The Distinct Effects of the Mitochondria-Targeted STAT3 Inhibitors Mitocur-1 and Mitocur-3 on Mast Cell and Mitochondrial Functions

Abstract

There is accumulating evidence that mitochondria and mitochondrial STAT3 are involved in the activation of mast cells. The mitochondria-targeted curcuminoids Mitocur-1 and Mitocur-3 have been suggested to reduce antigen-dependent mast cell activation by inhibiting mitochondrial STAT3. The aim of the current work was to investigate the mechanisms of action of these mitocurcuminoids on mast cells and mitochondrial functions. The pretreatment of rat basophilic leukemia cells RBL-2H3 with Mitocur-1 and Mitocur-3 decreased antigen-dependent degranulation but did not affect spontaneous degranulation. Both compounds caused mitochondrial fragmentation and increased mitochondrial ROS. Inhibition of Drp1 prevented mitochondrial fragmentation induced by Mitocur-3 but not by Mitocur-1. The antioxidant N-acetylcysteine inhibited mitochondrial fission induced by Mitocur-1 but not Mitocur-3. Mitochondrial fragmentation caused by Mitocur-3 but not Mitocur-1 was accompanied by activation of Drp1 and AMPK. These data suggest a distinct mechanism of action of mitocurcuminoids on the mitochondria of RBL-2H3 cells: Mitocur-3 stimulated AMPK and caused Drp1-dependent mitochondrial fragmentation, while Mitocur-1-induced mitochondrial fission was ROS-dependent. This difference may contribute to the higher toxicity of Mitocur-3 compared to Mitocur-1. The findings contribute to further drug development for inflammatory and allergic diseases.

Keywords: Mitocur-1; Mitocur-3; RBL-2H3 cells; mast cell; mitochondria; mitochondria-targeted curcuminoids.

Figure 1

Effect of different concentrations of mitochondria-targeted curcuminoids Mitocur-1 and Mitocur-3 on cell survival. The cells were incubated with 1–6 µM of mitocurcuminoids for 3 h. After washing in cell medium for 24 h, viability was measured by resazurin. The results are presented as the mean ± SD (n = 4). *** p ≤ 0.001, and **** p ≤ 0.0001 as compared to untreated cells calculated by one-way ANOVA, Dunnett’s test.

Figure 2

Effects of Mitocur-1 and Mitocur-3 on spontaneous and antigen-dependent degranulation. The cells were incubated with 1 µM of mitocurcuminoids for 3 h. To induce antigen-dependent degranulation, RBL-2H3 cells were sensitized by anti-DNP IgE and stimulated by DNP-BSA for 15 min. The level of degranulation was estimated by β-hexosaminidase release. The results are presented as the mean ± SD (n = 4). **** p ≤ 0.0001 as compared with the untreated DNP-BSA-stimulated cells calculated by one-way ANOVA, Dunnett’s test.

Figure 3

The effect of Mitocur-1 and Mitocur-3 on STAT3 serine 727 phosphorylation in RBL-2H3 cells. The cells were sensitized by anti-DNP IgE overnight, incubated in the presence of mitocurcuminoids for 3 h, stimulated with DNP-BSA for 20 min, and used for Western blot analysis. (A) Representative Western blots of cell lysates; (B) histograms denoting the relative amounts of proteins. The results are presented as the mean ± SD (n = 4). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, one-way ANOVA, Tukey’s test.

Figure 4

Effect of Mitocur-1 and Mitocur-3 on mitochondrial fragmentation. (A) Fluorescent microscopy of the mitochondria (labeled with MitoTracker Green) in the RBL-2H3 cells; (B) phase-contrast microscopy; (C) the percentage of cells with fragmented mitochondria. From 100 to 200 cells were analyzed in four independent experiments; (D) average mitochondrial length per cell. Twenty cells were analyzed in each group. The results are presented as the mean ± SD (n = 4). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001, one-way ANOVA, Tukey’s test.

Figure 5

The effect of Mitocur-1 and Mitocur-3 on the mitochondrial membrane potential of RBL-2H3 cells. Cells were treated with compounds for 3 h. After treatment, cells were incubated with 50 nM TMRM for 30 min to detect mitochondrial membrane potential. The fluorescence in the PE channel was detected by flow cytometry. The results are presented as the mean ± SD (n = 4). ** p ≤ 0.01, as compared with the control cells calculated by one-way ANOVA, Dunnett’s test.

Figure 6

The effect of Mitocur-1 and Mitocur-3 on the ROS production of RBL-2H3 cells. Cells were treated with compounds for 3 h. After treatment, cells were incubated with 500 nM MitoTracker Orange CM-H2TMRos for 30 min and fluorescence in the PE channel was measured by flow cytometry. The results are presented as the mean ± SD (n = 4). *** p ≤ 0.001 as compared with the untreated non-stimulated cells calculated by one-way ANOVA, Dunnett’s test.

Figure 7

The effect of Mitocur-1 and Mitocur-3 on cardiolipin peroxidation in RBL-2H3 cells. Cells were incubated with the mitocurcuminoids for 3 h, washed with medium and 12 h later stained with MitoCLox for 30 min. MitoCLox fluorescence in the FITC and PE channels was measured by flow cytometry. The results are presented as the mean ± SD (n = 4). # p ≤ 0.1,* p ≤ 0.05 as compared with the control cells calculated by one-way ANOVA, Dunnett’s test.

Figure 8

The effect of Mitocur-1 and Mitocur-3 on signaling molecules in RBL-2H3 cells. Cells were incubated in the presence of mitocurcuminoids for 3 h and used for Western blot analysis. (A) Representative Western blots of cell lysates; (B) histograms denoting the relative amounts of proteins. The results are presented as the mean ± SD (n ≥ 4). * p ≤ 0.05 when compared to untreated, non-stimulated cells, as determined by one-way ANOVA, Dunnett’s test.

Figure 9

The effect of Mitocur-1 and Mitocur-3 on the relative mitochondrial DNA (mtDNA) content (ratio of mtDNA to nuclear DNA) in RBL-2H3 cells. Cells were incubated with mitocurcuminoids for 3 h, washed with medium and used for qPCR analysis 24 h later. The results are presented as the mean ± SD (n = 4).