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Updated: Jun 6, 2026
Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms
Published on: December 9, 2015
Developing multidrug-resistant cells and exploring correlation between BCRP/ABCG2 over-expression and DNA
Nana Ji1, Jianhui Yuan, Jianjun Liu
1Shenzhen University, China.
Abstract:
Expression of breast cancer resistance protein/ATP-binding cassette sub-family G member 2 (BCRP/ABCG2) is the major cause of chemotherapy failure. It is important to establish and characterize the multidrug resistance cells and to investigate the mechanism of multidrug resistance. Multidrug-resistant cells expressing BCRP/ABCG2 based on human breast cancer MCF-7/wt cells were developed by gradually increasing application of low concentration of mitoxantrone. Real-time quantitative PCR, western blot, and immunofluorescence assay were employed to analyze BCRP mRNA and protein expression. Drug accumulation in the cells was measured by flow cytometry and DNA methyltransferases were analyzed by western blot. The results indicated that the inhibitory ratio of cell proliferative growth exhibited an exponential relation with the concentration of mitoxantrone. The IC₅₀ of MCF-7/wt cells to mitoxantrone was found to be 0.42 μM. 3-(4,5-Dimethylthlthiazol-2-YI)-2,5-Diphenyltetrazolium Bromide assay indicated that the mitoxantrone-resistant cells at different stages exhibited cross-resistance to adriamycin and taxol. BCRP/ABCG2 mRNA and protein levels in the mitoxantrone-resistant cells at different stages increased with increasing concentration of mitoxantrone. Intracellular accumulation of mitoxantrone in the cells decreased with the increase of the BCRP/ABCG2 expression levels. DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3a (DNMT3a) expressions in the cells at different stages decreased slightly, whereas DNA methyltransferase 3b (DNMT3b) expression decreases significantly. BCRP/ABCG2 overexpression and its drug-efflux function in the drug-resistant cells are the main factors to produce multidrug resistance. Our results suggest that multidrug resistance is related to overexpression of BCRP/ABCG2 and the decrease of DNA methyltransferases, especially DNMT3b.
Insights
Multidrug resistance in cancer cells is often caused by breast cancer resistance protein (BCRP/ABCG2) overexpression, which reduces chemotherapy drug accumulation. This study links BCRP/ABCG2 upregulation to decreased DNA methyltransferases, particularly DNMT3b, in resistant cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Breast cancer resistance protein/ATP-binding cassette sub-family G member 2 (BCRP/ABCG2) expression is a key factor in chemotherapy failure.
- Understanding multidrug resistance mechanisms is crucial for effective cancer treatment.
Purpose of the Study:
- To develop and characterize multidrug-resistant cells overexpressing BCRP/ABCG2.
- To investigate the relationship between BCRP/ABCG2 expression, drug accumulation, and DNA methyltransferase levels.
Main Methods:
- Development of mitoxantrone-resistant MCF-7 cells.
- Analysis of BCRP/ABCG2 mRNA and protein via real-time PCR and Western blot.
- Assessment of intracellular drug accumulation using flow cytometry.
- Evaluation of DNA methyltransferase expression by Western blot.
Main Results:
- Mitoxantrone-resistant cells showed cross-resistance to adriamycin and taxol.
- BCRP/ABCG2 mRNA and protein levels increased with mitoxantrone resistance.
- Intracellular mitoxantrone accumulation decreased as BCRP/ABCG2 expression rose.
- Significant decrease in DNA methyltransferase 3b (DNMT3b) expression was observed.
Conclusions:
- Overexpression of BCRP/ABCG2 and its drug-efflux activity are primary drivers of multidrug resistance.
- Multidrug resistance is associated with BCRP/ABCG2 upregulation and reduced DNA methyltransferase expression, especially DNMT3b.
