Research Of Synergistic Attenuated Effects Of Metformin On Arsenic Trioxide Induced Anti-Intrahepatic Cholangiocarcinoma Activity

Background:Arsenic trioxide is commonly used in the treatment of acute promyelocytic leukemia(APL),but does not benefit patients with solid tumors.High dose and drug resistance might mediate the failure of single ATO treatment in solid tumor.We first reported metformin amplified the inhibitory effect of arsenic trioxide on intrahepatic cholangiocarcinoma(ICC)cells more significantly than other agents,such as gemcitabine,5-FU and sorafenib.Intrahepatic cholangiocarcinoma(ICC)is the second most common type of primary liver cancer.The prognosis of ICC patients is very poor and the sensitivity of ICC to chemotherapy is also very low.To explore more effective agents with low toxicity for ICC patients is a difficult problem for clinicians.Moreover,arsenic trioxide often causes severe health hazards such as hepatotoxicity,dermatosis,neurotoxicity,nephrotoxicity and cardiotoxicity,which often limited the clinical use of arsenic trioxide.Liver is one of the major target organs of arsenic trioxide.The production of reactive oxygen species,(ROS)play a significant role in ATO-induced hepatotoxicity.In elegans,metformin-induced ROS could promote the expression of peroxiredoxin PRDX-2 and conse quently suppressed oxidative stress and increase the lifetime of elegans.Aims:1.Explore the suppression effect of metformin and arsenic trioxide combination on ICC cells.2.Explore the molecular mechanisms of the suppression effect of metformin and arsenic trioxide combination on ICC cells.3.Explore the protective effect of metformin on arsenic triode induced liver cell injury.Methods:1.The anti-proliferation effect was evaluated by cell viability,cell apoptosis,cell cycle,and intracellular-reactive oxygen species(ROS)assays.The in vivo efficacy was determined in nude mice with CCLP-1 xenografts.The Combination indices(CI)were calculated.2.The active status of AMPK,p38 MAPK,mTOR and ERK3 pathways was detected by western blot.Methods of genetic modulation and pharmacology were further used to demonstrate the relationship of the molecule.Seventy-three tumor samples from ICC patients were used to detect the expression of ERK3 by immunohistochemistry.The correlation between ERK3 and the clinical information of ICC patients were further analyzed.3.We used a normal mouse liver cell line,Alpha mouse liver(AML12)to evaluate the hepatotoxicity in vitro.The assay was based on a Kunming mouse model treated by arsenic trioxide and/or metformin.In AML12 cells,cell apoptosis,cell death and ROS were detected.In the mouse model,Histopathological variation,alanine aminotransferase,aspartate aminotransferase and cell apoptosis were detected.Gene expression screening revealed the gene express variation in the AML12 cellstreated by arsenic trioxide and/or metformin.By using a Seahorse system,we further explored the effects of metformin and/or trioxide,rotenone on the glucose metabolism of AML 12 cells.Results:1.Metformin and arsenic trioxide synergistically inhibited proliferation of ICC cells.Combined treatment with metformin and trioxide efficiently reduced ICC growth in an ICC xenograft model.2.Results of western blot confirm that metformin and trioxide cooperated to inhibited mTORC1.Metformin abrogated the activation of p38 MAPK induced by trioxide.Inactivation of p38 MAPK by SB203580 or specific short interfering RNA(siRNA)promoted the inactivation of mTORC1 in ICC cellstreated with metformin and trioxide.AMPK is a newfound positive regulator of ERK3 which is also a suppressor of mTORC1.Expression of ERK3(low)the independent risk factors for shorter overall survival time in ICC patients.3.Metformin protected AML12 cells from arsenic trioxide-induced apoptotic cell death.Metformin alleviated arsenic trioxide-induced liver injury.Glucose metabolism might be behind the metformin-induced protective effect on arsenic trioxide-treated AML 12 cells.Treatment with low glucose or rotenone,a mitochondrial respiratory chain complex I inhibitor,protected AML12 cells from ATO-induced apoptotic cell death.Metformin-induced inhibition of mitochondrial respiratory chain complex I mediated a protective effect on arsenic trioxide-induced apoptotic cell death in AML12 cells.Metformin increased the intracellular NADH/NAD+ ratio of AML12 cells.Conclusions:1.Combination of metformin and arsenic trioxide synergistically anti-proliferation of ICC cells,which is bridged by cell cycle arrest,cell apoptosis and ROS induction.2.Metformin and arsenic trioxidetargeted AMPK/p38 MAPK-ERK3/mTORCl pathways in ICC in vitro and in vivo.Expression of ERK3(low)the independent risk factors for shorter overall survival time in ICC patients.Detection of the p38 MAPK activation status and the expression of ERK3 are valuable for evaluating the sensitivity of ICC to metformin and arsenic trioxide.3.Metformin performed a protective effect for arsenic trioxide-induced liver injury by targeting mitochondrial complex I and increasing the NADH/NAD+ ratio,further reducing the ROS level,inhibit the apoptotic cell death.