GBM, the most common adult primary brain cancer, remains the deadliest of all forms of brain tumors despite the many clinical trials that have attempted to improve the dismal outcomes. GBM is characterized by resistance to apoptosis, which is largely responsible for the low effectiveness of the classical chemotherapeutic approaches based on apoptosis induction in cancer cells (Krakastad, Chekenya., 2010). For treating apoptosis-resistant tumors, the induction of non-apoptotic cell death could be an option.
Recently, natural products including curcumin (Yoon et al., 2010 & 2012), celastrol (Yoon et al., 2014), and paclitaxel (Rutkowski, Kaufman., 2007) reportedly induce paraptosis or paraptosis-like cell death in resistant malignant cancer cells, although the molecular basis of this cell death mode is not fully understood. Therefore, identification of the agents to induce paraptosis or paraptosis-like cell death and understating of their underlying mechanisms may provide an alternative therapeutic strategy for overcoming innate and acquired resistance to the current proapoptotic anticancer therapies. OP-A, a natural compound made by a fungus in order to attach plant cells, was found to have significant activity against apoptosis-resistant GBM cells through induction of paraptosis-like cell death (Bury et al., 2013a), but its underlying mechanism is largely unknown. In this study, I confirmed that OP-A induces paraptosis-like cell death in glioma cells based on the following morphological and biochemical characteristics: (a) OP-A induces progressive vacuolation prior to glioma cell death mainly via swelling and fusion of the ER without apoptotic features, such as formation of apoptotic bodies and the dependency on caspases (Figure 1-5). (b) OP-A-induced ER-derived vacuolation and cell death are effectively inhibited by CHX (Figure 7 and 8). (c) OP-A-induced ER-derived dilation and cell death is closely linked to ER stress (Figure 6 and 7).
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In the kingdoms of plant, fungi and Protista vacuoles are common intracellular organelles to maintain their life (Marty., 1999; Kilonsky, Herman, Emr., 1990; Plattner., 2015). But it is not normal to have vacuoles in most animal cells. Formation of large vacuoles (cytoplasmic vacuolization or vacuolation) in mammalian cells spontaneously or after exposure to bacterial or viral pathogens or various natural and artificial low-molecular-light compounds (Henics, Wheatley., 1999; Aki, Nara, Uemura., 2012; Chen et al., 2013). Irreversible vacuolization in mammalian cells indicates cytopathological or cytotoxic conditions leading to cell death. Moreover, irreversible vacuolation is induced by various natural and synthetic compounds of different chemical structure (Rogers-Cotrone et al., 2010;
Solano et al., 2013; Grandin et al., 2012; Korsnes et al., 2011; Zhang et al., 2010).
Sometimes irreversible vacuolization by several inducers causes known types of caspase-independent cell death including paraptosis or paraptosis-like cell death, necroptosis (Sperandio et al., 2004; Christofferson, Yuan., 2010). In addition, irreversible vacuolation can affect the ER and mitochondria and Golgi apparatus.
The most striking feature of paraptosis is a vacuolization derived from the ER and/or mitochondria. When I performed ICC to observe mitochondrial changes following OP-A treatment, mitochondrial aggregation rather than its dilation was detected (Figure 3). EM (electron microscopy) analysis showed the mitochondrial fragmentation but not megamitochondria formation due to fusion among swollen mitochondria, which was shown in curcumin- or celastrol-induced paraptosis (Yoon et al., 2012 & 2014). Therefore, OP-A-induced vacuolation may be derived from the dilation of the ER, but not mitochondria.
ER is composed of a series of continuous membranous structures including the rough ER, smooth ER in eukaryotic cells (Park, Blackstone., 2010). Each domain has different functions. For example, the rough ER comprise sheets and associated with polyribosomes for protein synthesis. Smooth ER is composed of tubules and associated with lipid synthesis and delivery, establishing contact with other
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organelles (Park, Blackstone., 2010). When I observed OP-A-induced cellular morphological changes by EM, ER expansion due to the fusion of swollen ER was progressed following OP-A treatment. Although free ribosomes were not discernably detected in our electron microscopic observation, I think the rough ER, rather than the smooth ER, has expanded possibly because of OP-A-induced disruption of proteostatic balance. Similar to our results, Kar et al. (2009) showed that 15d-PGJ2 induces cancer cell death accompanied by ER-derived vacuolation.
