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Comparative Study
. 2014 Mar 13;5(3):e1112.
doi: 10.1038/cddis.2014.85.

Stronger proteasomal inhibition and higher CHOP induction are responsible for more effective induction of paraptosis by dimethoxycurcumin than curcumin

M J Yoon  1 , Y J Kang  1 , J A Lee  1 , I Y Kim  1 , M A Kim  2 , Y S Lee  3 , J H Park  4 , B Y Lee  4 , I A Kim  5 , H S Kim  6 , S-A Kim  6 , A-R Yoon  7 , C-O Yun  7 , E-Y Kim  8 , K Lee  8 , K S Choi  9
Affiliations
Comparative Study

Stronger proteasomal inhibition and higher CHOP induction are responsible for more effective induction of paraptosis by dimethoxycurcumin than curcumin

M J Yoon et al. Cell Death Dis. .

Abstract

Although curcumin suppresses the growth of a variety of cancer cells, its poor absorption and low systemic bioavailability have limited its translation into clinics as an anticancer agent. In this study, we show that dimethoxycurcumin (DMC), a methylated, more stable analog of curcumin, is significantly more potent than curcumin in inducing cell death and reducing the clonogenicity of malignant breast cancer cells. Furthermore, DMC reduces the tumor growth of xenografted MDA-MB 435S cells more strongly than curcumin. We found that DMC induces paraptosis accompanied by excessive dilation of mitochondria and the endoplasmic reticulum (ER); this is similar to curcumin, but a much lower concentration of DMC is required to induce this process. DMC inhibits the proteasomal activity more strongly than curcumin, possibly causing severe ER stress and contributing to the observed dilation. DMC treatment upregulates the protein levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and Noxa, and the small interfering RNA-mediated suppression of CHOP, but not Noxa, markedly attenuates DMC-induced ER dilation and cell death. Interestingly, DMC does not affect the viability, proteasomal activity or CHOP protein levels of human mammary epithelial cells, suggesting that DMC effectively induces paraptosis selectively in breast cancer cells, while sparing normal cells. Taken together, these results suggest that DMC triggers a stronger proteasome inhibition and higher induction of CHOP compared with curcumin, giving it more potent anticancer effects on malignant breast cancer cells.

