Inonotus Obliquus Polysaccharides Induces Apoptosis Of Lung Cancer Cells And Alters Energy Metabolism Via The LKB1/AMPK Axis

Background:The incidence and mortality of lung cancer rank the first in global malignancies.Despite recent advances in the early diagnosis and combined therapies,however,the the treatment and prognosis of lung cancer patients remains unsatisfactory Chemotherapy is one of the most important treatments for patients with lung cancer,but it can cause serious side effects and drug resistance,which ultimately leads to treatment failure.Therefore,there is an urgent need to identify additional and effective methods as supplementary therapies for lung cancer.Natural products have received extensive attention in anti-cancer research because of their safety,effectiveness and low cost.Inonotus obliquus polysaccharides(IOP)is a mixture of polysaccharides extracted from the fruit body of Inonotus obliquus,which is one of the major components.IOP exhibits multiple biological activities including anti-tumor,antioxidant,antidiabetic and modulating immunity with low or no toxicity.Regardless that IOP is beneficial to human health and can be used as an alternative or supplemental medicine in the treatment of human diseases,the underlying mechanisms are poorly understood.Objective:To investigate the anti-lung cancer effect and its underlying molecular mechanism of IOP in vitro and in vivo,and provide experimental evidence for further clinical application of IOP.Method:(1)Murine Lewis lung cancer cell line LLC1 was treated with different concentrations of IOP(0,0.1,0.2,0.5,or 1.0 mg/ml)for 0-72 h,and the effect of IOP on cell proliferation was examined by CCK8 method and IC50 value was calculate;(2)LLC1 cells were treated with different concentrations of IOP(0,0.2,0.5,or1.0 mg/ml),and the effect of IOP on cell colony formation ability was examined by colony formation assay;(3)LLC1 cells were treated with IOP at different concentrations(0,0.2,0.5 or1.0 mg/ml)for 24 h.The effects of IOP on apoptosis and mitochondrial membrane potential were assessed by flow cytometry;(4)LLC1 cells,human lung cancer cells A549 and A549-LKB1 were treated with different concentrations of IOP(0,0.2,0.5,or 1.0 mg/ml),and the expression of proteins:Bax,Bcl-2,cleaved Caspase-3,Caspase-3,cleaved PARP,PARP,and p-AMPK-?(Thr172),AMPK-?,p-ACC(Ser79),and ACC were assessed by Western blot;(5)After treatment of LLC1 cells with the AMPK chemical inhibitor Compound C and IOP for 24 h,CCK8 assay was performed to explored cell proliferation,and the expression of Bax,Bcl-2,cleaved Caspase-3,Caspase-3,p-AMPK-?(Thr172),AMPK-?,p-ACC(Ser79),ACC were assessed by Western blot;(6)After treatment of LLC1 and A549 cells with IOP for 24 h,the effect of IOP on intracellular ATP content was detected by ATP detection kit;(7)After treatment of LLC1,A549 and A549-LKB1 cells with IOP for 24 h,the effects of IOP on glucose oxidation and glycolysis were analyzed by Seahorse Bioscience XF Analyzer using Seahorse XF Cell Mito Stress Test Kit;(8)The LLC1 cells were injected subcutaneously into C57BL/6J mice to establish a model of LLC1 tumor-bearing mice.Twenty-four hours after tumor implantation,experimental group were treated by intraperitoneal injection with 50mg/kg IOP,while the control group with the same volume of normal saline.Tumor was measured with a caliper every other days,tumor volume calculated using the formula V=1/2(width~2�length),and body weight recorded.At the end of 13 days,all mice were sacrificed,and the tumor tissues were removed and weighed.The apoptosis induction in the tumor tissues was examined by using TUNEL immunohistochemistry.The expression of apoptosis-related proteins and AMPK activation were examined by Western Blot.Results:(1)IOP significantly suppresseds the proliferation of LLC1 cells in a concentration-and time-dependent manner;(2)IOP suppresseds the colony-formation of LLC1 cells in a concentration-dependent manner;(3)IOP activated AMPK in a concentration-dependent manner and triggered apoptosis in LLC1,A549-LKB1 cells,IOP downregulated Bcl-2 protein,upregulates Bax,and enhances cleavage of Caspase-3 and PARP,but not in A549 cells;(4)Compound C,a chemical inhibitor of AMPK,prevents the effects of IOP on cell survival and apoptosis;(5)IOP diminished mitochondrial membrane potential in a dose-dependent manner,concurrent with decreases in oxidative phosphorylation and glycolysis,and inhibition of ATP production,which is dependent on LKB1/AMPK.(6)In vivo studies showed that injection of IOP i.p.at a dosage of 50 mg/kg every two days significantly inhibited LLC1 allograft tumor growth in C57BL/6 mice in association with activation of AMPK,increases of apoptosis,downregulation of Bcl-2 protein,upregulation of Bax,and elevation of cleavage of Caspase-3 and PARP.Conclusion:In vivo and in vitro experiments show that IOP triggers the apoptotic pathway of lung cancer cells through AMPK and decreases the mitochondrial membrane potential,resulting in inhibition of ATP production.Therefore,the study will provide a solid experimental basis for the use of IOP as a replacement or supplement in the treatment of lung cancer.