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. 2017 Nov 20;8(63):106740-106752.
doi: 10.18632/oncotarget.22537. eCollection 2017 Dec 5.

Ophiobolin A kills human glioblastoma cells by inducing endoplasmic reticulum stress via disruption of thiol proteostasis

In Young Kim  1   2 , MiRi Kwon  1   2 , Min-Koo Choi  3 , Dongjoo Lee  4 , Dong Min Lee  1   2 , Min Ji Seo  1   2 , Kyeong Sook Choi  1   2
Affiliations

Ophiobolin A kills human glioblastoma cells by inducing endoplasmic reticulum stress via disruption of thiol proteostasis

In Young Kim et al. Oncotarget. .

Abstract

Ophiobolin A (OP-A), a fungal sesterterpene from Bipolaris oryzae, was recently shown to have anti-glioma activity. We show here that OP-A induces paraptosis-like cell death accompanied by dilation of the endoplasmic reticulum (ER) in glioma cells, and that CHOP-mediated ER stress plays a critical role in this process. OP-A-induced ER-derived dilation and cell death were found to be independent of reactive oxygen species, but were effectively blocked by various thiol antioxidants. We observed that OP-A can react with cysteinyl thiols to form Michael adducts, suggesting that the ability of OP-A to covalently modify free sulfhydryl groups on proteins may cause protein misfolding and the accumulation of misfolded proteins, leading to paraptosis-like cell death. Taken together, these results indicate that the disruption of thiol proteostasis may critically contribute to the anti-glioma activity of OP-A.

