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. 2021 Nov 1;11:748698. doi: 10.3389/fonc.2021.748698

Figure 3.

Figure 3

Induction of cellular dicarbonyl stress by antitumor drugs and mechanism methylglyoxal-induced cytotoxicity. (A) Increase in cellular MG in HEK293 cells treated with antitumor drugs for 3 h. Control (no drug added) and ca. 2 × GC50 concentration in nontransfected cells: mechlorethamine (MCH, 4.8 µM), mitomycin C (MITC, 316 nM), doxorubicin (DOX, 6.0 nM), etoposide (ETOP, 323 nM), paclitaxel (PTX, 21 nM), methotrexate (METX, 7.6 nM), and BBGD (7.4 µM). Data are mean ± SEM (p < 0.001; one-way ANOVA; n = 3 except n = 4, 5, and 6 for DOX, METX, and MITC, respectively). (B) Flux of formation of D-lactate (surrogate for flux of MG) and (C) flux of glucose consumption incubated in vitro with and without investigational agent and drugs indicated. Cells were incubated for 24 h with and without 2 × GC50 concentration. Data are mean ± SEM (n = 3). (D–G) Effect of hypoxia on formation and metabolism of methylglyoxal and antiproliferative activity of BBGD. (D–F)) Flux of glucose consumption and formation of D-lactate and Glo1 activity of HEK293 cells incubated for 72 h in 20% and 3% oxygen, models of normoxia and hypoxia, respectively. Data are mean ± SEM (n = 3). (G) Dose-response curve for BBGD. The GC50 values for BBGD under normoxic and hypoxic conditions were 5.12 ± 0.33 µM and 0.085 ± 0.010 μM, respectively (N = 18). (H) Dose-response curve for MG-treated HEK293 cells. GC50 131 µM ± 19.1, with logistic regression coefficient n = 0.70 ± 0.05 (N = 18). (I) Effect of duration of exposure to MG on antiproliferative activity in HEK293 cells in vitro. Cells were incubated with 131 µM MG. Data are mean ± SD (n = 3). (J) Release of cytochrome c from mitochondrial in early-stage MG-induced toxicity. HEK293 cells were incubated with and without MG in vitro and cytosolic cytochrome c assayed by ELISA. Key: hollow bars, control; solid bars, +131 µM MG. Data are mean ± SD (n = 3). Significance: *p < 0.05, **p < 0.01, and ***p < 0.001 with respect to control (t-test). (K) Early-stage accumulation of glycated modified during MG-induced cytotoxicity of HEK293 cells in vitros. Key: open symbols, control; filled symbols, +131 µM MG. Data are mean ± SD (n = 3).