Using a novel computational drug-repositioning approach (DrugPredict) to rapidly identify potent drug candidates for cancer treatment

Abstract

Computation-based drug-repurposing/repositioning approaches can greatly speed up the traditional drug discovery process. To date, systematic and comprehensive computation-based approaches to identify and validate drug-repositioning candidates for epithelial ovarian cancer (EOC) have not been undertaken. Here, we present a novel drug discovery strategy that combines a computational drug-repositioning system (DrugPredict) with biological testing in cell lines in order to rapidly identify novel drug candidates for EOC. DrugPredict exploited unique repositioning opportunities rendered by a vast amount of disease genomics, phenomics, drug treatment, and genetic pathway and uniquely revealed that non-steroidal anti-inflammatories (NSAIDs) rank just as high as currently used ovarian cancer drugs. As epidemiological studies have reported decreased incidence of ovarian cancer associated with regular intake of NSAIDs, we assessed whether NSAIDs could have chemoadjuvant applications in EOC and found that (i) NSAID Indomethacin induces robust cell death in primary patient-derived platinum-sensitive and platinum- resistant ovarian cancer cells and ovarian cancer stem cells and (ii) downregulation of β-catenin is partially driving effects of Indomethacin in cisplatin-resistant cells. In summary, we demonstrate that DrugPredict represents an innovative computational drug- discovery strategy to uncover drugs that are routinely used for other indications that could be effective in treating various cancers, thus introducing a potentially rapid and cost-effective translational opportunity. As NSAIDs are already in routine use in gynecological treatment regimens and have acceptable safety profile, our results will provide with a rationale for testing NSAIDs as potential chemoadjuvants in EOC patient trials.

Figure 1

Schematic showing outline of DrugPredict approach.

Figure 2

DrugPredict ranked Non-steroidal anti-inflammatory (NSAID) significantly high. (a) Among a total of 6996 prioritized chemicals (including 1096 FDA-approved drugs), the 15 FDA-approved EOC drugs had a median ranking of 1.34% and mean ranking of 6.44% (b) Among a total of 882 fourth-level drug classes, 31 classes of drugs ranked significantly higher than random expectation. As expected, anticancer drugs (platinum compounds, pyrimidine analogs and other antineoplastic agents) ranked highest and anti-inflammatories were third highest. (c) List of the highly ranked NSAIDs reveal that celecoxib, nimesulide and indomethacin are the top 3.

Figure 3

Indomethacin decreases survival of both platinum-sensitive and platinum-resistant ovarian tumor cells. (a) 48 h MTT assay showing Indomethacin decreases viability in primary epithelial ovarian cancer cells (OV78: Stage IV HGSOC, OV81: Stage IIIC HGSOC, OV82: Stage IIIC low-grade serous EOC, OV84: Stage IIIC HGSOC). (b) Dose–response clonogenics assay on day 7 showing both OV81.2 and OV81.2-CP10 are sensitive to Indomethacin. (c) 48 h cell cycle analysis showing Indomethacin induces G1 arrest and increased cell death in patient-derived primary platinum-sensitive (OV81.2) and resistant (OV81.2-CP10) cell lines as well as established platinum-sensitive (A2780) and resistant (CP70) cells. (d) 48 h flow cytometry analysis of Annexin-V PI staining showing robust cell death induced in ovarian tumor cells upon Indomethacin treatment. (e) Western blots showing elevated cleaved-PARP and γ-H2AX protein levels by 24 h of Indomethacin treatment in CP10 and CP70 (*P<0.05, **P<0.005 and ***P<0.0005).

