1.CDDO-ME demonstrates a potent anti-cancer effect on breast cancer cells
2. CDDO-ME induces paraptosis-like cellular vacuolation prior to morphologies features of apoptosis in breast cancer cells
To understand the underlying mechanism of the potent cytotoxic effect of CDDO-ME on breast cancer cells, we first examined changes in the cellular morphologies of breast cancer cells following CDDO-ME treatment. Interestingly, we found that severe cellular vacuolation preceded the apoptotic morphologies, including cell shrinkage, cytoplasmic blebbing, and apoptotic bodies, commonly in MDA-MB 435S, MDA-MB 231 and MCF-7 cells (Figure 4). Since a double-membrane vesicle known as autophagosome were observed in cytoplasm during autophagy process, we next examined whether CDDO-ME-induced vacuolation and cell death was associated with autophagy. When we examined the knockdown effects of ATG5, Beclin-1, or LAMP2 on CDDO-ME-induced cellular responses, neither CDDO-ME-CDDO-ME-induced cellular vacuolation, which was observed at 12 h of CDDO-ME post-treatment, nor cell death, which was measured at 24 h of post-treatment, was affected by their knockdown (Figure 5A-5C). In addition, pretreatment with autophagy inhibitors, including 3-methyladenine (3-MA) and chloroquine (CQ), did not affect CDDO-ME-induced vacuolation and cell death (Figure 6A and 6B).Also, when we observed the expression of LC3, known as a autophagy marker, and p62, substrate proteins of autophagy (Johansen T et al. 2011), time-course experiment showed that the protein levels of both LC3B and p62 were significantly increased following CDDO-ME treatment (Figure 7). If CDDO-ME inducedautophagy,
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we could observe LC3 conversion from LC3-I to LC3-II and p62 degradation (Rogov V and Dötsch Vet al., 2014). However, we just observed accumulation of both LC3B and p62,suggesttingthat autophagy may not be associated with CDDO-ME-mediated cellular responses in these cells.
Next, we examined whether CDDO-ME-induced vacuoles were originated from the endoplasmic reticulum and/or mitochondria. For this purpose, we employed MDA-MB 435S sublines stably expressing fluorescence selectively in the ER (YFP-ER cells) or mitochondria (YFP-Mito cells). While the ER appeared as a reticular structure in untreated YFP-ER cells, ER fluorescence exactly co-localized with numerous vacuoles in YFP-ER cells treated with 1.5 µM CDDO-ME for 6 h. At 12 h of CDDO-ME treatment, the sizes of ER-derived vacuoles were further increased, but their numbers were decreased, possibly suggesting the fusion among these vacuoles (Figure 8). While mitochondria in untreated YFP-Mito cells showed a filamentous and elongated structure, mitochondrial fluorescence in the cells treated with CDDO-ME for 6 h revealed fragmented morphology or co-localized at very small vacuoles around the nuclei, but not at easily discernible vacuoles by the phase contrast microscopy. And then, most of mitochondria appeared to be fragmented in YFP-Mito cells treated with CDDO-ME for 12 h(Figure 8). Immunocytochemistry of protein disulfide-isomerase (PDI), an ER-resident protein, and the subunit A of succinate dehydrogenase (SDHA), a mitochondrial protein, showed that PDI expression of a reticulate structure and elongated SDHA expression were detected in untreated MDA-MB 435Scells (Figure 9),
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similar to ER and mitochondrial morphologies shown in YFP-ER and YFP-Mito cells (Figure 8). At 6 h of 1.5 µM CDDO-ME treatment, large rings of PDI expression and very small rings of SDHA expression were observed. SDHA-expressing mitochondria-derived vacuoles appeared to be localized near the nuclei, whereas PDI-expressing ER-derived vacuoles were peripheral to the mitochondria-ER-derived vacuoles. At 12 h of CDDO-ME treatment, the sizes of ER-derived vacuoles, but not mitochondria-derived vacuoles, were further increased. These results suggest that CDDO-ME-induced vacuolation is mainly resulted from the dilation of the ER in these breast cancer cells.
