Inhibition of lung cancer cells growth, motility and induction of apoptosis by Klotho, a novel secreted Wnt antagonist, in a dose-dependent manner

Abstract

Klotho (KL) is a transmembrane protein that can be shed, and act as a circulating hormone and modulate several signaling pathways. There also exists a splice variant of Klotho mRNA, which encodes a putative secreted protein (Klotho-S, KL-S) in both human and mouse. The potential anti-senescence gene Klotho has been recently found to participate in the progression of several different human cancers. In the current study, we undertook to study the expression and activity of Klotho in lung cancer cell line A549. Klotho expression was studied by using RT-PCR and western blotting. Effects of Klotho on cell growth and motility were assessed using MTT and scratch motility assay, and the apoptosis was assessed by TUNEL. Wnt signaling pathway activity was measured by western blotting. We established that the Klotho was endogenous expressed in A549 cells, but the expression level is lower compared with normal lung tissues. The overexpression of KL or KL-S could inhibit the cell proliferation, motility, and induce apoptosis in a dose-dependent manner. Also, we report KL could inhibit activation of Wnt -TCF/β-catenin signaling pathway, and it is involved in KL-induced growth inhibition. These studies indicate Klotho works as a potential tumor suppressor in lung cancer, and suggest that the Klotho tumor suppressive activities could be mediated through its KL-S isoform. These results suggest the use of Klotho or KL-S as potential strategy for the development of novel therapeutic interventions for lung cancers.

Figure 1. Endogenous expression of Klotho in A549 cells. (A) The KL transcripts detected by RT-PCR. (B) Western blot analysis of Klotho protein, HEK293 lysate transfected with Klotho as positive control. (C) Klotho mRNA levels were determined by quantitative RT-PCR in lung cancer clinical samples and adjacent normal tissue.

Figure 2. Klotho regulates the cell proliferation of A549. (A) A549 cells were transfected with Klotho or Klotho-S, and western blot was performed to verify similar expression levels of proteins in cell lysates. (B) Klotho in cell culture supernatants were immunodetected by Klotho antibodies. (C) Extracts from A549 cells transfected with si-Klotho were subjected to western blot and probed with antibodies to Klotho or β-tubulin (loading control). (D) MTT assay for A549 cells transfected with indicated vectors. Data are shown as the mean ± SEM from three independent experiments. *p < 0.05, KL group vs. control group; # < 0.05, KL-S vs. control group.

Figure 3. Induction of A549 cells apoptosis by Klotho expression. (A) Representative images of TUNEL assay. A549 cells were transfected with indicated constructs. (B) Data of TUNEL positive cells were summarized. Data are shown as the mean ± SEM from three independent experiments. *p < 0.05, one-way ANOVA.

Figure 4. Klotho regulates the cell motility of A549. (A) Scratch wound closure assay for motility of A549 cells transfected with indicated constructs. The photographs of wound closure from each group were taken and assessed every 24 h for 2 d. (B) Quantitative assays were performed by calculating cell numbers. The ratio of migrating cells was calculated into line chart. Data are shown as the mean ± SEM from three independent experiments. *p < 0.05, KL group vs. control group; # < 0.05, KL-S vs. control group. (C) Klotho suppresses A549 invasion activity. A549 cells (2 × 10⁴ cells) were seeded to the top chamber of a Matrigel invasion chamber in the absence of serum, and then subjected to invasion assay. Deteminations were made in triplicate. Data are shown as the mean ± SEM from three independent experiments. *p < 0.05, KL group vs. control group; ** < 0.01, KL-S vs. control group.

Figure 5. Inhibition of the Wnt/β-catenin pathway by Klotho-S in A549 and H1975 cells. (A) Western blot analysis of total β-catenin, and active β-catenin (A, B and C) in A549 cells transfected with either the Klotho/Klotho-S expression vector or the empty vector. (B) RT-PCR analysis of TCF/β-catenin target genes: c-MYC and cyclinD1 in A549 cells transfected with indicated constructs. (C) Western blot analysis of CyclinD1 in A549 cells transfected with either Klotho-S or co-transfected with Klotho-S andβ-cateninΔN. (D) MTT assay for A549 cells transfected with either Klotho-S or co-transfected with Klotho-S andβ-cateninΔN. (E), Western blot analysis of Klotho protein, HEK293 lysate transfected with Klotho as positive control. (F) Western blot analysis of total β-catenin, and active β-catenin (A, B and C) in H1975 cells transfected with either the Klotho-S expression vector or the empty vector. (G) RT-PCR analysis of c-MYC and cyclinD1 in H1975 cells transfected with indicated constructs. (H) MTT assay for H1975 cells transfected with either Klotho-S or co-transfected with Klotho-S and β-cateninΔN. Data are shown as the mean ± SEM from three independent experiments. *p < 0.05, one-way ANOVA.

Figure 6. Klotho regulates Wnt3a expression in A549 and H1975 cell lines. (A) RT-PCR analysis of human Wnt3a gene in A549 and H1975 cells transfected with indicated constructs. (B) Western blot analysis of Wnt3a protein in A549 andH1975 cells transfected with either the Klotho-S expression vector or the empty vector.