Jun Hee Lim

Quercetin sensitizes human hepatoma cells to TRAIL‐induced apoptosis via Sp1‐mediated DR5 up‐regulation and proteasome‐mediated c‐FLIPS down‐regulation

This study demonstrates that combined treatment with subtoxic doses of quercetin (3′,3′,4′,5,7‐pentahydroxyflavone), a flavonoid found in many fruits and vegetables, plus tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) induces rapid apoptosis in TRAIL‐resistant hepatocellular carcinoma (HCC) cells. Effective induction of apoptosis by the combined treatment with quercetin and TRAIL was not blocked by overexpression of Bcl‐xL, which is known to confer resistance to various chemotherapeutic agents. These results suggest that this combined treatment may provide an attractive strategy for treating resistant HCCs. While the proteolytic processing of procaspase‐3 by TRAIL was partially blocked in various HCC cells treated with TRAIL alone, co‐treatment with quercetin efficiently recovered TRAIL‐induced caspase activation. We found that quercetin treatment of HCC cells significantly up‐regulated the mRNA and protein levels of DR5, a death receptor of TRAIL, in a transcription factor Sp1‐dependent manner. Furthermore, treatment with quercetin significantly decreased the protein levels of c‐FLIP, an inhibitor of caspase‐8, through proteasome‐mediated degradation. Finally, administration of small interfering RNA against DR5 or overexpression of c‐FLIPS, but not c‐FLIPL, significantly attenuated quercetin‐stimulated TRAIL‐induced apoptosis. Collectively, these findings show that quercetin recovers TRAIL sensitivity in various HCC cells via up‐regulation of DR5 and down‐regulation of c‐FLIPS. J. Cell. Biochem. 105: 1386–1398, 2008.

NAG-1 up-regulation mediated by EGR-1 and p53 is critical for quercetin-induced apoptosis in HCT116 colon carcinoma cells

Quercetin, a flavonoid molecule ubiquitously present in nature, has multiple effects on cancer cells, including the inhibition of cell proliferation and migration. However, the responsible molecular mechanisms are not fully understood. We found that quercetin induces the expression of NAG-1 (Non-steroidal anti-inflammatory drug activated gene-1), a TGF-β superfamily protein, during quercetin-induced apoptosis of HCT116 human colon carcinoma cells. Reporter assays using the luciferase constructs containing NAG-1 promoter region demonstrate that early growth response-1 (EGR-1) and p53 are required for quercetin-mediated activation of the NAG-1 promoter. Overexpression of NAG-1 enhanced the apoptotic effect of quercetin, but suppression of quercetin-induced NAG-1 expression by NAG-1 siRNA attenuated quercetin-induced apoptosis in HCT116 cells. Taken together, the present study demonstrates for the first time that quercetin induces apoptosis via NAG-1, providing a mechanistic basis for the apoptotic effect of quercetin in colon carcinoma cells.

Superoxide anion and proteasomal dysfunction contribute to curcumin-induced paraptosis of malignant breast cancer cells.

Curcumin is considered a pharmacologically safe agent that may be useful in cancer chemoprevention and therapy. Here, we show for the first time that curcumin effectively induces paraptosis in malignant breast cancer cell lines, including MDA-MB-435S, MDA-MB-231, and Hs578T cells, by promoting vacuolation that results from swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). Inhibition of protein synthesis by cycloheximide blocked curcumin-induced vacuolation and subsequent cell death, indicating that protein synthesis is required for this process. The levels of AIP-1/Alix protein, a known inhibitor protein of paraptosis, were progressively downregulated in curcumin-treated malignant breast cancer cells, and AIP-1/Alix overexpression attenuated curcumin-induced death in these cells. ERK2 and JNK activation were positively associated with curcumin-induced cell death. Mitochondrial superoxide was shown to act as a critical early signal in curcumin-induced paraptosis, whereas proteasomal dysfunction was mainly responsible for the paraptotic changes associated with ER dilation. Notably, curcumin-induced paraptotic events were not observed in normal breast cells, including mammary epithelial cells and MCF-10A cells. Taken together, our findings on curcumin-induced paraptosis may provide novel insights into the mechanisms underlying the selective anti-cancer effects of curcumin against malignant cancer cells.

