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

Analysis of drug resistant profiles in MDR cells

We first examined the cytotoxic effects of several anticancer agents on MDR cells (MCF-7/ADR and HepG-2/ADM) and their parental cells. As shown in Table 1, the MDR cells showed significant resistance to these agents with 14.47, 17.62, 199.91, 442.42 multidrug resistance ratios (MRs) in MCF-7/ADR cells, and 108.94, 79.87, 116.74, 74.63 in HepG-2/ADM cells for doxorubin (Dox), epirubicin (EPI), daunorubicin (DNR) and paclitaxel, respectively. Since these anticancer agents are transported by P-gp [33], we measured the expression of P-gp in these cells and found that both MCF-7/ADR and HepG-2/ADM cells expressed higher levels of P-gp as compared with their parental cells (Figure 1A). Consistent with high P-gp expression, the intracellular accumulation of Rh123 (a specific fluorescent probe substrate for P-gp) in MDR cells was dramatically lower than in parental cells (Figure 1B, quantitative Rh123 accumulation results are shown in supporting information, Supplementary Figure S2). These results suggested that P-gp was overexpressed in MCF-7/ADR and HepG-2/ADM cells, which was consistent with our previous study [34]. Thus these two MDR cells were suitable target cells for studying the P-gp-mediated MDR reversal capability 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.

Asclepiasterol is not cytotoxic to MDR tumor and non-tumor cells

We next tested the cytotoxic effect of asclepiasterol on MDR cells (MCF-7/ADR, HepG-2/ADM) and their corresponding parental cell lines (MCF-7, HepG-2) using an MTT assay. As shown in Figure 2A and 2B, treatment with increasing concentrations of asclepiasterol between 0 and 10.0 μM for 48h did not inhibit the proliferation of these cells. More than 90% of cells were viable when treated with 5.0 μM of asclepiasterol. In addition, different cell lines, including HEK293, Chang, and LO2 cells, were also included in this study to evaluate the cytotoxicity of asclepiasterol on non-tumor cells. Asclepiasterol showed no obvious cytotoxic effect on these cells (Figure 2C). These results suggested that asclepiasterol was not cytotoxic, making it a candidate compound to investigate its MDR activity. Based on the above results, asclepiasterol at concentrations of 2.5 μM and 5.0 μM were used in subsequent experiments.

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.

Asclepiasterol reverses P-gp-mediated MDR in vitro

We next investigated asclepiasterol's modulating effect on MDR cells and the corresponding parental cell lines using an MTT assay. The IC₅₀values of chemotherapeutic agents, including Dox, EPI, DNR, paclitaxel and cisplatin, for sensitive and resistant cells in the presence or absence of asclepiasterol are shown in Table 1. Verapamil, a specific P-gp inhibitor at 10.0 μM was used in this study as a positive control. Asclepiasterol at 2.5 and 5.0 μM enhanced the cytotoxicity of these chemotherapeutic agents in a dose-dependent manner in MDR cells. Asclepiasterol at 5.0 μM showed 35.06-, 16.67-, 167.31-, 285.71-fold, and 62.41-, 33.48-, 24.29-, 85.09-fold, for the reversal of resistance to Dox, EPI, DNR and paclitaxel in MCF-7/ADR and HepG-2/ADM cells, respectively. These effects were stronger than those of 10.0 μM verapamil (the corresponding values were 34.71-, 5.03-, 58.39-, 11.79-fold and 38.44-, 7.43-, 56.81-, 78.11-fold for MCF-7/ADR and HepG-2/ADM, respectively). These results indicated that asclepiasterol specifically reversed P-gp-mediated MDR, and in most cases, asclepiasterol demonstrated stronger reversal effects than the positive control.

Asclepiasterol enhances the anticancer efficacy of chemotherapeutic agents in MDR cells

