Astragaloside IV reduces the expression level of P-glycoprotein in multidrug-resistant human hepatic cancer cell lines

Astragaloside is a saponin widely used in traditional Chinese medicine and has been reported to be a potent multidrug resistance (MDR) reversal agent. The present study investigated the role of astragaloside IV (ASIV) in the regulation of P-glycoprotein (P-gp, encoded by the mdr1 gene) and its effect on the reversal of MDR. The activity of ASIV was evaluated using human hepatic cancer cells Bel-7402 and the corresponding 5-fluorouracil (5-FU) resistant cells Bel-7402/FU. ASIV (0.08 mg/ml) potentiated the cytotoxicity of 5-FU which was demonstrated using the MTT assay on Bel-7402/FU cells. ASIV reduced the expression of P-gp as was revealed by immunocytochemistry. Accumulation and efflux studies with the P-gp substrate, rhodamine 123 (Rh123), demonstrated that ASIV inhibited P-gp-mediated drug efflux. Furthermore, it was demonstrated that ASIV enhanced the drug accumulation of 5-FU using a high performance liquid chromatography (HPLC) assay for drug resistant cells. Furthermore, ASIV may downregulate the expression of P-gp, which was examined using western blot analysis and polymerase chain reaction. In conclusion, the results of the present study indicated that ASIV reverses the drug resistance of Bel-7402/FU cells by downregulating the expression of mdr1. ASIV may represent a potent modulator of P-gp-mediated MDR in hepatic cancer therapy.


Introduction
Multidrug resistance (MDR) is considered a major cause of failure of anti-cancer chemotherapy. MDR is characterized by the simultaneous resistance to drugs that differ structurally and mechanistically (1). One of the major mechanisms of resistance in MDR mammalian cancer cells involves the increased expression of a 170 kDa transmembrane protein, P-glycoprotein (P-gp). P-gp, a member of the ATP binding cassette (ABC) transporter family, is encoded by mdr1 genes, also called ABCB1 and works in a similar manner to a pump to extrude anticancer drugs out of cells (2). P-gps expressed in the plasma membrane are mediators of MDR, actively effluxing a wide range of amphiphilic drugs irrespective of concentration gradient, thereby lowering intracellular concentrations to below therapeutic levels (3). The fact that P-gp is overexpressed in various cancer cells has prompted numerous research groups to search for effective inhibitors for this glycoprotein. Several compounds have been proposed as potential MDR modulators, including verapamil, PSC833 and XR9576 (4,5). Verapamil is one of the most extensively tested MDR modulators in the clinic and is used in conjunction with combination chemotherapy strategies. However, there has been limited success due to the cardiac toxicity associated with the high plasma levels required to effectively reverse MDR (6). To date, numerous natural compounds have been demonstrated to be capable of modulating P-gp transport, including rosmarinic acid, glaucine, gypenoside and oroxylin A (7)(8)(9)(10).
The present study aimed to determine whether ASIV reversed the MDR of the Bel-7402/FU cell line by mechanisms involving the P-gp/mdr1 gene.

Materials and methods
Extraction and isolation of ASIV. ASIV preparation was performed according to a previously published method (15). Bel-7402/FU cells were seeded at 1x10 4 cells/well and treated with 5-FU (0.025 mg/ml) alone, or in combination with ASIV (0.04 or 0.08 mg/ml) or 0.001 mg/ml (+)-verapamil (purity >99%; Sigma-Aldrich) with 5-FU. Subsequently, the cells were exposed to 5-FU, ASⅣ or verapamil continuously for 48 h and the cytotoxicity was assessed by MTT assay. The relative reversal fold (RRF) was calculated by dividing the inhibition rate of Bel-7402/FU cells treated with 5-FU and a modulator (ASⅣ or verapamil) by the inhibition rate of Bel-7402/FU cells treated with 5-FU.

Astragaloside Ⅳ reduces the expression level of P-glycoprotein in multidrug-resistant human hepatic cancer cell lines
Immunocytochemistry. The intracellular location and relative expression of P-gp was observed by immunocytochemistry. The cells (5x10 4 /ml) were exposed to 0.08 or 0.16 mg/ml ASⅣ or 0.001 mg/ml verapamil for 24 h. P-gp was detected using rabbit anti-P-gp monoclonal immunoglobulin G (IgG; Wuhan Boshide Biological Engineering Co., Ltd., Wuhan, Hubei, China) in a 1:200 dilution at 4˚C overnight. Subsequently, cells were rinsed three times with phosphate-buffered saline (PBS), and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L; Wuhan Boshide Biological Engineering Co., Ltd.) at 1:200 dilution. Cells were observed using fluorescence microscopy (BX51; Olympus, Center Valley, PA, USA).
Flow cytometric analysis of P-gp function. The cells (5x10 5 /ml) were incubated with or without ASIV (0.08 or 0.16 mg/ml) or

