Sonic Hedgehog activation is implicated in diosgenin-induced megakaryocytic differentiation of human erythroleukemia cells

Abstract

Differentiation therapy is a means to treat cancer and is induced by different agents with low toxicity and more specificity than traditional ones. Diosgenin, a plant steroid, is able to induce megakaryocytic differentiation or apoptosis in human HEL erythroleukemia cells in a dose-dependent manner. However, the exact mechanism by which diosgenin induces megakaryocytic differentiation has not been elucidated. In this study, we studied the involvement of Sonic Hedgehog in megakaryocytic differentiation induced by diosgenin in HEL cells. First, we showed that different elements of the Hedgehog pathway are expressed in our model by qRT-PCR. Then, we focused our interest on key elements in the Sonic Hedgehog pathway: Smoothened receptor, GLI transcription factor and the ligand Sonic Hedgehog. We showed that Smoothened and Sonic Hedgehog were overexpressed in disogenin-treated cells and that GLI transcription factors were activated. Then, we showed that SMO inhibition using siSMO or the GLI antagonist GANT-61, blocked megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, we demonstrated that Sonic Hedgehog pathway inhibition led to inhibition of ERK1/2 activation, a major physiological pathway involved in megakaryocytic differentiation. In conclusion, our study reports, for the first time, a crucial role for the Sonic Hedgehog pathway in diosgenin-induced megakaryocytic differentiation in HEL cells.

Figure 1. Effect of diosgenin on human Hedgehog signaling pathway gene expression after 24 and 48 h treatment.

Cells were treated or not (control) with 10 µM diosgenin for 24 and 48 h then total RNA was extracted. 1µg of total RNA were transcribed into cDNA and used for quantitative-PCR according to the RT² profiler PCR array « human hedgehog signaling pathway ». Relative levels of mRNA gene expression were calculated using the 2^−ΔΔCt method versus untreated cells. Each value represents the mean ± SEM of three separate experiments.

Figure 2. Effect of diosgenin on SHh, SMO and GLI1 gene expression during megakaryocytic differentiation in HEL and TF1a cell lines.

(A) Cells were treated or not with 10 µM diosgenin for 24, 48, 72 and 96 h then SHh, SMO and GLI1 genes expressions were evaluated. Total RNA was extracted and 2µg of total RNA were transcribed into cDNA and used for PCR. PCR resulting fragments were visualized by electrophoresis on a 1% agarose gel containing ethidium bromide. Quantification of SHh, SMO and GLI1 transcripts were normalized to 18S as an internal control. The agarose gels shown are representative of six separate experiments. (B) Cells were treated or not with 10µM diosgenin for 96 h and megakaryocytic differentiation was evaluated by analyzing nuclear ploidy. Cells were fixed and permeabilized in 70% ethanol in PBS at −20 °C overnight, washed in PBS, treated with RNase and stained with PI. Then, flow cytometric analyses (FC) were performed to analyze DNA content.

Figure 3. Effect of diosgenin on SHh-N production, SMO expression and GLI1 activation in HEL cells.

Cells were treated with 10 µM diosgenin for 12, 24, 48, 72 and 96 h. (A) SHh-N production was evaluated in cell culture supernatanst using the “Sonic Hedgehog Human ELISA Kit”. Each value represents the mean ± SD of three separate experiments, * P< 0.05 diosgenin vs. control. (B) SMO expression was evaluated by Western blot analysis after diosgenin treatment. GAPDH was used as a loading control. (C) GLI1 activation was evaluated by electromobility shift assay using the DIG Gel Shift Kit. The blots shown are representative of five separate experiments.

Figure 4. Effects of SMO and GLI1 inhibition on diosgenin-induced megakaryocytic differentiation in HEL cells.

(A) Cells were transfected with siSMO or pretreated with 5µM GANT-61 for 48 h (G5) then treated with 10 µM diosgenin for 12, 24, 48, 72 and 96 h.SMO expression was evaluated by Western blot. GAPDH was used as a loading control. (B) Cells were pretreated with 5µM GANT-61 (G5) then treated with 10 µM diosgenin for 12, 24, 48, 72 and 96 h. GLI1 activation was evaluated by electromobility shift assay using the DIG Gel Shift Kit. The blots shown are representative of five separate experiments. (C) Cells were transfected with siSMO or pretreated with 5µM GANT-61 (G5) then treated with 10 µM diosgenin for 96 h. Megakaryocytic differentiation was assessed by analyzing nuclear ploidy. (D) Cells were transfected with siSMO or pretreated with 5µM GANT-61 (G5) then treated with 10 µM diosgenin for 5, 20 min, 1, 3, 6, 12 and 24 h. ERK1/2 phosphorylation was quantified using DuoSet IC assay kit. Each value represents the mean ± SEM of three separate experiments, * P< 0.05 siSMO+diosgenin or GANT-61+ diosgenin vs. diosgenin, # P< 0.05 diosgenin vs. control.

Figure 5. Megakaryocytic differentiation mechanisms in diosgenin-treated cells in relation to the SHh pathway.

Diosgenin induced megakaryocytic differentiation in HEL cells. The SHh pathway is involved in diosgenin-induced megakaryocytic differentiation since its inhibition blocks this phenomenon. SMO inhibition by total silencing using siRNA or GLI1 inhibition using GANT-61, a specific inhibitor, blocked megakaryocytic differentiation induced by diosgenin. In addition, SHh pathway inhibition leads to ERK1/2 inhibition whose activation is absolutely required to induce megakaryocytic differentiation.