Since ER dilation rather than mitochondrial dilation was noted following CDDO-ME treatment, we next examined whether CDDO-CDDO-ME induces ER stress(Ron D et al., 2007; Huber AL et al., 2013; Szegezdi E et al., 2006; Bertolotti et al., 2000; Liu CY et al., 2003). Western blotting showed that CDDO-ME treatment markedly accumulated the protein levels of GRP78, phosphorylated eIF2α, ATF4, and CHOP (Figure 10).Interestingly, we found that caspase-4 was also cleaved from 12 h to 24 h by CDDO-ME treatment.We confirmed again that CDDO-ME induces ER stress by immunocytochemistry. When we performed immunocytochemistry of GRP78, ATF4 or CHOP, we observed that the expression levels increased in the nucleus (Figure 11).
These results indicated that CDDO-ME is an effective ER stress inducer in breast cancer cells.
To observe the changes in cellular organelles in more detail following CDDO-ME treatment, we performed electron microscopy. Untreated MDA-MB 435S cells
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possessed the ER structures with elongated sacs surrounded by one-layer membrane and mitochondria with intact cristae (Figure 12). In cells treated with CDDO-ME for 6 h, cellular space was mostly occupied with expanded structures of the ER and the fusion among the ER was further progressed after 12 h of treatment. When we quantitatively measured the sizes of the ER-derived vacuoles, their average width were 2.468 µm,
2.677 µm and 4.405 µm at 6, 12, and 24 h of CDDO-ME treatment, respectively. In contrast, swollen mitochondria were frequently observed at 6 h of treatment, but their sizes were rather reduced at 12 h. These results suggest that cells underwent a transient mitochondrial swelling in response to CDDO-ME treatment. Or mitochondria might undergo a fusion at 6 h of CDDO-ME treatment and then fission at 12 h. Very interestingly, the dilation of ER was accompanied by cytoplasmic blebbing and formation of apoptotic bodies at 12 h of post-treatment. At 24 h, chromatin condensation and DNA fragmentation were detected (Figure 12).
Interestingly, we observed that vacuolation induced by CDDO-ME is similar to vacuolation by paraptosis inducers, curcumin or celatrol(Yoon MJ et al., 2010; Yoon MJ and Lee AR et al., 2014). Electron microscopic images of breast cancer cells treated with CDDO-ME, curcumin or celastrol showed that all of these drugs induced dilation of the ER.Collectively, these results indicate that CDDO-ME induces paraptosis-like morphologies and subsequently apoptotic ones in these breast cancer cells (Figure 13).
Therefore, we next investigated whether paraptosis is also involved in CDDO-ME-induced cell death. Although the underlying mechanisms of paraptosis are not clearly
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understood, paraptosis is known to be inhibited by cycloheximide, because paraptosis required new gene transcription and translation(Sperandio Set al. 2004). When we tested whether CDDO-ME-induced cell death is affected by cycloheximide pretreatment, neither cell death nor ER-derived vacuolation induced by CDDO-ME was inhibited by cycloheximide pretreatment (Figure 14A and 14B). However, cells treated with curcumin and celastrol, inducing paraptosis, were completely blocked by cycloheximide.
In YFP-ER cells, although we pretreated with cycloheximide and further treated 1.5 µM CDDO-ME for 6 h, ER fluorescence still co-localized with numerous vacuoles. These results also suggested that CHX didn’t block ER dilation induced by CDDO-ME (Figure 15).
Chatellard-Causse C et al. reported that Alix interaction with endophilins can induce intracellular vacuole formation (Chatellard-Causse C et al. 2002). Alix was determined to be "the first specific inhibitor" of paraptosisby us and other groups (S. Sperandioet al.
2004; F. Valamaneshet al. 2007). Since Alix was reported a paraptosis inhibitor, we examined the changes in Alix protein levels following CDDO-ME treatment. We found that expression of Alix was not altered by CDDO-ME treatment, whereas it was markedly reduced by curcumin or celastrol, paraptosis inducers (Figure 16), indicating that CDDO-ME didn’t induce paraptosis.
