Elacridar Inhibits BCRP Protein Activity in 2D and 3D Cell Culture Models of Ovarian Cancer and Re-Sensitizes Cells to Cytotoxic Drugs
Abstract
1. Introduction
2. Results
2.1. The Drug Resistance Characterization of the A2780 Cell Line and TOP-Resistant Cell Lines
2.2. Characterization of BCRP Protein Expression in A2780 Cell Line and TOP-Resistant Cell Lines
2.3. BCRP Activity Analysis
2.4. Analysis of How Elacridar Affects the Resistance to Cytotoxic Drugs
2.5. BCRP Protein Expression After Elacridar Treatment
2.6. BCRP Protein Activity After Elacridar Treatment in 2D Culture
2.7. Analysis of Elacridar’s Effect in a 3D Model
3. Discussion
4. Materials and Methods
4.1. Reagents and Antibodies
4.2. Cell Lines and Cell Culture
4.3. Isolation of the RNA, cDNA Synthesis, and qPCR
4.4. Treatment of Cells with Elacridar
4.5. Protein Isolation and Western Blot Analysis
4.6. Immunofluorescence
4.7. Live-Cell Immunofluorescence (H33342 Accumulation)
4.8. Flow Cytometry Analysis
4.9. Analysis of MIT and H33342 Accumulation in Spheroids
4.10. 2D MTT Assay
4.11. 3D MTT Assay
4.12. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ABC | ATP-Binding Cassette |
ALS | Amyotrophic Lateral Sclerosis |
AML | Acute Myeloid Leukemia |
BCRP | Breast Cancer Resistance Protein |
CIS | Cisplatin |
CML | Chronic Myeloid Leukemia |
CNS | Central Nervous System |
cDNA | Complementary DNA |
DOX | Doxorubicin |
DPBS | Dulbecco’s Phosphate-Buffered Saline |
ECM | Extracellular Matrix |
EOC | Epithelial Ovarian Cancer |
FDA | Food and Drug Administration |
H33342 | Hoechst 33342 |
HCC | Hepatocellular Carcinoma |
MIT | Mitoxantrone |
MDR | Multidrug Resistance |
MSD | Membrane-Spanning Domain |
NBD | Nucleotide-Binding Domain |
P-gp | P-glycoprotein |
PAC | Paclitaxel |
PBS | Phosphate-Buffered Saline |
PhIP | 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine |
RNA | Ribonucleic Acid |
RT | Reverse Transcription |
SCLC | Small Cell Lung Cancer |
TM | Transmembrane |
TOP | Topotecan |
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Cell Line | TOP IC50 (ng/mL) | MIT IC50 (ng/mL) | CIS IC50 (ng/mL) |
---|---|---|---|
A2780 | 9.79 (3.93–17.4) 1.00 | 2.98 (1.61–4.99) 1.00 | 11,259 (5719–17,384) 1.00 |
A2780TR1 | 200.68 (140.79–241.43) 20.50 ↑** | 9.60 (8.52–11.8) 3.22 ↑*** | 12,213 (7330–17,583) 1.08 ↑ |
A2780TR2 | 132.00 (48.27–229.44) 13.48 ↑** | 9.65 (8.10–13.7) 3.24 ↑*** | 12,625 (5528–22,025) 1.12 ↑ |
Cell Line | Control TOP IC50 (ng/mL) | Elacridar 0.1 μM TOP IC50 (ng/mL) | Elacridar 1 μM TOP IC50 (ng/mL) | Elacridar 2 μM TOP IC50 (ng/mL) | Elacridar 5 μM TOP IC50 (ng/mL) |
---|---|---|---|---|---|
A2780 | 9.79 (3.93–17.43) 1.00 | 6.69 (4.53–18.49) 1.46 ↓ | 10.69 (4.85–17.28) 1.09 ↑ | 11.18 (5.21–15.3) 1.14 ↑ | 8.32 (5.19–9.77) 1.17 ↓ |
A2780TR1 | 204.68 (140.79–241.43) 1.00 | 18.81 (13.64–22.04) 10.88 ↓*** | 15.29 (8.27–21.4) 13.38 ↓*** | 14.99 (8.34–18.4) 13.65 ↓*** | 12.05 (4.86–18.85) 16.98 ↓*** |
A2780TR2 | 132.00 (48.27–229.44) 1.00 | 19.11 (9.29–35.64) 6.91 ↓** | 11.11 (6.78–15.54) 11.88 ↓*** | 10.02 (6.03–15.05) 13.17 ↓*** | 7.61 (6.23–9.52) 17.59 ↓*** |
Cell Line | Control MIT IC50 (ng/mL) | Elacridar 0.1 μM MIT IC50 (ng/mL) | Elacridar 1 μM MIT IC50 (ng/mL) | Elacridar 2 μM MIT IC50 (ng/mL) | Elacridar 5 μM MIT IC50 (ng/mL) |
---|---|---|---|---|---|
