Patrizia Portanova

JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways

The proteasome inhibitor bortezomib is an efficacious apoptotic agent in many tumor cells. This paper shows that bortezomib induced apoptosis in human hepatoma HepG2 cells associated with many modifications in the expression of survival or death factors. Although bortezomib increased the level of the protective factors HSP70 and HSP27, the effects of the drug that favour cell death were predominant. These events include accumulation of c-Jun, phospho-c-Jun and p53; increase in FasL level with activation of caspase-8; changes related to members of Bcl-2 family with increase in the level of pro-apoptotic members and decrease in that of anti-apoptotic ones; dissipation of mitochondrial potential with cytochrome c release and activation of caspase-3. In contrast, Chang liver cells exhibited a very low susceptibility to bortezomib-induced apoptosis, which was accompanied by modest modifications in the expression of apoptotic factors.In HepG2 cells bortezomib markedly increased AP-1 activity and the expression of its transcriptional targets such as c-Jun, FasL, BimEL, which are involved in apoptosis. Moreover, AP-1 induced its own production by increasing c-Jun content in the composition of the same AP-1 complex. In addition, bortezomib caused activation of JNK1, which in turn increased the level of phospho-c-Jun as well as stimulated the activation of caspase-3 and t-Bid, two fundamental apoptotic factors. Interestingly, siRNA silencing of c-Jun or JNK1 reduced HepG2 cell susceptibility to apoptosis and prevented the increase in AP-1 activity. Both JNK-1 and AP-1 thus exerted a crucial role in bortezomib-induced apoptosis. Differently, in Chang liver cells the different composition of AP-1 complex as well as the failure of JNK activation seemed to be responsible for the low susceptibility to apoptosis. Given the high susceptibility of hepatoma cells to bortezomib, our results suggest the potential application of this compound in clinical trials for liver cancers.

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

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.

Research paperApoptosis induced in HepG2 cells by the synthetic cannabinoid WIN: Involvement of the transcription factor PPARγ

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 Synthetic Cannabinoid WIN 55,212-2 Sensitizes Hepatocellular Carcinoma Cells to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis by Activating p8/CCAAT/Enhancer Binding Protein Homologous Protein (CHOP)/Death Receptor 5 (DR5) Axis

In this article, we demonstrate that the synthetic cannabinoid R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)-(1-naphthalenyl) methanone mesylate (WIN 55,212-2) sensitizes human hepatocellular carcinoma (HCC) cells to apoptosis mediated by tumor necrosis-related apoptosis inducing ligand (TRAIL). The apoptotic mechanism induced by treatment with WIN/TRAIL combination involved the loss of the mitochondrial transmembrane potential and led to the activation of caspases. In HCC cells, WIN treatment induced the up-regulation of TRAIL death receptor DR5, an effect that seemed to be related to the increase in the level of p8 and CHOP, two factors implicated in cellular stress response and apoptosis. This relationship was suggested by the observation that the down-regulation of p8 or CHOP by specific small interfering RNAs attenuated both WIN-mediated DR5 up-regulation and the cytotoxicity induced by WIN/TRAIL cotreatment. Moreover, WIN induced a significant decrease in the levels of some survival factors (survivin, c-inhibitor of apoptosis protein 2, and Bcl-2) and in particular in that of the active phosphorylated form of AKT. This event seemed to be dependent on the transcription factor peroxisome proliferator-activated receptor-γ whose level significantly increased after WIN treatment. Therefore, both the induction of DR5 via p8 and CHOP and the down-regulation of survival factors seem to be crucial for the marked synergistic effects induced by the two drugs in HCC cells. Taken together, the results reported in this article indicate that WIN/TRAIL combination could represent a novel important tool for the treatment of HCC.

Bortezomib induces in HepG2 cells IκBα degradation mediated by caspase-8

AbstactThe 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 IκBα level and a consequent increase in NF-κB 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 IκBα degradation occurred in conjunction with the activation of caspase-8; moreover, the decrease in IκBα 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 IκBα degradation. These findings suggest that caspase-8 can be involved in the control of IκBα 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-κB

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.