Ophiobolin A induces paraptosis-like cell death in glioma cells via dilation of

To investigate the underlying mechanism of OP-A-induced glioma cell death, I first examined the effect of various concentrations of OP-A on the viability of various glioma cells, including T98G, U373MG, U343, U251MG, U251N, A172 and U87MG cells. OP-A treatment dose-dependently reduced the viability of these cells, as assessed using calcein-AM and EthD-1 (Figure 1A and 1B). The sensitivity of glioma cells to OP-A was slightly different and A172 cells demonstrated the highest sensitivity against OP-A. Notably, OP-A-induced cell death was accompanied by a marked vacuolation (Figure 2). To identify the origin of OP-A-induced vacuolation, I examined the morphologies of the endoplasmic reticulum (ER) and mitochondria employing YFP-ER cells (T98G sublines that express the fluorescence specifically in the ER) and Mito-Tracker Red (red-fluorescent dye that specifically stains mitochondria). While the reticular patterns of ER and filamentous or elongated mitochondria were observed in untreated YFP-ER cells, the ER green fluorescence was detected within the vacuoles and aggregated mitochondria were observed beside the nuclei in YFP-ER cells treated with 2 µM OP-A for 12 h (Figure 3A). Immunocytochemistry of PDI, an ER resident protein, and COXII, a mitochondrial protein, showed that PDI was mainly expressed at the periphery of the extensively dilated vacuoles in the cytosol and COXII was expressed as aggregated forms beside the nuclei in T98G cells treated with 2 µM OP-A for 12 h (Figure 3B). Electron microscopy showed that ER cisternae were distended and mitochondria were shortened by treatment with 2 µM OP-A for 6 h

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in T98G cells (data not shown). At 12 h, further expansion and fusion of swollen ER were observed and a dramatic dilation of the perinuclear space was also detected. At time points beyond 12 h, fusion of the dilated ER was further progressed until the most of the cellular space was almost fully occupied by expanded ER-derived vacuoles. These results indicate that OP-A-induced vacuolation is mainly derived from the dilation of the ER. OP-A-induced vacuolation and cell death in T98G cells were not affected by pretreatment with z-VAD-fmk, a pan-caspase inhibitor (Figure 4A and 4B). In contrast, TRAIL-induced cell death in T98G cells were almost completely blocked by z-VAD-fmk pretreatment (Figure 4C). In addition, while treatment of T98G cells with TRAIL induced the cleavage of caspase-3 and PARP, a substrate of caspase-3, OP-A treatment did not. Additionally, both csaspase-3 and PARP were cleaved by TRAIL, but not by OP-A treatment (Figure 4D). Furthermore, OP-A induced cell death as well as vacuolation was not affected by pretreatment with necrostatin-1, a specific inhibitor of necroptosis (Figure 5A and 5B). Collectively, these results suggest that OP-A kills glioma cells via induction of paraptosis-like cell death.

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Figure 1. OP-A induces the cell death in various glioma cells. (A) Cellular viability was assessed using calcein-AM and EthD-1 after treatment with the indicated concentration of OP-A for 24 h. The percentage of live cells was normalized to that of untreated control cells (100%). Data represent the means ± SD. One-way ANOVA and Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01 vs.

untreated control. (B) The values of IC50 (the concentration of drug that is required to the viability of treated cell for 24 h to 50%) after the viability assay using Live and Dead assay were assessed.

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Figure 2. Ophiobolin A induces cytoplasmic vacuolation prior to cell death in diverse glioma cells. Cells treated with the indicated concentrations of OP-A for 24 h were observed under the phase-contrast microscope. Bar 10 μm.

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Figure 3. Ophiobolin A triggers ER swelling in T98G cells. (A) YFP-ER cells treated with 2 μM OP-A for 12 h were stained with 100 nM mitotracker-red (MTR) and observed under the phase-contrast and fluorescence microscope. Bar 20 μm. (B) T98G cells were treated with 2 μM OP-A for 12 h, fixed, and subjected for immunocytochemistry of COX II and PDI. Bar 20 μm.