Curcumin inhibits phorbol myristate acetate (PMA)-induced MCP-1 expression by inhibiting ERK and NF-κB transcriptional activity

Monocyte chemoattractant protein-1 (MCP-1) is a potent mediator of macrophage migration and therefore, plays an essential role in early events of inflammation. In the present study, we show the protein kinase C activator, phorbol myristate acetate (PMA), potently induced mRNA expression and secretion of the C-C chemokine MCP-1 in U937 cells. We found that curcumin, a natural biologically active compound extracted from rhizomes of Curcuma species, significantly inhibited the PMA-induced increase in MCP-1 expression and secretion. These effects of curcumin are dose dependent and correlate with the suppression of MCP-1 mRNA expression levels. Curcumin inhibited PMA-mediated activation of extracellular signal-regulated kinase (ERK) and NF-kappaB transcriptional activity. Therefore, one possible anti-inflammatory mechanism of curcumin may be to inhibit the secretions of inflammatory MCP-1 chemokine.

Activation of human cancer/testis antigen gene, XAGE-1, in tumor cells is correlated with CpG island hypomethylation

Expression of the XAGE‐1 antigen is restricted to germ cells of the testis and a variety of neoplastic tissues. To date, the molecular mechanism for regulating expression of this cancer/testis antigen gene has been unknown. To evaluate methylation as a potential mechanism for regulating expression of this gene, we first correlated gene methylation status (measured by sequencing of bisulfide‐modified DNA and COBRA) to expression of XAGE‐1 mRNA in normal and cancerous cells. This analysis revealed dense methylation of the CpG island in the XAGE‐1 gene promoter for the normal and cancerous cells that do not express this gene but loss of this methylation in normal testis, cancer cell lines and the primary gastric cancers where the gene is highly expressed. Further supporting the role of methylation in regulating expression of XAGE‐1 were observations that treatment of 2 heavily methylated cell lines, SNU620 and HT29, with 5′‐aza‐deoxycytidine resulted in demethylation of XAGE‐1 promoter and corresponding expression of this gene. Finally, we cloned various segments of the CpG‐rich XAGE‐1 gene promoter linked to a luciferase reporter construct and transiently transfected this construct into HCT116 cells. These experiments confirmed transcriptional regulatory activity for the promoter region that incorporates the CpG island and demonstrated that in vitro methylation of this island results in loss of promoter activity. Collectively, these studies indicate that XAGE‐1 expression in normal and cancerous tissues is regulated by methylation of the CpG island in the gene promoter.

Rottlerin induces heme oxygenase-1 (HO-1) up-regulation through reactive oxygen species (ROS) dependent and PKC δ-independent pathway in human colon cancer HT29 cells

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated by its substrate heme and diverse stimuli. The induction of HO-1 gene expression is one of the important events in cellular response to pro-oxidative and pro-inflammatory insults. In this study, the effect of rottlerin, a putative PKC delta inhibitor, on HO-1 expression in HT29 human colon cancer cells was investigated. Rottlerin-induced HO-1 at both protein and mRNA levels in a dose- and time-dependent manner. Rottlerin-mediated HO-1 induction was abrogated in the presence of N-acetylcysteine (NAC) or glutathione (GSH). Rottlerin induced nuclear translocation of NF-E2-related factor 2 (Nrf2) and increased antioxidant response element (ARE)-driven transcriptional activity. Additionally, rottlerin activated p38 mitogen-activated protein kinase (MAPK) and ERK. The pharmacological inhibition of ERK and p38 MAPK inhibited rottlerin-induced HO-1 up-regulation. However, suppression of protein kinase C delta (PKC delta) expression by siRNA or overexpression of WT-PKC delta did not abrogate the rottlerin-mediated induction of HO-1. These results suggest that rottlerin induces up-regulation of HO-1 via PKC delta-independent pathway. Taken together, the present study identified rottlerin as a novel inducer of HO-1 expression and identified the mechanisms involved in this process.