Paraptosis is known to lack caspase activation and is insensitive to caspase inhibitors (Gao X et al. 2011; Kim Y et al. 2002). When we examined the expression of caspases, treatment of MDA-MB 435S cells with 1.5 µM CDDO-ME induced the proteolytic
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processing of caspase-8 from 12 h and caspase-9 and -3 from 18 h. PARP, a substrate of caspase-3, was also cleaved from 18 h of CDDO-ME post-treatment (Figure 17).
Furthermore, pretreatment with z-VAD-fmk significantly and commonly inhibited CDDO-ME-induced cell death in MDA-MB 435S, MDA-MB 231, and MCF-7 cells (Figure 18). When we examined the location of cytochrome c following CDDO-ME treatment, we found that CDDO-ME treatment released cytochrome c from mitochondria into cytosol(Figure 19A). Cleaved PARP was detected in shrunken MDA-MB 435S cells treated with CDDO-ME for 24 h, but not in all the vacuolated cells treated with CDDO-ME for 24 h (Figure 19B). These results indicate that caspase-3 may be activated in parallel with cellular shrinkage. Finally, we sought to examine whether CDDO-ME-induced cell death was associated with changes in cell cycle distribution.MDA-MB 435S cells were treated with CDDO-ME and subjected to fluorescence activated cell sorting analysis, and the percentages of sub-G1cells were quantified (Figure 20). Treatment with CDDO-ME alter the cell cycle distribution. In cells treated with 1.5 μM CDDO-ME for 48 h, the percentage of sub-G1 cells was markedly increased, suggesting that CDDO-ME induced apoptotic cell death. Taken together, CDDO-ME triggers extensive vacuolation mainly derived from the ER, but ultimately kills breast cancer cells via caspase-mediated apoptosis.
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Figure 4. CDDO-ME induces extensive dilation prior to apoptotic cell death in MDA-MB 435S cells. Three breast cancer cells were treated with 1.5 µM CDDO-ME for the indicated time points and observed under the phase contrast microscopy.Bars, 20 μm.
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Figure 5. Knockdown of ATG5, beclin-1, LAMP2 does not block cell death andvacuolationinduced by CDDO-ME. (A) MDA-MB 435S cells were treated with the lentivirus encoding the control non-targeting RNA, ATG5, Beclin-1 or LAMP2 shRNA and further treated with or without 1.5 μM CDDO-ME for 24 h. Knockdown of these gene products was confirmed by Western blots. (B) Cellular viability was assessed using calcein-AM and EthD-1.(C) Cells were observed under a phase contrast microscope. Bar, 20 µm.
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Figure 6. Effects of autophagy inhibitors on CDDO-ME-induced vacuolation and cell death. (A) MDA-MB 435S cells were pretreated with 3-MA orchloroquine for 30 min and further treated with 1.5 µM CDDO-ME for 24 h. Cell were observed under a phase contrast microscope.Bar, 20 µm. (B)Cellular viability was assessed using calcein-AM and EthD-1.
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Figure 7. Autophagy-related proteins, LC3B and p62, were accumulated in MDA-MB 435S cells treated with CDDO-ME.MDA-MDA-MB435S cells were treated with 1.5 µM CDDO-ME for indicated time points. Whole cell extracts were prepared and subjected to western blotting using LC3B and p62 antibodies. β-actin was used as a loading control.The fold change of protein levels compared to 0 h was determined by a densitometric analysis.
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Figure 8. CDDO-ME induces dilation of the ER and mitochondrial fragmentation.YFP-Mito and YFP-ER cells were treated with 1.5 µM CDDO-ME for indicated time points and then observed under a fluorescence microscope. Bar, 20 μm.
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Figure 9. Morphological changes of the ER and mitochondria in CDDO-ME-treated breast cancer cells.MDA-MB 435S cells were CDDO-ME-treated with or without 1.5 µM CDDO-ME for 12 h. Immunocytochemistry using anti-SDHA (green) and anti-PDI (red) antibodies was performed and the representative phase contrast and fluorescence microscopic images of cells are shown.Bar, 20 µm.
