| Lung cancers are the leading cause of cancer incidence and mortality and are mainly classified as non-small cell lung cancer(NSCLC)which accounts for about 80% of all lung cancers.Now,the situation of resistance and relapse is extremely common.Thus,there is an urgent need to develop novel potential therapeutic agents for lung cancer treatment.Guaiazulene(1,4-dimethyl-7-isopropylazulene,GYZ),a dark blue crystalline hydrocarbon,is mainly extracted from the natural products,such as some corals and the oil of Guajacum ojficinale tree and chamomile.Previous reports have suggested that GYZ exhibited considerable anticancer properties.However,to date limited data exist indicating the efficacy of GYZ in the treatment of NSCLC and the potential mechanisms remain to be further clarified.Autophagy is a dynamic process during which damaged organelles and macromolecules are fused with lysosomes for degradation.Previous studies have indicated that there are four main functional forms of autophagy,including cytoprotective,nonprotective,cytostatic and cytotoxic autophagy in tumor cells in the context of chemotherapy or radiation.Figuring out the biologic function of autophagy in response to NSCLC anticancer therapy is scientifically worthwhile for providing potential novel therapeutic strategies.In the present study,we aimed to detect the antitumor effect of GYZ on NSCLC and investigate the underlying mechanism by using human NSCLC cell lines(A549,H1975,HCC827,PC9 and H1299)and A549 cell line-derived xenografts model.First of all,we found that GYZ could inhibit cell growth of NSCLC cells in vitro and the anti-proliferative effect of GYZ was independent of apoptosis.In addition,our data in A549 xenografts model further confirmed the antitumor effect of GYZ in vivo.Next,we found that GYZ treatment dramatically increased cellular ROS levels and ATP level was decreased within GYZ treatment.These results demonstrated that the inhibition of NSCLC cell growth might relate to the mitochondrial dysfunction and ROS accumulation induced by GYZ.As accumulating studies have emphasized the potential application of drug-induced autophagy in antitumor therapies,we investigated whether GYZ regulated autophagy in NSCLC cells.We conducted immunoblotting assay,immunofluorescence assay,immunohistochemistry assay and cell transfection assay,and found that GYZ induced the initiation process of autophagy in NSCLC cells both in vitro and in vivo and stimulated complete autophagic flux in NSCLC cells.To identify whether autophagy was involved in the anti-NSCLC effect of GYZ,combinational use of 3-MA or CQ with GYZ were treated in NSCLC cells.We found an obvious decrease in cell growth within co-treatment.In conclusion,our findings suggested that cytoprotective autophagy was induced in response to GYZ treatment and inhibition of autophagy augmented the antitumor activity of GYZ in NSCLC cells.In the end,to determine the mechanism of GYZ-induced autophagy in NSCLC cells,we conducted various molecular and cell biology techniques,and found that GYZ-induced autophagy was dependent on the suppression of Akt/mTOR pathway both in vitro and in vivo.In conclusion,our study revealed that GYZ inhibited the growth of NSCLC both in vitro and in vivo.GYZ induced mitochondrial dysfunction and increased the level of ROS which may be responsible for GYZ-induced growth inhibition.Furthermore,GYZ stimulated cytoprotective autophagy in NSCLC cells through suppression of the Akt/mTOR signaling pathway and co-treatment with autophagy inhibitors obviously enhanced the antitumor effects of GYZ.These findings provide profound insights into the antitumor efficacy of GYZ,which may provide a novel strategy for the treatment of NSCLC. |