Figure 4

Indomethacin exerts additive effect with cisplatin in ovarian tumor cells. (a) 48 h MTT analysis of OV81.2 and OV81.2-CP10 cells treated with increasing doses of cisplatin and cisplatin plus Indomethacin. (b) Isobologram analysis of the MTT data of Indomethacin cisplatin combination treatment in OV81.2 and OV81.2-CP10 cells showing additive effect of the combination of the two drugs. (c) Dose–response clonogenics assay on day 7 showing effect of Indomethacin and cisplatin combination on cell survival in OV81.2 and OV81.2-CP10. (d) 48 h Flow cytometry analysis of Annexin- V PI staining showing increased cell death upon Indomethacin and cisplatin combination treatment with the corresponding isobologram doses in OV81.2, OV81.2-CP10 and CP70. (e) Western blots showing elevated cleaved-PARP and γ-H2AX protein levels upon 24 h Indomethacin and cisplatin combination treatment with the corresponding isobologram doses in OV81.2-CP10 and CP70 (**P<0.005 and ***P<0.0005).

Figure 5

Indomethacin downregulates Wnt/β-catenin signaling in platinum-resistant ovarian tumor cells. (a) Western blot showing robust decrease in β-catenin protein level by 24 h upon treatment with Indomethacin in OV81.2-CP10 and CP70 cells and enhanced β-catenin downregulation in cisplatin plus Indomethacin combination treated OV81.2-CP10 and CP70 cells as compared with individual drug treatment alone 24 h. (b) Real-time PCR analysis 24 h showing Indomethacin decreases mRNA expression of β-catenin transcriptional targets AXIN2, TCF7, LEF-1 and LGR5 in OV81.2-CP10 and CP70. (c) Western blot showing overexpression of non-degradable β-catenin (β-S33Y) in OV81.2 (left) and effect of Indomethacin on β-catenin protein expression in OV81.2-β- S33Y cells (right). (d) clonogenics assay on day 7 showing β-S33Y overexpression partially rescues the effect of Indomethacin on cell survival in OV81.2. (e) 48 h Annexin-V PI staining flow cytometry analysis showing that β-S33Y overexpressing OV81.2 cells are more tolerant to Indomethacin induced cell death. (f) Western blot confirming lentiviral shRNA mediated β-catenin knockdown. (g) 48 h analysis of Annexin-V PI staining showing increased cell death upon Indomethacin treatment in OV81.2-CP10 and CP70 sh-β-catenin cells compared with sh-scram control (*P<0.05, **P<0.005 and. ***P<0.0005).

Figure 6

Indomethacin exerts additive effect with cisplatin in ALDH pos ovarian tumor-initiating cells (TICs). (a) Real-time PCR analysis showing increased mRNA expression of cancer stem cell markers EpCAM, CD133 and drug resistance marker ABCG2 in ALDH-positive CP70 cells (left) limiting dilution tumor sphere formation analysis showing increased tumor sphere formation ability in ALDH pos CP70 cells (right). (b) 48 h MTT analysis showing increased tolerance to cisplatin in ALDH pos CP70 cells. (c) PI staining cell cycle flow cytometry showing cell cycle arrest in G1 phase and increased SubG1 phase induced by Indomethacin in ALDH pos CP70 cells. (d) × 10 light microscopy images and 10 × 10 stitch imaging with integrated metamorph software analysis showing decreased tumor sphere formation by day 6 upon Indomethacin treatment. (e) Real-time PCR analysis 72 h showing Indomethacin decreases the mRNA expression of β-catenin driven cancer stem cell transcriptional targets in ALDH pos CP70 cells. (f) Isobologram analysis showing additive effect of Indomethacin cisplatin combination in ALDH pos CP70 cells. (g) 48 h Annexin-V PI staining flow cytomery analysis showing increased cell death upon Indomethacin plus cisplatin combo treatment as compared with individual drugs alone in both ALDH neg CP70 (left) and ALDH pos CP70 cells (right) clonogenics assay on day 7 showing greater decrease in cell survival upon Indomethacin plus cisplatin combo treatment as compared with individual drugs alone in both ALDH neg CP70 and ALDH pos CP70 cells (right). (h) Western blots showing increased cleaved-PARP and γ-H2AX protein levels and decreased β-catenin protein level in Indomethacin plus cisplatin combo treated cells as compared with individual drugs alone treated ALDH pos CP70 cells (*P<0.05, **P<0.005 and ***P<0.0005).