Based on the above results, we speculated that MDR cells might be more sensitive to combination treatment. To examine whether asclepiasterol in combination with the P-gp substrate anticancer agents had any influence on the chemotherapeutic agents mediated apoptosis, we treated MCF-7/ADR and HepG-2/ADM cells with (+) or without (−) 5.0 μM asclepiasterol or 10.0 μM VRP combined with 1.0 μM paclitaxel for 48h, and then measured the extent of apoptosis. Since the IC₅₀ values for paclitaxel had been previously determined for each cell line and the MDR cells were resistant to paclitaxel at more than 1.0 μM, paclitaxel in this experiment was tested at 1.0 μM. As shown in Figure 3, treatment of the MDR cells with 1.0 μM paclitaxel, asclepiasterol, or 10.0 μM VRP alone did not cause any apoptosis, while the apoptosis rate of the combination group was dramatically increased as compared with the control group or the treatment alone group (88.93%±2.34% and 81.33%±4.15% for asclepiasterol and VRP combination with paclitaxel, respectively, versus 4.2%±2.35% for paclitaxel alone in MCF-7/ADR and 65.77%±1.92% and 61.6%±1.7% for asclepiasterol and VRP combination with paclitaxel, respectively, versus 27.4%±2.5% for paclitaxel alone in HepG-2/ADM). In contrast, asclepiasterol could not increase paclitaxel-induced apoptosis in sensitive cells (Supplementary Figure S3, supporting information).

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.

Since the MDR cells with higher P-gp expression were reported to be more malignant and resistant to traditional chemotherapeutic agents [35, 36], we investigated the colony formation capacity of MCF-7/ADR and HepG-2/ADM treated with 3.0 μM Dox and its combination with asclepiasterol by soft agar assay. In this experiment, the dose of Dox was selected according to the cell growth assay and the MDR cells were shown to be resistant at this concentration (Table 1). As expected, the number of colonies formed in MCF-7/ADR and HepG-2/ADM cells was larger than those in the corresponding parental cells, suggesting that the MDR cells were more resistant to Dox treatment (Figure 4). Moreover, no difference was observed in colony size or number between the two MDR cells treated with asclepiasterol alone. In contrast, the combination of asclepiasterol with Dox decreased anchorage-independent cell growth capacity (Figure 4). The number of colonies formed in MCF-7/ADR and HepG-2/ADM were decreased 99.05% and 98.11%, respectively in the combination group. Taken together, these results indicated that asclepiasterol enhanced the efficacy of anticancer agents on the colony formation of MDR cancer cells. However, asclepiasterol did not influence the efficacy of Dox on colony formation in sensitive cells (Supplementary Figure S4, supporting information).

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.

Asclepiasterol increases the accumulation of Dox and Rh123 in MDR cells

The above results showed that asclepiasterol re-sensitized the overexpressing P-gp cells to chemotherapeutic agents which were P-gp substrates. We further examined the effect of asclepiasterol on Dox and Rh123 accumulation in P-gp overexpressing MCF-7/ADR and HepG-2/ADM cells and their corresponding parental cells, MCF-7 and HepG-2. After treatment with (+) or without (−) 2.5 and 5.0 μM asclepiasterol or 10.0 μM VRP (positive control), the cells were analyzed by flow cytometry. As shown in Figure 5A, both MDR cells treated with asclepiasterol accumulated more Dox in a dose-dependent manner as compared with treatment with Dox alone. Intracellular accumulation of Dox was increased by 9.58-, 37.54-, 8.40-fold in MCF-7/ADR, and 1.68-, 3.29-, 1.72-fold in HepG-2/ADM cells in the presence of 2.5 μM and 5.0 μM of asclepiasterol and 10.0 μM VRP, respectively (Figure 5A).

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.

We also tested the effect of asclepiasterol on accumulation of Rh123, a specific P-gp substrate. The fluorescence value of Rh123 was enhanced by 2.98-, 7.46- and 1.20-fold in MCF-7/ADR, and 2.23-, 4.75- and 1.39-fold in HepG-2/ADM cells in the presence of 2.5 μM and 5.0 μM of asclepiasterol and 10.0 μM VRP, respectively (Figure 5B). In contrast, the intracellular accumulation of Dox and Rh123 were not altered in the sensitive cells in the presence of asclepiasterol alone (Supplementary Figure S5, supporting information). We also measured the accumulation of Rh123 in MDR cells treated with asclepiasterol or VRP using fluorescence microscopy and found, consistent with the flow cytometry results, that the MDR cells treated with asclepiasterol or VRP had much higher cytosolic accumulation of Rh123 than the control group (Figure 5B). Both experimental results suggested that asclepiasterol promoted the accumulation of P-gp substrates in MDR cells and therefore re-sensitized them to chemotherapeutic agents.

