The Role Of Autophagy Induced By Phenethyl Isothiocyanate In Proliferation And Metastasis Of Lung Cancer Cells

Objectives:Lung cancer is the most common cancer worldwide,and is the most lethal cancer among China.Lung cancer metastasis is the main cause of treatment failure and death.However,the mechanism of metastasis is still need to be elucidated,and the development of novel anti-cancer drugs targeting metastasis is urgently needed.Recent studies indicate that the changes of autophagy status play important roles in carcinogenesis and development of tumor.Many researches show that autophagy act as a "double-edged sword" in cancer development and treatment.Isothiocyanates(ITCs)are naturally occurring small molecule that generated by the enzymic hydrolysis of glucosinolate in cruciferous vegetables.Our previous studies showed that benzyl isothiocyanate(BITC)induced protective autophagy in non-small cell lung cancer(NSCLC)cells.However,whether autophagy plays a role in the metastasis inhibition of phenethyl isothiocyanates(PEITC)is still unclear.In the present study,we investigated the effect of PEITC on autophagy status in lung cancer cells,and the role of autophagy in the inhibitory effect of PEITC on lung cancer cell metastasis potential,Which provides experimental basis and foundation for the clinical application and the development of joint program of isothiocyanates.Methods:In order to investigate the effects of PEITC on lung cancer cells,A549(adenocarcinoma),SK-MES-1(squamous cell carcinoma)and H661(large cell carcinoma)cells,which represent different pathological subtypes of lung cancer were selected.1.The cell viability was detected by the Cell Counting Kit-8 following the manufacture’s instruction.2.In order to investigated the effect of PEITC on autophagy status in lung cancer cells,the AVOs,the punctuate pattern of LC3,and the accumulation of LC3-Ⅱ and expression of Beclin-1by Western blot were all detected.3.Lung cancer cells were infected with Ad-m RFP-GFP-LC3 for 24 h,the effect of PEITC on autophagic flux was evaluated by using fluorescent LC3 puncta.4.We investigated the role of autophagy in the effect of PEITC on lung cancer cell metastasis potential by wound healing assay and the cell invasion by invasion chamber assay.5.We further knocked down Beclin-1 expression by RNAi technology or using autophagy inhibitor to investigate the role of autophagy induced by PEITC on proliferation and metastasis of lung cancer cells.6.In order to evaluate whether the role of autophagy in metastasis,we have tests by quantitative polymerase chain reaction and Western blot and so on.7.To determine the effect of PEITC on lung tumor growth and metastasis,and the role of autophagy in this matter,we performed animal experiments.Results:1.PEITC inhibited human lung cancer cells growth in a dose-depend manner.2.PEITC treatment induced autophagy in lung cancer cells.Lung cancer cells treated with PEITC results in significant increase in formation of yellow-orange AVOs,the punctuate pattern of LC3 and both LC3-II expression and Beclin-1 expression were increased compared with untreated cells.3.The autophagic flux was significantly increased after PEITC treatment.4.PEITC suppresses the metastatic and invasion potential of lung cancer cells.5.Inhibition of autophagy enhanced the inhibitory effect of PEITC on lung cancer cell proliferation,migration and invasion.6.Autophagy induced by PEITC preserves metastasis potential of lung cancer cells via JAK2/STAT3 pathway,and Inhibition of autophagy enhanced the inhibitory effect of JAK2/STAT3 pathway.7.Inhibition of autophagy enhanced the inhibitory effect of PEITC on lung cancer cell growth in vivo.PEITC inhibited the expression of metastasis-associated proteins and genes through JAK2/STAT3 pathway,and 3-MA/CQ enhanced this effect.Conclusions:1.PEITC treatment inhibited human lung cancer cells growth,and induced autophagy in NSCLC cells,which plays promote role in cell growth.2.PEITC suppresses the metastatic potential of lung cancer cells.Inhibition of autophagy enhanced the inhibitory effect of PEITC on lung cancer cell migration and invasion.3.PEITC inhibited JAK2/STAT3 pathway activate.Autophagy induced by PEITC preserves metastasis potential of lung cancer cells via JAK2/STAT3 pathway.4.PEITC effectively inhibited lung tumor growth and metastasis in vivo,and suppression of autophagy potentiated this effect.