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Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes.

Calcagno AM, Fostel JM, To KK, Salcido CD, Martin SE, Chewning KJ, Wu CP, Varticovski L, Bates SE, Caplen NJ, Ambudkar SV - Br. J. Cancer (2008)

Bottom Line: Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood.RNA interference analysis confirmed that ABCG2 confers drug resistance.This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, DHHS, Bethesda, MD 20892, USA.

ABSTRACT
Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.

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ABCG2 confers resistance to 21 nM single-step doxorubicin-selected clone. (A) Cytotoxicity assays using doxorubicin to evaluate the effect of inhibiting ABCG2 in the 21 nM single-step clone with 5 μM FTC. Dose–response curves were derived from six independent experiments using the CCK-8 assay for 21 nM cells with 5 μM FTC (▵) and without 5 μM FTC (▪). The mean values from six independent experiments are shown with error bars as s.e.m. (B) Western blotting analysis of ABCG2 protein using BXP-21 antibody following no treatment (lane 1), 50 nM negative siRNA treatment (lane 2) and 50 nM G2-2 siRNA treatment (lane 3). (C) Examination of two concentrations of siG2-2 siRNA on silencing of ABCG2. Levels of ABCG2 following siRNA treatment were analysed using the QuantiGene Reagent System (Panomics). Levels were normalised to cyclophilin B (PPIB) mRNA and results reflect the average and s.d. (n=5). (D) Cytotoxicity assays using mitoxantrone to evaluate the effect of silencing ABCG2 in the 21 nM single-step clone. Dose–response curves were derived from six independent experiments using the CCK-8 assay for 21 nM cells with 12.5 nM siG2-2 siRNA (□) and 21 nM cells with 12.5 nM siNeg (▴). The mean values from six independent experiments are shown with error bars as s.e.m.
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fig4: ABCG2 confers resistance to 21 nM single-step doxorubicin-selected clone. (A) Cytotoxicity assays using doxorubicin to evaluate the effect of inhibiting ABCG2 in the 21 nM single-step clone with 5 μM FTC. Dose–response curves were derived from six independent experiments using the CCK-8 assay for 21 nM cells with 5 μM FTC (▵) and without 5 μM FTC (▪). The mean values from six independent experiments are shown with error bars as s.e.m. (B) Western blotting analysis of ABCG2 protein using BXP-21 antibody following no treatment (lane 1), 50 nM negative siRNA treatment (lane 2) and 50 nM G2-2 siRNA treatment (lane 3). (C) Examination of two concentrations of siG2-2 siRNA on silencing of ABCG2. Levels of ABCG2 following siRNA treatment were analysed using the QuantiGene Reagent System (Panomics). Levels were normalised to cyclophilin B (PPIB) mRNA and results reflect the average and s.d. (n=5). (D) Cytotoxicity assays using mitoxantrone to evaluate the effect of silencing ABCG2 in the 21 nM single-step clone. Dose–response curves were derived from six independent experiments using the CCK-8 assay for 21 nM cells with 12.5 nM siG2-2 siRNA (□) and 21 nM cells with 12.5 nM siNeg (▴). The mean values from six independent experiments are shown with error bars as s.e.m.

Mentions: To further evaluate if ABCG2 was conferring resistance to doxorubicin in the 21 nM MCF-7 clone, we performed cytotoxicity assays in the presence and absence of fumitremorgin C (FTC) (Rabindran et al, 2000), a specific inhibitor of ABCG2. With the addition of 5 μM FTC, we were able to reverse the ABCG2-mediated resistance to doxorubicin (Figure 4A) to levels comparable to those of the parental MCF-7 cells (Table 1). The addition of FTC resulted in a 2.1-fold enhancement of the toxicity of doxorubicin on the 21 nM doxorubicin-selected MCF-7 cells. This indicates that the inhibition of ABCG2 results in greater toxicity of doxorubicin in these cells.


Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes.

