Hee Jung Um

Withaferin A sensitizes TRAIL-induced apoptosis through reactive oxygen species-mediated up-regulation of death receptor 5 and down-regulation of c-FLIP.

Withaferin A (Wit A) has reportedly shown cytotoxicity in a variety of tumor cell lines. Here, we show that cotreatment with subtoxic doses of Wit A and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in human renal cancer cells, Caki cells, but not in human normal mesangial cells. Moreover, the combined treatment with Wit A and TRAIL dramatically induces apoptosis in various cancer cell types, suggesting that this combined treatment might offer an attractive strategy for safely treating human cancers. Treatment of Caki cells with Wit A up-regulated death receptor 5 (DR5) in a C/EBP homologous protein (CHOP)-dependent manner. Interestingly, a Wit A-induced increase in ROS levels preceded the up-regulation of CHOP and DR5. The involvement of ROS in CHOP-mediated DR5 up-regulation was confirmed by the result that pretreatment with an antioxidant, NAC or catalase, inhibited Wit A-induced up-regulation of both CHOP and DR5. We also found that Wit A treatment down-regulated c-FLIP via NF-kappaB-mediated transcriptional control as well as ROS signaling pathways. Taken together, our results show that DR5 up-regulation and c-FLIP down-regulation contribute to the sensitizing effect of Wit A on TRAIL-mediated apoptosis in cancer cells.

Withaferin A inhibits JAK/STAT3 signaling and induces apoptosis of human renal carcinoma Caki cells.

Withaferin A, the active component of Withania somnifera, causes cytotoxicity in a variety of tumor cell lines. In this study, we show that withaferin A inhibits constitutive and IL-6-induced phosphorylation of STAT3 (on Tyr705), but not IFN-γ-induced STAT1 phosphorylation. Withaferin A-induced down-regulation of STAT3 activation is associated with a reduction in Janus-activated kinase 2 (JAK2) activity. Withaferin A also down-regulates the expression of STAT3 regulated genes such as Bcl-xL, Bcl-2, cyclin D1 and survivin. The apoptotic effect of withaferin A in Caki human renal cancer cells was investigated. Withaferin A induced dose-dependent apoptotic cell death in Caki cells, as measured by FACS analysis and PARP cleavage. Furthermore, overexpression of STAT3 attenuated withaferin A-induced apoptosis. Taken together, the present study provides strong evidence that down-regulation of the STAT3 signaling pathway mediates withaferin A-induced apoptosis.

Curcumin inhibits oxLDL-induced CD36 expression and foam cell formation through the inhibition of p38 MAPK phosphorylation.

The uptake of oxidized low density lipoprotein (oxLDL) via scavenger receptors transforms macrophages into foam cells, which are a hallmark of atherosclerosis. OxLDL markedly increases the expression of the CD36 scavenger receptor. Here, we investigated whether curcumin modulate CD36 expression in oxLDL-treated RAW 264.7 murine macrophages. Our results showed that curcumin dramatically inhibits CD36 expression and foam cell formation. Furthermore, oxLDL-induced expression and activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ), which is involved in CD36 expression, is also blocked in curcumin-treated cells. OxLDL activates the mitogen-activated protein kinase (MAPK) signaling transduction pathway, and p38 MAPK is associated with oxLDL-induced CD36 and PPAR-γ expression. Overexpression of dominant negative p38 MAPK blocks oxLDL-induced CD36 and PPAR-γ expression. Furthermore, curcumin markedly inhibits p38 MAPK phosphorylation. Taken together, our results suggest that curcumin modulates oxLDL-induced CD36 expression and foam cell formation via the inhibition of p38 MAPK phosphorylation in RAW 264.7 murine macrophages.

Melatonin sensitizes Caki renal cancer cells to kahweol-induced apoptosis through CHOP-mediated up-regulation of PUMA.

