ABCB1 (MDR1) induction defines a common resistance mechanism in paclitaxel- and olaparib-resistant ovarian cancer cells

Abstract

Background: Clinical response to chemotherapy for ovarian cancer is frequently compromised by the development of drug-resistant disease. The underlying molecular mechanisms and implications for prescription of routinely prescribed chemotherapy drugs are poorly understood.

Methods: We created novel A2780-derived ovarian cancer cell lines resistant to paclitaxel and olaparib following continuous incremental drug selection. MTT assays were used to assess chemosensitivity to paclitaxel and olaparib in drug-sensitive and drug-resistant cells±the ABCB1 inhibitors verapamil and elacridar and cross-resistance to cisplatin, carboplatin, doxorubicin, rucaparib, veliparib and AZD2461. ABCB1 expression was assessed by qRT-PCR, copy number, western blotting and immunohistochemical analysis and ABCB1 activity assessed by the Vybrant and P-glycoprotein-Glo assays.

Results: Paclitaxel-resistant cells were cross-resistant to olaparib, doxorubicin and rucaparib but not to veliparib or AZD2461. Resistance correlated with increased ABCB1 expression and was reversible following treatment with the ABCB1 inhibitors verapamil and elacridar. Active efflux of paclitaxel, olaparib, doxorubicin and rucaparib was confirmed in drug-resistant cells and in ABCB1-expressing bacterial membranes.

Conclusions: We describe a common ABCB1-mediated mechanism of paclitaxel and olaparib resistance in ovarian cancer cells. Optimal choice of PARP inhibitor may therefore limit the progression of drug-resistant disease, while routine prescription of first-line paclitaxel may significantly limit subsequent chemotherapy options in ovarian cancer patients.

Figure 1

Cross-resistance of paclitaxel- and olaparib-resistant ovarian cancer cell lines. Novel ovarian cancer A2780-derived paclitaxel (A2780pacR) and olaparib (A2780olapR) resistant cell lines were created as described in Materials and Methods section. MTT chemosensitivity assays were used to compare cell viability following exposure to serial dilutions of (A and D) paclitaxel and (B and C) olaparib in A2780 and A2780pacR (A and C) and A2780olapR (B and D) cells. Chemosensitivity values obtained from triplicate replicate experiments, expressed as IC₅₀ values, are summarised in panel E.

Figure 2

ABCB1 (P-glycoprotein) expression is increased in A2780pacR and A2780olapR cells. ABCB1 (A) mRNA expression, (B) copy number and (C and D) P-glycoprotein expression was compared in A2780, A2780pacR and A2780olapR cells by qRT-PCR analysis, Taqman-based copy number analysis, western blotting and immunohistochemical analysis, respectively, as described in Materials and Methods section.

Figure 3

Paclitaxel and olaparib resistance are reversible following combination treatment with the P-glycoprotein inhibitors verapamil and elacridar. MTT chemosensitivity assays were used as described in Materials and Methods section to compare cell viability following exposure to serial dilutions of paclitaxel and olaparib in A2780, A2780pacR and A2780olapR cells in the presence and absence of combination treatments (5 μM and 10 μM) with the P-glycoprotein inhibitors (A–C) verapamil and (D–F) elacridar.

Figure 4

P-glycoprotein-mediated drug efflux is altered in paclitaxel- and olaparib-resistant cells. (A) Vybrant Multidrug Resistance Assays were used, as described in Materials and Methods section, to compare P-glycoprotein-dependent efflux of the model substrate Calcein-AM in untreated A2780, A2780pacR and A2780olapR cells and (B) in the presence of 0–25 μM verapamil. Results were confirmed (C) using the P-glycoprotein-Glo Assay System, as described in Materials and Methods section, to demonstrate dose-dependent increases in luminescence following the incubation of paclitaxel or olaparib with P-glycoprotein-containing membranes.

Figure 5

Paclitaxel- and olaparib-resistant cells are cross-resistant to doxorubicin and rucaparib but not to AZD2461 or veliparib. MTT chemosensitivity assays were used as described in Materials and Methods section to compare cell viability in A2780, A2780pacR and A2780olapR cells following exposure to serial dilutions of (A and B) doxorubicin, (C and D) AZD2461, (E and F) veliparib and (G and H) rucaparib. Chemosensitivity values obtained from triplicate replicate experiments, expressed as IC₅₀ values, are summarised in Panel I.

Figure 6

The PARP inhibitors olaparib and rucaparib are P-glycoprotein substrates, while the alternative PARP inhibitors AZD2461 and veliparib are not actively effluxed. The P-glycoprotein-Glo Assay System was used, as described in Materials and Methods section, to investigate whether dose-dependent increases in luminescence were observed following the incubation of verapamil (positive control), olaparib, veliparib, rucaparib or AZD2461 with P-glycoprotein-containing membranes.