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).