Asclepiasterol decreases the expression of P-gp without stimulating or suppressing MDR1 mRNA expression

Reversal of P-gp-mediated MDR could be achieved by down-regulating P-gp expression in MDR cells apart from inhibiting its transport function. We therefore measured P-gp expression in MCF-7/ADR and HepG-2/ADM cells before and after asclepiasterol (2.5, 5.0 μM) treatment by Western blot analysis. As shown in Figure 6A, the expression of P-gp in MCF-7/ADR and HepG-2/ADM cells decreased in a dose-dependent manner by 4-7 fold in the presence of 2.5 or 5.0 μM asclepiasterol. We also examined the timing of asclepiasterol-mediated P-gp down-regulation. MCF-7/ADR and HepG-2/ADM cells were treated with 5.0 μM asclepiasterol for 4 to 48h. As shown in Figure 6B, P-gp expression began to decrease at 12h and this timing did not vary among the cell lines we tested.

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.

We next used RT-PCR to examine MDR1 mRNA expression in MDR cells in the presence or absence of asclepiasterol under the same conditions used for the Western blot experiment. Total RNA was extracted and RT-PCR was performed using MDR1 or GAPDH-specific oligonucleotides. As shown in Figure 6C, MDR1 mRNA levels were unchanged after asclepiasterol treatment at all concentrations in each of the MDR cells. From these data, we concluded that the effect of asclepiasterol in reducing the efflux of chemotherapeutic agents might be via the suppression of P-gp translation, since MDR1 mRNA levels had not been altered by asclepiasterol treatment while the protein level was decreased.

Asclepiasterol suppresses the phosphorylation of ERK1/2, but does not inhibit the phosphorylation of AKT in MDR cells

Many groups have reported that aberrant activation of AKT and ERK1/2 signaling pathways is important for the development of the MDR phenotype of tumor cells, and the suppression of ERK/AKT signaling is considered an effective therapeutic approach to overcome MDR [37, 38]. Thus we hypothesized that ERK/AKT pathways might be involved in asclepiasterol-mediated P-gp down-regulation. To test this hypothesis, we determined the total and phosphorylation levels of ERK1/2 and AKT in asclepiasterol-treated cells by Western blot. As shown in Figure 7A, phosphorylated ERK1/2 was increased in the P-gp overexpressing cells MCF-7/ADR and HepG-2/ADM as compared with the corresponding parental cells (MCF-7 and HepG-2). Asclepiasterol treatment led to a reduction in phosphorylated ERK1/2 particularly at 5.0 μM, without stimulating or suppressing the total ERK1/2 levels in the MDR cells. Meanwhile, P-gp expression was decreased in both MDR cells, and associated with the suppression of phosphorylation ERK1/2. However, the concentration of asclepiasterol we used in this experiment did not reduce phosphorylated AKT or total AKT levels in these cells. Taken together, these results suggested that the mechanism of asclepiasterol-mediated P-gp down-regulation was likely through a blockade of the ERK1/2 pathway.

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.

The above results indicated that asclepiasterol could suppress the phosphorylation of ERK1/2 and decrease P-gp expression in the MDR cells at the same time. This suggested that asclepiasterol might reverse P-gp-mediated MDR through the MEK-ERK signaling pathway. Therefore, the MEK1/2 inhibitor, U0126-EtOH, was used to test its effect on P-gp expression in MDR cells. Cells were treated with 10.0 μM U0126-EtOH for 12h and P-gp expression determined by Western blotting. U0126-EtOH suppressed the phosphorylation of ERK1/2, and the MDR cells treated with U0126-EtOH showed 2.94- and 2.25-fold lower levels of P-gp for MCF-7/ADR and HepG-2/ADM, respectively, as compared with the untreated cells (Figure 7B). These results suggested that the MEK-ERK pathway was crucial for the regulation of P-gp expression.

EGF activates the MEK-ERK pathway and reverses asclepiasterol-mediated P-gp down-regulation in MDR cells

Given that treatment with asclepiasterol and suppression of P-gp expression in MDR cells was associated with a blockade of the MEK-ERK pathway, we next investigated whether the activation of this pathway would enhance P-gp expression and reverse the effects of asclepiasterol. MCF-7/ADR and HepG-2/ADM cells were treated with (+) or without (−) asclepiasterol for 48h to decrease P-gp expression on the cell surface. Then we treated the cells with (+) or without (−) asclepiasterol combined with 100 μg/L EGF, an activator of the ERK1/2 pathway, for an additional 24h. P-gp and P-ERK1/2 expression were determined by Western blot analysis. The ERK1/2 pathway was activated in the presence of EGF with increased expression of phosphorylated ERK1/2, and the asclepiasterol-mediated down-regulation of phosphorylated ERK1/2 in the two MDR cells was suppressed (Figure 8A). There also was an obvious increase in P-gp expression in these two MDR cells, associated with the increased phosphorylated ERK1/2 expression by EGF.