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Figure 4. Apoptosis is not a major cell death mode induced by OP-A in glioma cells. (A, B) Various glioma cells pretreated with 20 µM z-VAD-fmk for 30 min

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and further treated with the indicated concentrations of OP-A for 24 h. (A) Cellular viability was assessed using calcein-AM and EthD-1. Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. * p < 0.05 vs. untreated control. (B) Treated cells were observed under the phase-contrast microscope. Bar 10 μm. (C) Cellular viability was assessed using calcein-AM and EthD-1. As a positive control of apoptosis, the effect of 20 ng/ml TRAIL was compared. Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. * p < 0.05 vs. untreated control, # p <0.05 vs TRAIL treatment. (D) T98G cells were treated with the indicated concentrations of OP-A for 12 h or 20 ng/ml TRAIL for 24 h. Cell extracts were prepared from the treated cells and Western blotting of was performed using anti-caspase 3 and anti-PARP antibody. α-tubulin was used as a loading control in Western blots.

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Figure 5. OP-A-induced cell death and vacuolation are not associated with necroptotic cell death. (A, B) Cells were pretreated with necroptosis inhibitors (Necrostatin-1) for 30 min and further treated with the indicated concentrations of OP-A for 24 h. (A) Cellular viability was assessed using calcein-AM and EthD-1 to detect live and dead cells, respectively. Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. * p <0.05 vs

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untreated control. (B) Treated cells were observed under a phase-contrast microscope. Bars, 10 μm.

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2. OP-A induces ER stress and CHOP plays a critical role in OP-A-induced paraptosis-like cell death

Previously, ER dilation was shown to be associated with the ER stress due to accumulation of misfolded proteins in the ER lumen (Lee et al., 2016; Mimnaugh et al., 2006). Therefore, I first examined whether OP-A affects the expression of the proteins related to ER stress. I found that OP-A treatment for 12 h dose-dependently upregulated the protein levels of Grp78, IRE1α, ATF4, and CHOP as well as the phosphorylation levels of PERK and eIF2α in T98G and U373MG cells (Figure 6A). Time-course experiment showed that 2 µM OP-A treatment dose- and time-dependently accumulated poly-ubiquitinated proteins.

Immunocytochemistry also revealed that treatment with 2 µM OP-A progressively accumulated the poly-ubiquitinated protein aggregates (Figure 6B), showing the disruption of proteostasis. Paraptosis or paraptosis-like cell death is known to require de novo protein synthesis (Sperandio et al., 2000). When I tested whether blocking of the protein synthesis affects OP-A-induced cellular responses, protein synthesis blocker cycloheximide (CHX) pretreatment almost completely inhibited OP-A-induced accumulation of poly-ubiquitinated proteins and CHOP (Figure 6C) as well as ER-derived vacuolation and cell death (Figure 7A and 7B) in T98G cells.

And pretreatment with Tauroursodeoxycholic acid (TUDCA), a chemical chaperone, slightly OP-A-induced ER-derived vacuolation and partially, but significantly, attenuated OP-A-induced cell death (Figure 7C and 7D). In addition,

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OP-A-induced vacuolation and cell death were commonly and effectively blocked by CHX in the tested other glioma cell lines (Figure 8A and 8B) suggesting that CHX, which arrests de novo protein synthesis, reduces the burden on homeostatic protein-folding mechanisms and significantly delayed OP-A-induced cell death response. Taken together, these results suggest that OP-A induces ER stress and dilation via accumulation of misfolded proteins, leading to paraptosis-like cell death in glioma cells.

Among the proteins related to the unfolded protein response, CHOP is involved in making the cell death decision associated with ER stress (Rutkowski, Kaufman., 2007). In addition, since OP-A-mediated CHOP induction was commonly observed in not only T98G and U373 MG cells (Figure 6A) but also in the tested other glioma cells (Figure 9A), I further asked whether OP-A-induced cytotoxicity is associated with CHOP induction. I found that the gene silencing of CHOP employing siRNA effectively inhibited OP-A-induced cell death (Figure 9B) and vacuolation (Figure 9C). Similar results were obtained using CHOP shRNA (Figure 9D and 9E). In addition, inhibition of OP-A-induced ER-derived vacuolation by CHOP knockdown was also confirmed by the fluorescence microscopy using YFP-ER cells (Figure 10A) and immunocytochemistry of PDI and CHOP (Figure 10B).

