| Lung cancer is one of the most common pulmonary malignancies menacing human’s health and lives severely, the non-small cell lung cancer accounts for about 80% of lung cancer. In the last five decades,many countries have reported that the morbidity and mortality of lung cancer have significantly increased. The primary cause of lung cancer is not entirely clear, but more and more literatures indicated smoking, ionizing radiation, atmospheric contamination, a history of chronic lung infection. Cisplatin(cis-diamminedichloroplatinum II, CDDP) is the most commonly used drug for the treatment of lung cancer. The mode of action for cisplatin is generally through its ability to crosslink with pyrimidine and purine bases on the DNA in cancer cells. Cisplatin induces cancer cell apoptosis by intervening in DNA damage repair processes and restraining DNA replication. However, cisplatin resistance and side effects are limiting the efficacy of lung cancer treatment during the prolonged, intense course of chemotherapy. Cisplatin has been widely applied in the current treatment of cancer and obtained very successful results. But patients tend to become tolerant to it during use, meanwhile, cisplatin-induced ototoxicity, neurotoxicity, renal toxicity restricts its clinical application. So it’s especially important for the analysis of the mechanisms of apoptosis induced by cisplatin. A growing body of literature shows that the process of cisplatin-induced lung cancer apoptosis has been closely associated with ER stress and autophagy.Endoplasmic reticulum(ER) is a membrane-bound organelle in eukaryotic cells and plays a role in proper folding and post-translational modifications of membrane proteins and secretion proteins. Moreover, ER also plays a key role in lipid biosynthesis, energy metabolism, intracellular Ca2+ homeostasis and redox balance. The function of protein folding in ER is sensitive to intracellular and extracellular stimulus, including ischemia reperfusion, glycosylation and altered calcium homeostasis. The accumulation of unfolded or misfolded proteins leads to ER stress and further activates unfolded protein response(UPR). UPR can strengthen ER capacity for protein folding and post-translational modification, weaken m RNA translation and degrade unfolded or misfolded proteins to maintain ER homeostasis by ER-associated degradation(ERAD) and autophagy. In the early stages of ER stress, the protein synthesis in the ER is reduced and protein translation and proper protein folding related gene can be activated, both of which play an important role in the maintenance of cell functions and survival of cells. But, when severe ER stress conditions persist for a long time or the function of UPR is destroyed, there is the accumulation of vast unfolded or misfolded proteins. And then, the pro-apoptotic signaling pathway can be activated. Until now, new research suggests that ER dysfunction in myocardial cell and pancreatic cell are considered to be a primary pathogenesis of heart and brain tissue ischemicinfarcts. Hence, ER stress not only provides the means of cellular survival, but also may be an important mechanism of inducing apoptosis.In the majority of eukaryotic cells, autophagy generally exists in all types of eukaryotic cells, also is an evolutionarily conserved process which can degrade cytoplasmic material and organelles via lysosome dependent pathway. Autophagy can degrade and recycle metabolites or organelles, and plays a vital role in maintenance of cellular homeostasis. Autophagy has duality influence on tumour cells, that is to say, on the one hand, it can maintain the homeostasis of intracellular environment and promote cancer cell survival by clearing damaged or defective proteins and organelles; on the other hand, overactive autophagy can activate non-apoptotic programmed cell death by inducing the damage of cytoplasmic organelle and the unbalance of normal intracellular homeostasis. An increasing number of studies have found that the actual role of autophagy in cancer depends on different phases of tumor growth and specific tumor types. Hence, it is of practical significance to the role of autophagy in cisplatin-induced lung cancer A549 and H460 cells apoptosis.This paper regards lung cancer A549 and H460 cells as research object, explored the role of ER stress-autophagic response in cisplatin-induced cells apoptosis, and helped to provide new idea and means for clinic treating lung cancer. Methods:(1) Cells were treated with various concentrations of cisplatin(5 μM, 10 μM, 20 μM, 40 μM and 80 μM) for 24 h. MTT assay was used to test the effect of cisplatin on viability of lung cancer A549 and H460 cells, LDH leakage(LDH%) was used to determine cell membrane damage of lung cancer A549 and H460 cells.(2) Cells were treated with various concentrations of cisplatin(0 μM, 20 μM and 40 μM) for 24 h. The flow cytometry and fluorescence spectrometer were used to the percentage of apoptosis cells and mitochondria membrane potential, respectively. Western blotting was used to detect the expression levels of cleaved caspase3, cleaved PARP, cytoplasmic Cyt c, Grp78, PERK and IRE1.(3) Cells were treated with cisplatin combined with ER stress inhibitors 4-PBA or TUDC. MTT assay was used to test the effect of cisplatin combined with ER stress inhibitors on viability of lung cancer A549 and H460 cells. Western blotting was used to detect the expression levels of cleaved caspase3 and Cyt c.(4) Western blotting was used to study the apoptotic and autophagic mechanisms involved in death of A549 and H460 cells by detecting LC3 and Beclin 1.(5) Cells were treated with cisplatin combined with autophagy inhibitors 3-MA or CQ. MTT assay was used to test the effect of cisplatin combined with autophagy inhibitors on viability of lung cancer A549 and H460 cells. Western blotting was used to detect the expression levels of cleaved caspase3 and Cyt c. Results:(1) Cisplatin inhibited A549 and H460 cells in dose-dependent manner, Similarly, cisplatin also increased LDH leakage in dose-dependent manner.(2) Cisplatin induced A549 and H460 cells apoptosis and decreases mitochondrial membrane potential, cisplatin enhanced the expression of cleaved caspase3, cleaved PARP, cytoplasmic Cyt c, Grp78, PERK and IRE1 in A549 and H460 cells.(3) Treatment combined with 4-PBA or TUDC increased the cytotoxic effects of cisplatin and enhanced apoptosis induced by cisplatin. Meanwhile, 4-PBA or TUDC increased the level of cleaved caspase-3 and cleaved Cyt c induced by cisplatin.(4) Cisplatin increased the ratio of autophagy associated protein LC3II/LC3 I and Beclin1 expression.(5) Treatment combined with 3-MA or CQ increased the cytotoxic effects of cisplatin and enhanced apoptosis induced by cisplatin. Meanwhile, 3-MA or CQ increased the level of cleaved caspase-3 and cleaved Cyt c induced by cisplatin. Conclusion:Our experiment data indicated that cisplatin induced A549 and H460 cells apoptosis. On one side, one the other side,cisplatin initiated mitochondria-mediated apoptotic pathway; one the other side, cisplatin activated ERS-associated apoptosis. Inhibition of ER stress dramatically enhanced apoptosis induced by The process of cisplatin induced A549 and H460 cells apoptosis was accompanied with autophagy activation. During the process of cisplatin induced A549 and H460 cells apoptosis, autophagy plays an important role in promoting cell survival. Inhibition of autophagy could obviously enhance the sensitivity of cisplatin in A549 and H460 cells. This research explored the role of ER stress-autophagic response in cisplatin-induced cells apoptosis, and further revealed the mechanism of anti tumor efficacy of cisplatin, as well as provided theoretical basis for lung cancer treatment. |
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