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.

To further confirm these findings, we next examined whether the effect of asclepiasterol-mediated accumulation of P-gp substrates in MDR cells could be reversed by EGF intervention through treatiment of the MDR cells with asclepiasterol in combination with EGF. As shown in Figure 8B, 100 μg/L EGF could completely block the effect of 2.5 μM asclepiasterol on Dox or Rh123 accumulation in the MDR cells, while those treated with 5.0 μM asclepiasterol were just partially reversed. These results further indicated that the MEK-ERK pathway was involved in asclepiasterol-mediated P-gp down-regulation.

Isolation and identification of asclepiasterol from A. curassavia

Asclepiasterol (scheme 1) was isolated from the ethyl acetate soluble fraction of 70% ethanol extract of the whole plant A. curassavica through silica gel column chromatography followed by preparative HPLC. The structure was ambiguously identified as a novel C₂₁ steroidal glycoside by a comprehensive analysis of the spectral data (UV, IR, MS, 1D and 2D-NMR). The detailed isolation and structural elucidation are shown in the supporting information (Supplementary Table S1 and Supplementary Figure S1).

Chemicals and reagents

Asclepiasterol was dissolved in dimethylsulfoxide (DMSO, Sigma Aldrich St. Louis MO) to make a 10 mmol/L stock solution and stored at −20°C. The purity of this compound was analyzed by high performance liquid chromatography and was found to be higher than 99%. The chemical structure was characterized by LC-MS and NMR.

Verapamil (VRP), paclitaxel, MTT, and rhodamine 123 (Rh123) were purchased from Sigma-Aldrich (Deisenhofer, Germany). Doxorubicin (Dox) and epirubicin (EPI) were obtained from Zhe-Jiang HISUN Pharmaceuticals Co (Zhejiang, China). Daunorubicin was supplied by National Institute for the Food and Drug Control (Beijing, China). Antibodies against P-gp/ABCB1, total AKT, total ERK, P-AKT^{(Thr 308/Ser473)} and P-ERK^(Tyr204) were products of Cell Signaling Technology. Alexa Fluor 488 goat anti-rabbit IgG (H+L) was purchased from Life Technologies. U0126-EtOH(2,3-bis(amino(2-aminophenylthio)methylene)succinonitrile,ethanol) was obtained from Selleckchem (Houston, TX) and dissolved in DMSO to make a 10μmol/L stock solution and stored at −20°.

Cell lines and cell culture

The breast cancer cell line MCF-7, Human hepatocellular carcinoma cell line HepG-2, and their Dox-selected P-glycoprotein (P-gp) overexpressing cells MCF-7/ADR and HepG-2/ADM were kindly provided by Cancer institute & Hospital Chinese Academy of Medical sciences (Beijing, China). HEK293, Chang, LO₂ cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Gibco^® RPIM media 1640 or DMEM supplemented with 10% fetal bovine serum, penicillin (100U/mL) and streptomycin (100 mg/ml) and were grown as monolayer cultures and maintained in a humidified atmosphere containing 5% CO₂ in air at 37°C. Penicillin, streptomycin, RPMI media 1640, and FBS were purchased from Life Technologies, Inc. (Grand Island, NY).

Cell proliferation assay

Cell proliferation was assessed by MTT assay. In brief, MCF-7, HepG-2, MCF-7/ADR, HepG-2/ADM, HEK293, Chang and LO₂ cells were seeded in 96-well plates at a density of 4×10³ cells per well and cultured overnight. Then cells were treated with various concentrations (0.005-50 μM) of doxorubicin, epirubicin, daunorubicin and paclitaxel in the presence or absence of asclepiasterol or VRP for 48h. 20μL MTT was then added into each well and the cells were incubated for an additional 4h. Finally, the purple formazan crystals formed were dissolved in 150 μL of DMSO and the absorbance was detected at 490 nm by multi-mode microplate reader (Bio-Rad laboratories, USA). The concentration required to inhibit cell growth by 50% (IC₅₀) was calculated by using Graph Pad Prism 5.0.

