Overexpression of hepatocyte growth factor protects chronic myeloid leukemia cells from apoptosis induced by etoposide

Abstract

Resistance to apoptosis induced by chemotherapy is still an obstacle for the treatment of chronic myeloid leukemia (CML). Numerous studies have indicated that upregulation of hepatocyte growth factor (HGF) protein expression reduced apoptosis induced by various factors. However, whether HGF has any effect on apoptosis induced by VP-16 (etoposide) in CML cells and its underlying mechanisms are unclear. HGF was overexpressed in the K562 cell line using transfection. The protein and mRNA expression levels, and the concentration of HGF were measured using western blot analysis, reverse transcription-quantitative (RT-qPCR) and ELISA respectively. Apoptosis in the K562 cell line was determined using flow cytometry and western blot analysis. Changes in cell viability were measured using a MTT assay. RT-qPCR and western blot analysis revealed that HGF was successfully upregulated at both the mRNA and protein expression levels in the K562 cell line, respectively. After VP-16 treatment, the number of apoptotic cells overexpressing HGF was lower compared with that in cells transfected with the empty vector. Mechanistic investigation revealed that overexpression of HGF led to the increase in Bcl-2 protein expression level and inhibition of caspase-3/9 activation. Furthermore, HGF overexpression resulted in activation of the PI3K/Akt signaling pathway. Therefore, the results of the present study revealed that targeting HGF could be used as a strategy to overcome VP-16 resistance in CML.

Keywords: CML; HGF; VP-16; apoptosis.

Figure 1.

Overexpression of HGF inhibits the cytotoxicity induced by VP-16 in the K562 cells. The K562 cells were transfected with pVITRO2-HGF or pVITRO2-mcs for 24 h, and (A) mRNA and (B) protein expression levels of HGF were analyzed using reverse transcription-quantitative PCR and western blot analysis, respectively. (C) The K562 cells were transfected with pVITRO2-HGF or pVITRO2-mcs for 24 h, treated with various doses of VP-16 for another 48 h, then cell viability was analyzed. The data are shown as the mean and SD from three independent experiments, performed in triplicate. *P<0.05, **P<0.01, ***P<0.001. HGF, hepatocyte growth factor; mcs, mock.

Figure 2.

Overexpression of HGF inhibits apoptosis induced by VP-16 in the K562 cells (A) The K562 cells were treated as indicated for 24 h, then apoptosis was measured using Annexin-V staining and flow cytometry. (B) The K562 cells were treated as indicated, then caspase-3/-9 activities were analyzed using a colorimetric assay kit. (C) The K562 cells were treated as indicated for 24 h, then the protein expression level of caspase-3/-9 was analyzed using western blot analysis. The data are shown as the mean and SD from three independent experiments, performed in triplicate. **P<0.01, ***P<0.001. HGF, hepatocyte growth factor; mcs, mock.

Figure 3.

Overexpression of HGF affects the expression level of Bcl-2 proteins in the K562 cells. The K562 cells were treated as indicated for 24 h, then the (A) mRNA and (B) protein expression levels of Bcl-2 and Bax were analyzed using reverse transcription-quantitative PCR. (C) The K562 cells were transfected with siRNA targeting Bcl-2 for 24 h, then treated with or without VP-16 for another 24 h. The protein expression levels of Bcl-2 and rate of apoptosis was analyzed. (D) The K562 cells were transfected as indicated for 24 h, then treated with or without VP-16 for another 24 h. The protein expression levels of Bax and the rate of apoptosis were analyzed. The data are shown as the mean and SD from three independent experiments, performed in triplicate. *P<0.05, **P<0.01. HGF, hepatocyte growth factor; mcs, mock, si, small interfering; ctrl, control.

Figure 4.

Overexpression of HGF leads to the activation of the PI3K/Akt signaling pathway in K562 cells. (A) The cells were transfected as indicated for 24 h, then treated with VP-16 for another 24 h. The expression level of proteins in the PI3K/Akt signaling pathway was analyzed using western blot analysis. (B) The cells were transfected with pVITRO2-HGF for 24 h, then treated with or without LY294002. The cells were treated with VP-16 for another 24 h, then the expression level of proteins in the PI3K/Akt signaling pathway was analyzed using western blot analysis. (C) The cells were transfected as indicated for 24 h with or without LY294002, then treated with or without VP-16 for another 24 h. Apoptosis was subsequently analyzed. The cells were transfected with pVITRO2-HGF for 24 h, treated with or without LY294002, treated with VP-16 for another 24 h, then the expression level of (D) caspase-3/-9 and (E) Bcl-2 and Bax was analyzed using western blot analysis. The data are shown as the mean and SD from three independent experiments, performed in triplicate. **P<0.01. HGF, hepatocyte growth factor; p, phosphorylated; mcs, mock.