A2780 | 2.98 (1.61–4.99) 1.00 | 2.56 (1.80–3.57) 1.16 ↓ | 3.08 (2.07–4.22) 1.03 ↑ | 2.54 (0.91–4.20) 1.17 ↑ | 2.55 (0.95–4.32) 1.17 ↑ |
A2780TR1 | 9.60 (8.52–11.8) 1.00 | 6.73 (5.20–8.12) 1.43 ↓* | 5.25 (4.09–6.29) 1.85 ↓*** | 2.44 (0.90–4.48) 3.93 ↓*** | 2.71 (0.93–4.81) 3.55 ↓*** |
A2780TR2 | 9.65 (8.10–13.7) 1.00 | 7.32 (6.48–8.19) 1.32 ↓** | 7.40 (6.08–8.39) 1.30 ↓* | 5.10 (0.98–8.19) 1.90 ↓* | 5.00 (1.37–8.32) 1.93 ↓* |
Cell Line | Control CIS IC50 (ng/mL) | Elacridar 0.1 μM CIS IC50 (ng/mL) | Elacridar 1 μM CIS IC50 (ng/mL) | Elacridar 2 μM CIS IC50 (ng/mL) | Elacridar 5 μM CIS IC50 (ng/mL) |
---|---|---|---|---|---|
A2780 | 11,259 (5719–17,384) 1.00 | 9228 (5174–16,051) 1.22 ↓ | 9470 (6001–14,572) 1.19 ↓ | 11,494 (6232–16,100) 1.02 ↑ | 11,089 (6489–14,751) 1.01 ↓ |
A2780TR1 | 12,213 (7330–17,583) 1.00 | 12,599 (8247–19,394) 1.03 ↑ | 11,430 (6418–19,332) 1.06 ↓ | 16,123 (8281–21,191) 1.32 ↑ | 13,686 (10,139–16,732) 1.12 ↑ |
A2780TR2 | 12,625 (5528–22,025) 1.00 | 13,744 (7392–18,169) 1.08 ↑ | 13,443 (6763–18,048) 1.08 ↑ | 11,971 (5840–15,251) 1.06 ↓ | 9045 (5325–11,274) 1.39 ↓ |
Cell Line | Control TOP IC50 (ng/mL) | Elacridar 1 μM TOP IC50 (ng/mL) | Elacridar 5 μM TOP IC50 (ng/mL) |
---|---|---|---|
A2780 | 17.61 (7.46–32.48) 1.00 | 11.71 (7.92–18.93) 1.49 ↓ | 10.19 (8.00–12.22) 1.73 ↓ |
A2780TR1 | 447.80 (159.01–774.23) 1.00 | 15.10 (9.26–20.36) 29.66 ↓** | 9.27 (8.70–9.97) 48.31 ↓** |
A2780TR2 | 404.38 (237.89–601.68) 1.00 | 62.63 (22.30–155.11) 6.46 ↓** | 16.02 (8.46–29.91) 25.24 ↓** |
Transcript | Sequence (5′–3′ Direction) Forward | Sequence (5′–3′ Direction) Reverse | ENST Number http://www.ensembl.org | Product Size (bp) |
---|---|---|---|---|
BCRP | TTCGGCTTGCAACAACTATG | TCCAGACACACCACGGATAA | 00000237612 | 128 |
GAPDH | GAAGGTGAAGGTCGGAGTCA | GACAAGCTTCCCGTTCTCAG | 00000229239 | 199 |
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Stasiak, P.; Sopel, J.; Płóciennik, A.; Musielak, O.; Lipowicz, J.M.; Rawłuszko-Wieczorek, A.A.; Sterzyńska, K.; Korbecki, J.; Januchowski, R. Elacridar Inhibits BCRP Protein Activity in 2D and 3D Cell Culture Models of Ovarian Cancer and Re-Sensitizes Cells to Cytotoxic Drugs. Int. J. Mol. Sci. 2025, 26, 5800. https://doi.org/10.3390/ijms26125800
Stasiak P, Sopel J, Płóciennik A, Musielak O, Lipowicz JM, Rawłuszko-Wieczorek AA, Sterzyńska K, Korbecki J, Januchowski R. Elacridar Inhibits BCRP Protein Activity in 2D and 3D Cell Culture Models of Ovarian Cancer and Re-Sensitizes Cells to Cytotoxic Drugs. International Journal of Molecular Sciences. 2025; 26(12):5800. https://doi.org/10.3390/ijms26125800
Chicago/Turabian StyleStasiak, Piotr, Justyna Sopel, Artur Płóciennik, Oliwia Musielak, Julia Maria Lipowicz, Agnieszka Anna Rawłuszko-Wieczorek, Karolina Sterzyńska, Jan Korbecki, and Radosław Januchowski. 2025. "Elacridar Inhibits BCRP Protein Activity in 2D and 3D Cell Culture Models of Ovarian Cancer and Re-Sensitizes Cells to Cytotoxic Drugs" International Journal of Molecular Sciences 26, no. 12: 5800. https://doi.org/10.3390/ijms26125800
APA StyleStasiak, P., Sopel, J., Płóciennik, A., Musielak, O., Lipowicz, J. M., Rawłuszko-Wieczorek, A. A., Sterzyńska, K., Korbecki, J., & Januchowski, R. (2025). Elacridar Inhibits BCRP Protein Activity in 2D and 3D Cell Culture Models of Ovarian Cancer and Re-Sensitizes Cells to Cytotoxic Drugs. International Journal of Molecular Sciences, 26(12), 5800. https://doi.org/10.3390/ijms26125800