Dox and Rh123 accumulation assay

Cells were seeded in the six-well plate at a density of 1×10⁵ cells per well and allowed to adhere overnight. Then the cells were treated with two concentrations of asclepiasterol (2.50 μM, 5.0 μM) or 10.0 μM VRP as positive control based on the previous assay for 48h at 37°C before incubating with 10.0 μM Dox or 10.0 μM Rh123 for an additional 2h in darkness at 37°C. After that, cells were harvested and washed 3 times with cold PBS. Finally, cells were re-suspended in PBS buffer and analyzed by Flow cytometry (BD Bioscience, San Jose, CA). Data were analyzed with FlowJo 7.6.1 software.

To further visualize the effect of asclepiasterol on the intracellular retention of Rh123, 5×10⁵ cells per well were seeded in the 6-well plate for overnight. The cells were treated with asclepiasterol (2.5, 5.0 μM) or 10.0 μM VRP for 48h, and were then incubated with 10.0 μM Rh123 alone or 10.0 μM Rh123 combination with asclepiasterol or VRP in the fresh RPMI 1640 medium for 2h in darkness at 37°C. After that cells were washed 3 times with cold PBS and images were acquired by fluorescence microscopy.

Annexin V/PI staining

For detection of apoptotic cells, HepG-2/ADM and MCF-7/ADR cells were treated with (+) or without (−) 2.5 or 5.0 μM asclepiasterol combined with 1.0 μM paclitaxel for 48h. Cells were then harvested and washed 3 times with binging buffer. After that, cells were incubated with fluorescein isothicocyannte (FITC)-conjugated annexin V reagent and PI in binging buffer for 15 min at room temperature as described by the manufacture (BD Bioscience, San Jose, CA) and finally analyzed by Flow cytometry. Data were analyzed with FlowJo 7.6.1 software.

Soft agar colony formation assay

Soft agar colony formation was assessed according to Cifone and Fidler [60]. In brief, the MDR cells MCF-7/ADR and HepG-2/ADM were treated with asclepiasterol for 24h. Then the cells were re-suspended and plated into six-well plates containing DMEM plus 10% FBS in 0.4% agar above a layer of 0.6% agar at a density of 1000 cells per well. Colonies were counted under a phase contrast microscope after 14 days. All experiments were done in triplicate and repeated three times.

Western blotting analysis

Western blotting analysis was performed as previously described [61]. Cells (1×10⁶) were incubated with 2.5 or 5.0 μM asclepiasterol for 48h. For immunoblotting, cells were pelleted by centrifugation at 320 ×g for 10 min and suspended in lysis buffer (20 mM Tris-HCl pH 7.4, 2 mM EDTA, 500 mM sodium orthovanadate, 1% Triton X-100, 0.1% SDS, 10 mM NaF, 10 mg/mL leupeptin, and 1 mM PMSF). Aliquots (20 mg) of the lysates were separated on a 4–12% SDS-polyacrylamide gel and transferred into a PVDF membrane (Millipore, USA). Blots were blocked for 2 h in blocking buffer (5% non-fat dry milk in PBST buffer (10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4)) and incubated with primary antibodies (1:1000) overnight at 4°C. After washing with PBST, the appropriate HRP-conjugated secondary antibody (1:2000) was added to the preparation. The blot was incubated at 37°C for 1 h and developed using an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology, China).

Real-time PCR

MDR-1 mRNA expression was examined by RT-PCR. Cells were treated with asclepiasterol under the same condition used for Western blot analysis. Total cellular RNA was extracted by using the Trizol reagent (Takara, Japan). Complementary DNA (cDNA) corresponding to 0.8 μg of total RNA was used for per reaction (20μL) in a real-time quantitative PCR reaction performed on a Roche Light cycler (Manncheim, Germany) using power SYBER Green Master Mix (Takara, Japan).

Statistical analysis

Each experiment was performed at least three times and the data were presented as mean ± standard deviation (SD). Statistical significance of the results was calculated by one-way analysis of variance (ANOVA) or two-way ANOVA using Prism 5 (GraphPad, La Jolla, CA) and the prior significance level was set at P < 0.05, P <0.01, P <0.001.

The multidrug resistance ratio (MR) was defined to evaluate the extent of cell resistance to the anti-cancer drugs:

MR = IC_{50 (the P-gp overexpressing cell lines)}/IC_{50 (the corresponding parental cell lines)}

The reversal ratio (RR) was defined to evaluate the ability of a reversing agent to reverse the MDR:

RR = IC_{50 (the P-gp overexpressing cell lines)}/IC_{50 (the presence of reverser the P-gp overexpressing cell lines)}