Notch inhibition restores TRAIL-mediated apoptosis via AP1-dependent upregulation of DR4 and DR5 TRAIL receptors in MDA-MB-231 breast cancer cells.

Notch is a family of transmembrane receptors whose activation through proteolytic cleavage by γ-secretase targets genes which participate in cell development, differentiation and tumorigenesis. Notch signaling is constitutively activated in various cancers, including breast cancer and its upregulation is usually related with poor clinical outcomes. Therefore, targeting Notch signaling with γ-secretase inhibitors (GSIs) is considered a promising strategy for cancer treatment. We report that the γ-secretase inhibitor-I (GSI-I) sensitizes human breast cancer cells to apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The antiproliferative GSI-I/TRAIL synergism was stronger in ER-negative MDA-MB-231 breast cancer cells compared with ER-positive MCF-7 cells. In MDA-MB-231 cells, GSI-I treatment induced upregulation of DR4 and DR5 TRAIL receptors. This effect seemed to be related to the activation of the transcription factor AP1 that was a consequence of Notch inhibition, as demonstrated by Notch-1 silencing experiments. Combined treatment induced loss of the mitochondrial transmembrane potential and activation of caspases. GSI-I alone and/or GSI-I/TRAIL combination also induced a significant decrease in the levels of some survival factors (survivin, c-IAP-2, Bcl-xL, BimEL and pAKT) and upregulation of pro-apoptotic factors BimL, BimS and Noxa, enhancing the cytotoxic potential of the two drugs. Taken together, these results indicate for the first time that GSI-I/TRAIL combination could represent a novel and potentially effective tool for breast cancer treatment.

Synthesis, characterization, cellular uptake and interaction with native DNA of a bis(pyridyl)-1,2,4-oxadiazole copper(II) complex.

The copper(II) complex of 3,5-bis(2'-pyridyl)-1,2,4-oxadiazole was synthesized and characterized. X-Ray crystallography revealed that the complex consists of a discrete [Cu(3,5-bis(2'-pyridyl)-1,2,4-oxadiazole)(2)(H(2)O)(2)](2+) cation and two ClO(4)(-) anions. The Cu(II) coordination sphere has a distorted octahedral geometry and each ligand chelates the copper ion through the N(4) nitrogen of the oxadiazole ring and the nitrogen of one pyridine moiety. The coordinated water molecules are in cis position and each of them is H-bonded to the 5-pyridyl nitrogen of the oxadiazole ligand and to an oxygen of the perchlorate anion. Biological assays showed that, despite the free ligand not being effective, [Cu(3,5-bis(2'-pyridyl)-1,2,4-oxadiazole)(2)(H(2)O)(2)](2+) reduced the vitality of human hepatoblastoma HepG2 and colorectal carcinoma HT29 cells in a dose- and time-dependent manner. The interaction of the cationic copper complex with native DNA was investigated by variable-temperature UV-vis spectroscopy, circular dichroism, viscosity and gel electrophoresis, indicating that it is a groove binder with binding constant K(b) = 2.2 × 10(4) M(-1).

Apoptosis induced in HepG2 cells by the synthetic cannabinoid WIN: involvement of the transcription factor PPARgamma.

It has recently been shown that cannabinoids induce growth inhibition and apoptosis in different tumour cell lines. In the current study, the effects of WIN 55,212-2 (WIN), a synthetic and potent cannabinoid receptor agonist, are investigated in hepatoma HepG2 cells and a possible signal transduction pathway is proposed. In these cells, WIN induces a clear apoptotic effect which was accompanied by up-regulation of the death-signalling factors Bax, Bcl-X(S), t-Bid and down-regulation of the survival factors survivin, phospho-AKT, Hsp72 and Bcl-2. Moreover, WIN-induced apoptosis is associated with JNK/p38 MAPK pathway activation and mitochondrial depolarisation demonstrated by a cytofluorimetric assay. The results also show that in HepG2 cells WIN markedly increases the level of the transcription factor PPARgamma in a dose- and time-dependent manner. The addition of the PPARgamma antagonists GW9662 and T0070907 significantly reduces the effects of the drug on both cell viability and the levels of survivin, phospho-AKT and phospho-BAD, suggesting that PPARgamma plays a key role in WIN-induced apoptosis. Altogether, the results seem to indicate a potential therapeutic role of WIN in hepatic cancer treatment.

The role of oxidative stress in apoptosis induced by the histone deacetylase inhibitor suberoylanilide hydroxamic acid in human colon adenocarcinoma HT-29 cells.

