Saikosaponin b2 enhances the hepatotargeting effect of anticancer drugs through inhibition of multidrug resistance-associated drug transporters

Abstract

Aims: Vinegar-baked Radix Bupleuri (VBRB) potentiates the activity of anticancer drugs in the liver by increasing their hepatic distribution. However, this phenomenon may be associated with drug transporters. We investigated the effect of saikosaponin b2 (SSb2; the main component of VBRB) on the activity and expression of different drug transporters in both normal cells and those that overexpress the transporter.

Main methods: The activities of transporters were analyzed by concentration of their cellular substrates. Concentrations of colchicine (substrate of Pgp and MRP1) and cisplatin (substrate of OCT2 and MRP2) were determined by high-performance liquid chromatography (HPLC). The concentration of rhodamine B was determined by flow cytometry. The expression of transporter gene and protein were determined by qRT-PCR and Western blotting analysis.

Key findings: SSb2 increased colchicine efflux in HEK293 cells by primarily increasing Mrp1 activity, independent of gene and protein expression. SSb2 enhanced Mrp2 function and increased cisplatin efflux in BRL3A cells by upregulating Mrp2 gene expression, with a marginal effect on Pgp in normal cells. SSb2 increased OCT2 activity in OCT2-HEK293 cells by increasing the expression of OCT2 protein and mRNA; however, SSb2 inhibited MRP2 activity in MRP2-HEK293 cells by decreasing MRP2 protein expression, and decreased Pgp and MRP1 activity in Pgp- and MRP1-HEK293 cells.

Significance: SSb2 might potentially be the key active component of VBRB that enhances the hepatotargeting of anticancer drugs through the inhibition of multidrug resistance-associated drug transporters (Pgp, MRP1, and MRP2) in an environment-dependent manner.

Keywords: Drug transporters; MRP; OCT2; Pgp; Saikosaponin b2.

Fig. 1

The structure of saikosaponin b2.

Fig. 2

Effect of saikosaponin b2 on Mrp1 and Pgp in HEK293 cells. Colchicine uptake was determined by high performance liquid chromatography after 48-h treatment of cells with saikosaponin b2 (A), MK571 or MK571 plus saikosaponin b2 (B), verapamil and verapamil plus saikosaponin b2 (C), followed by the addition of 50 μM colchicine for an additional 1 h. Protein expressions of Pgp (D) and Mrp1 (F) in HEK293 cells treated with saikosaponin b2 for 48 h were determined by Western blotting analysis. The mRNA expressions of Pgp (E) and Mrp1 (G) were measured by qRT-PCR analysis after 48-h treatment of cells with saikosaponin b2. GAPDH was used as the loading control. Relative protein and mRNA expressions were calculated by sample/control. Mean + SD values from three independent experiments are presented. *P < 0.05, **P < 0.01 versus control, ^#P < 0.05 versus verapamil-control.

Fig. 3

Effect of saikosaponin b2 on Mrp2 and Oct2 in BRL cells. Cisplatin uptake was determined by high performance liquid chromatography after 24-h treatment of cells with saikosaponin b2, t, followed by the addition of 50 μg/mL cisplatin for another 4 h (A). Protein expressions of Oct2 (B) and Mrp2 in cells treated with saikosaponin b2 were analyzed by Western blotting analysis. The mRNA expressions of Oct2 (C) and Mrp2 (E) were measured by qRT-PCR analysis after 24-h treatment of cells with saikosaponin b2. The relative protein and mRNA expressions were calculated by sample/control. Mean + SD values from three independent experiments are presented. *P < 0.05, **P < 0.01 versus control.

Fig. 4

Effect of Saikosaponin b2 on OCT2 activity and expression in OCT2-HEK293 cells. (A) Cisplatin uptake was analyzed by high performance liquid chromatography after 24-h treatment of cells with saikosaponin b2, followed by incubation with 50 μg/mL cisplatin for another 4 h. (B) OCT2 protein expression was determined by Western blotting analysis after 24-h treatment of cells saikosaponin b2. GAPDH was used as the loading control. (C) OCT2 mRNA expression was measured by qRT-PCR analysis after 24 h-treatment of cells with saikosaponin b2. The relative protein and mRNA expressions were calculated by sample/control. Mean + SD values from three independent experiments are presented. *P < 0.05, **P < 0.01 versus control.

Fig. 5

Effect of Saikosaponin b2 on MRP2 activity and expression in MRP2-HEK293 cells. (A) Cisplatin uptake was analyzed by high performance liquid chromatography after 24-h treatment of cells with saikosaponin b2 or MK571, followed by incubation with 50 μg/mL cisplatin for another 4 h. (B) MRP2 protein expression was determined by Western blotting analysis after 24-h treatment of cells with saikosaponin b2 or MK571. GAPDH was used as the loading control. (C) MRP2 mRNA expression was measured by qRT-PCR analysis after 24-h treatment of cells with saikosaponin b2 or MK571. The relative protein and mRNA expressions were calculated by sample/control. Mean + SD values from three independent experiments are presented. *P < 0.05 versus control.

Fig. 6

Effect of Saikosaponin b2 on Pgp activity and expression in Pgp-HEK293 cells. (A) Rhodamine B uptake was determined by flow cytometry after 24-h treatment of cells with saikosaponin b2 or verapamil, followed by treatment with rhodamine B for another 30 min. (B) Pgp protein expression was determined by Western blotting analysis after 24-h treatment of cells with saikosaponin b2. GAPDH was used as the loading control. (C) Pgp mRNA expression was measured by qRT-PCR analysis after 24-h treatment of cells with saikosaponin b2. Relative protein and mRNA expressions were calculated by sample/control. Mean + SD values from three independent experiments are presented. *P < 0.05, **P < 0.01 versus control.

Fig. 7

Effect of Saikosaponin b2 on MRP1 activity and expression in glutathione (GSH)-stimulated HEK293 cells. (A) Colchicine uptake was determined by high performance liquid chromatography after 24-h treatment of cells with GSH, GSH plus saikosaponin b2, and GSH plus MK571, followed by incubation with colchicine for an another 1 h. (B) MRP1 protein expression was determined by Western blotting analysis after 24-h treatment of cells with GSH, GSH plus saikosaponin b2 and GSH plus MK571. GAPDH was used as the loading control. (C) MRP1 mRNA expression was measured by qRT-PCR analysis after 24-h treatment of cells with GSH, GSH plus saikosaponin b2 and GSH plus MK571. Relative protein and mRNA expressions were calculated by samples/glutathione-control. Mean + SD values from three independent experiments are presented. *P < 0.05, **P < 0.01 versus glutathione-control.