Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression

Abstract

Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0μM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants.

Keywords: P-glycoprotein; asclepiasterol; multidrug resistance (MDR); phosphorylation of ERK1/2 (P-ERK); steroid.

Scheme 1

Chemical structure of asclepiasterol

Figure 1. P-glycoprotein (P-gp) expression in MCF-7/ADR and HepG-2/ADM cells in comparison to corresponding parental cell lines, MCF-7 and HepG-2

A. Western blot analysis of proteins extracted from MDR cells and their parental cells with P-gp antibody. GAPDH was used as loading control. B. Fluorescence microscope detection of the accumulation of rhodamine123 (Rh123) in MDR cells and their parental cells. Images were acquired at 488nm extraction and 535nm emission wavelengths for Rh123.

Figure 2. Cytotoxic effects of asclepiasterol

The MTT cytotoxicity assay was assessed in pairs of multidrug resistant cell lines and their corresponding parental cell lines, as well as in non-tumor cell lines. Cells were treated with or without increasing concentrations of asclepiasterol for 48h and cell viability was determined. A. MCF-7 and MCF-7/ADR cells. B. HepG-2 and HepG-2/ADM cells. C. HEK293, LO₂ and Chang cells. Inhibition of asclepiasterol on cell proliferation was calculated based on the absorbance ration between treatment and control. Values represent mean ± SD (n=6) of three independent experiments.

Figure 3. Asclepiasterol sensitizes MDR cells to apoptosis

MDR cells were treated with paclitaxel alone (1.0 μM), asclepiasterol alone (5.0 μM), VRP alone (10.0 μM), or their combination for 48h. The IC₅₀ values for paclitaxel had been previously determined for each cell line. Apoptosis was analyzed by flow cytometry as the percentage of cells labeled by Annexin V and PI. A representative set of data from three independent experiments is shown. MDR cells were resistant to paclitaxel, and asclepiasterol or VRP increased cell apoptosis induced by paclitaxel. All experiments were repeated three times and data are presented in histogram as means±SD. *P < 0.05, **P < 0.01, compared with doxorubicin treatment alone.

Figure 4. Colony formation assay of MDR cells treated with Dox (3.0 μM) in the absence or presence of Asclepiasterol (2.5, 5.0 μM)

Cells were re-suspended and plated in 6-well plates containing DMEM plus 10% FBS in 0.4% agar above a layer of 0.6% agar at a density of 10000 cells per well. Colonies were counted under a phase contrast microscope after 14 days. Representative images of colonies (magnification ×20) are shown. Summary of colony formation assay data from three independent experiments is shown. *P < 0.05, **P < 0.01, compared with doxorubicin treatment alone.

Figure 5. Effect of asclepiasterol at different concentrations on the accumulation of doxorubicin and Rh123

VRP at 10.0 μM was used as positive control. A. Flow cytometry analysis of the accumulation of Dox (10.0 μM) and Rh123 (10.0 μM) in MDR cells and their parental cells with or without asclepiasterol or VRP treatment. Intracellular fluorescence was analyzed by flow cytometry. B. Fluorescence microscope detection of the accumulation of rhodamine123 (Rh123) in MDR cells after asclepiasterol treatment. Cells were treated with 2.5, 5.0 μM asclepiasterol and 10.0 μM VRP for 48h, and then Rh123 at 10.0 μM was added and incubated for an additional 2h in the dark. Images were taken at 488 nm extraction and 535 nm emission wavelength after washing the cells with cold PBS three times.

Figure 6. The effect of asclepiasterol on P-gp protein and MDR1 mRNA expression in MDR cells

A. MDR cells were treated with asclepiasterol at concentrations of 2.5 and 5.0 μM for 48h. P-gp expression was analyzed by Western blotting. GAPDH was used as a loading control. The summary from three independent experiments is shown under the representative blot image. B. Western blot analysis of P-gp expression after a time-course of asclepiasterol treatment. MDR cells were treated with 5.0 μM asclepiasterol for 4 to 48h, P-gp expression was analyzed by Western blotting. GAPDH was used as a loading control. C. RT-PCR analysis of MDR1 mRNA expression in MDR cells after treatment with asclepiasterol at 2.5 and 5.0 μM for 48h. Total RNA was extracted from the cells and the mRNA levels of MDR1 and GAPDH were analyzed by RT-PCR. Independent experiments were performed three times and the summary of the results shown. *P < 0.05, **P < 0.01, compared with the untreated control.

Figure 7. Effect of asclepiasterol on the blockade of ERK1/2 and AKT phosphorylation in MDR cells

A. Western blot analysis of P-gp expression and ERK1/2, Akt phosphorylation status in MDR cells treated with 2.5 or 5.0 μM asclepiasterol for 48h. GAPDH was used as a loading control. MCF-7 and HepG-2 cells were used as negative controls for detection. B. Western blot analysis of P-gp expression and ERK1/2 phosphorylation status in MDR cells treated with 10.0 μM U0126-EtOH for 12h. Relative P-gp and P-ERK expression of MDR cells from three independent experiments are shown under each Western blot image, *P < 0.05, **P < 0.01, compared with the untreated control.

Figure 8. Effect of EGF on asclepiasterol-mediated P-gp down-regulation and P-ERK suppression

A. Western blot analysis P-gp expression and ERK1/2 phosphorylation after treatment of MDR cells with EGF alone, asclepiasterol alone (2.5, 5.0 μM), or their combination. MDR cells were treated with (+) or without (−) asclepiasterol for 48h to down-regulate P-gp expression on the cell surface and then were treated with (+) or without (−) asclepiasterol combined with 100μg/L EGF for an additional 24h. P-gp and P-ERK1/2 expression were determined by Western blot analysis. GAPDH was used as loading control. Relative P-gp and P-ERK expression of MDR cells from three independent experiments are shown. B. Flow cytometry analysis of the accumulation of Dox (10.0 μM) and Rh123 (10.0 μM) in MDR cells treated with asclepiasterol (2.5, 5.0 μM) in combination with 100μg/L EGF. MDR cells were treated under the same conditions as in (A), and were incubated with Dox (10.0 μM) or Rh123 (10.0 μM) for a further 2h. Intracellular fluorescence was analyzed by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the untreated control.