Effects of Astragalus polysaccharides on P-glycoprotein efflux pump function and protein expression in H22 hepatoma cells in vitro

Abstract

Background: Astragalus polysaccharides (APS) are active constituents of Astragalus membranaceus. They have been widely studied, especially with respect to their immunopotentiating properties, their ability to counteract the side effects of chemotherapeutic drugs, and their anticancer properties. However, the mechanism by which APS inhibit cancer and the issue of whether that mechanism involves the reversal of multidrug resistance (MDR) is not completely clear. The present paper describes an investigation of the effects of APS on P-glycoprotein function and expression in H22 hepatoma cell lines resistant to Adriamycin (H22/ADM).

Methods: H22/ADM cell lines were treated with different concentrations of APS and/or the most common chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine. Chemotherapeutic drug sensitivity, P-glycoprotein function and expression, and MDR1 mRNA expression were detected using MTT assay, flow cytometry, Western blotting, and quantitative RT-PCR.

Results: When used alone, APS had no anti-tumor activity in H22/ADM cells in vitro. However, it can increase the cytotoxicity of certain chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine, in H22/ADM cells. It acts in a dose-dependent manner. Compared to a blank control group, APS increased intracellular Rhodamine-123 retention and decreased P-glycoprotein efflux function in a dose-dependent manner. These factors were assessed 24 h, 48 h, and 72 h after administration. APS down regulated P-glycoprotein and MDR1 mRNA expression in a concentration-dependent manner within a final range of 0.8-500 mg/L and in a time-dependent manner from 24-72 h.

Conclusion: APS can enhance the chemosensitivity of H22/ADM cells. This may involve the downregulation of MDR1 mRNA expression, inhibition of P-GP efflux pump function, or both, which would decrease the expression of the MDR1 protein.

Figure 1

IC50 of APS combined with ADM, DDP, VCR, 5-FU, VP-16 or CTX in H22/ADM cells.^★P < 0.05, vs. control group.

Figure 2

Flow cytometry analysis of P-gp activity with Rh-123 in H22/ADM cells. A: Rh-123 fluorescence intensity curve in H22/ADM cells after exposure to chemotherapeutic agents at 24 h. B: Rh-123 fluorescence intensity curve in H22/ADM cells after exposure to chemotherapy agents at 48 h. C: Rh-123 fluorescence intensity curve in H22/ADM cells after exposure to chemotherapy agents at 72 h.

Figure 3

P-GP expressions in H22/ADM cells after exposure to the different chemotherapy agents at different time points (24 h, 48 h, and 72 h).