Aspirin Induces Mitochondrial Ca2+ Remodeling in Tumor Cells via ROS‒Depolarization‒Voltage-Gated Ca2+ Entry

Figures

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  Figure 1. Aspirin and salicylate reduce cell viability in melanoma cells in a reactive oxygen species (ROS)-dependent manner. (A) A375, (B) A2058, and (C) human dermal fibroblasts (HDF) cells in DMEM supplemented with 10% fetal bovine serum (FBS) (FBS/DMEM) were treated with the indicated concentrations of aspirin (ASA) for 72 h at 37 ˚C and analyzed for their viability by the WST-8 assay. Data represent the mean ± SD (n = 3). # p < 0.05; ## p < 0.01; ### p < 0.001; n.s., not significant, vs. control. (D) A375 cells were treated with aspirin or salicylate in the absence or presence of MnTBaP (30 µM) and NAC (2 mM) for 72 h at 37 °C and analyzed for their viability as described above. Data represent the mean ± SD (n = 3). ### p < 0.001 vs. control. * p < 0.05; *** p < 0.001. (E) A2058 and HOS cells in FBS/DMEM were cultured in 6-well plates for 24 h and then exposed to aspirin (ASA, 5 mM), salicylate (SA, 5 mM) or NAC alone or in combination for 15 min. Cells were labeled with DCFH-DA FITC antibody and observed with a fluorescence microscope.

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  Figure 2. Aspirin and salicylate induce melanoma cell death. (A) A2058 cells treated with aspirin (ASA) or salicylate (SA) (5 mM) and TRAIL (100 ng/mL) alone or in combination for 24 h at 37 ˚C were observed under a BZX-710 all-in-one biological microscope and analyzed using the BZ-H3A application software. Scale bars, 10 µm. (B) Cells were treated with the indicated concentrations of aspirin (ASA) and TRAIL (100 ng/mL) alone or in combination with the absence or presence of Z-VAD-FMK (10 µM; ZVAD) for 72 h at 37 °C and were analyzed for viability by the WST-8 assay. Data represent the mean ± SD (n = 3). ### p < 0.001 vs. control. * p < 0.05; *** p < 0.001; n.s., not significant.

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  Figure 3. Aspirin evokes rapid and persistent depolarization in a ROS-dependent manner. (A‒D) (A,B) A375 and (C,D) A2058 cells loaded with DiBAC4(3) were washed, resuspended in HBSS, and treated with the indicated concentrations of aspirin (ASA) or TRAIL (100 ng/mL). (E) A375 cells loaded with the same probe were treated with the indicated concentrations of ASA in the absence or presence of NAC and catalase. The cells were measured for their fluorescence for 3 min in a microplate fluorescence reader with excitation and emission at 485 and 538 nm, respectively. The trace with the vehicle alone is considered as a baseline. Data are shown as F/F0, where F and F0 are the sample’s fluorescence at each time point and time zero, respectively. Graphs in (B,D,E) represent the mean ± SD of the average F/F0 (n = 3). ### p < 0.001; n.s., not significant vs. baseline. * p < 0.05; *** p < 0.001.

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  Figure 4. Ca2+ regulates the anti-melanoma effect of aspirin. (A,B) (A) A375 and (B) A2058 cells were treated with the indicated concentrations of aspirin (ASA) in the absence or presence of BAPTA-AM (30 µM) and EGTA (0.2 mM) for 72 h and analyzed for viability by the WST-8 assay. Data represent the mean ± SD (n = 3). ### p < 0.001; n.s., not significant vs. control. * p < 0.05; *** p < 0.001. (C,D) (C) A375 and (D) A2058 cells were treated with aspirin in the absence or presence of BAPTA-AM (30 µM), EGTA (0.2 mM), and nifedipine or verapamil (1 µM) for 72 h and analyzed for viability as described above. Data represent the mean ± SD (n = 3). *** p < 0.001.

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  Figure 5. Aspirin modulates the intracellular Ca2+ dynamics in melanoma cells. (A,B) A2058 cells were loaded with Fluo4-AM, treated with the indicated concentrations of aspirin (ASA), and immediately measured for their fluorescence for 3 min with excitation and emission at 485 and 538 nm, respectively, in a microplate reader. Data are shown as F/F0, where F and F0 are the sample’s fluorescence and control, respectively, and represent the mean ± SD (n = 3). *** p <0.001; n.s., not significant vs. control. (C,D) Cells were loaded with dihydrorhod 2-AM, treated with the indicated concentrations of ASA or salicylate (SA) and TRAIL (100 ng/mL), and measured for their fluorescence for 3 min with excitation and emission at 542 and 592 nm, respectively. In (C), data are shown as F/F0, where F and F0 are the fluorescence of the sample and control, respectively. In (D), data are shown as fluorescence intensity (arbitrary units, [A.U.]) and represent mean ± SD (n = 3). *** p < 0.001 vs. unstimulated control.

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  Figure 6. Cav1.2 downregulation modulates the effect of aspirin on Ca2+m. (A‒D) (A,B) Control (scrambled control siRNA-transfected) and (C,D) Cav1.2 knockdown A375 cells were treated with the indicated concentrations of aspirin (ASA), salicylate (SA), and TRAIL and [Ca2+]m was measured as described in the legend of Figure 2. In (A) and (C), data are shown as F/F0, where F and F0 are the fluorescence of the sample and control, respectively. In (B) and (D), data are shown as fluorescence intensity (arbitrary units, [A.U.]) and represent the mean ± SD (n = 3). *** p < 0.001 vs. unstimulated control.

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  Figure 7. A schematic summary of the present study. Both aspirin and salicylate induce rapid and persistent depolarization, and ROS mediate the effect. The ROS may be primarily generated by the electron transport chain through loss of ΔΨm, as we previously showed with electron transport chain inhibitors, rotenone, antimycin A, and FCCP [20]. In turn, the depolarization without repolarization leads to excessive activation of VGCCs, including Cav1.2, thereby leading to Ca2+m deregulation, mitochondrial dysfunction, and cell death.