Phenformin Influence The Biological Phenotype Of Intrahepatic Cholangiocarcinoma And The Mechanisms Related With LKB1-AMPK Pathway

Background and AimsAccording to their different occurring sites,Cholangiocarcinoma can be divided into intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma.In which the incidence of intrahepatic cholangiocarcinoma is lower than the extrahepatic cholangiocarcinoma.But intrahepatic cholangiocarcinoma is insidious onset,and early symptoms are often not obvious,many patients may have lost the opportunity to lead to radical surgery when diagnosed.The intrahepatic cholangiocarcinoma have poor sensitivity to the current conventional chemotherapy drug,and patients have poor tolerance to the drug.Radiation therapy is also just a palliative treatment too.Thus,discover a mild and effective new drug is very necessary.Biguanides are commonly used in the treatment of diabetes,it is used for years and the security is reliable.Epidemiological data show thatthe morbidity and mortalityof diabetes and cancer are related.Cross-sectional study and retrospective study also suggests that for type 2 diabetes patients,taking metformin can reduce the prevalence of cancer and improve the response to chemotherapy and reduce their mortality.Like metformin,phenformin also belongs to biguanides,it is also used in the treatment of diabetes,but because of its side effects of lactate accumulation,it was fobidden.However,in terms of tumor therapy,phenformin have better effects and the standard of cancer treatment and the treatment of diabetes are also different.Thus phenformin is a better choice for tumor therapy.AMPK is a target of biguanides,which indicates that the expression of LKB1 is likely to have a significant impact on biguanides' anti-tumor effects.Therefore we hope to know the anti-tumor effects of phenformin for intrahepatic cholangiocarcinoma and it's mechanisms related with LKB1-AMPK pathway.And we also want to know how the state of LKB1 expression affect the anti-tumor effects of phenformin.Methods1.Use CCK-8 assay to test different concentrations of phenformin inhibit intrahepatic cholangiocarcinoma RBE,HuH-28 cell line proliferation,and calculate the IC 50.Test the same concentration of phenformin inhibit RBE,HuH-28 cell proliferation at different duration.2.Treating RBE,HuH-28 cells with different concentrations of phenformin and test the apoptosis rate by flow cytometry.3.Treating RBE cells with phenformin and detect LKB1,AMPK,p-AMPK protein expression by Western Blot.4.Treating RBE,HuH-28 cells with different concentrations of phenformin and detecte the PH value,changes of glucose,lactose content in both cell culture medium by microplate reader.5.Culture RBE,HuH-28 cells with three kinds of culture medium(high sugar,low sugar,low sugar plus lactate)and treat with phenformin,find how the culture medium affect the efficacy of phenformin.6.Knockdown LKB1 gene of RBE cell by LKB1 SiRNA,using Western Blot method to detect knock-down effect.7.Use Ki67 immunofluorescence to test how LKB1 gene knockdown affect proliferation of RBE8.Treating the wild-type and LKB1 knockdown RBE cells with phenformin,detecte the effect of phenformin on both cell proliferation by CCK-8 assay.9.Treating the wild-type and LKB1 knockdown RBE cells with phenformin,detecte the effect of phenformin on both cell's apoptosis rate.10.Treating the wild-type and LKB1 knockdown RBE cells with phenformin,detecte the effect of phenformin on the expression of p-AMPK by western-Blot.11.Treating the wild-type and LKB1 knockdown RBE cells with phenformin,detecte the effect of phenformin on the expression of Bax?cleaved-caspase3 in both cells by western-Blot.Results1.With the increase of the concentration of phenformin,extension of time,the viability of RBE,HuH-28 cell decreased.The IC 50 of RBE,is 1138?M,the IC 50 of HuH-28 is 854?M.2.With the increase of the concentration of phenformin,BE,HuH-28 cell apoptosis rate increased gradually.3.After treating RBE cells with phenformin(500?M)for 12,24,36 h,there was no significant change in the expression of AMPK,the expression of LKB1 slightly increased,while the expression of p-AMPK increased significantly.4.With the increase of the concentration of phenformin,both cell culture medium were acidified,sugar content decreased,lactate levels were increased.5.To the control group(without phenformin),no matter which culture medium(high-sugar,low-sugar or low-sugar + lactate)there is no significant differences in survival rates,while the experimental group(phenformin intervention)in the high-sugar,low-sugar,low-sugar + lactate three media,cell viability decreased sequentially.6.Western Blot show that the LKB1 expression was significantly decreased in LKB1 SiRNA treated RBE cells.7.The LKB1 knockdown group have more obvious cell proliferation.8.When there is no phenformin intervention,LKB1 knockdown increase cell proliferation.After phenformin intervention,the proliferation rate of LKB1 knockdown RBE cells was significantly lower than the wild type RBE cells.9.LKB1 knockdown RBE cells have lower apoptosis rate compared with wild-type cells.After phenformin intervention the apoptosis rate of both cells increased,but the LKB1 knockdown cells more obvious.10.When there is phenformin intervention,LKB1 knockdown cells have less p-AMPK expression than wild type RBE cells.11.Both cells increase the expression of Bax,cleaved-caspase3 gene under phenformin intervention,but LKB1 knockdown cells increased more significantly.ConclusionThrough our experiment,we studied the influence of phenformin on intrahepatic cholangiocarcinoma proliferation,apoptosis,energy metabolism and the mechanisms related with LKB1-AMPK pathway.In addition,we found that LKB1 gene can influence the effect of phenformin to cholangiocarcinoma,and briefly discusses the possible mechanisms of it.We conclude:1.Phenformin can inhibit cholangiocarcinoma RBE,HuH-28 cell proliferation,and increased apoptosis rate,and this is dose/duratin depedent.2.Phenformin can activate AMPK,making it to be phosphorylated AMPK(pAMPK),the anti-tumor effects may related with LKB1-AMPK pathway.3.With the increasing concentration of phenformin,cellular anabolism inhibited,catabolism and glycolysis increased,and the ccumulation of metabolites,make the culture medium acidized,we speculated that phenformin make tumor cells to enter state similar to sress.The over enhanced energy metabolism in turn,makes the cells more sensitive to phenformin in low-sugar and metabolites accumulated microenvironment.4.LKB1 is a tumor suppressor gene,knockdown LKB1 accelerate cell proliferation.5.Cells with lower LKB1 expression level are more sensitive to phenformin.The increased apoptosis rate while LKB1 knockdown RBE cells treated with phenformin may be due to the increased expression of Bax,cleaved-caspase3.