They argued that the origins of 15d-PGJ2-induced vacuoles might be rough ER from their electron microscopy. To further clarify the origins of ER-derived vacuoles, immunocytochemistry of Climp63, an ER sheet marker protein, and Reep-5, an ER tubule marker protein were performed. Our lab observed that Climp63 is present in Eeyarestain 1 (another paraptosis inducer)-induced ER vacuoles.
As a cause of ER-derived vacuolation, Mimnaugh et al. (2006) proposed the accumulation of misfolded and ubiquitinated proteins within the ER lumen.
They suggest that buildup of misfolded proteins within the ER lumen could induce an osmotic force, leading to an influx of water from the cytoplasm to the ER lumenal space.
The ER is the subcellular organelle in which secretory and membrane proteins are folded, stabilized by disulfide bonds, post-translationally modified, oligomerized, and ultimately exported. This process is tightly monitored by ER quality control mechanisms that sense any disruption and retain unfolded protein in the ER, triggering ER stress. The unfolded protein response (UPR) is triggered by accumulating misfolded proteins in the ER lumen and attempts to restore homeostasis. This is a general notion that ER stress-induced cell death is triggered by terminal UPR when early UPR fails to restore ER functions (Sano, Reed., 2013).
I found that OP-A treatment progressively accumulated the aggregates of poly-ubiquitinated proteins and the protein levels of GRP78 and IRE1αin T98G and
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U373MG cells. The phosphorylation levels of PERK and eIF2αas well as ATF4 expression increased with a peak at 6 h and then decreased by OP-A treatment (Figure 6). In contrast, CHOP protein levels were also upregulated from 6 h of 2
M OP-A treatment and but fairly sustained afterwards, suggesting that ER stress may contribute to OP-A cytotoxicity in these glioma cells. I also found that CHOP knockdown effectively attenuated OP-A-induced ER-derived vacuolation and subsequent cell death, suggesting a crucial role of CHOP in OP-A-induced paraptosis-like cell death glioma cells (Figure 9 and 10). Han et al. (2013) recently reported that ATF4- and CHOP-mediated transcriptional regulation increases proteins synthesis, critically contributing to ER-stress-induced cell death. Therefore, further work is warranted to determine whether the induction of CHOP observed in the present work contributes to ER dilation through the transcriptional control of specific protein(s) or a global increase in protein synthesis.
Previously, our lab reported that ROS play a key role in paraptosis induced by curcumin, DMC (dimethylated curcumin), celastrol. Therefore, I investigated whether ROS are also importantly involved in OP-A induced cell death. However, measurement of ROS using DCF-DA revealed that ROS levels were not increased by Ophiobolin A (Figure 12). In addition, when I tested the effects of various antioxidants, various thiol antioxidants, including NAC, GSH, and NMPG, but not other ROS scavengers, including MnTBAP, ascorbic acid and tiron, very effectively blocked Ophiobolin A-induced vacuolation and cell death (Figure 11).
In addition, I found that ER stress markers, such as accumulation of poly-ubiquitinated proteins as well as upregulation of CHOP, induced by Ophiobolin A was effectively inhibited by thiol antioxidants, but not by other ROS scavengers (Figure 14). Taken together, these results indicate that Ophiobolin A induces paraptosis-associated cell death via disruption of sulfhydryl homeostasis rather than ROS generation. Similar to OP-A, both 15d-PGJ2 (Kar et al., 2009) and manumycin A (Singha et al., 2013) were shown to induce cancer cell death
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accompanied by ER-derived vacuolation. Like our results, the cytoplasmic vacuolation and cell death induced by 15d-PGJ2 or manumycin A were effectively blocked by thiol antioxidants, but not by other ROS scavengers (Kar et al., 2009).
