RhoA inhibits apoptosis and increases proliferation of cultured SPCA1 lung cancer cells

Abstract

The Rho kinase pathway has previously been reported to possess a close relationship with the growth, migration and invasion of lung cancer cells. However, the molecular mechanisms underlying the effects of this pathway on lung cancer cells are still elusive. The aim of the present study was to investigate the effects and underlying molecular mechanisms of Ras homolog family member A (RhoA) on the proliferation and apoptosis of SPCA1 lung carcinoma cells. Stable SPCA1 lung cancer cell lines, in which RhoA expression was silenced by small interfering RNA, were isolated following Geneticin screening. Inhibition of RhoA expression significantly decreased the proliferation of SPCA1 lung cancer cells, whereas apoptosis was significantly increased (P<0.01) as determined by the MTS tetrazolium assay and flow cytometry analysis, respectively. At the molecular level, knockdown of RhoA resulted in the significant activation of caspase‑3 (P<0.01), and a significant reduction in the levels of phosphorylated signal transducer and activator of transcription (phospho‑STAT3; P<0.01), as determined by western blotting. The results suggested that RhoA knockdown prevents cell proliferation and induces apoptosis in SPCA1 lung cancer cells. Furthermore, the underlying mechanisms responsible for these effects may include the activation of caspase‑3 and the reduction of phospho‑STAT3 levels.

Figure 1.

Generation of stable RhoA siRNA-transfected SPCA1 lung cancer cells and western blot analysis of RhoA protein expression. (A) Sequence analysis of recombinant plasmids and synthesized siRNA oligonucleotides. (B) RhoA siRNA-transfected SPCA1 cell lines under a fluorescence microscope (magnification, ×50). (C) Western blot analysis of RhoA protein expression. Lane 1, control cells; lane 2, RhoA siRNA-transfected cells. Rho, Ras homolog family member A; siRNA, small interfering RNA.

Figure 2.

Effects of RhoA siRNA on the proliferation of cultured SPCA1 lung cancer cells. The proliferation of RhoA siRNA-transfected SPCA1 lung cancer cells was measured over the course of seven consecutive days using an MTS assay. Following RhoA knockdown, a significant decrease in the proliferation of SPCA1 cells was observed when compared with the control cells. **P<0.01 vs. control. RhoA, Ras homolog family member A; siRNA, small interfering RNA.

Figure 3.

Level of apoptosis in cultured RhoA siRNA-transfected SPCA1 lung cancer cells. (A and B) RhoA siRNA-transfected cells and controls were collected and stained with annexin V (apoptosis stain) and PI (nuclear stain), and analyzed by flow cytometry. (C) The results from one of three independent assays are presented. The level of apoptosis in RhoA siRNA-transfected cells was significantly greater when compared with the control cells. **P<0.01 vs. control. RhoA, Ras homolog family member A; siRNA, small interfering RNA; UR, upper right; LR, lower right; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 4.

Activation of caspase-3 and caspase-9. (A) Western blot analysis of casp-3, casp-9 and β-actin protein expression in RhoA siRNA-transfected SPCA1 cells and untreated controls using anti-casp-3, anti-casp-9 and β-actin antibodies. β-actin was used as a loading control. (B) A significant increase in the active casp-3 form was observed in RhoA siRNA-transfected cells when compared with the control cells. By contrast, RhoA knockdown did not significantly alter casp-9 activation. **P<0.01 vs. control. RhoA, Ras homolog family member A; siRNA, small interfering RNA; casp-3, caspase-3; casp-9, caspase-9.

Figure 5.

Protein expression levels of STAT3 and p-STAT3 in RhoA siRNA-transfected SPCA1 cells. (A) STAT3 and p-STAT3 (Tyr 705) protein expression levels were measured by western blotting and subsequent densitometry analysis (mean ± standard error; n=3). (B) Quantification of STAT3 and p-STAT3 protein expression levels. Following RhoA knockdown, the level of p-STAT3 decreased by 31.5% in RhoA siRNA-transfected cells, whereas RhoA siRNA did not significantly affect STAT3 protein levels. **P<0.01 vs. control. STAT3, signal transducer and activator of transcription 3; p-STAT3, phosphorylated-STAT3; RhoA, Ras homolog family member A; siRNA, small interfering RNA.