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. 2019 Feb 22;10(3):187.
doi: 10.1038/s41419-019-1360-4.

Gambogic acid triggers vacuolization-associated cell death in cancer cells via disruption of thiol proteostasis

Min Ji Seo  1   2 , Dong Min Lee  1   2 , In Young Kim  2 , Dongjoo Lee  3 , Min-Koo Choi  4 , Joo-Youn Lee  5 , Seok Soon Park  6 , Seong-Yun Jeong  6 , Eun Kyung Choi  7 , Kyeong Sook Choi  8   9
Affiliations

Gambogic acid triggers vacuolization-associated cell death in cancer cells via disruption of thiol proteostasis

Min Ji Seo et al. Cell Death Dis. .

Abstract

Gambogic acid (GA), a xanthonoid extracted from the resin of the tree, Garcinia hanburyi, was recently shown to exert anticancer activity in multiple studies, but the underlying action mechanism remains unclear. Here, we show that GA induces cancer cell death accompanied by vacuolation in vitro and in vivo. This GA-induced vacuolation in various cancer cells was derived from dilation of the endoplasmic reticulum (ER) and mitochondria, and was blocked by cycloheximide. These findings suggest that GA kills cancer cells by inducing paraptosis, a vacuolization-associated cell death. We found that megamitochondria formation, which arose from the fusion of swollen mitochondria, preceded the fusion of ER-derived vacuoles. GA-induced proteasomal inhibition was found to contribute to the ER dilation and ER stress seen in treated cancer cells, and megamitochondria formation was followed by mitochondrial membrane depolarization. Interestingly, GA-induced paraptosis was effectively blocked by various thiol-containing antioxidants, and this effect was independent of ROS generation. We observed that GA can react with cysteinyl thiol to form Michael adducts, suggesting that the ability of GA to covalently modify the nucleophilic cysteinyl groups of proteins may cause protein misfolding and subsequent accumulation of misfolded proteins within the ER and mitochondria. Collectively, our findings show that disruption of thiol proteostasis and subsequent paraptosis may critically contribute to the anti-cancer effects of GA.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. GA induces cell death accompanied by vacuolation in vitro and in vivo.
a, c Cells were treated with the indicated concentrations of GA for 24 h. Cellular viability was assessed using IncuCyte as described in Materials and Methods. Data represent the means ± SEM (n = 3). Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. *p < 0.01 vs. untreated control. b, d Cells treated with GA at the concentrations around the IC50, (MDA-MB 453 (1.5 μM), MDA-MB 468 (2.35 μM), MDA-MB 435S (1.33 μM), BxPC-3 (1.68 μM), NCI-H460 (2.35 μM), SNU-449 (1.62 μM), and SNU-668 (1.41 μM)), which were calculated using GraphPad Prism, were observed by phase-contrast microscopy. MCF-10A cells treated with GA at the indicated concentrations for 24 h were observed by phase-contrast microscopy. Bars, 40 μm. e Athymic nude mice of 6–8 weeks old were xenografted with MDA-MB 435S cells and injected with vehicle, 4 mg/kg GA, and 8 mg/kg GA as described in Materials and Methods. Tumor sizes were measured every 2–3 days after the beginning of vehicle or GA injection and plotted for growth curve. Data represent the means ± SD. Kruskal-Wallis test was performed followed by Dunn’s test. *p < 0.05 vs. vehicle-treated mice. f treated mice and tumors isolated from those mice were photographed on the 14th day. g The results of H&E staining in tumor tissues of the mice treated with 4 mg/kg GA. Bars, 20 μm
Fig. 1
Fig. 1. GA induces cell death accompanied by vacuolation in vitro and in vivo.
a, c Cells were treated with the indicated concentrations of GA for 24 h. Cellular viability was assessed using IncuCyte as described in Materials and Methods. Data represent the means ± SEM (n = 3). Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. *p < 0.01 vs. untreated control. b, d Cells treated with GA at the concentrations around the IC50, (MDA-MB 453 (1.5 μM), MDA-MB 468 (2.35 μM), MDA-MB 435S (1.33 μM), BxPC-3 (1.68 μM), NCI-H460 (2.35 μM), SNU-449 (1.62 μM), and SNU-668 (1.41 μM)), which were calculated using GraphPad Prism, were observed by phase-contrast microscopy. MCF-10A cells treated with GA at the indicated concentrations for 24 h were observed by phase-contrast microscopy. Bars, 40 μm. e Athymic nude mice of 6–8 weeks old were xenografted with MDA-MB 435S cells and injected with vehicle, 4 mg/kg GA, and 8 mg/kg GA as described in Materials and Methods. Tumor sizes were measured every 2–3 days after the beginning of vehicle or GA injection and plotted for growth curve. Data represent the means ± SD. Kruskal-Wallis test was performed followed by Dunn’s test. *p < 0.05 vs. vehicle-treated mice. f treated mice and tumors isolated from those mice were photographed on the 14th day. g The results of H&E staining in tumor tissues of the mice treated with 4 mg/kg GA. Bars, 20 μm