Since these compounds also commonly harbor sulfhydryl (-SH)-reactive, , β-unsaturated carbonyl group(s), their covalent modification of sulfhydryl groups and a resultant protein misfolding were proposed to be the cause of ER-derived vacuolation (Kar et al., 2009; Oliva et al., 2003). Xinhe et al. (2006) suggested that disruption of disulfide bonding by thiol-Michael adduct formation lead to protein misfolding and ER stress. As the action mechanism OP-A in animal cells, Dasari et al. (2015) proposed that the reaction of the 1,4-dicarbonyl moiety of OP-A with the primary amines of intracellular proteins to lead covalent modification is important for its toxicity. Chidley et al. (2016) recently identified phosphatidylethanolamin (PE) as the molecular target of OP-A and proposed that formation of PE-OP-A adducts directly causes the observed cytotoxicity of OP-A through membrane destabilization. Based on these reports, I guessed that OP-A reacts with cysteinyl thiols to form Michael adduct. And I suggest that the ability of OP-A to covalently modify free sulfhydryl groups on proteins may cause protein misfolding and their accumulation, leading to paraptosis-like cell death. Structurally, the highly electrophilic character of the , β-unsaturated ketone substructure of OP-A allows it to react with the thiol groups of NAC or GSH to form a covalent Michael adduct, such as NAC-OP-A and GSH-OP-A conjugate, respectively (data not shown). I further investigated the effect of the interaction between OP-A and NAC on OP-A-mediated cytotoxicity. I preincubated the mixture of 2 M OP-A and different doses of NAC for different time points at room temperature in the tube to allow the chemical adduct formation before its treatment to T98G cells for 24 h. I found that increased preincubation time of the mixture of OP-A and NAC diminished the cytotoxic effect of OP-A and lower doses of NAC were required to alleviate the cytotoxicity of OP-A at the prolonged preincubation (data not shown). Furthermore,
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OP-A-induced cell death is blocked by various thiol antioxidants, but not by other ROS scavengers, not only in many GBM cells but also in many cancer cells of different tissue origins (Figure 11, 13 and 21). These results strongly suggest that NAC blocked OP-A cytotoxicity by eliminating its ability to form Michael adducts, particularly with their nucleophilic thiol groups. Disulfide bonds are formed in the ER through a series of exchange reactions between cysteinyl proteins and ER thiol-disulfide oxidoreductases (Oka, Bulleid., 2013; Vlashi et al., 2010). Disulfide bonds are also formed during the folding process of secretory proteins. The potential of OP-A to covalently modify the free sulfhydryl groups of proteins leading to protein adduct formation could disrupt the formation of correct disulfide bond and cause protein misfolding. Therefore, this disruption of sulfhydryl homeostasis by OP-A may lead to the accumulation of misfolded and subsequently ubiquitinated proteins. This could then trigger cytoplasmic vacuolation derived from ER dilation and subsequent cell death.
Paraptosis requires transcription and de novo protein synthesis (Sperandio et al., 2000 &2004). In this study, OP-A induced vacuolation and cell death were completely inhibited by the protein synthesis blocker, CHX (Figure 7A and 7B).
Probably, since continued protein synthesis may aggravate accumulation of misfolded proteins by adduct formation with OP-A, CHX pretreatment might protect cancer cells from OP-A-induced ER-derived vacuolation and death by reducing the overall protein load within the ER. In addition, newly synthesized proteins may be more sensitive than mature proteins for covalent modification by OP-A.
Very recently, Morrison et al. (2017) investigated the effects of OP-A on various cancer cell lines with different tissue origins and they showed that OP-A induces apoptosis or necrosis depending on the cell line analyzed. I also analyzed the death modes induced by OP-A in 6 different cancer cell lines using z-VAD-fmk (an apoptosis inhibitor), necrostatin-1 (a necroptosis inhibitor), and cycloheximide
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(a paraptosis inhibitor). I found that OP-A-induced vacuolation and cell death in HeLa, Huh-7 cells were effectively protected by CHX, but not by either z-VAD-fmk or necrostatin-1, suggesting that paraptosis or paraptosis-like cell death may be a major cell death in these cancer cells. In contrast, OP-A-induced vacuolation and cell death in MDA-MB 435S, SNU-449, and U2OS cells were effectively blocked by necrostatin-1, compared to z-VAD-fmk or cycloheximide, suggesting that OP-A demonstrates cytotoxicity via induction of necroptosis as a main cell death mode.
OP-A-induced cell death in MDA-MB 468 and U2OS cells significantly attenuated by z-VAD-fmk, but not by necrostatin-1 or cycloheximide. In addition, notable vacuolation was not induced by OP-A in MDA-MB 468 cells, suggesting that apoptosis may be a major cell death mode in these cancer cells (Figure 18-20).