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Figure 10. CDDO-ME increases the protein levels of ER stress-associated proteins in MDA-MB 435S cells. Cells were treated with 1.5 µM CDDO-ME for the indicated time points or indicated doses of CDDO-ME for 24 h and then Western blotting was performed. β-actin was used as a loading control in Western blots. The fold change of protein levels compared to β-actin was determined by a densitometric analysis.
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Figure 11. CDDO-ME induces ER stress in MDA-MB 435S cells.
Immunocytochemistry of GRP78, ATF4 and CHOP was performed in MDA-MB 435S cells treated with 1.5 µM CDDO-ME for 6 h. Representative fluorescence microscopic images of cells are shown.Bar, 20 µm
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Figure 12. Electron microscopic observation of MDA-MB 435S cells treated with CDDO-ME. MDA-MB 435S cells were treated with 1.5 µM CDDO-ME for the indicated time points and observed by transmission electron microscopy. The average widths of the vacuoles originated from the ER were measured in MDA-MB 435S cells treated with 1.5 μM CDDO-ME for the indicated time points using AxioVision Rel.
4.8 software (Zeiss). White arrow heads, ER; Black arrow heads, mitochondria; White arrows, apoptotic bodies; Black arrows, blebbing. Bars, 2 µm.
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Figure 13. Electron microscopic images of MDA-MB 435S cells treated with curcumin or celastrol. MDA-MB 435S cells were treated with 40 µM curcumin for 24 h or 2 µM celastrol for 12 h and observed by transmission electron microscopy; White arrow heads, ER; Black arrow heads, mitochondria. Bars, 2 µm.
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Figure 14. Cycloheximide, a translation inhibitor, does not block CDDO-ME-induced cell death and vacuolation. (A) MDA-MB 435S cells were pretreated with the indicated concentrations of CHX for 30 min and further treated with 1.5 µM CDDO-ME for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. (B) Cells were observed under a phase contrast microscope. Bar, 20 µm
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Figure 15. CDDO-ME-induced ER dilation is not blocked by cycloheximide.YFP-ER cells pretreated with CHX and further treated with or without 1.5 μM CDDO-ME for 6 h were stained with Mitotracker Red and then observed using phase contrast and fluorescence microscopy. Bar, 20 µm
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Figure 16. Change in protein expression of Alix.MDA-MB 435S cells were treated with 1.5 µM CDDO-ME for the indicated time points or 40µM curcumin or 2µM celastrol for 24 h and then Western blotting was performed. β-actin was used as a loading control in Western blots. The fold change of protein levels compared to β-actin was determined by a densitometric analysis.
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Figure 17. Activation of caspases in MDA-MB 435S cells treated with CDDO-ME.MDA-MB 435S cells were treated with indicated doses of CDDO-ME for 24 h.
Whole cell extracts were prepared from the treated cells and subjected to Western blotting. β-actin was used as a loading control in Western blots. The fold change of protein levels compared to β-actin was determined by a densitometric analysis.
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Figure 18. Inhibition of caspase activation is critically involved in CDDO-ME-induced cell death. MDA-MB 435S, MDA-MB 231, MCF-7 cells were pretreated with the indicated concentrations of z-VAD-fmk for 30 min and further treated with 1.5 µM CDDO-ME for 24 h. Cellular viability was assessed using calcein-AM and EthD-1.
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Figure 19. CDDO-ME induces chromatin condensation and release of cytochrome c. (A) DAPI staining were performed in MDA-MB 435S cells untreated or treated with 1.5 µM CDDO-ME for 24 h. Representative fluorescence microscopic images of cells are shown. (B) Immunocytochemistry of the cytochrome c and the subunit I of cytochrome c oxidase (COX IV) was performed in MDA-MB 435S cells treated with 1.5 µM CDDO-ME for 24 h, as described in Materials and Methods. Representative fluorescence microscopic images of cells are shown. Bars, 20 μm.
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Figure 20. Effect of CDDO-ME on MDA-MB 435S cell cycle progression.MDA-MB 435S cells were treated with or without 1.5 µM CDDO-ME for 48 h. Cells were stained with propidium iodide and FACS analysis.
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