Determination of intracellular drug concentration.
Bel-7402/FU cells (5x10 5 /ml) were exposed to 0.025 mg/ml 5-FU in the presence or absence, of 0.08 mg/ml ASIV or 0.001 mg/ml verapamil for 24 h at 37˚C. Following trypsinization, the cells were extracted with 500 µl of methanol by ultrasonication and centrifuged at 12,000 x g for 30 min at 4˚C. The supernatant was filtered and dried with nitrogen gas. Subsequently, the mobile phase was added to achieve a metered volume of 0.5 ml for the quantitative analysis. Analysis was performed using the Agilent 1100 high performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA) comprised of a quaternary pump, an autosampler and a UV detector. A C18 column (250 x 4.6 mm, 5 µm, Diamonsil; Dikma, Lake Forest, CA, USA) was used for the separation. The flow rate was 1 ml/min with methanol:water (10:90, v/v) as the mobile phase. Peak areas were determined at 265 nm for 5-FU.
Western blot analysis. Bel-7402 and Bel-7402/FU cells (5x10 5 /ml) were treated with 0.08 mg/ml or 0.16 mg/ml ASIV or 0.001 mg/ml verapamil. Cells were lysed in a radioimmunoprecipitation assay buffer (Sigma-Aldrich). Cell lysates were boiled at 100˚C and cytosolic proteins were separated

Determination of MDR.
The MTT assay demonstrated that Bel-7402/FU cells were resistant not only to 5-FU but also to adriamycin and mitomycin. Bel-7402/5-FU cells were 19.64-fold more resistant than the control Bel-7402 cells to 5-FU (Table Ⅰ).
Cytotoxicity assay of ASIV. The intrinsic toxicity of ASIV was evaluated in order to ascertain the ability of the modulator to reverse the resistance at nontoxic concentrations. ASIV inhibited the proliferation of Bel-7402 and Bel-7402/FU cells in a dose-dependent manner (Fig. 2). As determined by the dose-effect curve, 0.04 and 0.08 mg/ml of ASIV were not cytotoxic (inhibition rate <5%). Thus, the concentration of 0.04 or 0.08 mg/ml ASIV was used as the dose for the reversal effect of MDR.

ASIV reverses the resistance of Bel-7402/FU cells to 5-FU.
The in vitro MDR reversing activity of ASIV was studied by determining the cytotoxicity of 5-FU in Bel-7402/FU cells (Table Ⅱ). In the presence of 0.08 mg/ml ASIV, the Bel-7402/FU cells exhibited a significantly increased sensitivity to 5-FU. The potency of ASIV was comparable with that of verapamil. The results demonstrated that ASIV was able to reverse MDR in vitro.
ASIV decreases P-gp expression. In order to observe the intracellular location of P-gp and assess the P-gp levels, immuncytochemistry was performed (Fig. 3). Using fluorescence microscopy, the positive response of P-gp was indicated by brown-yellow staining, mainly located in the cytoplasm   and cytomembrane, demonstrating a uniform fine granular distribution. As the expression of the Bel-7402/FU group increased, the color darkened. Following the treatment of Bel-7402/FU cells with 0.08 or 0.16 mg/ml ASIV, P-gp expression decreased, the color became lighter and the number of brown-yellow granules in the cytoplasm reduced significantly.
ASIV decreases the transport activity of P-gp. The ability of ASIV to inhibit P-gp-mediated transport was investigated using the P-gp substrate rhodamine 123 (Rh123). Fig. 4 illustrates that Rh123 accumulation in Bel-7405/FU cells was markedly lower than that found in Bel-7402 cells. Bel-7402/FU cells preincubated with ASIV for 48 h exhibited an increase in the intracellular accumulation of fluorescent Rh123.
ASIV enhances the intracellular accumulation of 5-FU. Intracellular 5-FU accumulation was determined by incubation of Bel-7402/FU cells with 5-FU (0.025 mg/ml) in the presence or absence of (0.08 mg/ml) ASIV by HPLC. Fig. 5 demonstrates that ASIV increased the intracellular accumulation of 5-FU in Bel-7402/FU cells.
ASIV downregulates mdr1 expression. Whether ASIV affected mdr1 mRNA and P-gp expression was examined using qPCR and western blot analysis. As shown in Fig. 6, mdr1 gene expression was markedly increased in Bel-7402/FU cells compared with Bel-7402 cells. The levels of mdr1 mRNA were decreased by 0.08 mg/ml ASIV and completely deregulated by 0.16 mg/ml ASIV. As shown in Fig. 7, 0.08 mg/ml ASIV decreased the P-gp levels in Bel-7402/FU cells.

