Sonic hedgehog pathway mediates genistein inhibition of renal cancer stem cells

Abstract

Cancer stem cells (CSCs) have been implicated in the genesis, progression and recurrence of renal cancer. The sonic hedgehog (Shh) pathway serves a critical role in maintaining the stemness of CSCs. Genistein, a major isoflavone component extracted from soybeans and soy products, has been demonstrated to possess anticancer activity. However, the effects of genistein on renal CSCs and its underlying mechanisms remain to be fully elucidated. The aim of the present study was to investigate the role of the Shh pathway in genistein inhibition of renal CSCs. The results of the present study demonstrated that expression levels of renal CSC markers were markedly upregulated in the sphere-forming cells, which were isolated and enriched from 786-O and ACHN cells in a tumor sphere formation assay, and more cells were arrested at the G₀/G₁ phase instead of the S₁ phase compared with the adherent cells. Furthermore, the present study demonstrated that genistein could effectively diminish the activity of renal CSCs by suppressing tumor sphere formation, decreasing renal CSCs markers, inhibiting proliferation and inducing apoptosis. Additionally, the downregulation of Shh pathway activity could inhibit renal CSCs. Genistein exhibited an inhibitory effect on renal CSCs by attenuating the activation of the Shh pathway. In conclusion, the results illustrated the role of the Shh pathway in regulating renal CSC traits and the intervention of renal CSCs by genistein, which could provide novel insights into the molecular mechanisms of renal CSC intervention.

Keywords: genistein; inhibition; renal cancer stem cells; renal carcinoma; sonic hedgehog pathway.

Figure 1.

Tumor sphere-formation assay. 786-O and ACHN cells were cultured in SSM and SFM for 5 days. (A) Cell images were captured using a light microscope. Scale bar, 100 µm. (B) Reverse transcription-quantitative PCR was used to analyze the mRNA levels of renal CSC markers, including CD133, CD44, ALDH1A1, Oct4 and Nanog. (C) Western blotting was used to quantitatively measure the expression levels of corresponding renal CSC markers. (D) Flow cytometry was used to assess cell cycle phase distribution. Data are presented as the mean ± standard deviation from three independent experiments. **P<0.01 vs. SSM. ALDH1A1, aldehyde dehydrogenase 1 family member A1; CSC, cancer stem cell; SFM, serum-free medium; SSM, serum-supplemented medium.

Figure 2.

Genistein diminishes renal CSC characteristics. 786-O and ACHN sphere-forming cells were treated with different concentrations of genistein (0, 30, 60 and 90 µM) for 5 days. (A) Representative images. Scale bar, 100 µm. (B) Numbers of tumor spheres were counted and normalized to the control group. (C) mRNA expression levels of renal CSC markers were measured by reverse transcription-quantitative PCR. (D) Corresponding protein expression levels of renal CSCs markers were measured by western blotting. (E) Immunofluorescence staining images of 786-O tumor spheres were captured to determine CD44 expression. Scale bar, 100 µm. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. genistein 0 µM. ALDH1A1, aldehyde dehydrogenase 1 family member A1; CSC, cancer stem cell.

Figure 3.

Genistein inhibits the proliferation and induces the apoptosis of renal CSCs. 786-O and ACHN sphere-forming cells were treated with different concentrations of genistein (0, 30, 60 and 90 µM) for 5 days. Western blotting was used to analyze the expression levels of various (A) proliferation-associated (PCNA and cyclin D1) and (B) apoptosis-associated (Bcl-2, Bax, cleaved caspase 8, cleaved caspase 9 and cleaved caspase 3) proteins. Apoptotic populations among ACHN sphere-forming cells were detected using flow cytometry. (C) Representative images. (D) Percentage of apoptotic cells among ACHN sphere-forming cells. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. genistein 0 µM. CSC, cancer stem cell; PCNA, proliferating cell nuclear antigen.

Figure 4.

Genistein blocks the activity of the Shh pathway. (A) 786-O and ACHN sphere-forming cells were treated with the Smo inhibitor vismodegib (15 µM) for 5 days, and the expression levels of renal CSC markers were assessed by western blotting. (B) 786-O and ACHN sphere-forming cells were treated with different concentrations of genistein (0, 30, 60 and 90 µM) for 5 days. The expression levels of critical components of the Shh pathway were measured by western blotting. (C) Immunofluorescence staining images of 786-O tumor spheres were captured to determine Smo expression. Scale bar, 100 µm. (D) 786-O and ACHN sphere-forming cells were treated with 90 µM genistein in the presence or absence of 1 µM purmorphamine for 5 days. Representative images of sphere-forming cells. Scale bar, 100 µm. (E) Numbers of sphere-forming cells were counted and normalized to the control group (genistein 0 µM and purmorphamine 0 µM). *P<0.05, **P<0.01 vs. genistein 0 µM. ^##P<0.01 vs. genistein 90 µM. ALDH1A1, aldehyde dehydrogenase 1 family member A1; CSC, cancer stem cell; Gli, glioma-associated oncogene; Shh, sonic hedgehog; Smo, smoothened.

Figure 5.

Genistein diminishes renal CSC characteristics by suppressing the Shh pathway. 786-O and ACHN sphere-forming cells were treated with 90 µM genistein in the presence or absence of 1 µM purmorphamine for 5 days. (A) Western blot analysis of renal CSC markers. (B) Western blot analysis of the critical components of the Shh pathway. Western blotting was used to analyze the expression levels of (C) proliferation-associated proteins (PCNA and cyclin D1) and (D) apoptosis-associated proteins (Bcl-2, Bax, cleaved caspase 8, cleaved caspase 9 and cleaved caspase 3). *P<0.05, **P<0.01 vs. genistein 0 µM. ^#P<0.05, ^##P<0.01 vs. genistein 90 µM. ALDH1A1, aldehyde dehydrogenase 1 family member A1; CSC, cancer stem cell; Gli, glioma-associated oncogene; PCNA, proliferating cell nuclear antigen; Shh, sonic hedgehog; Smo, smoothened.