c-FLIP L downregulation plays a critical role in CDDO-ME-induced apoptotic cell death, but not in vacuolation

To clarify the possible contributing factor(s) that lead to caspase-dependent apoptosis by CDDO-ME, we tested whether CDDO-ME modulates the expression of caspase antagonists. Among tested IAPs, protein levels of c-IAP2 were not altered in MDA-MB 435S cells by 1.5 µM CDDO-ME treatment, although those of XIAP and c-IAP1 protein levels were slightly reduced from 12 or 24 h of CDDO-ME post-treatment (Figure 34). In contrast, the protein levels of c-FLIPL, the caspase-8 inhibitor (Kataoka T et al. 2005; Safa AR. et al. 2012), were rapidly and strikingly reduced from 3 h of CDDO-ME treatment, although c-FLIPS protein levels were not detected in the absence or presence of CDDO-ME. In addition, CDDO-ME treatment commonly and dose-dependently reduced the protein levels of c-FLIPL in MDA-MB 435S, MDA-MB 231 and MCF-7 cells (Figure 35).

When we analyzed whether CDDO-ME affected the protein stability of c-FLIPL by blocking of protein synthesis, c-FLIPL protein levels were reduced from 3 h of cycloheximide (Figure 36A). In contrast, those of c-FLIPL were decreased from 1 h by treatment with cycloheximide plus CDDO-ME, indicating that CDDO-ME treatment reduces the protein stability of c-FLIPL. In addition, we found that CDDO-ME progressively dowregulated c-FLIPL mRNA levels by RT-PCR analysis (Figure 36B).

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Taken together, these results suggest that CDDO-ME potently downregulates c-FLIPL

both at the transcriptional and post-transcriptional control.

Since the expression of c-FLIP was previously reported to be modulated by proteasome activity (Fukazawa Tet al. 2001; Zhong Q et al. 2005), we further tested the effect of the proteasome inhibitors on the protein levels of c-FLIPL. We found that CDDO-ME-induced reduction of c-FLIPL protein levels were effectively restored by pretreatment with either MG132 or bortezomib (Figure 37), suggesting that proteasome-mediated degradation of c-FLIPL protein may be involved in CDDO-ME-induced downregulation.

To investigate the functional significance of c-FLIP downregulation during CDDO-ME-induced apoptosis, we next investigated whether exogenously expressed c-FLIPL

could block ME-induced cell death. When we examined the effect of CDDO-ME using MDA-MB 435S sublines overexpressing c-FLIPL, we found that c-FLIPL

overexpression significantly attenuated CDDO-ME-induced cell death (Figure 38A and 38B). Interestingly, c-FLIPL overexpression did not affect CDDO-ME-induced vacuolation, but inhibited cellular shrinkage and formation of apoptotic bodies, which were detected in the control MDA-MB 435S sublines treated with CDDO-ME for 24 h (Figure 38C).In addition, c-FLIPL overexpression blocked CDDO-ME-induced cleavage of PARP, similar to the effect of z-VAD-fmk (Figure 38A), suggesting that c-FLIP downregulation may play a critical role in CDDO-ME-mediated apoptotic cell death.Similar to these results, pretreatment with z-VAD-fmk also blocked apoptotic morphologies, which were detected in the control cells treated with CDDO-ME for 24 h,

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but not vacuolation, which were observed in cells treated with CDDO-ME for 6 h(Figure 39A and 39B).

We further investigated the relationship between Ca²⁺, ROS and c-FLIP downregulation in the progression of CDDO-ME-induced cell death. We found that pretreatment with neither BAPTA plus BAPTA-AM nor NAC did block induced downregulation of c-FLIP (Figure 40). These results suggest that CDDO-ME-induced c-FLIP downregulation is independent of ER-derived vacuolation that is mainly controlled by the increase in intracellular Ca²⁺ and ROS. However, CDDO-ME-induced c-FLIPL downregulation may contribute to switch the balance of these breast cancer cells.

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Figure 34. Change in protein levels of caspase antagonists in 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 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 35. CDDO-ME downregulates c-FLIPL in breast cancer 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 36. CDDO-ME modulates c-FLIPL in both transcriptional and post-transcriptional level.(A) MDA-MB 435S cells were treated with or without 1.5 μM CDDO-ME in the presence of CHX (2 μM) for the indicated time periods. The band intensities of c-FLIPL were measured by a densitometric analysis. (B) The mRNA expression levels of c-FLIPL were determined by RT-PCR. The level of GAPDH was used as loading controls.

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Figure 37. CDDO-ME downregulates c-FLIPL expression by proteasome system.

Cells were pretreated with 1 µM MG132 or 10 nM bortezomib and further treated with 1.5 µM CDDO-ME for 24 h followed by Western blotting. β-actin was used as a loading control.

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Figure 38.c-FLIP downregulation is critical for apoptotic cell death but not for vacuolation by CDDO-ME.(A) Vector cells (MDA-MB 435S/Vec) and c-FLIPL

overexpressed cells (MDA-MB 435S /c-FLIP) were treated with 1.5 µM CDDO-ME for 24 h. Overexpression of c-FLIP was confirmed by Western blotting using anti-c-FLIPL

antibody. β-actin was used as a loading control in Western blots. Cellular viability was assessed using calcein-AM and EthD-1. (B) Cells were observed under the phase contrast microscopy. Bars, 20 µm.

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Figure 39.Overexpression of c-FLIP has similar effectsto z-VAD-fmk. (A)MDA-MB 435S cells were pretreated with 10 µM z-VAD-fmk and further treated with 1.5 µM CDDO-ME for the indicated time points and observed under the phase contrast microscopy. Bars, 20 µm. (B) MDA-MB 435S cells were pretreated with 10 µM z-VAD-fmk and further treated with 1.5 µM CDDO-ME for 24 h followed by Western blotting. β-actin was used as a loading control in Western blots.

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Figure 40.c-FLIP downregulation is not associated with increase of both intracellular Ca²⁺ and ROS levels.MDA-MB 435S cells were pretreated with NAC or BAPTA+BAPTA-AM and further treated with 1.5 µM CDDO-ME for 24 h followed by Western blotting of c-FLIPL. β-actin was used as a loading control in Western blots.

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6. Higher increase in intracellular Ca

and ROS levels as well as