Calcagno AM, Fostel JM, To KK, Salcido CD, Martin SE, Chewning KJ, Wu CP, Varticovski L, Bates SE, Caplen NJ, Ambudkar SV - Br. J. Cancer (2008)

ABCG2 confers resistance to 21 nM single-step doxorubicin-selected clone. (A) Cytotoxicity assays using doxorubicin to evaluate the effect of inhibiting ABCG2 in the 21 nM single-step clone with 5 μM FTC. Dose–response curves were derived from six independent experiments using the CCK-8 assay for 21 nM cells with 5 μM FTC (▵) and without 5 μM FTC (▪). The mean values from six independent experiments are shown with error bars as s.e.m. (B) Western blotting analysis of ABCG2 protein using BXP-21 antibody following no treatment (lane 1), 50 nM negative siRNA treatment (lane 2) and 50 nM G2-2 siRNA treatment (lane 3). (C) Examination of two concentrations of siG2-2 siRNA on silencing of ABCG2. Levels of ABCG2 following siRNA treatment were analysed using the QuantiGene Reagent System (Panomics). Levels were normalised to cyclophilin B (PPIB) mRNA and results reflect the average and s.d. (n=5). (D) Cytotoxicity assays using mitoxantrone to evaluate the effect of silencing ABCG2 in the 21 nM single-step clone. Dose–response curves were derived from six independent experiments using the CCK-8 assay for 21 nM cells with 12.5 nM siG2-2 siRNA (□) and 21 nM cells with 12.5 nM siNeg (▴). The mean values from six independent experiments are shown with error bars as s.e.m.
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fig4: ABCG2 confers resistance to 21 nM single-step doxorubicin-selected clone. (A) Cytotoxicity assays using doxorubicin to evaluate the effect of inhibiting ABCG2 in the 21 nM single-step clone with 5 μM FTC. Dose–response curves were derived from six independent experiments using the CCK-8 assay for 21 nM cells with 5 μM FTC (▵) and without 5 μM FTC (▪). The mean values from six independent experiments are shown with error bars as s.e.m. (B) Western blotting analysis of ABCG2 protein using BXP-21 antibody following no treatment (lane 1), 50 nM negative siRNA treatment (lane 2) and 50 nM G2-2 siRNA treatment (lane 3). (C) Examination of two concentrations of siG2-2 siRNA on silencing of ABCG2. Levels of ABCG2 following siRNA treatment were analysed using the QuantiGene Reagent System (Panomics). Levels were normalised to cyclophilin B (PPIB) mRNA and results reflect the average and s.d. (n=5). (D) Cytotoxicity assays using mitoxantrone to evaluate the effect of silencing ABCG2 in the 21 nM single-step clone. Dose–response curves were derived from six independent experiments using the CCK-8 assay for 21 nM cells with 12.5 nM siG2-2 siRNA (□) and 21 nM cells with 12.5 nM siNeg (▴). The mean values from six independent experiments are shown with error bars as s.e.m.
Mentions: To further evaluate if ABCG2 was conferring resistance to doxorubicin in the 21 nM MCF-7 clone, we performed cytotoxicity assays in the presence and absence of fumitremorgin C (FTC) (Rabindran et al, 2000), a specific inhibitor of ABCG2. With the addition of 5 μM FTC, we were able to reverse the ABCG2-mediated resistance to doxorubicin (Figure 4A) to levels comparable to those of the parental MCF-7 cells (Table 1). The addition of FTC resulted in a 2.1-fold enhancement of the toxicity of doxorubicin on the 21 nM doxorubicin-selected MCF-7 cells. This indicates that the inhibition of ABCG2 results in greater toxicity of doxorubicin in these cells.

Bottom Line: Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood.RNA interference analysis confirmed that ABCG2 confers drug resistance.This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, DHHS, Bethesda, MD 20892, USA.

ABSTRACT
Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.

Show MeSH
Related in: MedlinePlus