Sapitinib Reverses Anticancer Drug Resistance in Colon Cancer Cells Overexpressing the ABCB1 Transporter

Abstract

The efficacy of anti-cancer drugs in patients can be attenuated by the development of multi-drug resistance (MDR) due to ATP-binding cassette (ABC) transporters overexpression. In this in vitro study, we determined the reversal efficacy of the epidermal growth factor receptor (EFGR) inhibitor, saptinib, in SW620 and SW720/Ad300 colon cancer cells and HEK293/ABCB1 cells which overexpress the ABCB1 transporter. Sapitinib significantly increased the efficacy of paclitaxel and doxorubicin in ABCB1 overexpressing cells without altering the expression or the subcellular location of the ABCB1 transporter. Sapitinib significantly increased the accumulation of [³H]-paclitaxel in SW620/AD300 cells probably by stimulating ATPase activity which could competitively inhibit the uptake of [³H]-paclitaxel. Furthermore, sapitinib inhibited the growth of resistant multicellular tumor spheroids (MCTS). The docking study indicated that sapitinib interacted with the efflux site of ABCB1 transporter by π-π interaction and two hydrogen bonds. In conclusion, our study suggests that sapitinib surmounts MDR mediated by ABCB1 transporter in cancer cells.

Keywords: ABCB1; colon cancer; drug resistance; reversal; sapitinib.

Figure 1

The in vitro cytotoxicity of different compounds in ABCB1-overexpressing cells and their corresponding parental cell. (A) The structure of sapitinib. (B) Cell availability of different treatment on SW620 and SW620/Ad300 cells. (C) Cell availability of different treatment on HEK293/pcDNA3.1 and HEK293/ABCB1 cells. *p < 0.05 versus no inhibitor group.

Figure 2

The effect of sapitinib on the in vitro intracellular accumulation of [³H]-paclitaxel in SW620 and SW620/Ad300 cancer cells. (A) The intracellular accumulation of [³H]-paclitaxel in SW620 cancer cells following incubation with sapitinib or verapamil. (B) The intracellular accumulation of [³H]-paclitaxel in SW620/Ad300 cancer cells following incubation with sapitinib and verapamil. The data represent the mean ± SD. *P < 0.05 compared to the control group.

Figure 3

The effect of the incubation of sapitinib on ABCB1 expression in SW620/Ad300 cancer cells. (A) Western blots. (B) Quantification of the Western blot bands.

Figure 4

The effect of sapitinib on the cellular location of ABCB1 in SW620/Ad300 cancer cells. ABCB1-overexpressing SW620/Ad300 cells were seeded in 24-well plate and incubated with or without sapitinib (5 µM) for 0, 24, 48 and 72 h. The cells were then fixed using 4% paraformaldehyde at 37°C for 5 min. Subsequently, the cells were blocked using 6% BSA at 37°C for 1 h and co-cultured with the monoclonal antibody C219 at 4°C overnight. The secondary antibody, Alexa Fluor 488 was added to co-culture with the cells and incubated at 37°C for 1 h. After washing with ice-cold PBS, the nuclei were counterstained with DAPI at 37°C for 10 min. An EVOS^® FL Auto fluorescence microscope was used to detect and capture the immunofluorescence images. SW620 cells were used as a negative control.

Figure 5

The effect of sapitinib on the ATPase activity of ABCB1 transporter and the growth of multicellular tumor spheroids. (A) Effect of sapitinib (0.625–40 μM) on the ATPase activity of ABCB1 transporter in membrane vesicles of High Five insect cells. (B) The effect of the incubation of sapitinib, paclitaxel and the paclitaxel + sapitinib on the growth of SW620/Ad300 MCTSs (sapitinib: 5 μM; paclitaxel: 1 μM). Scale bar = 300 μm. (C) The corresponding relative volume change (V/V0) from control ***P < 0.001 versus Paclitaxel.

Figure 6

An illustration of the interaction between sapitinib and the human ABCB1 transporter protein based on docking analysis.