Characterization of SN38-resistant T47D breast cancer cell sublines overexpressing BCRP, MRP1, MRP2, MRP3, and MRP4
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Hee-Jeong Lee1, Cheol-Hee Choi2,3
1Department of Hemato-Oncology, Chosun University Hospital, Gwangju, 501-759, Korea.
Background:
Although several novel resistant breast cancer cell lines have been established, only a few resistant breast cancer cell lines overexpress breast cancer resistance proteins (BCRP). The aim of this study was to establish new resistant breast cancer cell lines overexpressing BCRP using SN38 (7-ethyl-10-hydroxycamptothecin), an active metabolite of irinotecan and was to discover genes and mechanisms associated with multidrug resistance.
Methods:
SN38-resistant T47D breast cancer cell sublines were selected from the wild-type T47D cells by gradually increasing SN38 concentration. The sensitivity of the cells to anti-cancer drugs was assessed by 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Expression profiles of the resistance-related transporters were examined using RT-qPCR, and western blot analysis. Intracellular fluorescent dye accumulation in the resistant cells was determined using flow cytometry.
Results:
The SN38-resistant T47D breast cancer cell sublines T47D/SN120 and T47D/SN150 were established after long-term exposure (more than 16 months) of wild-type T47D cells to 120 nM and 150 nM SN38, respectively. T47D/SN120 and T47D/SN150 cells were more resistant to SN38 (14.5 and 59.1 times, respectively), irinotecan (1.5 and 3.7 times, respectively), and topotecan (4.9 and 12 times, respectively), than the wild-type parental cells. Both T47D/SN120 and T47D/SN150 sublines were cross-resistant to various anti-cancer drugs. These resistant sublines overexpressed mRNAs of MRP1, MRP2, MRP3, MRP4, and BCRP. The DNA methylase inhibitor 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A increased the expression levels of BCRP, MRP1, MRP2, MRP3, and MRP4 transcripts in T47D/WT cells. Fluorescent dye accumulation was found to be lower in T47D/SN120 and T47D/SN150 cells, compared to that in T47D/WT cells. However, treatment with known chemosensitizers increased the intracellular fluorescent dye accumulation and sensitivity of anti-tumor agents.
Conclusion:
T47D/SN120 and T47D/SN150 cells overexpressed MRP1, MRP2, MRP3, MRP4, and BCRP, which might be due to the suppression of epigenetic gene silencing via DNA hypermethylation and histone deacetylation. Although these resistant cells present a higher resistance to various anti-cancer drugs than their parental wild-type cells, multidrug resistance was overcome by treatment with chemosensitizers. These SN38 resistant T47D breast cancer cell sublines expressing resistance proteins can be useful for the development of new chemosensitizers.
Insights
New breast cancer cell lines resistant to SN38 were developed, overexpressing multiple drug resistance proteins (MRP1-4 and BCRP). These cells show cross-resistance to various drugs but can be resensitized with chemosensitizers.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Few established breast cancer cell lines overexpress breast cancer resistance proteins (BCRP).
- Developing cell lines with BCRP overexpression is crucial for studying multidrug resistance mechanisms.
Purpose of the Study:
- To establish new SN38-resistant breast cancer cell lines overexpressing BCRP.
- To identify genes and mechanisms underlying multidrug resistance in breast cancer.
Main Methods:
- SN38-resistant T47D breast cancer cell sublines were generated by gradual SN38 exposure.
- Drug sensitivity was assessed using MTT assays; transporter expression was analyzed via RT-qPCR and Western blot.
- Intracellular dye accumulation was measured using flow cytometry.
Main Results:
- Two SN38-resistant sublines, T47D/SN120 and T47D/SN150, were established, exhibiting significant resistance to SN38, irinotecan, and topotecan.
- These sublines showed cross-resistance to multiple anti-cancer drugs and overexpressed MRP1, MRP2, MRP3, MRP4, and BCRP.
- Epigenetic modifiers increased transporter expression, and chemosensitizers restored drug sensitivity and dye accumulation.
Conclusions:
- Established T47D sublines overexpress MRP1-4 and BCRP, potentially due to suppressed epigenetic silencing.
- The developed cell lines demonstrate significant multidrug resistance, which can be overcome by chemosensitizers.
- These BCRP-overexpressing, SN38-resistant cell lines are valuable tools for developing novel chemosensitizers.
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