Keywords: endoplasmic reticulum stress; ophiobolin A; paraptosis-like cell death; proteostasis; thiol.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Neither apoptosis nor necroptosis is involved in OP-A-induced cell death in various glioma cells
(A, B) Cells were treated with the indicated concentrations of OP-A for 24 h. (A) Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means ± SD (n = 7). One-way ANOVA and Bonferroni’s post hoc test. *P < 0.01, **P < 0.001 vs. untreated control. IC50s were calculated using GraphPad Prism. (B) Phase-contrast microscopy. Bar 20 μm. (C, D) Cells were pretreated with z-VAD-fmk (C) or necrostatin-1 (D) for 30 min and further treated with the indicated concentrations of OP-A for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means ± SD (n = 7). One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 vs. untreated control.
Figure 2
Figure 2. OP-A induces paraptosis-like cell death in various glioma cells
(A, B) Cells were untreated or treated with 2 μM OP-A for 12 h. (A) YFP-ER Cells were stained with Mito-tracker red (MTR) and observed under the phase-contrast and fluorescence microscope. Bar 20 μm. (B) Cells were subjected for immunocytochemistry of COX II and PDI. Bar 20 μm. (C) Transmission electron microscopy of T98G cells treated with 2 μM OP-A. Bar 20 μm.
Figure 3
Figure 3. OP-A induces ER stress in glioma cells
(A) T98G or U373 MG cells were treated with the indicated concentrations of OP-A for 12 h or 2 µM OP-A for the indicated time points and Western blotting of the indicated proteins was performed. α-tubulin was used as a loading control in Western blots. (B) T98G cells treated with 2 µM OP-A for the 12 h were fixed, immunostained using anti-ubiquitin antibody (green), and subjected to immunocytochemistry.
Figure 4
Figure 4. OP-A-induced vacuolation and cell death in various glioma cells are inhibited by CHX pretreatment
(A) T98G cells were untreated or pretreated with 1 µM CHX and further treated with 2 µM OP-A for 12 h. Western blotting of ubiquitin, CHOP, and α-tubulin was performed. (B) Cells were pretreated with the indicated concentrations of CHX and further treated with the indicated concentrations of OP-A for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means ± SD (n = 7). Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. *P < 0.001 vs. untreated control, #P < 0.05 vs. OP-A treatment. (C) Cells pretreated with 1 µM CHX and further treated with the indicated concentrations of OP-A for 24 h were observed under the phase-contrast microscope. Bar, 20 μm. (D) YFP-ER cells were untreated or pre-treated with 2 μM CHX and further treated with 2 μM OP-A for 12 h. Cells were observed under the phase-contrast and fluorescence microscopy. Bar, 20 µm.
Figure 5
Figure 5. CHOP upregulation plays a critical role in OP-A-induced paraptosis-like cell death
(A) U343, U251N, U251MG and A172 cells were treated with indicated concentration of OP-A for 12 h and Western blotting of CHOP and α-tubulin was performed. (B–E) T98G cells transfected with the non-targeting siRNA (siNT) or CHOP siRNA were further treated with 2 μM OP-A for 16 h. (B) CHOP knockdown was confirmed by western blotting (left). Cellular viability was assessed using calcein-AM and EthD-1 (right). Data represent the means ± SD (n = 7). One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 vs. untreated control, #P < 0.05 vs. OP-A treatment. (C) Treated cells were observed under the phase-contrast microscope. Bar, 20 µm. (D) YFP-ER cells were transfected with siNT or CHOP siRNA and further treated with 2 μM OP-A for 16 h. Phase-contrast and fluorescence microscopy was performed. (E) Treated cells were subjected to immunocytochemistry of PDI and CHOP. Representative pictures of cells are shown. Bar 20 μm.
Figure 6
Figure 6. OP-A-induced cellular responses in T98G cells are effectively blocked by thiol antioxidants, but not by other ROS scavengers
(A) Cellular viability was assessed using calcein-AM and EthD-1 in T98G cells pretreated with the indicated concentrations of various antioxidants and further treated with 2 µM OP-A for 24 h. Data represent the means ± SD (n = 7). One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 vs. untreated control, #P < 0.05 vs. OP-A treatment. (B, C) T98G (B) or YFP-ER (C) cells were pretreated with 1 mM NAC, 1 mM GSH, 0.2 mM NMPG, 0.25 mM ascorbic acid, 200 µM MnTBAP, or 1 mM Tiron and further treated with 2 µM OP-A for 16 h. Cells were observed under the phase-contrast microscope (B) or fluorescence microscopy (C). Bar 20 μm. (D) T98G cells were treated with 2 µM OP-A or 10 mM H2O2 for the indicated time points and then incubated with DCF-DA and subjected for flow cytometry. (E) T98G cells treated with 10 mM H2O2 for 30 min or 2 µM OP-A for 10 h were observed under the phase-contrast and fluorescence microscope. Bar, 20 μm.
Figure 7
Figure 7. OP-A-induced cellular responses in various glioma cells are effectively blocked by NAC, but not by Tiron
(A) Cells were pretreated with the indicated concentrations of NAC or Tiron and further treated with 2 µM OP-A for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means ± SD (n = 7). One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 vs. untreated control, #P < 0.05 vs. OP-A treatment. (B) Cells pretreated with 1 mM NAC or 1 mM Tiron and further treated with the indicated concentrations of OP-A for 24 h were observed under the phase-contrast microscope. Bar, 20 μm.
Figure 8
Figure 8. The thiol reactivity of OP-A is critical for its ability to induce paraptosis-like cell death in glioma cells
(A) T98G cells were pretreated with the indicated concentrations of the respective antioxidant and further treated with 2 µM OP-A for 12 h. Cell extracts were subjected to Western blotting for the indicated proteins. (B) Proposed chemical structures of the OP-A-GSH and OP-A-NAC adducts. (C) Full-scan product ion scan spectra and the expected structures of OP-A, OP-A-NAC, and OP-A-GSH adducts formed upon Michael addition of NAC or GSH. The m/z values of the OP-A-NAC adduct represent NAC at 164, OP-A at 401, and the adduct form at 564. The m/z values of the OP-A-GSH adduct represent GSH at 308, OP-A at 401, and the adduct form at 708. (D) Increasing concentrations of NAC were pre-incubated with 2 µM OP-A in serum-free medium for the indicated time durations at room temperature, and these mixtures were used to treat T98G cells for 24 h. The relative cell viability was measured using calcein-AM and EthD-1. Data represent the means ± SD (n = 7). One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001, **P < 0.0001 vs. OP-A-treated cells. (E) T98G cells were treated with the indicated concentrations of NAC and/or OP-A for 4 h. As a positive control to reduce intracellular protein-SH levels, IAM was used. T98G cells were treated with 50 µM IAM for 4 h. Protein-SH levels were measured using the dibromobimane assay, as described in the Materials and Methods. Data represent the means ± SD. Kruskal-Wallis test was performed followed by Dunn’s test. *P < 0.001 vs. untreated control, #P < 0.001 vs. OP-A treatment.
Figure 9
Figure 9. Hypothetical model for the underlying mechanism of OP-A-induced paraptosis-like cell death
See this image and copyright information in PMC

References

    1. Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, Hahn WC, Ligon KL, Louis DN, Brennan C, Chin L, DePinho RA, Cavenee WK. Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev. 2007;21:2683–2710. - PubMed
    1. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomized phase III study: 5-year analysis of the EORTCNCIC trial. Lancet Oncol. 2009;10:459–466. - PubMed
    1. Yoon MJ, Kim EH, Lim JH, Kwon TK, Choi KS. Superoxide anion and proteasomal dysfunction contribute to curcumin-induced paraptosis of malignant breast cancer cells. Free Radic Biol Med. 2010;48:713–726. - PubMed
    1. Yoon MJ, Kim EH, Kwon TK, Park SA, Choi KS. Simultaneous mitochondrial Ca2+ overload and proteasomal inhibition are responsible for the induction of paraptosis in malignant breast cancer cells. Cancer Lett. 2012;324:197–209. - PubMed
    1. Yoon MJ, Lee AR, Jeong SA, Kim YS, Kim JY, Kwon YJ, Choi KS. Release of Ca2+ from the endoplasmic reticulum and its subsequent influx into mitochondria trigger celastrol-induced paraptosis in cancer cells. Oncotarget. 2014;5:6816–31. https://doi.org/10.18632/oncotarget.2256. - DOI - PMC - PubMed