Collectively, these results suggest that CHOP upregulation plays a crucial role in OP-A-induced paraptosis-like cell death in glioma cells.

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Figure 6. Ophiobolin A induces ER stress by accumulation of misfolded protein aggregates in glioma cells. (A) Cell extracts were prepared from the cells treated with 2 µM OP-A for the indicated time points or indicated concentrations of OP-A for 12 h. 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 20 h were fixed, immunostained using anti-ubiquitin antibody (red) and subjected to immunocytochemistry. (C) T98G cells were untreated or pretreated with 1 µM CHX and further treated with 2 µM OP-A for 12 h. Western blotting of

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ubiquitin and CHOP was performed.

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Figure 7. CHX pretreatment effectively blocks the ER dilation and cell death by OP-A in T98G cells. (A, C) YFP-ER cells pre-treated with or without 2 μM CHX (A) or 20 μM TUDCA (C) and further treated with 2 μM OP-A for 12 h.

Cells were observed under the phase-contrast and fluorescence microscopy. (B, D) T98G cells were pretreated with the indicated concentrations of CHX (B) or TUDCA (D) and further treated with 2 µM OP-A for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. * p < 0.05 vs.

untreated control, # p <0.05 vs OP-A treatment.

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Figure 8. OP-A-induced vacuolation and cell death were commonly blocked by CHX in the tested other glioma cells. (A, B) Cells untreated or pretreated with 2 μM CHX for 30 min and further treated with the indicated concentrations of OP-A for 24 h. (A) Cellular viability was assessed using calcein-AM and EthD-1 to detect live and dead cells, respectively. Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. * p < 0.05 vs.

untreated control, # p <0.05 vs OP-A treatment. (B) Treated cells were observed under a phase-contrast microscope. Bars, 10 μm.

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Figure 9. Upregulation of CHOP plays a critical role in Ophiobolin A-induced ER dilation and cell death. (A) Cell extracts were prepared from the cells treated with the indicated concentrations of OP-A for 12 h. Western blotting of the indicated proteins was performed. α-tubulin was used as a loading control in Western blots. (B, C) T98G cells transfected with the non-targeting siRNA (siNT) or CHOP siRNA were further treated with 2 μM OP-A for 24 h. (B) Cellular viability was assessed using calcein-AM and EthD-1. Knockdown of CHOP was confirmed by Western blotting. α-tubulin expression was analyzed to confirm equal loading of the protein samples. Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. * p < 0.05 vs. siNT untreated control, # p <0.05 vs siNT OP-A treatment. (C) Treated cells were observed under the phase-contrast microscope. Bars, 10 μm. (D, E) T98G cells were incubated with

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the lentivirus containing non-targeting (NT) shRNA or a CHOP-targeting shRNA (CHOP shRNA) and further treated with 2 μM OP-A for 24 h. (D) Cellular viability was assessed using calcein-AM and EthD-1. shRNA-mediated knockdown of CHOP was confirmed by Western blotting. To confirm successful siRNA- or shRNA-mediated knockdown, Western blotting of the proteins of interest was performed. Statistical significance was determined using one-way ANOVA followed by Bonferroni’s post hoc tests. * p < 0.05 vs. siNT untreated control, # p

<0.05 vs siNT OP-A treatment. (E) Treated cells were observed under the phase-contrast microscope. Bars, 10 μm.

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Figure 10. CHOP is critically involved in OP-A-induced ER dilation. (A) YFP-ER cells were transfected with siNT or CHOP siRNA and further treated with 2 μM OP-A for 8 h. Phase-contrast and fluorescence microscopy was performed. Bar 20 μm. (B) T98G cells transfected with siNT or CHOP siRNA were further treated with 2 μM OP-A for 8 h. Cells were subjected to immunocytochemistry of PDI and CHOP. Representative pictures of cells are shown. Bar 20 μm.

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3. Thiol-antioxidants but not other ROS scavengers block OP-A-induced