Histone deacetylase inhibitors (HDACIs) activate genes that promote cell cycle arrest and apoptosis in a number of tumor cells. This study showed that suberoylanilide hydroxamic acid (SAHA), a potent and commonly used HDACI, induced apoptosis in human colon adenocarcinoma HT-29 cells in a time- and dose-dependent manner. This effect was accompanied by the induction of oxidative stress, dissipation of mitochondrial transmembrane potential and activation of executioner caspases. Moreover, SAHA increased the levels of phosphorylated active forms of p38 and JNK. The addition of either the antioxidant N-acetylcysteine or the specific inhibitor of NADPH oxidase diphenylene iodonium chloride reduced the cytotoxic effects of SAHA in HT-29 cells, suggesting that the induction of oxidative stress represents a crucial event in the apoptotic mechanism. In addition, SAHA up-regulated the death receptor DR5, inducing the activation of caspase-8 with the consequent cleavage of Bid. Furthermore, SAHA down-regulated FLIPL and Akt, two proteins which exert an inhibitory role in apoptosis.

Hsp72 controls bortezomib-induced HepG2 cell death via interaction with pro-apoptotic factors.

The proteasome inhibitor bortezomib is an efficacious inducer of apoptosis in the hepatoma HepG2 cell line. This study shows that bortezomib increased in these cells the level of the survival factor Hsp72 in a time- and dose-dependent manner. In a first phase of treatment, Hsp72 rapidly increased so that at 24 h of incubation with 50 nM bortezomib its level was approximately five-fold higher than the control. In this phase Hsp72 seemed to play a role in preventing HepG2 cell death, since it interacted with and sequestered the pro-apoptotic factors p53, AIF, Bax and Apaf-1. During a second day of treatment, although the nuclear levels of Hsp72, p53 and AIF increased, the interaction of Hsp72 with these factors diminished. In addition, bortezomib induced the activation of caspases, which stimulated Hsp72 degradation. In conclusion, in the second day of treatment with bortezomib the protective ability of Hsp72 decreased thus favouring the appearance of apoptosis.

Bortezomib induces in HepG2 cells IkappaBalpha degradation mediated by caspase-8.

The present paper demonstrates that the proteasome inhibitor bortezomib, which behaves as an apoptotic agent in hepatoma HepG2 cells, caused in these cells a decrease in IkappaBalpha level and a consequent increase in NF-kappaB activity. The effect already appeared at 4 h of treatment and preceded the onset of apoptosis which was observed at 24 h. Our results demonstrate that bortezomib-induced IkappaBalpha degradation occurred in conjunction with the activation of caspase-8; moreover, the decrease in IkappaBalpha level was prevented in a dose-dependent manner by the addition of z-IETD, a specific inhibitor of caspase-8. Bortezomib caused the same effects in non-tumor Chang liver cells, which were not susceptible to the apoptotic effect of the drug. Our results also show that other proteases, such as caspase-3 and calpains, exerted only a limited effect on IkappaBalpha degradation. These findings suggest that caspase-8 can be involved in the control of IkappaBalpha level. In addition, the activation of caspase-8 can exert, at least in the first phase of treatment with bortezomib, a protective effect in both HepG2 and Chang liver cells, favouring the activation of the survival factor NF-kappaB.

Anandamide-induced apoptosis in Chang liver cells involves ceramide and JNK/AP-1 pathway.

In the present study we demonstrate that anandamide, the most important endogenous cannabinoid, markedly induced apoptosis in Chang liver cells, an immortalized non-tumor cell line derived from normal liver tissue, while it induced only modest effects in a number of hepatoma cell lines. The apoptotic effect was reduced by methyl-beta-cyclodextrin, a membrane cholesterol depletor, suggesting an interaction between anandamide and the membrane microdomains named lipid rafts. Anandamide effects were mediated by the production of ceramide, as demonstrated by experiments performed with the sphingomyelinase inhibitor, desipramine, or with the sphingomyelinase activator, melittin. This conclusion was confirmed by the observation that exogenous C2-ceramide induced a remarkable apoptotic effect in the same cells. Anandamide-induced apoptosis in Chang liver cells involved oxidative stress and activation of p38/JNK pathway, which was accompanied by a remarkable increase in AP-1 DNA-binding activity. Moreover, the levels of both c-Jun and JunB, two components of the AP-1 complex, and those of FasL and Bim, two transcriptional targets of AP-1, also increased during anandamide treatment. In addition, anandamide increased the level of Bax and caused degradation of full-length Bid with the production of the active truncated form. These effects were accompanied by dissipation of mitochondrial transmembrane potential with the consequent activation of both caspase-3 and caspase-6. On the contrary, in hepatoma cells, anandamide did not induce apoptotic effects and it was not possible to observe any increase in p38/JNK pathway and AP-1 activity after drug treatment. Our results suggest that the induction of cell death in non-tumor Chang liver cells by anandamide was mediated by ceramide, JNK and AP-1 and was dependent on the activation of both the extrinsic and intrinsic pathways of apoptosis.