The inhibition of ABCB1/MDR1 or ABCG2/BCRP enables doxorubicin to eliminate liver cancer stem cells

Abstract

Two ATP-binding cassette transporters, ABCB1/MDR1 and ABCG2/BCRP, are considered the most critical determinants for chemoresistance in hepatocellular carcinoma. However, their roles in the chemoresistance in liver cancer stem cells remain elusive. Here we explored the role of inhibition of MDR1 or ABCG2 in sensitizing liver cancer stem cells to doxorubicin, the most frequently used chemotherapeutic agent in treating liver cancer. We show that the inhibition of MDR1 or ABCG2 in Huh7 and PLC/PRF/5 cells using either pharmacological inhibitors or RNAi resulted in the elevated level of intracellular concentration of doxorubicin and the accompanied increased apoptosis as determined by confocal microscopy, high-performance liquid chromatography, flow cytometry, and annexin V assay. Notably, the inhibition of MDR1 or ABCG2 led to the reversal of the chemoresistance, as evident from the enhanced death of the chemoresistant liver cancer stem cells in tumorsphere-forming assays. Thus, the elevation of effective intracellular concentration of doxorubicin via the inhibition of MDR1 or ABCG2 represents a promising future strategy that transforms doxorubicin from a traditional chemotherapy agent into a robust killer of liver cancer stem cells for patients undergoing transarterial chemoembolization.

Figure 1

Expression of MDR1 and ABCG2 in the liver cancer stem cell (LCSC) population of Huh7 and PLC/PRF/5 cells. (a) The gating of viable cells using forward scatter (FSC) and side scatter (SSC). (b) The percentage of MDR1⁺ and ABCG2⁺ expression in the bulk tumor population. (c1,d1) The percentage of LCSC markers EpCAM and CD133 in the bulk tumor population. The cells shown in (c1) and (d1) were divided into four quadrants (Q1, Q2, Q3 and Q4). (c2–c5,d2–d5) The expression of MDR1 and ABCG2 in the cells from Q1, Q2, Q3 and Q4 of (c1) and (d1) was determined. The Huh7 and PLC/PRF/5 cells were stained with IgG isotype-matched control antibodies of (e) EpCAM and CD133 and (f) MDR1 and ABCG2. Horizontal and vertical axes denote expression intensity. One representative experiment of three is shown.

Figure 2

Robust extrusion of doxorubicin (DOX) by EpCAM⁺–CD133⁺ population of Huh7 and PLC/PRF/5 cells. The Huh7 or PLC/PRF/5 cells were treated with 200 nM or 100 nM of DOX for 24 h, respectively. The percentage of DOX fluorescence-positive cells (a), as well as the intracellular DOX fluorescence expressed as the median fluorescence intensity (b) in the bulk and the EpCAM⁺–CD133⁺ population of Huh7 and PLC/PRF/5 cells, were determined using flow cytometry. Data shown are means ± SD, (n = 3). ****p < 0.0001; ***p < 0.001; **p < 0.01; compared with the bulk population of Huh7 and PLC/PRF/5 cells respectively.

Figure 3

Involvement of MDR1 and ABCG2 in doxorubicin (DOX) efflux as determined by pharmacological inhibitors. The Huh7 or PLC/PRF/5 cells were treated with 200 nM or 100 nM of DOX in the presence or absence of MDR1 inhibitor valspodar (1 µM) or ABCG2 inhibitor ko143 (1 µM) for 24 h followed by flow cytometric analysis. (a) Percentage of DOX-positive cells; (b) DOX intensity followed by indicated treatments. Data shown are means ± SD, (n = 3). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; compared with DOX treatment.

Figure 4

The downregulation of ABCG2 or MDR1 via RNAi and the intracellular concentration of doxorubicin (DOX). Cells were treated with siRNAs (20 nM) against MDR1 or ABCG2 for 6 h followed by a further 42 h culture in the fresh culture medium. Then the Huh7 cells or PLC/PRF/5 cells were treated with 200 nM or 100 nM of DOX for 24 h. The intracellular retention of DOX measured as (a) the percentage of DOX-positive cells or (b) their intracellular DOX fluorescence intensity was determined via flow cytometry in the bulk Huh7 and PLC/PRF/5 cells as well as in their EpCAM⁺–CD133⁺ counterparts. Data shown are means ± SD, (n = 3). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; compared with DOX treatment.

Figure 5

The effect of inhibition of MDR1 and ABCG2 on DOX-induced apoptosis. The cells were treated with 1 µM of MDR1 inhibitor valspodar or 1 µM of ABCG2 inhibitor ko143, and doxorubicin (DOX) (200 nM for Huh7 and 100 nM for PLC/PRF/5 cells, respectively) for 24 h. Alternatively, the cells were treated with 20 nM siRNA to MDR1 or ABCG2 for 6 h, followed by a further 42 h culture in the fresh culture medium. Then the Huh7 cells or PLC/PRF/5 cells were treated with 200 nM or 100 nM of DOX for 24 h. The total percentage of apoptotic cells as defined by the combination of 7-AAD⁻/Annexin V⁺ and 7-AAD⁺/Annexin V⁺ cells are shown for (a) bulk Huh7 cells, (b) bulk PLC/PRF/5 cells, (c) EpCAM⁺–CD133⁺ Hun7 cells as well as (d) EpCAM⁺-CD133⁺ PLC/PRF/5 cells. Data shown are mean ± SD, n = 3. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; compared with DOX-only treatment.

Figure 6

Effect of the inhibition of MDR1 and ABCG2 on the capability of doxorubicin (DOX) in the elimination of cancer stem cells in vitro. Huh7 or PLC/PRF/5 cells were treated with either doxorubicin (DOX) alone or treated with inhibitors or siRNA to MDR1 or ABCG2 first, followed by DOX treatment as described in “Materials and methods” section. Cells were then plated in ultra-low attachment plates at a density of 10, 20, or 50 cells/well and incubated for 7 days under the condition of in vitro limiting dilution assay to assess the self-renewal capacity of treated cells. The frequency of tumorsphere-forming cells for Huh7 (a) or PLC/PRF/5 cells (b) under indicated treatment is shown. Data presented are mean ± SD, n = 3. *p < 0.05; ***p < 0.001.