive under varying oxygen tension,which is fatal to cells that rely on oxidative phosphorylation（OXPHOS）to produce ATP,because the hemodynamics of distant blood vessels are variable.Second,lactic acid,as the main end product of aerobic glycolysis,can produce an acidic environment that is in favor of tumor invasion and inhibition of anti-cancer immune effects.Epigallocatechin gallate（EGCG）,it is the main component of green tea polyphenols and belongs to catechins monomer.Clinical studies have shown that tea polyphenols not only have"health function",but also have a good"pharmacological function".This project mainly studied whether EGCG inhibits pancreatic cancer by inhibiting the glycolysis and proliferation of pancreatic cancer cell.Method:1.Pancreatic cancer cells were treated with EGCG under hypoxic conditions（1%O₂,5%CO₂and 94%N₂）for four hours,The cell population was measured using the CCK-8 kits.Biochemical kits were used to detect the rate of glucose consumption and lactic acid production in cancer cells.2.Real-time PCR was performed to measure the expression of the genes of Glut-1、PFK-1、HK-Ⅱ、PKM2 and LDHA at the m RNA level.Western blotting was performed to measure the expression of the genes of HIF-1α,Glut-1、PFK-1 and HK-Ⅱat the protein level.3.Western blotting method was used to detect the expression level of HIF-1αprotein.To investigate the molecular biology of EGCG on HIF-1αexpression by the following experiments:a.Under normal oxygen conditions,Mia Pa Ca-2 cells were treated with EGCG for four hours,and the expression levels of proteins that can stimulate the HIF-1αsignaling pathway were detected by western blotting.b.Under normal oxygen conditions,Mia Pa Ca-2 and PANC-1 cells were co-incubated with EGCG and proteasome inhibitor（MG132,20μM）for four hours,and the expression levels of P564 and P402 hydroxylated HIF-1αwere detected by western blotting.c.Two types of pancreatic cancer cells were incubated with EGCG（50μM）under anoxic conditions for four hours.CHX（100μg/ml）was rapidly added and treated under normal oxygen conditions for 0、5 and 10 minutes.The HIF-1αprotein levels were measured by western blotting and the degradation rate was observed.4.The tumor-bearing model of nude mice was established and Mia Pa Ca-2 cells were used as tumor seed cells.The tumor-bearing cells were divided into the tumor-bearing control group and the EGCG group.The EGCG group was given 50 mg/kg of EGCG intraperitoneal injection,and the control group was given equal volume of PBS intraperitoneal injection for 5 times/week for 8 weeks.At the end of the experiment,blood was taken from the eyeball to measure the tumor weight and length of the meridians.The apoptosis status of intratumoral cells was detected by apoptosis staining,and the influence of HIF-1αprotein,tumor intimal receptor and downstream signaling pathway in the tumor was detected by Western blotting method.Result:1.EGCG could effectively inhibit the proliferation of Mia Pa Ca-2 and PANC-1 cells.Under normoxic conditions,EGCG can inhibit glucose consumption and lactic acid generation of pancreatic cancer cells.2.The EGCG decreased the m RNA expression of some glycolysis-related proteins of glycolysis,i.e.Glut-1、PFK-1、HK-Ⅱ、PKM2 and LDHA.EGCG could inhibit the expression of HIF-1αprotein,EGCG could inhibit glycolysis-related proteins,e.g.Glut-1、PFK-1 and HK-Ⅱ.3.EGCG inhibits the expression of HIF-1αprotein.a.EGCG can inhibit the expression levels of two membrane receptors IR/IGF1R and downstream phosphorylated proteins in the upstream signaling pathway of HIF-1α.b.EGCG can inhibit the expression level of HIF-1αprotein hydroxylated by P564 and P402,indicating that EGCG can inhibit the generation of HIF-1αin normal oxygen environment.c.Degradation experiments have shown that EGCG can also accelerate the degradation of HIF-1α.4.In vivo experiments showed that:a.EGCG inhibited tumor growth in nude mice and reduced tumor weight and tumor volume.b.Western blotting method detected that EGCG can inhibit the expression levels of HIF-1αand downstream target proteins in tumors.c.EGCG could inhibit the protein expression levels of two membrane receptors IR/IGF1R and their downstream signaling pathways in tumors.Conclusion:In vitro studies showed that EGCG inhibited the proliferation of pancreatic cancer cells,and inhibited glycolysis by inhibiting the expression of two receptors IR/IGF1R and its two downstream signaling pathways,PI3K/Akt,MEK/ERK and HIF-1α,respectively.In vivo studies have shown that EGCG can inhibit the growth of tumors in vivo,as well as the protein expression of HIF-1α,target protein,IR/IGF1R receptor and downstream signaling pathways in tumors.