Abstract:  Melatonin has recently gained attention as a regulator of biologic processes in addition to its effects on circadian rhythms. The mechanisms whereby melatonin regulates the apoptotic program remain poorly understood. In this study, we investigated the combined effect of melatonin and kahweol on apoptosis of cancer cells, but not in most normal human cell types, thus presenting an attractive novel strategy for cancer treatment. In our experiments, treatment with a combination of melatonin and kahweol induced apoptosis, stimulated DEVDase activity, and DNA fragmentation. Co‐treatment with melatonin and kahweol induced up‐regulation of p53‐upregulated modulator of apoptosis (PUMA) while down‐regulation of PUMA expression using small interfering RNAs attenuated melatonin plus kahweol‐induced apoptosis. In addition, co‐treatment with kahweol and melatonin induced PUMA up‐regulation through endoplasmic reticulum stress‐mediated C/EBP homologous protein induction and the p53‐independent pathway. Our results collectively demonstrate that up‐regulation of PUMA contributes to the sensitizing effect of melatonin plus kahweol on apoptosis in cancer cells.

The coffee diterpene kahweol sensitizes TRAIL-induced apoptosis in renal carcinoma Caki cells through down-regulation of Bcl-2 and c-FLIP.

Kahweol, a coffee-specific diterpene, found in the beans of Coffea arabica, has potent anti-carcinogenic, anti-tumor, and anti-inflammatory properties. TRAIL is a potential anti-cancer compound that induces apoptosis in a wide variety of cancer cells, but not in most normal human cell types. In the present study, we show that kahweol sensitizes human renal cancer cells, but not normal human mesangial cells, to TRAIL-mediated apoptosis. Moreover, treatment with a combination of kahweol and TRAIL induces significant apoptosis in various cancer cell types, thus presenting an attractive novel strategy for cancer treatment. Our experiments show that treatment with a combination of kahweol and TRAIL-induced apoptosis, and stimulated of DEVDase activity, DNA fragmentation, and cleavage of PARP, which was prevented by pretreatment with z-VAD, indicative of cell death via a caspase-dependent pathway. Kahweol-induced down-regulation of Bcl-2 and ectopic expression of Bcl-2 led to attenuation of kahweol plus TRAIL-mediated apoptosis, indicative of Bcl-2 involvement in the apoptotic process. In addition, the c-FLIP and caspase signal pathways seem to play a crucial role in apoptosis triggered by the combination of kahweol and TRAIL in Caki cells. Our results collectively demonstrate that down-regulation of Bcl-2 and c-FLIP contributes to the sensitizing effect of kahweol on TRAIL-mediated apoptosis in cancer cells.

Protective effect of melatonin on oxaliplatin-induced apoptosis through sustained Mcl-1 expression and anti-oxidant action in renal carcinoma Caki cells.

Abstract:  Melatonin is an indolamine initially found to be produced in the pineal gland but now known to be synthesized in a variety of other tissues as well. The mechanisms whereby melatonin regulates the apoptotic program remain only partially understood. Anti‐/pro‐apoptotic effects of exogenous melatonin on various stimuli‐mediated apoptosis were investigated in this report. We investigated the combined effect of melatonin and death receptor–mediated ligands (TNF‐α, TRAIL, and anti‐Fas antibody) or endoplasmic reticulum (ER) stress‐inducing agents (thapsigargin, brefeldin A, and tunicamycin) on apoptosis of cancer cells. Death receptor– or ER stress–induced apoptosis was not significantly influenced by melatonin treatment. However, pretreatment with melatonin significantly inhibited DNA damage–induced apoptosis and glutathione (GSH) depletion, suggesting the reactive oxygen species mediate oxaliplatin/etoposide‐induced apoptosis. Interestingly, we also found the involvement of myeloid cell leukemia‐1 (Mcl‐1) downregulation in oxaliplatin‐induced apoptosis; thus, pretreatment with melatonin inhibited Mcl‐1 downregulation, and ectopic expression of Mcl‐1 attenuated oxaliplatin‐induced apoptosis. Taken together, the results demonstrate that melatonin attenuates oxaliplatin‐induced apoptosis in cancer cells by inhibition of GSH depletion and Mcl‐1 downregulation.