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Figures

Figure 1
Figure 1
DMC demonstrates more potent anticancer effects than curcumin in vitro and in vivo. (a) Chemical structures of curcumin and DMC. (b) Effects of curcumin and DMC on the viability of various breast cancer cells in vitro. Cells were treated with curcumin or DMC at the indicated concentrations for 24 h and their cellular viabilities were assessed using calcein-AM and EthD-1. (c) Dose- and time-dependent effects of curcumin and DMC on the long-term survival of MDA-MB 435S cells. MDA-MB 435S cells seeded on six-well plates were treated with DMC or curcumin at the indicated concentrations for 12 h and then media were replaced with drug-free media. Following the subsequent incubation for 9 days, cells were stained with 0.5% crystal violet. Representative dishes after clonogenic assay are shown and colony-forming units were enumerated and expressed as the percentages of control cells. (d) Effects of curcumin and DMC on the tumor sizes of the nude mice with xenograft. Athymic nude mice of 6–8 weeks old were xenografted with MDA-MB 435S cells and injected with vehicle, 25 mg/kg curcumin, 50 mg/kg curcumin, 25 mg/kg DMC or 50 mg/kg DMC as described in Materials and Methods section. Tumor sizes were measured every 2 days after the beginning of vehicle (filter-sterilized PBS containing 0.25% DMSO), curcumin or DMC injection. (e) MDA-MB 435S/Luc cells were injected into the left thigh of athymic mice. Xenografted mice were treated with vehicle, 50 mg/kg curcumin or 50 mg/kg DMC as described in Materials and Methods section. Tumor progression was evaluated by bioluminescent imaging at day 20 after the beginning of the indicated treatments
Figure 2
Figure 2
Dilation of mitochondria and ER precedes DMC-induced cell death in breast cancer cells. (a) Cells were treated with 20 μM DMC for 12 h and observed under a phase contrast microscope. Bars, 20 μm. (b) Hematoxylin and eosin-stained sections of MDA-MB 435S xenografts treated with vehicle or 25 mg/kg DMC at intervals of 2 days for 20 days. Vacuoles are indicated by arrows. (c) MDA-MB 435S cells were pre-treated with the indicated specific inhibitors of autophagy (3-MA; bafiolmycin A1 (Bafilo. A1); CQ) and further treated with 20 μM DMC for 24 h. Cell viability was assessed using calcein-AM and EthD-1. (d) MDA-MB 435S or MDA-MB 231 cells were treated with 20 μM DMC for the indicated time points and western blotting of autophagy-related proteins were performed. Western blotting of β-actin served as the loading control of protein samples. (e) MDA-MB 435S cells were transfected with LC3 siRNA and further treated with or without 20 μM DMC for 24 h. Knockdown of LC3 was confirmed by western blotting using anti-LC3 antibody. Western blotting of β-actin was served as a loading control (upper panel). Cellular viability was assessed using calcein-AM and EthD-1 (lower panel). (f) MDA-MB 435S cells were transfected with LC3 siRNA and further treated with or without 20 μM DMC for 16 h. Cellular morphologies were observed under a phase contrast microscope. Bar, 20 μm
Figure 3
Figure 3
DMC induces the dilation of mitochondria and the ER. (a) MDA-MB 435S sublines (YFP-Mito/435S or YFP-ER/435S) expressing the fluorescence selectively in mitochondria or the ER were treated with 20 μM DMC for the indicated time points and observed under the fluorescent and phase contrast microscope. Bars, 20 μm. (b) The average widths of the vacuoles originated from mitochondria or the ER were measured in YFP-Mito cells or YFP-ER cells treated with 20 μM DMC for the indicated time points using AxioVision Rel. 4.8 software (Zeiss). Marked increase in the width of the ER-derived vacuoles was observed following treatment with 20 μM DMC. Results were repeated in three other independent experiments. In each experiment, 50 cells were scored. Bar, 20 μm. (c) MDA-MB 435S cells were untreated or treated with 20 μM DMC for 16 h, fixed and subjected for immunocytochemistry of COX IV and PDI. Representative pictures of cells are shown. Bars, 20 μm. (d) MDA-MB 435S cells were treated with 20 μM DMC for the indicated durations and electron microscopy was performed as described in the Materials and Methods section. White arrows, megamitochondria; black arrows, swollen and fused ER. Bars, 2 μm. (e) MDA-MB 435S cells were treated with the indicated concentrations of DMC or curcumin for 16 h. Representative pictures of cells are shown. Bar, 20 μm
Figure 4
Figure 4
DMC activates paraptotic signals in malignant breast cancer cells. (a) MDA-MB 435S cells were pre-treated with CHX at the indicated concentrations and further treated with 20 μM DMC for 24 h. Cell viability was assessed using calcein-AM and EthD-1. (b) MDA-MB 435S cells were pre-treated with or without 2 μM CHX and further treated with 20 μM DMC for 16 h. Cellular morphologies after treatments were observed under a phase contrast microscope. Bar, 20 μm. (c) MDA-MB 435S cells were treated with 20 μM DMC or 40 μM curcumin for the indicated time points. Cell extracts were prepared for western blotting. Compared with control (untreated cells), the fold change of protein levels was determined. (d) MDA-MB 435S cells were pre-treated with the indicated specific inhibitors (PD98059, U0126, L-JNKI and SB203580) and further treated with 20 μM DMC for 24 h. Cell viability was assessed using calcein-AM and EthD-1. (e) MDA-MB 435S cells treated with 20 μM DMC or 40 μM curcumin for the indicated time points were subjected to western blotting of the indicated proteins. Western blotting of β-actin was served as a loading control. (f) MDA-MB 435S cells were pre-treated with the indicated concentrations of various antioxidants and further treated with 20 μM DMC for 24 h. Cell viability was assessed using calcein-AM and EthD-1. (g) MDA-MB 435S cells were treated with 20 μM DMC for the indicated time periods or treated with DMC at the indicated concentrations for 4 h and stained with MitoSOX-Red. Treated and stained cells were processed for FACS analysis. (h) MDA-MB 435S cells were treated with DMC or curcumin at the indicated concentrations for 4 h and stained with MitoSOX Red. Treated and stained cells were processed for FACS analysis. (i) MDA-MB 435S cells treated with 20 μM DMC for the indicated time points were subjected to western blotting of the indicated proteins. Western blotting of β-actin was served as a loading control. Compared with control (untreated cells), the fold change of protein levels was determined (j) MDA-MB 435S cells were pre-treated with or without 2 μM CHX and further treated with 20 μM DMC for 24 h. Cell extracts were prepared for western blotting. Compared with control (untreated cells), the fold change of protein levels was determined
