Melatonin Enhances Cisplatin and Radiation Cytotoxicity in Head and Neck Squamous Cell Carcinoma by Stimulating Mitochondrial ROS Generation, Apoptosis, and Autophagy

Figure 2

Combined treatment with melatonin and IR or CDDP increases apoptotic cell death in the HNSCC cell line Cal-27. Apoptosis was analyzed by flow cytometry. (a–g) Representative plots showing the redistribution of phosphatidylserine (annexin V staining) in the presence of propidium iodide (PI). The bottom right quadrant represents the percentage of early apoptotic cells (annexin V+/PI-), whereas the top right quadrant represents the percentage of late apoptotic cells (annexin V+/PI+). Statistical analysis of early and late apoptosis of cells exposed to IR (b) and CDDP (h), respectively. Western blot analysis (f–l) and densitometric quantification of Bax (c–i) and Bcl-2 (d–j) and the Bax/Bcl-2 ratio (e–k) in cells exposed to IR or CDDP, respectively. Treatment groups include the control (vehicle), IR (8 Gy), CDDP 10 μM, melatonin (aMT) 1000 or 1500 μM, and CDDP or IR plus aMT 100, 500, 1000, or 1500 μM. per group. Data are presented as . , , and vs. the control; ^#, ^##, and ^### vs. the IR- or CDDP-treated group; ^δ, ^δδ, and ^δδδ vs. IR+aMT 100; and ^$, ^$$, and ^$$$ vs. IR+aMT 500.