Interleukin-1β promotes the expression of monocyte chemoattractant protein-1 in human aorta smooth muscle cells via multiple signaling pathways

Monocyte chemoattractant protein-1 (MCP1) plays a key role in monocyte/macrophage infiltration to the sub-endothelial space of the blood vessel wall, which is a critical initial step in atherosclerosis. In this study, we examined the intracellular signaling pathway of IL-1β-induced MCP1 expression using various chemical inhibitors. The pretreatment of a phosphatidylcholine (PC)-specific PLC (PC-PLC) inhibitor (D609), PKC inhibitors, or an NF-κB inhibitor completely suppressed the IL-1β-induced MCP1 expression through blocking NF-κB translocation to the nucleus. Pretreatment with inhibitors of tyrosine kinase or PLD partially suppressed MCP1 expression and failed to block nuclear NF-κB translocation. These results suggest that IL-1β induces MCP1 expression through activation of NF-κB via the PC-PLC/PKC signaling pathway.

RU486, a glucocorticoid receptor antagonist, induces apoptosis in U937 human lymphoma cells through reduction in mitochondrial membrane potential and activation of p38 MAPK

RU486 (mifepristone) exerts an anticancer effect on cancer cells via induction of apoptosis. However, the molecular mechanisms are not fully understood. Here, we investigated the effect of RU486 on the apoptosis of U937 human leukemia cells. RU486 markedly increased apoptosis in U937 cells as well as in MDA231 human breast carcinoma, A549 human lung adenocarcinoma epithelial and HCT116 human colorectal carcinoma cells. RU486 increased dose-dependent release of mitochondrial cytochrome c, and reduced the mitochondrial membrane potential (MMP, Δψm) in RU486-treated U937 cells. We also found that overexpression of Bcl-2 completely blocked RU486-mediated apoptosis. However, reactive oxygen species signaling had no effect on RU486-induced apoptosis. RU486 increased the phosphorylation of p38 MAPK and JNK, but p38 MAPK only was associated with RU486-mediated apoptosis. Taken together, RU486 induces apoptosis through reduction in the mitochondrial membrane potential and activation of p38 MAPK in U937 human leukemia cells.

Angelicin potentiates TRAIL-induced apoptosis in renal carcinoma Caki cells through activation of caspase 3 and down-regulation of c-FLIP expression

Preclinical Research & Development

The coffee diterpene kahweol enhances sensitivity to sorafenib in human renal carcinoma Caki cells through down-regulation of Mcl-1 and c-FLIP expression

Sorafenib is approved for the treatment of hepatocellular carcinoma (HCC) and advanced renal cell carcinoma (RCC). However, low tumor response and side effects have been widely reported. Therefore, to improve the efficacy of sorafenib, we investigated whether combined treatment with sorafenib and kahweol, the coffee-specific diterpene, has a synergistic effect on apoptotic cell death. Combined treatment with sorafenib and kahweol markedly induced caspase-mediated apoptosis in renal carcinoma Caki cells. Combined treatment with sorafenib and kahweol induced down-regulation of Mcl-1 and c-FLIP expression. We found down-regulation of Mcl-1 and c-FLIP expression was modulated by the ubiquitin-proteasome pathway. Ectopic expression of Mcl-1 inhibited sorafenib plus kahweol-induced apoptosis. Interestingly, combined treatment with sorafenib and kahweol induced apoptotic cell death in c-FLIP overexpressed cells. In addition, combined treatment with sorafenib and kahweol markedly induced apoptosis in human lung carcinoma (A549) and breast carcinoma (MDA-MB-361) cells, but not in human normal mesangial cells and human skin fibroblast cells (HSF). Collectively, our study demonstrates that combined treatment with sorafenib and kahweol induces apoptotic cell death through down-regulation of Mcl-1 expression.