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Novel anti-cancer agent myrtucommulone-A and thymoquinone abrogate epithelial–mesenchymal transition in cancer cells mainly through the inhibition of PI3K/AKT signalling axis

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Abstract

Epithelial–mesenchymal transition (EMT) plays a prominent role in cancer progression and metastasis. Inhibition of EMT-associated regulators may hold a huge promise for cancer therapy. Although TGF-β signalling has a pivotal role in the induction of EMT, alterations during the EMT process are usually initiated and controlled by the cross-talk of multiple signalling pathways, and in most cases this is context-dependent. In the present study, we aimed at identifying the molecular mechanisms during the inhibition of EMT by novel anti-cancer agent myrtucommulone-A (MC-A) and thymoquinone (TQ). We used epithelial cancer cells to study the effects of MC-A and TQ on EMT. We first showed the functional inhibition of EMT by MC-A or TQ using migration assays and confirmed the EMT inhibition by analysing the expression of EMT markers with RT-PCR, immunocytochemistry and Western blotting. We evaluated the changes in intracellular dynamics by Western blotting and compared the effects of MC-A and TQ with the effects of selective inhibitors of PI3K (LY294002), ERK 1/2 (U0126) and TGF-βR (SB431542). We demonstrate that both MC-A and TQ treatment negatively regulate the EMT process through modulation of signalling pathways in cancer cells. MC-A and TQ treatment inhibited phosphorylation of multiple proteins in a context-dependent manner. Novel anti-cancer agent MC-A and TQ regulate distinct signalling pathways for the repression of EMT which emphasises the significance of combinational therapies in cancer treatment. MC-A and TQ could be considered as candidate molecules for combinational therapies with their ability to interfere signalling pathways regulating cancer cell behaviour.

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Acknowledgments

We are grateful to Prof. Dr. Johann Jauch and Dr. Maël Charpentier (Institut für Organische Chemie der Universität des Saarlandes in Saarbrücken, Germany) for providing MC-A.

Grant support

This study was supported by the grants from the The Scientific and Technological Research Council of Turkey (No: 115S042, No: 114S542 and No: 113S927).

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Correspondence to Banu Iskender.

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The authors declare no conflicts o interest.

Electronic supplementary material

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11010_2016_2697_MOESM1_ESM.pdf

Electronic Supplementary Material_1. Dose dependent anti-proliferative effects of MC-A or TQ in cancer cell lines. (A) The percentages of BrdU positive cells at 1, 2.5, 5, 10, 25 μM concentrations of MC-A-treated cells for 24 h. MC-A showed dose-dependent anti-proliferative effect on MDA –MB-231 cells. (B) The percentages of BrdU positive cells at 1, 5, 10, 25, 50 μM concentrations of TQ treated cells for 24 h. TQ showed dose-dependent antiproliferative effect on MDA-MB-231 cells. (C) The percentages of BrdU positive cells at 1, 5, 10, 25, 50 μM concentrations of TQ treated cells for 24 h were measured. TQ showed dose-dependent antiproliferative effect on HTB-9 cancer cells 24 h. (PDF 487 kb)

11010_2016_2697_MOESM2_ESM.pdf

Electronic Supplementary Material_2. Dose dependent cytotoxic effects of MC-A or TQ in cancer cell lines. The percentages of viable MDA –MB-231 cells after 48 h treatment with MC-A (A) and TQ (B) at 1, 2.5, 5, 10 μM concentrations. MC-A and TQ showed dose-dependent cytotoxic effect on MDA –MB-231 cells. The percentages of viable HTB-9 cells after 48 h treatment with MC-A (C) and TQ (D) at 1, 2.5, 5, 10 μM concentrations. MC-A and TQ showed dose-dependent cytotoxic effect on HTB-9 cells. (PDF 483 kb)

11010_2016_2697_MOESM3_ESM.pdf

Electronic Supplementary Material_3. Inhibitory effects of different doses of MC-A or TQ on TGF-β induced migration in MDA-MB-231 cells. A confluent monolayer of MDA-MB-231 cells were serum starved for 12 h, scratched and pretreated with MC-A (12.5, 25, 50 µM) and TQ (25, 35, 50 µM). The cultures were then treated with TGF-β (5 ng/ml) for 24 h. Wound areas were photographed just after scratching (time zero), after 8 h and the experiments were finalised at 24 h. Migration inhibition was scored by measurement of the cell-free area in the pictures. The quantified value was normalised against the value at time zero. Data is representative of two independent experiments (PDF 25910 kb)

11010_2016_2697_MOESM4_ESM.pdf

Electronic Supplementary Material_4. Inhibitory effects of different doses of MC-A or TQ on TGF-β induced migration in HTB-9 cells. A confluent monolayer of HTB-9 cells were serum starved for 12 h, scratched and pretreated with MC-A (2.5, 10, 12.5 µM) and TQ (12.5, 50, 100 µM). The cultures were then treated with TGF-β (5 ng/ml) for 24 h. Wound areas were photographed just after scratching (time zero), after 8 h and the experiments were finalised at 24 h. Migration inhibition was scored by measurement of the cell-free area in the pictures. The quantified value was normalised against the value at time zero. Data is representative of two independent experiments. (PDF 14147 kb)

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Iskender, B., Izgi, K. & Canatan, H. Novel anti-cancer agent myrtucommulone-A and thymoquinone abrogate epithelial–mesenchymal transition in cancer cells mainly through the inhibition of PI3K/AKT signalling axis. Mol Cell Biochem 416, 71–84 (2016). https://doi.org/10.1007/s11010-016-2697-y

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