Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: Comparative analysis of the response of A2780 and A2780ADR cells to treatment with anticancer drugs (Doxo, Eto and CisPt) and effect of mechanisms of drug transport. Logarithmically growing parental A2780 and A2780ADR variant cells were treated with the anticancer drugs (A) Doxo, (B) Eto and (C) CisPt at the indicated concentrations. At 72 h after drug addition, viability was monitored by use of the AlamarBlue assay as described in methods. Data shown are the mean ± SD from three independent experiments each performed in biological quadruplicates (n=3; n=4). Dashed lines indicate inhibitory concentrations (IC 20 and IC 50 ). For viability data (IC 50 ) after 24 and 72 h of treatment also see and . (D) Comparative analysis of the mRNA expression of selected transporters in A2780 and A2780ADR. Data shown are the mean ± SD from triplicate determinations. mRNA expression of transporters was normalized to GAPDH mRNA levels and set to 1.0 in the parental A2780 cells. The dashed lines indicate changes in mRNA levels of ≥2.0 and ≤0.5, which are considered as biologically relevant. (E) Comparative analysis of the protein expression of representative drug transporters under basal situation and after 24 h treatment with Doxo (0.1 and 1.0 μ M). Data shown are from a representative western blotting using ERK2 protein levels as loading control. Data obtained after 72 h are presented in (left panel). (F) Intracellular Doxo fluorescence was measured by flow cytometry-based methods after 2 h Doxo pulse-treatment (0.25 and 1.0 μ M) and was taken as indicative of drug import. To measure drug export, Doxo pulse-treated cells were post-incubated for 6 h in the absence of the drug before fluorescence was monitored. Data shown in the left panel are representative results obtained from flow cytometry analyses. C, control; I, import, E, export. The histogram in the right panel depicts quantitative data obtained from n=3 independent experiments each performed in biological triplicates (n=3). *** P≤0.001. (G) Analysis of basal mRNA expression of topoisomerase II isoforms TOP2A, TOP2B and TopBP1. Data shown are the mean ± SD from triplicate determinations. Relative mRNA level in A2780 cells was set to 1.0. The dashed lines indicate changes in mRNA levels of ≥2.0 and ≤0.5, which are considered as biologically relevant. (H) Comparative analysis of the protein expression of TOP2A and TopBP1 under basal situation and after 24 h treatment with Doxo (0.1 μ M, 1.0 μ M). Data shown are from a representative western blotting using ERK2 as protein loading control. Data obtained after 72 h Dox treatment are presented in (right panel). Doxo, doxorubicin; Eto, etoposide; CisPt, cisplatin; SD, standard deviation; Nd, not detectable TOP2A, topoisomerase IIα; TOP2B, topoisomerase IIβ; TOPBP1, topoisomerase binding protein 1; MDR1, multi-drug resistance gene 1; ATP7A, copper transporting ATPase; OCT2, organic cation transporter-2; CTR1, copper uptake protein 1; Topo IIa, topoisomerase IIα; ERK2, extracellular regulated kinase 2.

Article Snippet: The following primary antibodies were used: ATP7A (cat. no. PA5-103110; Invitrogen; Thermo Fisher Scientific; Inc.), Caspase-7 cleaved (Asp198; cat. no. 9491S; Cell Signaling Technology, Inc.), Chk1phospho (Ser345; cat. no. 2341, Cell Signaling Technology, Inc.), Chk2phosphoT68 (Y171; cat. no. ab32148; Abcam), CTR1/SLC31A1 (EPR7936; cat. no. ab129067; Abcam), Cyclin B1 (cat. no. 4138, Cell Signaling Technology, Inc.), ERK2 [PA5-32396, Invitrogen/Thermo Fisher Scientific; Carlsbad, USA], Galactosidase beta (E2U2I) (cat. no. 27198, Cell Signaling Technology, Inc.), GAPDH (14C10; cat. no. 2118S; Cell Signaling Technology, Inc.), H2AX phospho (Ser139, cloneJBW301; cat. no. 05-636, Merck KGaA), MDR1/ABCB1 (D3H1Q; cat. no. 12683; Cell Signaling Technology, Inc.), OCT2 (cat. no. MBS9600162, Biozol Diagnostics Vertrieb GmbH), P16 (F-12; cat. no. sc-1661; Santa Cruz Biotechnology, Inc.), p21 (C-19; cat. no. sc-397; Santa Cruz Biotechnology, Inc.), P53 phospho (S15; cat. no. 9284S; Cell Signaling Technology, Inc.), PARP (cat. no. 9542S, Cell Signaling Technology, Inc.), Rad51 (cat. no. ab63801; Abcam), RPA32 phospho (S4/S8; cat. no. ICH-00422; Bethyl Laboratories Inc.), TopBP1 (D8G4L; cat. no. 14342, Cell Signaling Technology, Inc.), Topoisomerase II alpha (D10G9; cat. no. 12286, Cell Signaling Technology, Inc.).

Techniques: Drug Transport Assay, Variant Assay, Alamar Blue Assay, Expressing, Western Blot, Control, Fluorescence, Flow Cytometry, Incubation, Standard Deviation, Binding Assay