Effects Of β-PGG On Human Pancreatic Cancer Cells And Cachexia In Tumor-carrying Mice

Background Pancreatic cancer is a common malignant tumor of the digestive system,with a high degree of malignancy.Due to the difficulty in early diagnosis,low surgical resection rate and poor response to non-surgical treatment of advanced pancreatic cancer,the 5-year survival rate of pancreatic cancer is less than 1%.Pancreatic cancer cells provide energy mainly through glycolysis.Insulin and insulin-like growth factor-1(IGF-1)play an important role in this process.Insulin and IGF-1 not only bind specifically to their own receptors,namely IR and IGF1 R,but also cross-bind to each other’s receptors,thus promoting the growth of pancreatic cancer cells.Therefore,interfering with the downstream signaling pathway of insulin and its receptor may play a regulatory role in the development of pancreatic cancer.Due to the rapid growth of pancreatic cancer cells,the tumor is often in a relatively anoxic state,so hypoxia-inducible factor-1α(HIF-1α)can express and form HIF-1 in pancreatic cancer cells.High expression of HIF-1 in tumor cells can also activate downstream caveolae protein(caveolin-1,cav-1).HIF-1α and cav-1 can increase cell growth,glycolysis and tumor angiogenesis,which support the rapid growth of tumor cells.HIF-1α is an important target to support the occurrence and development of pancreatic cancer.Moreover,even under the condition of sufficient oxygen,pancreatic cancer cells will still increase glycolytic key enzymes(HK-Ⅱ and PFK-1),and this phenomenon is named as “Warburg effect”.Warburg effect can provide energy to tumor cells,promote gluconenogenesis in liver,degrade skeletal muscle,and accelerate lipolysis.Finally,cancer cachexia is caused by weight loss and negative nitrogen balance.Hence,inhibition of the Warburg effect on tumor cells is a potential target for the treatment of pancreatic cancer cachexia.Pentagalloylglucose(PGG)is a natural polyphenol compound from plants.PGG is a ligand of IR,which can bind to IR/IGF1 R and play an important role in regulating glucose metabolism in cells.PGG is used to treat many malignant tumors,such as breast cancer,prostate cancer,colon cancer and liver cancer.Its mechanism includes regulating cell cycle of tumour cells,the expression of HIF-1α,and cell nutrition metabolism,etc.Objectives The purpose of this study is to investigate whether β-PGG attenuates the Warburg effect in pancreatic cancer cells through decreasing the activity of IR/IGF1 R and the expression of HIF-1α.We also studied whether β-PGG relieves cachexia in athymic mice carrying the pancreatic cancer cells.Methods Part Ⅰ: 1)Pancreatic cancer cell line MiaPaCa-2 was stimulated with different concentrations of insulin(100 p M-1mM),and the experiments were conducted under both normoxic(95% air)and hypoxic(1% oxygen)conditions.Western blot was used to detect the phosphorylation of IR and IGF1 R in MiaPaCa-2 under normoxic and hypoxic conditions.The activation of PI3K-Akt and MEK-ERK1/2 signaling pathways,the changes of HIF-1α and cav-1,and the key enzymes of cell glycolysis(HK-Ⅱ and PFK-1)were observed in pancreatic cancer cells.2)β-PGG(5,13,25,50mM)was used in MiaPaCa-2 and PANC-1 cells.The phosphorylation levels of IR,IGF1 R,ERK and Akt,the expression of HIF-1α,cav-1,HK-Ⅱ and PFK-1 were detected by Western blot.3)MiaPaCa-2 cells were treated with β-PGG(25 and 50m M),and MTT assay and colony formation assay were performed to detect the proliferation capacity of pancreatic cells.Meanwhile,migration assay and scratch assay were applied to determine the migration capacity of cells.Part Ⅱ: A tumor-bearing mouse model of pancreatic cancer was established with MiaPaCa-2 cells.Forty athymic mice were randomly divided into four groups(10animals per group).The control group was group I(intact athymic mice),the tumor carriers that were untreated otherwise were group U,β-PGG treated tumor-bearing group was group P,and rhein treated tumor-bearing group was group R.The tumor tissue mass(2 mm3)was implanted subcutaneously in one flank.After one week,mice in group P were given 20 mg/kg β-PGG once a day by gavage.Mice in group R were given 100 mg/kg rhein once a day.PBS was given in group U.Tumor size and body weight were measured weekly.After eight weeks of administration,the animals were sacrificed under anaesthesia.The tumor was analyzed by using morphological measurement and histological staining.In order to determine the treatment effect ofβ-PGG on pancreatic cancer,the samples of plasma,skeletal muscle,fat and liver were analyzed for the evaluation of metabolic state,and the phosphorylation levels of IR,IGF1 R,ERK and Akt,the expression of HIF-1α,cav-1,HK-Ⅱ and PFK-1 were detected by Western blot.As regulators of enery homeostasis,PCB and G-6-Pase in liver,Atrogin-1and Mu RF-1 in skeletal muscle,and ATGL in fat were determined to evaluate the effect of β-PGG on cancer cachexia.Results For in vitro:1)When insulin stimulated MiaPaCa-2 cells,the phosphorylation of IR/IGF1 R,PI3K-Akt and MEK-ERK1/2 signaling pathways were increased,the expression of HIF-1α,cav-1 and the key enzymes of glycolysis(HK-Ⅱ and PFK-1)were increased.2)β-PGG decreased the phosphorylation of IR/IGF1 R,PI3K-Akt and MEK-ERK1/2 signaling pathways both in MiaPaCa-2 and PANC-1 cells,and reduced the expression of HIF-1α,cav-1,HK-Ⅱ and PFK-1.β-PGG significantly inhibited the proliferation and migration of MiaPaCa-2 cells.For in vivo:3)β-PGG significantly attenuated the activation of IR/IGF1 R,PI3K-Akt and MEK-ERK1/2,and reduced the the expression of HIF-1α,cav-1 and the key enzymes of glycolysis in tumor tissues.β-PGG significantly decreased the weight,volume and cross-sectional area of the tumor,and promoted the apoptosis of pancreatic cancer cells.It significantly inhibited the tumor growth in pancreatic cancer tumor-bearing mice.4)β-PGG decreased the levels of PCB and G-6-Pase in liver,Atrogin-1and Mu RF-1in skeletal muscle,and ATGL in fat of the tumor-bearing mice.Conclusions For pancreatic cancer,insulin can enhance glycolysis activity and the Warburg effect by activating the phosphorylation of IR/IGF1 R,PI3K-Akt and MEK-ERK1/2signaling pathways,and upregulating the expression of HIF-1α and cav-1.All above processes promote the occurrence and development of pancreatic cancer.β-PGG can significantly inhibit the activity of IR/IGF1 R and its downstream signaling pathways in pancreatic cancer cells.Metnwhile,β-PGG can significantly attenuate the activation of HIF-1α pathway and the Warburg effect of pancreatic cancer cells,as well as the degree of liver glycogenesis,muscle decomposition and fat hydrolysis in cachexic tumor-bearing mice.In summary,β-PGG inhibits the growth of pancreatic cancer cells,alleviates the development of pancreatic cancer cachexia,and delays the pathological process of pancreatic cancer.