Dimeric procyanidin B2 inhibits constitutively active NF-kappaB in Hodgkin's lymphoma cells independently of the presence of IkappaB mutations

Due to long-term toxicity of current Hodgkin's lymphoma (HL) treatment, the present challenge is to find new therapies that specifically target deregulated signaling cascades, including NF-kappaB, which are involved in Hodgkin (H) and Reed-Sternberg (RS) cell proliferation and resistance to apoptosis. We previously presented evidence that dimeric procyanidin B2 (B2) can interact with NF-kappaB proteins inhibiting the binding of NF-kappaB to DNA. Herein, we investigated if B2, acting at a late event in NF-kappaB signaling cascade, could be effective in inhibiting NF-kappaB in H-RS cells with different mechanisms of constitutive NF-kappaB activation. B2 caused a concentration-dependent inhibition of NF-kappaB-DNA binding to a similar extent (41-48% inhibition at 25 microM B2) in all the tested H-RS cell lines (L-428, KM-H2, L-540, L-1236 and HDML-2). This was associated with the inhibition of NF-kappaB-driven gene expression, including cytokines (IL-6, TNFalpha and RANTES) and anti-apoptotic proteins (Bcl-xL, Bcl-2, XIAP and cFLIP). The finding of similar amounts of RelA and p50 proteins in the nucleus, but decreased NF-kappaB-DNA binding, even in those H-RS cells characterized by mutations in the inhibitory IkappaB proteins, supports that B2 acts by preventing the binding of NF-kappaB to DNA. B2 did not inhibit AP-1 and STAT3 constitutive activation in H-RS cells, indicating that the moderate effects of B2 on cell viability are due to the complex signaling aberrations in HL. Thus, several signaling pathways should be targeted when designing therapeutics for HL. In this regard, the capacity of B2 to inhibit NF-kappaB could be valuable in a multi-drug approach.     

Role of reactive oxygen species, glutathione and NF-kappaB in apoptosis induced by 3,4-methylenedioxymethamphetamine ("Ecstasy") on hepatic stellate cells

"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA), is a derivative of amphetamine with hepatotoxic effects that has been shown to induce apoptosis of cultured liver cells. In the present work, we studied the role played by oxidative stress in the apoptotic response caused by MDMA on a cell line of hepatic stellate cells (HSC). MDMA-treatment provoked oxidative stress determined as reactive oxygen species (ROS) accumulation and decrease of intracellular reduced glutathione levels. Pre-treatment with the antioxidant pyrrolidine dithiocarbamate blocked ROS production but did not prevent MDMA-induced apoptosis of HSC. The pro-oxidant menadione induced in HSC ROS production and apoptosis that were prevented by pyrrolidine dithiocarbamate, showing HSC to be susceptible to oxidative stress-induced apoptosis. Addition of exogenous GSH or its precursor NAC potentiated the apoptotic action of MDMA but blocked apoptosis induced by menadione. Pre-treatment of HSC with the cytochrome P450 inhibitor quinine diminished the extent of apoptosis caused by MDMA, suggesting the involvement of a metabolic derivative of MDMA on its apoptotic effect. Nuclear factor NF-kappaB was activated by MDMA in a oxidative stress independent fashion and played a protective role in the apoptotic response, since inhibition of NF-kappaB by treatment with parthenolide or by viral infection with a dominant-negative form of NIK (Ad5dnNIK) resulted in an increase of MDMA-induced cell death. In summary, MDMA-induced apoptosis of HSC is accompanied, but not caused by oxidative stress; a metabolic derivative of the drug is responsible for the apoptotic effect of MDMA, which is partially blocked by NF-kappaB activation.     

The changes of intracellular H2O2 are an important factor maintaining mitochondria membrane potential of antimycin A-treated As4.1 juxtaglomerular cells

We investigated an involvement of ROS, such as H2O2 and O2- and GSH in the As4.1 cell death by antimycin A and examined whether ROS scavengers rescue antimycin A-induced As4.1 cell death and its mechanism. Levels of intracellular H2O2 and O2- were markedly increased in antimycin A-treated cells. Antimycin A reduced the intracellular GSH content. A ROS scavenger, Tiron down-regulated the production of intracellular H2O2. However, the reduction of intracellular H2O2 level did not change the apoptosis parameters, such as sub-G1 DNA content and annexin V binding. Interestingly, treatment of Tiron could partially prevent the loss of mitochondrial transmembrane potential (DeltaPsi(m)). Treatment of SOD and catalase also reduced the intracellular H2O2 and loss of mitochondrial transmembrane potential (DeltaPsi(m)) without reducing O2- level and apoptosis in antimycin A-treated As4.1 cells. All the ROS scavengers, SOD and catalase did not inhibit GSH depletion induced by antimycin A, resulting in failure of preventing the apoptosis. In addition, all the reagents including antimycin A did not induce any specific phase arrest of cell cycle in As4.1 cells. In summary, these results demonstrate that antimycin A generates potently ROS, H2O2 and O2- and induces the depletion of GSH content in As4.1 JG cells, and that Tiron, SOD and catalase inhibited partially the loss of mitochondrial transmembrane potential (DeltaPsi(m)) via the reduction of intracellular H2O2 level.     

