Disruption of thiol homeostasis by OP-A critically contributes to OP-

Next, I examined whether inhibition of OP-A-induced cell death by thiol antioxidants occurs also in various cancer cell lines, but not is restricted in glioma cells. Recently, Morrison et al. (2017) have shown that OP-A induces different mechanisms of cell death in mammalian cells depending on the cancer cell origin.

When I examined the effects of OP-A on various cancer cells, including MDA-MB 468 and MDA-MB 435S breast cancer cells, Huh-7 and SNU-449 hepatocellular carcinoma cells (HCC), and U20S osteosarcoma cells, and HeLa cervical cancer cells, OP-A dose-dependently reduced their viabilities with a slight difference in its cytotoxicity (Figure 16A and 16B). When I examined the effect of different doses of OP-A on cellular morphologies, treatment with 3 µM OP-A for 24 h induced cellular vacuolation and cell death in HeLa, U2OS, Huh-7, and SNU-449 cells (Figure 17). Treatment with 2 µM OP-A induced a marked blebbing and swelling in MDA-MB468 and MDA-MB 435S cells together with cellular vacuolation. In an attempt to understand OP-A-mediated cell death mechanism, I tested the effects of z-VAD-fmk (an inhibitor of apoptosis), necrostatin-1 (an inhibitor of necroptosis), and CHX (an inhibitor of paraptosis). I found that OP-A-induced cell death was not affected by z-VAD-fmk in MDA-MB 435S, Huh-7, SNU-449, and HeLa cells. However, it was very effectively blocked by z-VAD-fmk in MDA-MB 468 cells and slightly but significantly inhibited in U2OS cells (Figure 18A and 18B). OP-A-induced cell death was not altered by necrostatin-1 in MDA-MB 468, Huh-7, and HeLa cells (Figure 19A and 19B). In contrast, it was effectively blocked in MDA-MB 435S and SNU-449 cells and partially blocked in U2OS cells.

OP-A-induced cell death was effectively blocked by CHX in all the tested cancer cells (Figure 20A and 20B). These results suggest that paraptosis may be a major contributor to OP-A-mediated toxicity in HeLa and Huh-7 cells, whereas both

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paraptosis and necroptosis contribute to it in SNU-449 and MDA-MB 435S cells.

In addition, both paraptosis and apoptosis are involved in MDA-MB 468 treated with OP-A, mixed cell death including paraptosis, necroptosis, and apoptosis occurs in OP-A-treated U2OS cells. Collectively, these results suggest that depending on the cancer cell types, possibly depending on the genetic backgrounds, OP-A may induce different cell death modes but paraptosis may be an important death mechanism to explain OP-A-mediated toxicity in various cancer cells.

Finally, I investigated whether the dependency of OP-A-mediated cytotoxicity on cellular thiol modulation in these cancer cells. Very interestingly, NAC pretreatment, but not Tiron pretreatment, potently blocked OP-A-induced cell death commonly in all the tested cancer cells (Figure 21A and 21B). These results suggest that disruption of thiol homeostasis may be the key underlying mechanisms involved in the anti-tumor effect of OP-A in various cancer cells, irrespective the ultimate cell death mode(s) induced by OP-A.

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Figure 16. OP-A induces the cell death in various cancer cells. (A) Various cancer cells were treated with OP-A at the indicated concentrations for 24 h.

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, ** 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 17. Ophiobolin A induces cytoplasmic vacuolation prior to cell death in diverse cancer 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 18. OP-A-induced cell death and vacuolation have a different effect by apoptosis inhibitor (z-VAD-fmk) depending on the cancer cell types. (A, B) Cells were pretreated with apoptosis inhibitors (z-VAD-fmk) 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

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vs OP-A treatment. (B) Treated cells were observed under a phase-contrast microscope. Bars, 10 μm.

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Figure 19. OP-A-induced cell death and vacuolation have a different effect by necroptosis inhibitor (Necrostatin-1) depending on the cancer cell types. (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 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 20. OP-A-induced vacuolation and cell death were commonly blocked by CHX in the tested various cancer cells. (A, B) Cells untreated or pretreated with paraptosis inhibitor (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 21. Ophiobolin A-induced vacuolation and cell death is blocked by thiol antioxidants, but not by other radical scavengers in various cancer cells. (A)

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Diverse cancer cells were pretreated with the indicated concentrations of NAC, Tiron and further treated with the indicated concentrations of 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. (B) Cells were untreated or pretreated with with 1 mM NAC or 1 mM Tiron and further treated with 1 μM or 2 μM OP-A for 24 h. Cells were observed under the phase-contrast microscope. Bar 10 μm.

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