Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells

Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 μg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2α and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells. Jin and Song contributed equally to this article.     

An intact NEDD8 pathway is required for Cullin-dependent ubiquitylation in mammalian cells

Skp1-Cdc53/Cul1-F-box (SCF) complexes constitute a class of E3 ubiquitin ligases. Recently, a multiprotein complex containing pVHL, elongin C and Cul2 (VEC) was shown to structurally and functionally resemble SCF complexes. Cdc53 and the Cullins can become covalently linked to the ubiquitin-like molecule Rub1/NEDD8. Inhibition of neddylation inhibits SCF function in vitro and in yeast and plants. Here we show that ongoing neddylation is likewise required for VEC function in vitro and for the degradation of SCF and VEC targets in mammalian cells. Thus, neddylation regulates the activity of two specific subclasses of mammalian ubiquitin ligases.     

NF-kappaB pathway is involved in griseofulvin-induced G2/M arrest and apoptosis in HL-60 cells

Griseofulvin (GF), an oral antifungal agent, has been shown to exert antitumorigenesis effect through G2/M cell cycle arrest in colon cancer cells. But the underlying mechanisms remained obscure. The purpose of this study is to test the cytotoxic effect of GF on HL-60 and HT-29 cells and elucidate its underlying molecular pathways. Dose-dependent and time-course studies by flow cytometry demonstrated that 30 to 60 microM GF significantly induced G2/M arrest and to a less extend, apoptosis, in HL-60 cells. In contrast, only G2/M arrest was observed in HT-29 cells under similar condition. Pretreatment of 30 microM TPCK, a serine protease inhibitor, completely reversed GF-induced G2/M cell cycle arrest and apoptosis in HL-60 cells but not in HT-29 cells. The GF-induced G2/M arrest in HL-60 cells is reversible. Using EMSA and super-shift analysis, we demonstrated that GF stimulated NF-kappaB binding activity in HL-60 cells, which was completely inhibited by pretreatment of TPCK. Treatment of HL-60 with 30 microM GF activated JNK but not ERK or p38 MAPK and subsequently resulted in phosphorylation of Bcl-2. Pretreatment of TPCK to HL-60 cells blocked the GF-induced Bcl-2 phosphorylation but not JNK activation. Time course study demonstrated that activation of cdc-2 kinase activity by GF correlated with Bcl-2 phosphorylation. Taken together, our results suggest that activation of NF-kappaB pathway with cdc-2 activation and phosphorylation of Bcl-2 might be involved in G2/M cell cycle arrest in HL-60 cells.     

Cordycepin enhances hyperthermia-induced apoptosis and cell cycle arrest by modulating the MAPK pathway in human lymphoma U937 cells

Molecular Biology Reports - Hyperthermia induces cancer cell death. However, the cytotoxic effect of hyperthermia is not sufficient. Cordycepin can also induce apoptosis in cancer cells and enhance...     

Blockade of histone deacetylase inhibitor-induced RelA/p65 acetylation and NF-kappaB activation potentiates apoptosis in leukemia cells through a process mediated by oxidative damage, XIAP downregulation, and c-Jun N-terminal kinase 1 activation

NF-kappaB activation is reciprocally regulated by RelA/p65 acetylation and deacetylation, which are mediated by histone acetyltransferases (HATs) and deacetylases (HDACs). Here we demonstrate that in leukemia cells, NF-kappaB activation by the HDAC inhibitors (HDACIs) MS-275 and suberoylanilide hydroxamic acid was associated with hyperacetylation and nuclear translocation of RelA/p65. The latter events, as well as the association of RelA/p65 with IkappaBalpha, were strikingly diminished by either coadministration of the IkappaBalpha phosphorylation inhibitor Bay 11-7082 (Bay) or transfection with an IkappaBalpha superrepressor. Inhibition of NF-kappaB by pharmacological inhibitors or genetic strategies markedly potentiated apoptosis induced by HDACIs, and this was accompanied by enhanced reactive oxygen species (ROS) generation, downregulation of Mn-superoxide dismutase and XIAP, and c-Jun N-terminal kinase 1 (JNK1) activation. Conversely, N-acetyl L-cysteine blocked apoptosis induced by Bay/HDACIs by abrogating ROS generation. Inhibition of JNK1 activation attenuated Bay/HDACI lethality without affecting NF-kappaB inactivation and ROS generation. Finally, XIAP overexpression dramatically protected cells against the Bay/HDACI regimen but failed to prevent ROS production and JNK1 activation. Together, these data suggest that HDACIs promote the accumulation of acetylated RelA/p65 in the nucleus, leading to NF-kappaB activation. Moreover, interference with these events by either pharmacological or genetic means leads to a dramatic increase in HDACI-mediated lethality through enhanced oxidative damage, downregulation of NF-kappaB-dependent antiapoptotic proteins, and stress-related JNK1 activation.     

Enhancement of esculetin on arsenic trioxide-provoked apoptosis in human leukemia U937 cells

In order to overcome chemotherapy resistance, many laboratories are searching for agents that increase the sensitivity of cancer cells to anticancer drugs. Arsenic trioxide (As₂O₃) is widely used in treating human acute polymyelocytic leukemia (APL). However, solid tumors and other leukemia cells such as U937 promonocytic leukemia cells are insensitive to As₂O₃. Esculetin, a coumarin derivative, has previously induced cell cycle arrest and apoptosis of HL-60 cells as well as enhanced taxol-induced apoptosis in HepG2 cells, thereby displaying anticancer potential. In this study, esculetin inhibited proliferation and mitogen activated protein kinases (MAPKs) activation in human leukemia U937 cells. Since inhibitors of MAPKs have modulated the GSH-redox state and enhanced the sensitivity of leukemia cells to As₂O₃-provoked apoptosis, we monitored the effect of combining esculetin and As₂O₃ (2.5 μM) on the GSH level. Our study showed that esculetin, PD98059 (MEK/ERK inhibitor), and SP600125 (JNK inhibitor) similarly enhanced the As₂O₃-induced GSH depletion. We found that the As₂O₃ (2.5 μM) treatment slightly induced apoptosis and the pretreatment of esculetin enhanced the As₂O₃-provoked apoptosis significantly. In addition, esculetin enhanced the effect of As₂O₃ on caspase activation in U937 cells. We compared the combined esculetin and As₂O₃ treatment to the As₂O₃ treated alone. The combined esculetin and As₂O₃ treatment increased Bid cleavage, Bax conformation change and cytochrome C release. The study also indicated that esculetin enhanced the As₂O₃-induced lysosomal leakage and apoptosis. Furthermore, pretreatment with N-acetylcysteine (NAC) reduced these enhanced effects. Based on these studies, esculetin enhances the As₂O₃-provoked apoptosis by modulating the MEK/ERK and JNK pathways and reducing intracellular GSH levels. GSH depletion led to higher oxidative stress which activated lysosomal-mitochondrial pathway of apoptosis.