EGCG Suppresses Pancreatic Cancer Growth Through ROS-dependent Glycolysis:Enhanced Efficacy When Combined With Gemcitabine

In recent years,the morbidity of pancreatic cancer keeps at a high level and has become one of the top five factors causing death among all the cancer types.Due to the lack of diagnosis methods,patients with pancreatic cancer are usually diagnosed at an advanced stage and almost have no chance to be cured.Epigallocatechin-3-gallate（EGCG）,one of the essential components in tea,possesses excellent anti-cancer effect.However,the specific mechanisms of EGCG on pancreatic cancer still keeps elusive,thus in this work,we explored the effect and the interior mechanisms of EGCG on pancreatic cancer in vitro and in vivo.The results are as follows,Different pancreatic cancer cell lines have different sensitivity to EGCG.The cell growth rate of 6 human pancreatic cancer cell lines,1 murine pancreatic cancer cell line were tested together with a normal human pancreas cell line HPNE.Cell growth of all cell lines were inhibited after treating with EGCG.It is worth noting that HPNE seemed to be more resistant to EGCG compared with those pancreatic cancer cell lines.After double-staining cells with Annexin V/FITC and PI,the results showed that EGCG induced cell apoptosis in Panc-1 and MIA PaCa-2 cells by increasing the ratio of Annexin V/FITC⁺cells.However,EGCG showed less effect on cell cycle arrest,only inducing a slight arrest of S and G2 phase in Panc-1 cells,indicating the main effect of EGCG on cell growth was related to induce cell apoptosis.What’s more,EGCG strongly inhibited pancreatic cancer cell migration and invasion by affecting Epithelial-Mesenchymal Transition（EMT）.In vivo,tumor of the mice grow slowly if injected intraperitoneally with either 10 mg/kg/d or 20 mg/kg/d EGCG while EGCG didn’t showed any toxicity to the mice.Glycolysis plays a key role to produce energy to support cell growth.In pancreatic cancer cell lines,EGCG significantly reduced glycolysis rate and the cell toxicity induced by EGCG increased under the condition of glucose deprivation and2-Deoxy-D-Glucose（2-DG）treatment.Thus the suppression of EGCG on glycolysis is somehow related to its inhibition on cell growth.Phosphofructokinase（PFK）and pyruvate kinase（PK）are two rate-limiting enzymes in glycolysis.After treating with EGCG,both the activity and the protein expression of these two enzymes were significantly decreased.We also found the inhibition effect of EGCG on cell growth was further enhanced after silencing PFK.EGCG inhibited glycolysis by producing ROS.Detected by H2DCFDA and Amplex^?Red probe,the level of ROS and H₂O₂significantly increased in EGCG groups.Moreover,EGCG enhanced the levels of mitochondrial superoxide anion in a concentration-and time-dependent manner in Panc-1 and MIA PaCa-2 cells.In both cell lines,catalase mostly abrogated the inhibitory effect of EGCG on the expression of platelet-type phosphofructokinase（PFKP）and pyruvate kinase M2（PKM2）,suggesting that the effect of EGCG on glycolytic enzymes is dependent on the ability of EGCG to induce ROS.Meanwhile,catalase could also partly prevent the reduction of cell growth induced by EGCG.EGCG sensitized pancreatic cancer cells to gemcitabine.After we treated Panc-1 and MIA PaCa-2 cells with EGCG alone or in combination with five commonly used chemotherapeutics,we found EGCG together with gemcitabine have a stronger inhibition effect on pancreatic cancer cell growth.By modulating the expression level of cell-cycle related protein and affecting the apoptotic protein Caspases and their downstream effector PARP,EGCG further promoted cell cycle arrest,and induced more cell apoptosis than gemcitabine alone.In vivo,gemcitabine and EGCG alone decreased the tumor weight by 51%and 41%,separately,while the combination group further decreased the tumor weight by 67%.Meanwhile,EGCG also enhanced the inhibition effect of gemcitabine on the activity and expression of PFK and PK.Akt pathway is an essential moderator of drug resistance,and also plays a critical role in modulating glycolysis.By promoting the protein degradation and inhibiting transcription and translation,EGCG strongly decreased the protein expression of Akt.Overexpression of Akt1 weakened the growth inhibition effect of EGCG on pancreatic cancer cells.Co-treated cells with gemcitabine and EGCG,the inhibition effect of gemcitabine on Akt pathway was further enhanced.Above all,this study evaluated the effect and interior mechanisms of EGCG on pancreatic cancer cells in vitro and in vivo,and found its growth inhibition effect is related to affect glycolysis modulating by the ROS.Also,EGCG showed a strong combination effect with gemcitabine by further acting on glycolysis and Akt pathway.