Data showing the circumvention of oxaliplatin resistance by vatalanib in colon cancer

Abstract

We have recently reported that vatalanib, an orally active small molecule multi-tyrosine kinase inhibitor (Hess-Stumpp et al., 2005 [1]), can sensitize multidrug resistant (MDR) colon cancer cells to chemotherapy under hypoxia by inhibiting two MDR transporters ABCB1 and ABCG2 (To et al., 2015 [2]). This data article describes the possible circumvention of resistance to specifically platinum (Pt)-based anticancer drugs by vatalanib via inhibition of two other efflux transporters ABCC2 and ATP7A. Data from the flow cytometric transporter efflux assay showed specific inhibition of ABCC2 activity by vatalanib in stable transfected cells and ABCC2-overexpressing oxaliplatin-resistant colon cancer cells HCT116/Oxa. We also performed the transporter ABCC2 ATPase assay and showed an increase in ATP hydrolysis by ABCC2 in the presence of vatalanib. ATP7A mRNA expression was also shown to be upregulated in HCT116/Oxa cells. Vatalanib was shown to suppress this upregulated ATP7A expression. Data from the cellular Pt accumulation assay showed a lower Pt accumulation in HCT116/Oxa cells than the parental sensitive HCT116 cells. Vatalanib was shown to increase cellular Pt accumulation in a concentration-dependent manner. Combination of oxaliplatin and vatalanib was shown to restore the suppressed apoptosis in HCT116/Oxa cells.

Keywords: Chemoresistance; Oxaliplatin; Platinum drugs; Tyrosine kinase inhibitor; Vatalanib.

Fig. 1

Vatalanib sensitized oxaliplatin-resistant HCT116 to oxaliplatin-induced apoptosis. (A) HCT116 parental cell line and its oxaliplatin-resistant subline were exposed to oxaliplatin (10 μM), vatalanib (2 μM) alone, or their combination for 48 h before harvest for apoptosis assay. A representative set of data from three independent experiments is shown. (B) Summary of apoptosis assay data from three independent experiments. Data are presented in histogram as means±SD.

Fig. 2

mRNA expression of ABCC2 (A) and ATP7A (B) in parental HCT116 and oxaliplatin-resistant HCT116/Oxa cells treated with or without vatalanib (1 or 2 μM, 24 h). Relative mRNA expression of the transporters are shown after normalization with β-actin. The expression in the parental HCT116 cells was set as 10 for comparison. * p<0.05, compared with untreated cells. (C) Real-time RT-PCR analysis of the downregulation of ATP7B in HCT116/Oxa cells by vatalanib with or without a 4-h pretreatment of actinomycin D (5 μg/mL).

Fig. 3

mRNA expression of other transporters implicated in Pt resistance: ABCC6 (A), ATP7B (B) and LRP (C) in parental HCT116 and oxaliplatin-resistant HCT116/Oxa cells treated with or without vatalanib (1 or 2 μM, 24 h). Relative mRNA expression of these efflux transporters are shown after normalization with β-actin. The expression in the parental HCT116 cells was set as 10 for comparison. No statistical significant difference was observed between the treatment groups.

Fig. 4

Inhibition of ABCC2-mediated efflux of fluorescent probe substrate CDCF by vatalanib in ABCC2-overexpressing HCT116/Oxa cells (A) and ABCC2 stably-transfected HEK293/ABCC2 cells (B). Data obtained in the HCT116 parental cells and the backbone vector transfected HEK293/pcDNA3 cells is also shown as control for comparison. Top panel: Representative efflux histogram from three independent experiments are shown. Bottom panel: The relative fluorescent probe retention was also quantified by setting the value in transporter-overexpressing cells in the absence of vatalanib as 10.

Fig. 5

Effect of vatalanib on the ATPase activity of ABCC2. The vanadate-sensitive ATPase activity of ABCC2 in the recombinant transporter proteins obtained from cell membrane fraction was determined at different concentrations of vatalanib. ATP hydrolysis was monitored by measuring the amount of inorganic phosphate released using a colorimetric assay. In the presence of vatalanib, all of the stimulated ABCC2 ATPase activity was significantly different from the basal level (p<0.05).

Fig. 6

Cellular Pt accumulation in oxaliplatin (100 μM, 4 h)-incubated HCT116 and HCT116/Oxa cells in the presence of 1 or 2 μM vatalanib. In pilot experiments, drug accumulation was found to be increased in a linear manner with increasing drug concentration up to at least 400 μM. Cell lysates obtained were subjected to concentrated HNO₃ digestion before analysis by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). The absorbance of Pt at 265.95 nm was used for quantification. Protein concentration of the cell lysate was measured separately by the Bradford method for normalization purpose.