Small Molecules Identified from a Quantitative Drug Combinational Screen Resensitize Cisplatin's Response in Drug-Resistant Ovarian Cancer Cells

Abstract

Drug resistance to chemotherapy occurs in many ovarian cancer patients resulting in failure of treatment. Exploration of drug resistance mechanisms and identification of new therapeutics that overcome the drug resistance can improve patient prognosis. Following a quantitative combination screen of 6060 approved drugs and bioactive compounds in a cisplatin-resistant A2780-cis ovarian cancer cell line, 38 active compounds with IC₅₀s under 1 μM suppressed the growth of cisplatin-resistant ovarian cancer cells. Among these confirmed compounds, CUDC-101, OSU-03012, oligomycin A, VE-821, or Torin2 in a combination with cisplatin restored cisplatin's apoptotic response in the A2780-cis cells, while SR-3306, GSK-923295, SNX-5422, AT-13387, and PF-05212384 directly suppressed the growth of A2780-cis cells. One of the mechanisms for overcoming cisplatin resistance in these cells is mediated by the inhibition of epidermal growth factor receptor (EGFR), though not all the EGFR inhibitors are equally active. The increased levels of total EGFR and phosphorylated-EGFR (p-EGFR) in the A2780-cis cells were reduced after the combined treatment of cisplatin with EGFR inhibitors. In addition, a knockdown of EGFR mRNA reduced cisplatin resistance in the A2780-cis cells. Therefore, the top active compounds identified in this work can be studied further as potential treatments for cisplatin-resistant ovarian cancer. The quantitative combinational screening approach is a useful method for identifying effective compounds and drug combinations against drug-resistant cancer cells.

Figure 1

Platinum drug-resistant ovarian cancer cells and quantitative combination drug screens. (A and B) Concentration-response curves showing the inhibition effect of cisplatin and carboplatin treatment on the viability of both sensitive (A2780) and resistant (A2780-cis) ovarian cancer cells. (C) The primary screens of 6060 compounds from the Library of Pharmacologically Active Compounds (LOPAC), the National Center for Advancing Translational Sciences (NCATS) Chemical Genomics Center Pharmaceutical Collection (NPC), and the Mechanism Interrogation PlatE (MIPE) library were carried out in A2780-cis cells using an ATP content viability assay. Each compound was tested at five concentrations in combination with 1 μM cisplatin. A group of 383 hits from the primary screen were selected for confirmation in the same assay, in the presence of vehicle, 6 μM, 12 μM, or 18 μM cisplatin; 11 various concentrations of cisplatin were further combined with the 12 compounds at IC₂₅, IC_50, or IC₇₅ for evaluation of combinational effects. Lastly, the above combinational studies were validated for the five candidates in a secondary alamarBlue® viability assay. All values represent the mean ± the standard error of the mean (SEM) (n = 3 replicates).

Figure 2

Distribution of known drug indications and targets and/or pathways of 38 newly identified potent compounds against cisplatin-resistant ovarian cancer. (A) Number of active compounds in each drug class. If a compound has more than one indication, it is counted once by the following order: anticancer, antibiotic, antifungal, or others. (B) Number of active compounds in each known drug targets/pathways; some compounds have more than one designation.

Figure 3

Five clinically relevant potent anticancer hits are confirmed. Chemical structures (A) and dose–response curves (B) in ATP content viability assays showing the anticancer activities of GSK-923295, SNX-5422, AT-13387 AU, PF-05212384, and SR-3306 against cisplatin-resistant ovarian cancer cell. All values represent the mean ± SEM (n = 3 replicates).

Figure 4

Combinational anticancer activities of cisplatin and 500 hits. (A) Heatmap showing the change of IC₅₀ of hits in the presence of 0, 6, 12, or 18 μM of cisplatin. (B) Magnified heatmap showing the five compounds selected for follow-up studies. (C-G) Chemical structures and dose–response curves in ATP content viability assays showing the improved anticancer activities of cisplatin against drug-resistant ovarian cancer cells in combination with CUDC-101 (C), OSU-03012 (D), Oligomycin A (E), VE-821 (F), and Torin2 (G). All values represent the mean ± SEM (n = 3 replicates).

Figure 5

Confirmation of combinational anticancer activities of cisplatin and five hits in alamarBlue® viability assays. Dose–response curves in viability assays showing the improved anticancer activities of cisplatin against resistant ovarian cancer cell in the combination with CUDC-101 (A), OSU-03012 (B), Oligomycin A (C), VE-821 (D), and Torin2 (E). All values represent the mean ± SEM (n = 3 replicates).

Figure 6

Inhibitory effects of CUDC-101 on EGFR, p-EGFR, and HER2 in cisplatin-resistant ovarian cancer cells. (A) Western blot of EGFR, p-EGFR, and HER2 expressions in both cisplatin sensitive ovarian cancer cells and resistant ovarian cancer cells. (B) Western blot of EGFR, p-EGFR (Tyr1068), and HER2 expressions after treatment with cisplatin, CUDC-101, or a combination of both in cisplatin-resistant ovarian cancer cells. All experiments are repeated at least three times with a representative blot shown.

Figure 7

Combinational effects of cisplatin with other EGFR inhibitors in resistant ovarian cancer cells. (A-F) Dose–response curves showing the inhibition effect of cisplatin in combination with WZ4002, varlitinib, and canertinib on the viability of resistant ovarian cancer cells. All values represent the mean ± SEM (n = 3 replicates).

Figure 8

Improved response to cisplatin in EGFR knock-down resistant ovarian cancer cells. (A) Western blot of EGFR, p-EGFR, and HER2 expressions after treatment with three individual EGFR-siRNA in cisplatin-resistant ovarian cancer cells. (B-D) Quantitation of EGFR (B), p-EGFR (C), and HER2 (D) expression change after treatment with EGFR-siRNA in cisplatin-resistant ovarian cancer cells. (E) The results showed the EGFR-siRNA-3 transfection can decrease the EGFP, p-EGFP and HER2 expression, and the EGFR-siRNA-2 transfection can decrease the p-EGFP expression, and the EGFR-siRNA-1 transfection has no effect. Dose–response curves showing the inhibitory effect of cisplatin on the viability of both sensitive and resistant ovarian cancer cells with/without EGFR-siRNA treatment. All values represent the mean ± SEM (n = 3 replicates).

Figure S1

Dose-response curves showing the inhibitory effect of adriamycin, topotecan, etoposide, and paclitaxel treatment on the viability of both sensitive (A2780) and resistant (A2780-cis) ovarian cancer cells. All values represent the mean ± SEM (n = 3 replicates).

Figure S2

Western blot of HDAC (Ach3) expressions in both cisplatin sensitive ovarian cancer cells and resistant ovarian cancer cells. Three individual experiments were performed with a representative blot shown.