Klotho inhibits growth and promotes apoptosis in human lung cancer cell line A549.
BACKGROUND
Klotho, as a new anti-aging gene, can shed into circulation and act as a multi-functional humoral factor that influences multiple biological processes. Recently, published studies suggest that klotho can also serve as a potential tumor suppressor. The aim of this study is to investigate the effects and possible mechanisms of action of klotho in human lung cancer cell line A549.


METHODS
In this study, plasmids encoding klotho or klotho specific shRNAs were constructed to overexpress or knockdown klotho in vitro. A549 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 quantitative reverse transcription-polymerase chain reaction (qRT-PCR).


RESULTS
Overexpression of klotho reduced the proliferation of lung cancer A549 cells, whereas klotho silencing in A549 cells enhanced proliferation. Klotho did not show any effects on HEK-293 cells. 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
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 A549 cells.
Chen
Bo
B
Department of Geriatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu, China.
Wang
Xueli
X
Zhao
Weihong
W
Wu
Jianqing
J
D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D005434:Flow Cytometry; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D000090265:Klotho Proteins; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007328:Insulin; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D005966:Glucuronidase; D000090265:Klotho Proteins