Discussion
Intrinsic and acquired resistance of malignant cells to cytotoxic agents is a major cause of treatment failure during chemotherapy (16). One of the well-established mechanisms of resistance is the MDR process, due to increased ABC transporter expression (17). Inhibition of drug transporters and modulating MDR are among the most important strategies in the field of cancer chemotherapy. Previous studies have reported that astragaloside Ⅱ (ASII) may be capable of reversing hepatoma MDR in vitro by downregulating the expression of the mdr1 gene and P-gp. However, ASII may also inhibit the mitogen-activated protein kinase (MAPK) signal transduction pathway (15). ASIV has been proven to be a novel anti-inflammatory agent and has been suggested as a potential agent for the treatment of cardiovascular diseases (18,19). However, few studies have reported on the effects of ASIV on the reversal of MDR and its molecular mechanisms. In the present study, the in vitro potency of ASIV was evaluated with several assays using human hepatic cancer Bel-7402 and Bel-7402/FU cells. Cytotoxicity assays of several anticancer drugs (5-FU, adriamycin and mitomycin) demonstrated that Bel-7402/FU cells were 19.64-fold more resistant to 5-FU than Bel-7402 cells. In order to investigate the reversal effect of ASIV on drug resistant cells, ASIV was administered to cells at the nontoxic concentrations of 0.04 or 0.08 mg/ml, which increased the sensitivity of Bel-7402/FU cells to 5-FU by 1.87-fold. Therefore, ASIV partially reversed the MDR of Bel-7402/FU cells. P-gp is expressed in a cell-and tissue-specific manner, with high levels detectable in the kidney, liver and intestine (20). The immuncytochemistry assay demonstrated that ASIV significantly inhibited P-gp expression.
The overexpression of P-gp on the surface of tumor cells leads to MDR. This protein acts as an energy-dependent drug efflux pump, reducing the intracellular concentration of structurally unrelated drugs. In the study of accumulation and efflux, sensitive hepatic cancer Bel-7402 cells acted as a negative control and accumulated the most Rh123 compared with Bel-7402/FU cells which accumulated the least (21). However, Bel-7402/FU cells treated with ASIV accumulated more Rh123 than untreated cells. Further confirmation of ASIV inhibiting P-gp-mediated drug efflux was provided by the fact that the modulator increased the intracellular accumulation of 5-FU in Bel-7402/FU cells, according to HPLC analysis. These studies indicated that the reversal of MDR by ASIV was through the enhancement of drug uptake and the inhibition of P-gp mediated drug efflux. Similarly, the reversal of P-gp-mediated MDR with milbemycins correlated with an increase in the accumulation of adriamycin and Rh123 via the inhibition of P-gp efflux in MCF-7/adr cells (22).
The inhibition of P-gp function or inhibition of its expression was able to prevent the P-gp-mediated MDR phenotype and improve the effectiveness of chemotherapy (23).
qPCR and western blot analyses were performed in order to determine the interaction of ASIV with the mdr1 gene and P-gp. The qPCR assay revealed that ASIV downregulated mdr1 mRNA expression in Bel-7402/FU cells. Furthermore, western blot analysis revealed that ASIV downregulated P-gp expression in Bel-7402/FU cells. Thus, the downregulation of the mdr1 gene and P-gp expression by ASIV may be involved in the reversal of MDR.
The MAPK pathway is an important signal transduction pathway activated by various stimuli. Previous reports have demonstrated that modulators of the MAPK pathway may affect the drug transport activity of P-gp in certain multidrug-resistant cell lines (24,25). Adenovirus-mediated enhancement of c-Jun NH 2 -terminal kinase (JNK) reduces the levels of P-gp and reverses P-gp-mediated MDR in human gastric carcinoma resistant cell lines (26). BIRB796, an active inhibitor of p38 MAPK, reverses P-gp-mediated MDR by directly inhibiting its transport function (27). Previous studies have reported that ASⅡ suppressed the phosphorylation of extracellular signal regulated kinase1/2, p38 and the c-Jun NH 2 -terminal kinase. Whether ASIV downregulates P-gp expression via the MAPK pathway requires to be elucidated by future studies.
In conclusion, ASIV has the potential to be used as a P-gp-mediated MDR reversal agent and may be a potential adjunctive agent for human hepatic cancer chemotherapy.