Figure 5
Figure 5
DMC more potently inhibits proteasomal activity than curcumin. (a) MDA-MB 435S cells treated with the indicated concentrations of DMC or curcumin for 24 h were subjected to western blotting. Protein poly-ubiquitination and free ubiquitin levels were examined by western blot analysis using an anti-ubiquitin antibody. β-Actin was examined to verify equal loading (left). Quantitation of the free ubiquitin and ubiquitinated protein levels. Compared with control (untreated cells), the fold change of protein levels was determined. Two independent western blots were normalized to β-actin band in the same sample (right). (b) MDA-MB 435S cells treated with 20 μM DMC or curcumin for 16 h were fixed, immunostained using anti-ubiquitin antibody (green) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Processed cells were observed under the fluorescence and phase contrast microscope. Bar, 20 μm. (c) Cellular proteasomal activity in MDA-MB 435S cells treated with curcumin or DMC. MDA-MB 435S cells were treated with curcumin or DMC at the indicated concentrations for 6, 12 and 24 h. The activities of 20S proteasome, including trypsin-like, chymotrypsin-like and PGPH-like activities, in the cell lysates were measured as described in Materials and Methods section. (d) The effects of curcumin or DMC on proteasome hydrolytic activities. Purified 20S proteasome (70 ng) was incubated with the indicated concentrations of curcumin or DMC for 2 h. Proteasomal trypsin-like, chymotrypsin-like and PGPH-like activities were measured as described in Materials and Methods section. (e) MDA-MB 435S cells treated with 20 μM DMC for the indicated time points were subjected to western blotting of the indicated proteins. Western blotting of β-actin was served as a loading control. (f) MDA-MB 435S cells treated with the indicated concentrations of DMC or curcumin for 24 h and cell extracts were prepared for western blotting of the indicated proteins
Figure 6
Figure 6
CHOP, but not Noxa, is critically involved in DMC-induced ER dilation and subsequent cell death. (a) MDA-MB 435S cells transfected with CHOP or Noxa siRNA were further treated with 20 μM DMC for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. (b) MDA-MB 435 cells were transfected with CHOP or Noxa siRNA and their knockdown was confirmed by western blotting of CHOP or Noxa. Effect of CHOP or Noxa knockdown on ubiquitinated proteins was examined by western blotting using anti-ubiquitin antibody. β-Actin expression was analyzed to confirm equal loading of the protein samples. (c) The sublines expressing the fluorescence selectively in ER (YFP-ER cells/435S) or mitochondria (YFP-Mito cells/435S) were transfected with CHOP siRNA and further treated with 20 μM DMC for 16 h. Cells were observed under a fluorescence microscope. Bars, 20 μm. (d) The changes in the widths of mitochondria-derived vacuoles and the ER-derived vacuoles by CHOP knockdown were quantitatively measured in YFP-Mito cells and YFP-ER cells treated with 20 μM DMC for 16 h using AxioVision Rel. 4.8 software. CHOP knockdown significantly reduced the DMC-induced increase in the width of the ER. Results were repeated in three other experiments. In each experiment, 50 cells were scored as described in Materials and Methods section. (e) MDA-MB 435S cells were infected with the lentivirus containing non-targeting (NT) shRNA or a CHOP-targeting shRNA (CHOP shRNA) and then treated with 20 μM DMC for 16 h. Treated cells were processed for immunocytochemistry of CHOP, PDI and COX IV. Bars, 20 μm. (f) Tumors in nude mice treated with DMC (50 mg/kg) or curcumin (50 mg/kg) were collected after 25 days of treatments and tissue extracts were prepared for western blotting using anti-ubiquitin and anti-CHOP antibody. Western blotting of α-tubulin was examined to verify equal loading
Figure 7
Figure 7
DMC does not induce cell death in normal breast cells. (a) MCF-10A or HMECs were treated with DMC or curcumin at the indicated concentrations for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. (b) MCF-10A cells or HMECs were treated with 20 μM DMC for 16 h and observed under a phase contrast microscope. Bars, 20 μm. (c) HMECs or MDA-MB 435S cells were treated with DMC at the indicated concentrations for 24 h. Cells extracts were prepared for western blotting of the indicated proteins. Western blotting of β-actin was served as a loading control. (d) HMECs or MDA-MB 435S cells were treated with 20 μM DMC for 16 h. Immunocytochemistry using anti-ubiquitin antibody or anti-CHOP antibody was performed and the representative images of cells are shown. Bars, 20 μm. (e) HMECs or MDA-MB 435S cells were treated with DMC at the indicated concentrations for 24 h. Cells extracts were prepared for western blotting of the indicated proteins. Western blotting of β-actin was served as a loading control. (f) HMECs or MDA-MB 435S cells were treated with or without 20 μM DMC for 4 h and FACS analysis to detect mitochondrial superoxide using MitoSOX-Red was performed. The representative histograms are shown
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