Effects And Possible Mechanisms Of Klotho In Human Lung Adenocarcinoma A549 Cells

Background:Lung cancer is the leading cause of cancer deaths worldwide because of its highest incidence and mortality. Although the development of conventional therapy is enormous, overall survival rates of lung cancer patients are still very low, with 5-year survival rates less than 15%. Thus, it is urgent to explore a novel approach which is safe and effective. Gene therapy is one of promising modality whose target is the"abnormal gene"which is recognized as the origin of carcinogenesis, and some are being used for clinical therapy. The target abnormal gene is the foundation stone of gene therapy, which influence the efficiency and safety of therapy. Therefore, an ideal target gene is very important for gene therapy.Aging is the greatest risk factor for cancer, and there are great associations between aging and cancer. With the aging people increasing in nowadays and future, cancer is a huge challenge we have to face. In addition to its role in aging, klotho can also involve in multiple cell signal pathways with complex roles. It involves in aging, carcinogenesis and regulating immunity, which may be a key role between aging and cancer.A recently published research suggests that klotho serves as a potential tumor suppressor and identify it as an inhibitor of the insulin-like growth factor-1 (IGF-1) pathway and activator of the fibroblast growth factor (FGF) pathway in human breast cancer. There are many common mechanisms of carcinogenesis between lung cancer and breast cancer, of which IGF-1 signaling is just the very one we found.Objective:(1) To investigate the effects and possible mechanisms of action of klotho in A549 cells;(2) To explore an ideal target gene for lung cancer gene therapy.Methods:We compared A549 cells with HEK-293 cells using as a control group to evaluate expression levels of klotho through quantitative reverse transcription-polymerase chain reaction (qRT-PCR); The target cells were respectively treated with pCMV6-MYC-KL or klotho specific shRNAs. The MTT assay was used to evaluate the cytotoxic effects of klotho and flow cytometry was utilized to observe and detect the apoptosis of A549 cells induced by klotho. The activation of IGF-1/insulin signal pathways in A549 cells treated by pCMV6-MYC-KL or shRNAs were evaluated by western blotting. The expression levels of bcl-2 and bax transcripts were evaluated by qRT-PCR.Results:Expression levels of klotho in A549 cells were much lower than that in HEK-293 cells; Overexpression of klotho reduced the proliferation of lung cancer A549 cells (P<0.01), whereas klotho silencing in A549 cells enhanced proliferation (P<0.05). Klotho did not show any effects on HEK-293 cells (P>0.05); Klotho overexpression in A549 cells was associated with reduced IGF-1/insulin-induced phosphorylation of IGF-1R (IGF-1 receptor)/IR (insulin receptor) (P<0.01); Overexpression of klotho can promote the apoptosis of A549 cells (P<0.01). Overexpression of klotho, a bcl family gene bax, was found up-regulated and bcl-2, an anti-apoptosis gene, was found down-regulated (P<0.01). In contrast, bax and bcl-2 were found down-regulated (P<0.05) and up-regulated (P<0.01), respectively when silencing klotho using shRNAs.Conclusions:A549 cells do have low levels of klotho expression; Klotho can inhibit proliferation and increase apoptosis of A549 cells, this may be partly due to the inhibition of IGF-1/insulin pathways and involving regulating the expression of the apoptosis-related genes bax/bcl-2. Thus, klotho can serve as a potential tumor suppressor in lung adenocarcinoma A549 cell line.