Studies On Molecular Mechanism Of Cell Death Induced By Cryptococcus Heimaeyensis S20 Exopolysaccharide CHEPS In Non-small Cell Lung Cancer

The morbidity and mortality of lung cancer rank the first of cancer in the world.Non-small cell lung cancer accounts 80% of lung cancer,with very low survival rate.Chemotherapy,targeted therapy and immunotherapy are the main therapies for advanced lung cancer.However,that limited efficacy and significant toxicity of these methods decline the quality of patients' lives.Therefore,it is of great significance to develop innovative antitumor drugs with high efficiency and low toxicity.In this study,exopolysaccharide of Cryptococcus heimaeyensis S20 isolated from Antarctic was extracted,and its antitumor activity and mechanism in NSCLC were studied.Cryptococcus heimaeyensis is widely distributed in cold and dry regions and protects itself mainly by producing exopolysaccharides.Cryptococcus heimaeyensis S20 was isolated from Antarctica.So far,the structure of its exopolysaccharide(CHEPS)and the relevant bioactivity have not been researched systematically.In this study,we found that CHEPS effectively inhibited the viability of NSCLC cells,and had a weaker inhibitory effect on normal human embryonic lung fibroblasts WI-38 and MRC-5.Further studies showed that CHEPS effectively induced S-and G2/M-phase arrest in NSCLC cells by up-regulating the expression of cycle regulatory proteins p53 and p21 and down-regulating the expression of Cyclin B1,CDK1,Cyclin A2 and CDK2.CHEPS did not induce S-and G2/M-phase arrest in WI-38 cells to the same extent as in NSCLC cells.Furthermore,CHEPS did not induce cell death by apoptosis pathway,manifested by that it could not induce phosphatidylserine valgus and up-regulation of Bax and cleave of Caspase 3 and PARP.Through the EGFP-LC3 fluorescence puncta formation assay,LC3 conversion and turnover experiment,electron microscopy assay and the detection of m RNA and protein levels of autophagy related genes,we proved that CHEPS induced autophagy flux in NSCLC and WI-38 cells.Early autophagy inhibitor 3-MA treatment and protein expression of autophagy gene ATG5 interfered by sh RNA both partly rescued CHEPS-induced cell death in NSCLC cells.However,3-MA could not restore CHEPS-induced cell death in WI-38 cells.These results suggest that CHEPS induced autophagic cell death in NSCLC cells.Although CHEPS induced autophagy in WI-38 cells,autophagy did not cause WI-38 cell death.PI3K/AKT,AMPK,MAPK are three important signals for regulating autophagy.Our study showed that CHEPS did not inhibit the phosphorylation and activation of AKT,indicating that CHEPS did not induce autophagy through PI3K/AKT signaling.CHEPS activated AMPK signaling,but activated AMPK failed to stimulate autophagy and death in NSCLC cells.CHEPS induced phosphorylation and activation of p38,ERK,and JNK in the MAPK pathway,of which p38 and ERK effectively promoted CHEPS-induced NSCLC cell death;and inhibitions of p38 and ERK partly recused CHEPS-induced NSCLC cell death.CHEPS did not effectively activate MAPK signaling in WI-38 cells.Further experiments showed that phosphorylation and activation of ERK promoted S-and G2/M-phase arrest induced by CHEPS.Both p38 and ERK promoted CHEPS-induced NSCLC cell autophagy,and the expression of LC3-II induced by CHEPS was partly down-regulated by their inhibitors and RNA interference experiments.The accumulation of reactive oxygen species(ROS)is an important molecular mechanism by which some chemotherapeutic drugs induce tumor cell death.CHEPS treatment induced ROS production.ROS scavenger NAC partly inhibited the phosphorylation levels of p38 and ERK,cell autophagy and cell death induced by CHEPS in NSCLC cells.These results suggested that ROS accumulation promoted p38-and ERK-regulated autophagic cell death.ROS had no effect on Sand G2/M-arrest induced by CHEPS in NSCLC cells.CHEPS did not induce ROS accumulation in WI-38 cells.Besides in vitro cell experiments,we also established an A549 lung cancer cell xenograft tumor model of nude mouse to study the effect of CHEPS on lung cancer cells in vivo.100mg/kg CHEPS effectively inhibited the growth of A549 xenograft tumor without causing body weight loss and organ toxicity of liver,spleen,lung and kidney in nude mice.Consistent with the results in vitro,CHEPS also induced ROS production,activated p38 and ERK pathways,and stimulated autophagy in vivo.In summary,our research demonstrates,for the first time in vivo and in vitro,the antitumor activity of Cryptococcus heimaeyensis S20 exopolysaccharide CHEPS in non-small cell lung cancer and explore the potential mechanisms.CHEPS effectively induces S and G2/M arrest and autophagic cell death in NSCLC cells through the activation of ROS/p38/ERK signaling.CHEPS has little or no toxicity to normal embryonic lung cells in vitro or vital organs in vivo,indicating the safety and efficacy of CHEPS as a natural active polysaccharide against cancer in vivo.CHEPS may become a new anticancer drug candidate with great potential and prospect in the therapy of non-small cell lung cancer.