Rottlerin induces pro-apoptotic endoplasmic reticulum stress through the protein kinase C-δ-independent pathway in human colon cancer cells

Rottlerin, a compound reported to be a PKC δ-selective inhibitor, has been shown to induce growth arrest or apoptosis of human cancer cell lines. In our study, rottlerin dose-dependently induced apoptotic cell death in colon carcinoma cells. Treatment of HT29 human colon carcinoma cells with rottlerin was found to induce a number of signature ER stress markers; phosphorylation of eukaryotic initiation factor-2α (eIF-2α), ER stress-specific XBP1 splicing, and up-regulation of glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein-homologous protein (CHOP). However, suppression of PKC δ expression by siRNA or overexpression of WT-PKC δ and DN-PKC δ did not abrogate the rottlerin-mediated induction of CHOP. These results suggest that rottlerin induces up-regulation of CHOP via PKC δ-independent pathway. Furthermore, down-regulation of CHOP expression using CHOP siRNA attenuated rottlerin-induced apoptosis. Taken together, the present study thus provides strong evidence to support an important role of ER stress response in mediating the rottlerin-induced apoptosis.

Expression of cyclin D3 through Sp1 sites by histone deacetylase inhibitors is mediated with protein kinase C‐δ (PKC‐δ) signal pathway

The histone deacetylase (HDAC) inhibitors are an exciting new class of drugs that are targeted as anti‐cancer agents. These compounds can induce growth arrest, apoptosis, and/or terminal differentiation in a variety of cancers. The inhibition of HDACs shifts toward hyper‐acetylation, thereby driving transcriptional activation. In present study, HDAC inhibitor apicidin was used to elucidate the effect on expression of cell cycle related proteins and the molecular mechanism for transcriptional regulation of cyclin D3 in response to HDAC inhibitors in human colon cancer cells. We found that apicidin increases the transcriptional activity of cyclin D3 gene, which results in accumulation of cyclin D3 mRNA and protein. Apicidin‐induced cyclin D3 expression is mediated by Sp1 sites within the cyclin D3 promoter. Apicidin‐mediated cyclin D3 expression is attenuated by rottlerin, a specific protein kinase C‐δ (PKC‐δ) inhibitor, but not mitogen‐activated protein kinases (MAPKs) inhibitors. Furthermore, suppression of PKC‐δ expression by transfection with its siRNA prominently attenuated apicidin‐induced cyclin D3 expression. These results indicate that the cyclin D3 induction caused by apicidin was associated with PKC‐δ signaling pathway not MAPKs signaling pathways. Taken together, these results suggest that the activation of cyclin D3 transcription by HDAC inhibitor apicidin was mediated through Sp1 sites and pointed to the possible participation of PKC‐δ. J. Cell. Biochem. 101: 987–995, 2007.

Rottlerin induces apoptosis of HT29 colon carcinoma cells through NAG-1 upregulation via an ERK and p38 MAPK-dependent and PKC δ-independent mechanism

Rottlerin, a selective inhibitor of novel isoforms of protein kinase C δ (PKC δ), has been shown to exert multiple effects on cancer cells, including inhibition of cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We found that rottlerin dramatically induced non-steroidal anti-inflammatory drug activated gene-1 (NAG-1) expression in both p53 wild-type and p53-null cancer cell lines, suggesting that NAG-1 upregulation is a common response to rottlerin that occurs independently of p53 in multiple cell lines. Although rottlerin is known to inhibit PKC δ, PKC δ siRNA and overexpression of dominant-negative (DN)-PKC δ did not affect rottlerin-mediated induction of NAG-1. These results suggest that rottlerin induces NAG-1 upregulation via a PKC δ-independent pathway. We also observed that CHOP protein levels were significantly increased by rottlerin, but CHOP siRNA did not affect rottlerin-induced NAG-1 expression. In addition, we demonstrated the involvement of the mitogen-activated protein kinase (MAP kinase) signal transduction pathway in rottlerin-induced NAG-1 expression. Inhibitors of MEK (PD98059) and p38 MAP kinase (SB203580) prevented rottlerin-induced NAG-1 expression. Furthermore, we found that down-regulation of NAG-1 attenuated rottlerin-induced apoptosis. Collectively, the results of this study demonstrate, for the first time, that upregulation of NAG-1 contributes to rottlerin-induced apoptosis in cancer cells.