Ex Vivo treatment of coronary artery endothelial cells with serum post-exercise training offers limited protection against in vitro exposure to FEC-T chemotherapy

Figures

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  FIGURE 1. Representative gating strategy of endothelial cells by morphology (A), singlets (B), and CD31 expression as displayed by FITC histogram (C). Histograms of, Annexin-V (D), Cleaved Caspase-3 (E) expression are displayed as a function of their respective lasers (PerCp-Cy.5, V450, respectively).

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  FIGURE 2. Schematic representation of the protocol for serum preconditioning of HCAECs at 0-h. For flow cytometry analysis, FEC-T chemotherapy exposure occurred 24-h after serum preconditioning. HCAECs were re-incubated and washed with PBS and fresh media added at corresponding time points (Tx), respective to each individual drug condition. HCAECs were then analysed by flow cytometry at corresponding time points (Ty), respective to each individual drug condition. For the wound healing assay, FEC-T drugs were added immediately after the monolayer was scratched. HCAECs were re-incubated and washed at corresponding time points (Tz), respective to each individual drug condition. HCAECs were imaged using Primovert Axiocam ERc5s microscope (ZEISS, Germany) using ×4 magnification at 0-, 3-, 4-, 6-, 12-, 24-, and 48-h.

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  FIGURE 3. Dose-response effects of combined 5-fluorouracil, epirubicin, cyclophosphamide (FEC; (A,B) and docetaxel (C,D) on human coronary artery endothelial cells (HCAEC) phosphatidylserine (A,C) and cleaved caspase-3 expression (B,D) across a range of physiological concentrations at 12- (A,B) and 48-h (C,D) post-exposure. Data shown are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. FEC1 = 5-fluorouracil: 1 µM, epirubicin: 0.003 µM, cyclophosphamide: 19 µM; FEC2 = 5-fluorouracil: 1.5 µM, epirubicin: 0.006 µM, cyclophosphamide: 38 µM; FEC3 = 5-fluorouracil: 2 µM, epirubicin: 0.009 µM, cyclophosphamide: 57 µM.

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  FIGURE 4. Representative images of human coronary artery endothelial cells (HCAEC) gap closure over time (0-, 3-, 6-, and 24- hours) with and without FEC drug exposure (5-fluorouracil: 1.5 µM, epirubicin: 0.006 µM, cyclophosphamide: 38 µM), and drug exposure with pre- or post-exercise intervention serum conditioning. Images are ×4 magnification. Scale bar is 250 µm.

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  FIGURE 5. Area under the human coronary artery endothelial cells (HCAEC) gap closure curve with exposure to 1.5 µM 5-fluorouracil (A), 0.006 µM epirubicin (B), 38 µM cyclophosphamide (C), FEC combined (D); 5-fluorouracil: 1.5 µM, epirubicin: 0.006 µM, cyclophosphamide: 38 µM, and 6 µM docetaxel (E) treatments with dimethyl sulfoxide (DMSO), drug without serum, pre-training serum with drug and post-training serum with drug. Data shown are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  FIGURE 6. Comparison of pre- and post-exercise intervention serum preconditioning on the effects of FEC (A,B): 5-fluorouracil: 1.5 µM, epirubicin: 0.006 µM, cyclophosphamide: 38 µM) and 6 µM docetaxel (C,D) exposure on human coronary artery endothelial cells (HCAEC) phosphatidylserine (A,C) and cleaved caspase-3 expression (B,D) at 4- and 48-h post-exposure, respectively. Data shown are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.