Fig. 2
Fig. 2. GA induces the paraptotic morphologies in cancer cells.
a, b YFP-ER or GFP-Sec61β cells (a) and YFP-Mito cells (b) and treated with 1 μM GA for the indicated time points were observed under the confocal microscope. Representative pictures of cells are shown. Bars, 20 μm. c Time-lapse imaging results of YFP-Mito cells treated with GA under the confocal microscope. Representative pictures of cells are shown. Bars, 20 μm. d Cells were treated with 1 μM GA for 12 h, fixed, and subjected to the immunocytochemistry of PDI and SDHA. Bars, 20 μm. e Transmission electron microscopy of MDA-MB 435S cells treated with 1 μM GA for 24 h. Bars, 2 μm
Fig. 3
Fig. 3
(See legend on next page.)
Fig. 4
Fig. 4. ROS levels are not noticeably increased in spite of the MMP loss in GA-induced paraptosis.
a MDA-MB 435S cells pretreated with or without 2 μM CHX and further treated with 1 μM GA for the indicated time points. Cell extracts were prepared for Western blotting of the indicated proteins. Western blotting of β-actin was used as a loading control. b, c YFP-Mito cells (b) or MDA-MB 435S cells (c) were treated with 1 μM GA for the indicated time points or 20 μM CCCP for 12 h were incubated with TMRM. Samples were subjected for confocal microscopy (b) or flow cytometry (c). d YFP-ER cells were treated with 1 μM GA for the indicated time points or 20 μM CCCP for 12 h. Treated cells were incubated with MTR and observed under the confocal microscope. e Cells treated with GA (1 μM for MDA-MB 435S; 2 μM for MDA-MB 453; 3 μM for MDA-MB 468 and NCI-H460 cells) for 16 h or 5 mM H2O2 for 10 min were incubated with CM-H2DCF-DA (DCF-DA) and subjected for the fluorescence microscopy. Bars, 40 μm
Fig. 5
Fig. 5
(See legend on next page.)
Fig. 5
Fig. 5
(See legend on next page.)
Fig. 6
Fig. 6. The activity of GA to bind to thiol-containing proteins may be critical for its paraptosis-induced ability in cancer cells.
a Proposed chemical structures of the GA-GSH and GA-NAC adducts. b Full-scan product ion scan spectra and the expected structures of GA, GA-GSH, and GA-NAC adduct formed upon Michael addition of GSH or NAC. The m/z values of the GA-GSH adduct represent GSH at 308, GA at 629, and the adduct form at 936. The m/z values of the GA-NAC adduct represent NAC at 164, GA at 651, and the adduct form at 814. c Increasing concentrations of NAC were pre-incubated with 1 μM GA in serum-free medium for the indicated time durations at room temperature, and these mixtures were used to treat MDA-MB 435S cells for 24 h. The cell viability was measured using IncuCyte. Data represent the means ± SD. Kruskal-Wallis test was performed followed by Dunn’s test. *p < 0.05; **p < 0.01 vs. GA-treated cells. d MDA-MB 435S cells were treated with the indicated concentrations of GA or 10 μM iodoacetamide (IAM; a positive control to reduce intracellular protein-SH levels) for 12 h. Protein-SH levels were measured using the dibromobimane (dBrB) assay, as described in the Materials and Methods. Data represent the means ± SD. Kruskal-Wallis test was performed followed by Dunn’s test. *p < 0.01 vs. untreated control. e Cell extracts were prepared from the cells pretreated with the antioxidants and further treated with GA (1 μM for MDA-MB 435S and 2 μM for MDA-MB 453 cells) for the indicated concentrations for Western blotting. β-actin was used as a loading control. f MDA-MB 435S cells pretreated with 2 mM NAC and further treated with 1 μM GA for the indicated time points were incubated with TMRM. Samples were subjected for flow cytometry
Fig. 7
Fig. 7
Hypothetical model for the underlying mechanisms of GA-induced paraptosis
See this image and copyright information in PMC

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