Collectively, these results suggest that depending on the cell types and the used OP-A concentrations, the contribution of the respective cell death mode may be different. The final cell death mode induced by OP-A may be determined by the genotypes of cancer cells. However, OP-A progressively induced cellular vacuolation in these cells, albeit not in every cell, suggesting that paraptosis-like cell death critically contribute to OP-A-induced cytotoxicity in these cancer cells.
It is interesting to speculate how OP-A induces paraptosis-like cell death commonly in glioma cells as a main cell death mode. OP-A induced glioma cell death was not affected by z-VAD-fmk (an apoptosis inhibitor) or necrostatin-1 (a necroptosis inhibitor), differently from their effects on other types of cancer cells.
These results suggest that glioma cells may be more vulnerable to induction of OP-A-induced paraptosis-like cell death, whereas OP-A induces mixed types of cell death modes in other cancer cells. In addition, lower OP-A concentrations were required to effectively kill glioma cells in the present study, compared to the effects of OP-A on other cancer cell types in the results by Morrison et al. (2017). These results suggest that glioma cells may be more sensitive to the anti-cancer effects of OP-A than cancer cells originated from other tissues.
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Notably, OP-A-induced cell death in all the tested cells was completely blocked by NAC (Figure 11 and Figure 21). Therefore, disruption of thiol homeostasis due to the activity of OP-A to react with thiol-containing proteins and a resultant induction of ER stress may be a common mechanism to explain the anti-cancer effect of OP-A, which is not restricted to GBM cells. It is also intriguing to speculate whether OP-A specifically or non-specifically forms adduct with intracellular thiol-containing proteins. Recent studies have reported dysregulated potassium channel expression in human cancer (Huang X, Jan LY., 2014). For example, overexpression of the voltage-gated potassium channel Kv1.1 marks a subgroup of medulloblastoma (Taylor et al., 2012); elevated Kv1.3 expression is detected in a number of human malignancies including breast, colon, and prostate cancer (Comes et al., 2013); high Kv11.1 (HERG) expression marks both solid and blood cancer (Pillozzi et al., 2002; Jehle et al., 2011); and Overexpression of a specific splice isoform of the BK channel correlates with the malignancy grade of glioma (Liu et al., 2002). Huang X, Jan LY. (2014) summarized the overexpression of the specific types of potassium channel in cancer. They showed that many potassium channel genes are overexpressed in glioma cells. Especially, the KCNA5, channel gene expression is inversely correlated with tumor malignancy and clinical aggressiveness in glioma (Preussat et al., 2003) and lymphoma (Bielanska et al., 2009). Moreover, Previously, Bury et al. (2013a) proposed that the decrease in BK channel activity may be involved in Ophiobolin A-induced paraptosis-like cell death in glioblastoma cells, although its functional significance was not examined yet. Therefore, we cannot exclude the possibility that the cysteinyl residues of BKC channels, which are overexpressed in glioma cells, can be much more easily targeted by OP-A, contributing to effective induction of paraptosis-like cell death.
The present study clearly showed that CHOP plays a critical role in the context of ER dilation. However, it is not clear how the activity of CHOP was not affected by OP-A without the adduct formation. CHOP is a prominent resident of
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the ER and intracellular thiols such as cysteine, homocysteine, and glutathione play critical roles in the regulation of ER protein synthesis and folding (Kumar et al., 2006). Interestingly, CHOP has no cysteine in its protein structure, suggesting that CHOP can be still active, because it is not targeted by OP-A to form adduct leading to its inactivation. Vinay et al. (2012) reported that free L-cysteine, but not D-cysteine and homoD-cysteine and glutathione, changes the global conformation in response to the cellular redox state and/or ER stress. Therefore, it remains to be clarified whether sequestration of cysteinyl thiols by OP-A to form adduct may affect the activity of CHOP, which is relatively upregulated at later phase of ER stress, by modulating its conformation.
In summary, the activity of OP-A to covalently modify free sulfhydryl groups on proteins may cause accumulation of misfolded proteins to induce ER stress and ER dilation, ultimately leading tor paraptosis-like cell death (Figure 15).
Therefore, the ability of OP-A to disrupt sulfhydryl proteostasis may provide an effective therapeutic strategy against human glioblastoma, a deadliest malignancy which are resistant to various anti-cancer treatments.
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