Combined Effects Of Klotho And Soluble CD40 Ligand On A549 Lung Cancer Cells

Background:Lung cancer is one of common malignant tumors. Its morbidity is first in all kinds of malignant tumors, with 5-year survival rate approximately 10% for non-small cell lung cancer (NSCLC). Although the traditional treatment has made great progress, the overall survival rate of patients with lung cancer has not improved (1). It is urgent to find a new treatment. Now more and more evidences prove that combined gene therapy on tumers has become a hotspot (2, 3). Klotho gene was identified as an"anti-aging"gene in mice. Recently, klotho was identified as a potential tumer suppressor. sCD40L, belonging to TNF superfamily, has been first identified on CD4~+ T cells. Interaction of CD40L-CD40 is crucial for adaptive immune responses. In addition to its expression in normal lymphoid cells, CD40 is also found in a variety of malignant cells. sCD40L-CD40 can inhibit tumor cell growth and increase apoptosis, with the direct mechanism or indirect mechanism. Since both klotho and sCD40L gene can inhibit the development of lung cancer, and both of them can regulate the cellular immunity and humoral immunity, we hypothesized that the klotho gene combined with sCD40L can suppress tumor growth better than Klotho gene or sCD40L alone.Objective:This study was designed to investigate biological effects of Klotho combined sCD40l on lung cancer cell line A549 (CD40 positive ) and its possible mechanism .Methods: Quantitative real-time fluoresence polymerase chain reaction (RT-PCR) was used to determine the effect of overexpression of klotho in A549 and 293T cells after transfected with klotho; MTT assay and flow cytometry were employed to determine the changes of cell proliferation inhibition,cell cycle and apoptosis of A549 cells; Expression change of Bcl-2 and Bax were observed by quantitative real-time fluoresence polymerase chain reaction (RT-PCR) .Results:(1) Compared with the group transfected with the klotho gene or treated with sCD40l only ,the cell proliferation inhibition, the apoptosis rate and cell cycle inhibition increased in A549 cells treated with Klotho gene combined sCD40l(p﹤0.05). (2) Compared with the group transfected with the klotho gene or treated with sCD40l only , Bcl-2 gene expression decreased in A549 cells treated with Klotho combined sCD40l(p﹤0.05); Bax gene expression upregulated after transfecting klotho gene ,but no significant change were shown in the group treated with sCD40l(p≥0.05).Conclusions:In this study, we found that klotho, soluble CD40 ligand and their combination can increase cell proliferation inhibition and the apoptosis rate in A549 cell line by inhibiting cell cycle, upregulating Bax gene expression and (or) downregulating Bcl-2 gene expression; and their combination has a stronger effect on A549 cells apoptosis and proliferation inhibition compared with klotho or sCD40L alone.