BBA, a derivative of 23-hydroxybetulinic acid, potently reverses ABCB1-mediated drug resistance in vitro and in vivo

Abstract

23-O-(1,4'-Bipiperidine-1-carbonyl)betulinic acid (BBA), a synthetic derivative of 23-hydroxybetulinic acid (23-HBA), shows a reversal effect on multidrug resistance (MDR) in our preliminary screening. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCG2, and ABCC1 has been reported in recent studies to be a major factor contributing to MDR. Our study results showed that BBA enhanced the cytotoxicity of ABCB1 substrates and increased the accumulation of doxorubicin or rhodamine123 in ABCB1 overexpressing cells, but had no effect on non ABCB1 substrate, such as cisplatin; what's more, BBA slightly reversed ABCG2-mediated resistance to SN-38, but did not affect the ABCC1-mediated MDR. Further studies on the mechanism indicated that BBA did not alter the expression of ABCB1 at mRNA or protein levels, but affected the ABCB1 ATPase activity by stimulating the basal activity at lower concentrations and inhibiting the activity at higher concentrations. In addition, BBA inhibited the verapamil-stimulated ABCB1 ATPase activity and the photolabeling of ABCB1 with [(125)I] iodoarylazidoprazosin in a concentration-dependent manner, indicating that BBA directly interacts with ABCB1. The docking study confirmed this notion that BBA could bind to the drug binding site(s) on ABCB1, but its binding position was only partially overlapping with that of verapamil or iodoarylazidoprazosin. Importantly, BBA increased the inhibitory effect of paclitaxel in ABCB1 overexpressing KB-C2 cell xenografts in nude mice. Taken together, our findings suggest that BBA can reverse ABCB1-mediated MDR by inhibiting its efflux function of ABCB1, which supports the development of BBA as a novel potential MDR reversal agent used in the clinic.

Figure 1.

(A) The chemical structure of BBA. BBA inhibited the function of ABCB1. (B) The effect of BBA on the accumulation of DOX (top) and rhodamine123 (bottom) detected by laser scanning confocal microscope. Original amplification, 63; bar, 10 μm. (C) The accumulation of DOX (left) and rhodamine123 (right) in HepG2/ADM cells was analyzed by flow cytometry. (D) The effect of BBA on the efflux of rhodamine123 in HepG2/ADM cells. The mean values are plotted, and the error bars depict standard of deviation from at least three independent experiments performed in triplicate.

Figure 2.

BBA affected the ATP activity, and partly inhibited the photoaffinity labeling of ABCB1 with [I¹²⁵]IAAP. (A) BBA did not affect the mRNA (top) and protein expression (bottom) of multidrug-resistant proteins in HepG2/ADM cells. (B) The localization of ABCB1 at membrane and cytosol of HepG2/ADM cells. Immunofluorescence staining of cells with primary antibody against ABCB1 and Alexa Fluor 488-conjugated secondary antibody was observed by confocal microscope. Original amplification, 63; bar, 10 μm. No immunostaining occurred when the primary antibody was omitted from the procedure (data not shown). (C) The Vi-sensitive ATPase activity of ABCB1 was determined in membrane vesicles as a function of concentration of BBA in the absence (left) and presence of 5 μM verapamil (right). The values and the error bars represent the mean values and standard of deviation from three independent experiments in plot A, while the values in plot B are averages of three independent experiments. (D) The top panel shows the autoradiogram of incorporation of IAAP into the ABCB1 band from a representative experiment. The radioactivity associated with the ABCB1 band was quantified and represented in the lower panel using a phosphorimager and the curve-fitting software GraphPad PRISM. The mean values are plotted, and the error bars depict standard of deviation from at least three independent experiments.

Figure 3.

BBA was not a transported substrate of ABCB1 and had persistent reversal activity. (A) Cytotoxicity of BBA on drug-sensitive HepG2 and drug-resistant HepG2/ADM cells. (B) Effect of verapamil and cyclosporine A on the sensitivity of BBA in ABCB1-overexpressing HepG2/ADM cells. Cells were treated with various concentrations of BBA in the presence or absence of verapamil (5 μM) or cyclosporine A (5 μM) for 48 h, and cytotoxicity of BBA was detected by MTT assay. The mean values are plotted, and the error bars depict standard of deviation from at least three independent experiments. (C) Duration of BBA reversing DOX resistance in HepG2/ADM cells. Cells were treated with or without BBA (1.25 and 5 μM) or verapamil (5 μM) for 24 h before being washed out, then treated with DOX for 48 h. Cytotoxicity of DOX was detected by MTT assay. The mean values are plotted, and the error bars depict standard of deviation from at least three independent experiments.

Figure 4.

Docking analysis of BBA, verapamil, and IAAP toward ABCB1. (A) The binding mode and position of BBA, verapamil, and IAAP with ABCB1. (B) The residual amino acid around BBA. (C) The residual amino acid around BBA, verapamil, and IAAP; key residues were labeled.

Figure 5.

The MDR reversal effect of BBA on KB-C2 xenograft model. Potentiation of antitumor effects of paclitaxel by BBA in ABCB1 overexpressing KB-C2 xenograft model is shown. (A) A representative picture of the excised KB-C2 tumor sizes from different mice is shown on the 18th day after implantation. (B) Changes in tumor volume over the time course of the experiment in ABCB1 overexpressing KB-C2 xenograft model are shown. The treatments were as follows: (a) control (saline, q3d × 6), (b) BBA (15 mg/kg, po, q3d × 6), (c) paclitaxel (18 mg/kg, ip, q3d × 6), and (d) BBA (15 mg/kg, po, q3d × 6) + paclitaxel (18 mg/kg, ip, q3d × 6), BBA was given 2 h before paclitaxel administration. Points represent mean of tumor volume for each group (n = 6) after implantation. Each point on line graph represents the mean of tumor volume (mm³) at a particular day after implantation, and each bar represents SD. *, P < 0.05 versus the control group; #, P < 0.05 versus paclitaxel alone group. (C) The bar graph represents the mean of tumor weights (mice, n = 6) of the excised KB-C2 tumor from different mice. Each column represents the mean of determinations, and the bar represents SD. *, P < 0.05 versus the control group; #, P < 0.05 versus the paclitaxel alone group. (D) Changes in the mean of body weight over the time course of the experiment in ABCB1 overexpressing KB-C2 xenograft model are shown. One pilot study and two independent experiments were carried out using athymic (nu/nu) NCR nude mice implanted sc with KB-C2 cells. A representative result is shown.