Abstract
Hepatocellular carcinoma is highly chemoresistant, and ATP-binding cassette subfamily G member 2 (ABCG2) is thought to play a critical role in this drug resistance. The present study aims to develop effective therapeutic strategies to decrease ABCG2 expression level and to surmount drug resistance in hepatocellular carcinoma chemotherapy. First, we verified a positive correlation between the ABCG2 protein level and the drug resistance of hepatocellular carcinoma cell lines. ABCG2 was preferentially expressed in highly chemoresistant hepatocellular carcinoma cancer stem cells (CSC) enriched with CD133. In addition, ABCG2 was N-linked glycosylated in hepatocellular carcinoma cells, and this modification was involved in sustaining its protein stability. The N-linked glycosylation (NLG) inhibitor tunicamycin dramatically reduced ABCG2 expression, altered its subcellular localization, and reversed its drug efflux effect in multiple hepatocellular carcinoma cell lines. Furthermore, tunicamycin reduced the expression levels of several CSC markers and suppressed the tumorigenicity of CD133(+) CSCs. Tunicamycin combined with cisplatin (CDDP) inhibited proliferating cell nuclear antigen (PCNA) expression and increased the cleavage of PARP; this effect was partially rescued by the overexpression of ABCG2 or Akt-myr. The combination therapy more effectively suppressed tumor growth in xenograft mice than did single-agent therapy with either drug. Finally, the CDDP treatment combined with UDP-GlcNAc-dolichol-phosphate N-acetylglucosamine-1 phosphate transferase (DPAGT1) knockdown recapitulated the effect observed when CDDP was used in combination with tunicamycin. In summary, our results suggest that tunicamycin may reverse the drug resistance and improve the efficacy of combination treatments for hepatocellular carcinomas by targeting the DPAGT1/Akt/ABCG2 pathway.
Highlights
Resistance to chemotherapy is common in malignant tumors and leads to unsuccessful treatment [1]
Bank; PLC/PRF/5 and HEK 293T were purchased from the American Type Culture Collection; SMMC-7721 was obtained from the Cell Bank of the Institute of Biochemistry and Cell Biology, China Academy of Sciences (Shanghai, China); and human HCC-LY5 was a primary hepatocellular carcinoma cell line established in our laboratory. (This cell line was routinely screened for Mycoplasma species with Mycoplasma Stain Kit purchased from Beyotime; and its purity was confirmed with STR genomic analysis.) All above cell lines used were always less than 40 passages from the stocks tested for purity
Following digestion with the glycosidase PNGase F, Western blotting using an antiABCG2 antibody revealed the appearance of a new band of approximately 60 kDa, showing that ATP-binding cassette subfamily G member 2 (ABCG2) is glycosylated in hepatocellular carcinoma cells (Fig. 1A and B)
Summary
Resistance to chemotherapy is common in malignant tumors and leads to unsuccessful treatment [1]. Hepatocellular carcinoma is the fifth most common cancer worldwide. Hepatocellular carcinoma is highly chemoresistant, leaving patients with this disease with no effective therapeutic options. Numerous lines of evidence imply that a small cellular subpopulation with stem cell–like. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Characteristics, commonly referred to as cancer stem cells (CSC), is critical for tumorigenicity and tumor maintenance [2]. CSCs appear to be protected from routine chemotherapeutic drugs through various mechanisms, such as high expression levels of ATP-binding cassette (ABC) drug transporters [3]. Drug-resistant CSCs have been shown to survive treatment with several widely used chemotherapeutic agents [4, 5]
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