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. 2021 Apr 23;13(9):2039. doi: 10.3390/cancers13092039

Figure 1.

Figure 1

A disintegrin and metalloprotease 17 (ADAM17) inhibition reduces cell viability and enhances cisplatin-induced apoptosis in 2D monolayers. Ovarian cancer (OvCa) cells (A2780, Igrov-1, HEY, SKOV-3, and OVCAR-8) were seeded as 2D monolayers and treated with the indicated concentrations of cisplatin (cis) or NaCl solvent control and 3 µM of either GI254023X (ADAM10 selective), GW280264X (ADAM17 and ADAM10 selective) or the solvent control DMSO. Following 48 h of treatment, the ApoLive-Glo™ Multiplex Assay was used to quantify (a) cell viability as relative fluorescence units (RFU) and (b) caspase3/7 activation as relative luminescence units (RLU). Data were normalized to control (DMSO and NaCl). The mean (± standard error of the mean (SEM)) of at least 3 biological replicates is displayed. Curve fitting was performed using GraphPad Prism. The half minimal (50%) inhibitory concentration (IC50) or the half maximal effective concentration (EC50) values were calculated from each curve DMSO, GI254023X (GI), and GW280264X (GW), setting the response of each individual inhibitor (0 µM cis) as the upper baseline value for calculation (i.e., SKOV-3: GI254023X, 85%; GW280264X, 48%). Thus, IC50 and EC50 values represent the combinatorial effects of cisplatin and inhibitors. Based on the Shapiro–Wilk normality test, IC50 and EC50 values of the biological replicates were analyzed by ANOVA following Tukey´s multiple comparison test (normally distributed), or Friedman’s test followed by Dunn´s multiple comparison test (not normally distributed). Comparison of IC50 or EC50 values between DMSO and GW; ns: not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Inhibition of GI254023X did not show significant differences compared with the DMSO control. (a) ADAM17 inhibition leads to a strong reduction in cell viability even without the application of cisplatin and a significant reduction in IC50 compared with the control (DMSO). (b) The combined effect of GW280264X and cisplatin in cisplatin-sensitive cells (HEY) was most pronounced with lower concentrations (1–5 µM) of cisplatin compared with intermediate-sensitive SKOV-3 and OVCAR-8 cells (5–20 µM cisplatin). (c,d) Drug reduction index (DRI50) at 50% effectiveness. As an example, DRI50 represents the fraction of cisplatin concentration required in combination with GW compared with cisplatin alone (to reach 50% effectiveness). DRI50 can be applied to describe the strength and direction of drug interaction into synergistic (DRI50 < 1), additive (DRI50 = 1), or antagonistic (DRI50 > 1) effects.