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

Wang Yin,Yingchun Hou,Yinglu Han,Phuong H.-L Tran,Liang Qiao,Cuong V Pham,Guoqin Jiang,Wei Duan,Dongxi Xiang,Yimin Zhu,Yumei Zhang,Tao Wang,Shufeng Zhou

doi:10.1038/s41598-021-89931-9

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.

Highlights

Abbreviations HCC Hepatocellular carcinoma liver cancer stem cells (LCSCs) Liver cancer stem cells CSC Cancer stem cells DOX Doxorubicin ATP binding cassette (ABC) transporter ATP‐binding cassette transporter ABCB1/MDR1 ATP-binding cassette sub-family B member 1/multidrug resistance protein 1 ABCG2/BCRP ATP-binding cassette sub-family G member 2/breast cancer resistance protein forward scatter (FSC) Forward scatter
There is a paucity of experimental evidence on the overexpression of ABC superfamily transporters in LCSCs, which can be phenotypically defined as the cells expressing both EpCAM and CD133 ( EPCAM+–CD133+) in Huh[7] and PLC/PRF/5 human HCC cells[21,22,23,24,25,26,27]
We explored the possibility of transforming a traditional chemotherapy drug into a CSC killer via the inhibition of MDR1 and ABCG2 in HCC cells, with Huh[7] and PLC/PRF/5 cells as a model

Summary

Introduction

Abbreviations HCC Hepatocellular carcinoma LCSC Liver cancer stem cells CSC Cancer stem cells DOX Doxorubicin ABC transporter ATP‐binding cassette transporter ABCB1/MDR1 ATP-binding cassette sub-family B member 1/multidrug resistance protein 1 ABCG2/BCRP ATP-binding cassette sub-family G member 2/breast cancer resistance protein FSC Forward scatter. DOX used in both systemic and local therapies for HCC has limited efficacy as it kills the tumor bulk but fails to eliminate the cancer stem cell population of HCC. Two HCC cell lines Huh[7] and PLC/PRF/5 were used to represent HCC cells with different expression patterns of MDR1 and ABCG2, as well as the differential expression of LCSC markers (epithelial adhesion molecule (EpCAM) and CD133). In addion, these two cell lines represent different pathological backgrounds in that PLC/PRF/5 cells were derived from a patient infected with HBV whereas Huh[7] cells are HBV-free. As long as sufficient intracellular concentration is achieved, DOX is able to eliminate LCSCs, in sharp contrast to the popular belief that chemotherapeutic agents are generally unable to eradicate C SC18,19

Methods

Results

Conclusion