doi: 10.1038/s41419-018-1102-z.
The thiosemicarbazone Me2NNMe2 induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition
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- PMID: 30323190
- PMCID: PMC6189190
- DOI: 10.1038/s41419-018-1102-z
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The thiosemicarbazone Me2NNMe2 induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition
Cell Death Dis.
.
Abstract
Due to their high biological activity, thiosemicarbazones have been developed for treatment of diverse diseases, including cancer, resulting in multiple clinical trials especially of the lead compound Triapine. During the last years, a novel subclass of anticancer thiosemicarbazones has attracted substantial interest based on their enhanced cytotoxic activity. Increasing evidence suggests that the double-dimethylated Triapine derivative Me2NNMe2 differs from Triapine not only in its efficacy but also in its mode of action. Here we show that Me2NNMe2- (but not Triapine)-treated cancer cells exhibit all hallmarks of paraptotic cell death including, besides the appearance of endoplasmic reticulum (ER)-derived vesicles, also mitochondrial swelling and caspase-independent cell death via the MAPK signaling pathway. Subsequently, we uncover that the copper complex of Me2NNMe2 (a supposed intracellular metabolite) inhibits the ER-resident protein disulfide isomerase, resulting in a specific form of ER stress based on disruption of the Ca2+ and ER thiol redox homeostasis. Our findings indicate that compounds like Me2NNMe2 are of interest especially for the treatment of apoptosis-resistant cancer and provide new insights into mechanisms underlying drug-induced paraptosis.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
a Time-dependent cell viability of SW480 and HCT-116 cells treated with either Triapine or Me2NNMe2, determined by MTT assay after 24, 48, and 72 h. Values given in the graph are the mean ± standard deviation of triplicates from one representative experiment out of three, normalized to the untreated control of the same time-point. IC50 values (µM) ± standard deviations (SD) are given in the table . b Morphological changes in SW480 cells induced by 24 and 48 h treatment with the indicated concentrations of Triapine or Me2NNMe2. Cytoplasmic vacuoles were mainly seen with Me2NNMe2 (arrows). Scale bar: 100 µm. c Increase in cell size of SW480 and HCT-116 cells treated with the indicated concentrations of Triapine and Me2NNMe2 for 48 h
a Representative fluorescence microscopy images and overlaid differential interference contrast images of the ER lumen of ER-YFP-transfected SW480 cells treated with 10 µM Me2NNMe2 for 24 h (scale bar: 50 µm). b Life-cell fluorescence imaging of ER-located YFP-transfected SW480 cells treated with 1 µM Me2NNMe2. Time after treatment is indicated as hh:mm (scale bar: 10 µm). c Representative fluorescence microscopy images of mitochondria (MitoTracker) showing no overlap with vesicles in ER-YFP-transfected SW480 cells treated with 10 µM Me2NNMe2. (scale bar: 50 µm). d Raman microspectroscopy of SW480 cells treated with 10 µM Me2NNMe2 for 24 h. Principal component analysis (PCA) of Raman spectra can differentiate between background (black), cell (green) and vesicles (red). CLS fitting of Me2NNMe2 Raman spectrum to the spectral map of the cell revealed accumulation of the drug inside the vesicles
a Mitochondrial membrane potential depolarization measured by the percentage of cells with decreased JC-1 fluorescence (red). SW480 or HCT-116 cells were treated with the indicated concentrations of Triapine or Me2NNMe2 for 24 h. Values given are the mean ± standard deviation of three independent experiments. b Fluorescence microscopy of increased calcium levels (Rhod-2 AM in red) specifically in the mitochondria (MitoTracker in green) after thapsigargin (Tg, 1 µM), Me2NNMe2 (0.1 µM) or Triapine (1 µM) treatment of SW480 cells for 48 h (scale bar: 50 µm). White arrows indicate co-localization. c Cell viability of HCT-116 wild-type (wt) and BAX knockout (KO) cells measured by MTT after 72 h treatment with indicated concentrations of Triapine or Me2NNMe2. Values given are the mean ± standard deviation of triplicates of one representative experiment out of three. d Western blot analysis of BAX and Bcl-xL expressed by SW480 and HCT-116 cells treated with Triapine or Me2NNMe2 for 24 or 48 h. The ratio of BAX to Bcl-xL is given below the respective bands. β-actin was used as a loading control. Significance was calculated to control with one-way (a) and to wt cells with two-way (c) ANOVA and Bonferroni’s multiple comparison test (***p < 0.001, **p ≤ 0.01, *p ≤ 0.05)
a Cell viability measured by MTT assay of SW480 and HCT-116 cells after 72 h treatment with the indicated concentrations of Triapine (full lines) or Me2NNMe2 (dotted lines) alone or in combination with 10 µM z-VAD-FMK (VAD, gray lines). Values given are the mean ± standard deviation of triplicates from one representative experiment out of three. b Phase-contrast microscopy images of SW480 cells treated with 10 µM Me2NNMe2 or 25 µM z-VAD-FMK as well as the combination (scale bar: 100 µm). c Percentage of annexin V-positive (AV+) and/or PI+ SW480 or HCT-116 cells detected by flow cytometry after 48 h of Triapine or Me2NNMe2 treatment in combination with 10 µM z-VAD-FMK (VAD). Values given are the mean ± standard deviation of three independent experiments. For calculation of significance AV+ cell fractions (AV+/PI−, AV+/PI+) were added. Significance to control was calculated by two-way (a) or one-way (c) ANOVA and Bonferroni’s multiple comparison test using GraphPad Prism software (***p < 0.001, **p ≤ 0.01, *p ≤ 0.05)
