Study On Signal Transduction Pathway Of Apoptosis Of Human Non - Small Cell Lung Cancer A549 Cells Induced By

lung cancer as the leading cause of cancer-related death in the world is becoming the highest incidence of cancer cases in China., despite intensive research in diagnosis and therapy, but the anticancer drugs have sever side effects and toxicity, besides the overall 5-year survival rate in many countries is still less than 15%. So the need for 1 researching novel antitumour drugs with low toxicity and side effects are urgent. Costunolide, a sesquiterpene lactone, was isolated from many plant species such as Saussurea lappa. Accumulated data indicated that costunolide has potent anticancer activity, but it has not been reported the anticancer effect of costunolide on A549 cells. In this study, we employed human lung cancer A549 cells to evaluate the inhibitory effect of costunolide and explored the possible molecular mechanisms. From the study, the main results are as follows:1. MTT assay showed costunolide inhibited the growth of A549、HepG2、HeLa in dose-dependent manner, and A549 cells were more sensitive to costunolide treatment which indicated that various tumor cells exhibit different sensitivities to costunolide treatment.2、To determine whether apoptosis is responsible for costunolide-induced A549 cell growth inhibition, flow cytometry assay was employed. The results showed that costunolide induce cell apoptosis in a concentration-dependent manner.3、Costunolide treatment could significantly enhance the level of intracellular ROS levels and activated ER stress which accompanied by elevated calcium levels in the cytoplasm, and up-regulation the levels of the ER stress marker GRP78 as well as UPR sensors IREla,CHOP etc. Knockdown of IREla using IREla siRNA significantly attenuated costunolide-induced apoptosis in A549 cells, further confirming the role of ER stress in costunolide-induced apoptosis. Further studies showed that IRE1a-ASK1-JNK pathway was activated to phosphorylate Bcl-2, leading to the inactivation of the anti-apoptotic actions of this protein. JC-1 staining showed the integrity of the mitochondrial membrane was destructed by costunolide treatment. NAC, a ROS scavenger, can reverse the costunolide-induced inhibition of cell proliferation, mitochondrial membrane potential and apoptosis. All this results indicated that ROS participated in costunolide-induced apoptosis as a upstream signal molecular.4、A549 cells were treated with 10μM、20μM,30μM for 24h, the caspase activity analysis showed that costunolide treatment significantly increase the activities of caspase3 and 9. Moreover, Z-VAD-FMK, a specific caspase inhibitor, can effective recovery the cell viability.. Western blot analysis showed costunolide treatment up-regulated the Bax/Bcl-2 ratio and released the cytochrome c from mitochondria to cytosol, activated caspase 3 and cleaved PARP. All this results suggested mitochondrial apoptotic pathway was involved in the process of costunolide-induced apoptosis.This study first elucidated that costunolide induced A549 cells apoptosis through the IRE1α-JNK signaling pathway medicated by ER stress. These findings further reveal the anti-tumor mechanism of costunolide and contribute to develop costunolide to be a promising anti-caner drug.