a GSEA from whole-genome gene expression data revealed significant enrichment of genes in the “MAPK signaling pathway” gene set in SW480 cells treated with 0.1 or 1 µM Me2NNMe2 compared to untreated cells. Normalized enrichment score (NES) and false discovery rate (FDR) are given. b Illustration of genes up- (red) or down-regulated (blue) in the KEGG-derived “MAPK signaling pathway” of Me2NNMe2 (1 µM)-treated compared to untreated SW480 cells using whole-genome gene expression data. c Western blot analysis of MEK1/2 and ERK1/2 as well as their phosphorylated protein levels in SW480 and HCT-116 cells treated with indicated concentrations of Triapine and Me2NNMe2 for 24 h. β-actin was used as a loading control
a Impact of MEK1/2 inhibitors U0126 (5 µM), PD98058 (5 µM), selumetinib (50 nM) or trametinib (100 nM) on viability of Triapine- or Me2NNMe2-treated SW480. Change in viability is given as mean fold IC50 value increase ± standard deviation compared to Triapine or Me2NNMe2 treatment alone, measured by three independent MTT viability experiments. b Representative phase-contrast microscopy images of SW480 cells treated with Me2NNMe2 (10 µM) and U0126 (20 µM) or PD98058 (10 µM) as well as the combinations for 48 h (scale bar: 100 µm). c Percentage of vacuolated cells counted from phase-contrast microscopy images seen in b. Values given are the mean ± standard deviation of three images with at least 30 cells in total. Significance to single treatment was calculated by one-way ANOVA and Bonferroni’s multiple comparison test (***p < 0.001, **0.001 ≥ p ≤ 0.01, *0.01 ≥ p ≤ 0.05). d Protein expression detected by Western blot of MEK2, MEK1/2, and ERK1/2 in SW480 and HCT-116 cells after 48 h gene silencing with scrambled (siSCR) or MEK2 (siMEK2) siRNA. β-actin was used as a loading control. e Representative images of SW480 cells transfected with siSCR or siMEK2 and treated with 10 µM Me2NNMe2 for 24 h (scale bar: 100 µm). f Percentage of cell vacuolization of SW480 or HCT-116 cells transfected with siSCR or siMEK2 and treated with the indicated concentrations of Me2NNMe2 for 24 h. Values given are the mean ± standard deviation of several regions of two experiments. Significance to siSCR was calculated by Student's T-test (***p < 0.001, **p ≤ 0.01)
a Quantification of immunofluorescence intensities in the nucleus of the ER stress marker CHOP in SW480 and HCT-116 cells treated with 1 µM thapsigargin (Tg), 1 µM Triapine or 0.1 and 1 µM Me2NNMe2 for 24 h. Values given are the mean intensities ± the interquartile range and 10 and 90 percentile whiskers of one representative experiment out of three. b Western blot analysis of various ER stress proteins expressed by SW480 cells treated with indicated concentrations of Triapine and Me2NNMe2 for 24 h. β-actin was used as a loading control and Tg (1 µM) as positive control for ER stress. c mRNA expression levels for PDI (P4HB) and ero1L-α (ERO1A) in treated (1 µM Me2NNMe2) or untreated SW480 cells were assessed by whole-genome gene expression microarrays. Two independent P4HB oligonucleotides were spotted on the array and gave comparable results. Data for oligonucleotide A_23_P107412 is shown. Normalized values of four replicates indicate upregulation of PDI and ero1L-α mRNA in treated compared to untreated cells. d PDI reduction activity in the presence of Triapine, Me2NNMe2 as well as their copper complexes was measured by PROTEOSTAT PDI assay kit. Bacitracin (1 mM) was used as a positive PDI inhibition control. Values given are the mean ± standard deviation of triplicates of one representative experiment out of two. e Cell viability measured by MTT assay of SW480 or HCT-116 cells after 72 h treatment of indicated concentrations of Me2NNMe2 alone as well as in combination with N-acetylcysteine (NAC) or 1-thioglycerol. Values given are the mean ± standard deviation of triplicates of one representative experiment out of three. f Superoxide production measured by flow cytometry of DHE fluorescence in HL-60 cells treated with indicated concentrations of Triapine and Me2NNMe2 for 45 min. Antimycin A (AMA) was used as positive control. g Detection of total and oxidized glutathione (GSSG) by fold increase to control of luminescence in SW480 cells treated with indicated concentrations of Triapine and Me2NNMe2 for 24 h. Significance to control (or CuCl2) was calculated with one-way ANOVA and Dunnett’s multiple comparison test (***p < 0.001, **p ≤ 0.01, *p ≤ 0.05)
Me2NNMe2 accumulates in the ER, where it inhibits the reductive potential of PDI. This leads to the disruption of the ER thiol redox homeostasis, which in turn activates PERK signaling and release of Ca2+ ions from the ER. While PERK activation is followed by CHOP translocation into the nucleus and increased transcription of PDI and ero1L-α, released Ca2+ ions are taken up by mitochondria. Prolonged Ca2+ imbalance initiates organelle swelling and mitochondrial membrane depolarization. NAC and 1-thioglycerol can ameliorate thiol redox imbalances. MAPKs further regulate Ca2+ and thiol redox homeostasis, which can be inhibited by U0126
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