Pyridine N-oxide derivatives inhibit viral transactivation by interfering with NF-kappaB binding

Pyridine N-oxide derivatives represent a new class of anti-HIV compounds for which some members exclusively inhibit HIV-1 RT, whereas other members act, additionally or alternatively, at a post-integrational event in the replicative cycle of HIV. A prototype pyridine N-oxide derivative, JPL-32, inhibited tumor necrosis factor alpha (TNF-alpha)-induced HIV-1 expression in latently HIV-1-infected OM-10.1 and U1 cells, which could be reversed by the addition of N-acetyl-L-cysteine (NAC). The reversal of the antiviral activity of JPL-32 by NAC suggested the possible role of a redox-sensitive factor as target of inhibition. Indeed, when nuclear extracts of TNF-alpha-stimulated OM-10.1 and U1 cells cultured in the presence of JPL-32 were analyzed by an electrophoretic mobility shift assay (EMSA), a dose-dependent inhibition of DNA binding of nuclear NF-kappaB was observed, which could be reversed by the addition of NAC. JPL-32 did not inhibit the release and subsequent degradation of IkappaBalpha, nor did JPL-32 affect the nuclear translocation of NF-kappaB. EMSA revealed that the inhibition of the NF-kappaB DNA binding activity by JPL-32 could be reversed by the addition of reducing agents such as dithiothreitol or beta-mercaptoethanol. Moreover, JPL-32 was able to directly oxidize the thiol groups on the purified p50 subunit of recombinant NF-kappaB. The oxidative modification of the thiol groups on NF-kappaB by JPL-32 could be ascribed to the intracellular pro-oxidant effect of JPL-32. Consequently, JPL-32 was able to increase the intracellular glutathione (GSH) levels and to induce apoptosis in a dose-dependent way.     

Aggravation of L-DOPA-induced neurotoxicity by tetrahydropapaveroline in PC12 cells

Tetrahydropapaveroline (THP) is formed in Parkinsonian patients receiving L-DOPA therapy and is detected in the plasma and urine of these patients. In this study, we have investigated the effects of THP on L-DOPA-induced neurotoxicity in cultured rat adrenal pheochromocytoma, PC12 cells. Exposure of PC12 cells up to 10 microM THP or 20 microM L-DOPA after 24 or 48 hr, neither affected the cell viability determined by MTT assay, nor induced apoptosis by flow cytometry and TUNEL staining. However, at concentrations higher than 15 microM, THP showed cytotoxicity through an apoptotic process. In addition, THP at 5-15 microM for both incubation time points significantly enhanced L-DOPA-induced neurotoxicity (L-DOPA concentration, 50 microM). Exposure of PC12 cells to THP, L-DOPA and THP plus L-DOPA for 48 hr resulted in a marked increase in the cell loss and percentage of apoptotic cells compared with exposure for 24hr. The enhancing effects of THP on L-DOPA-induced neurotoxicity were concentration- and treated-time-dependent. THP, L-DOPA and THP plus L-DOPA produced a significant increase in intracellular reactive oxygen species generation and decrease in ATP levels, supporting the involvement of oxidative stress in THP- and L-DOPA-induced apoptosis. The antioxidant N-acetyl-L-cysteine strongly inhibited changes in apoptosis, decreases in cell viability and ROS generation induced by THP associated with L-DOPA. These results suggest that THP aggravates L-DOPA-induced oxidative neurotoxic and apoptotic effects in PC12 cells. Therefore, Parkinsonian patients treated with L-DOPA for long-term need to be monitored for the relationship between plasma concentration of THP and the symptoms of neurotoxicity.     

Theophylline inhibits NF-κB activation and IκBα degradation in human pulmonary epithelial cells

Previous studies demonstrated that theophylline modulates NF-kappaB activation in mast cells and pulmonary epithelial cells. We examined whether or not this modulation of NF-kappaB activation by theophylline is due to inhibition of the degradation of the IKBalpha protein, which suppresses NF-kappaB activation. TNF-alpha-induced NF-kappaB activation in a human pulmonary epithelial cell line (A549) was evaluated by Western blotting and a chloramphenicol acetyltransferase (CAT) assay. Expression of the IkappaBalpha protein was evaluated by Western blotting. Western blotting of nuclear extracts of A549 cells demonstrated that theophylline suppresses NF-kappaB-p65 nuclear translocation. The CAT assay indicated that NF-kappaB-dependent reporter gene expression is inhibited in A549 cells pretreated with theophylline. Western blotting of cytoplasmic extracts of A549 cells revealed that this inhibition was linked to theophylline-induced protection of expression of the IkappaBalpha protein. Moreover, theophylline inhibited interleukin-6 production induced by TNF-alpha in A549 cells. These findings are consistent with the idea that theophylline suppresses the production of proinflammatory cytokines via inhibition of NF-kappaB activation through protection of the IkappaBalpha protein.     

Regulation of mastoparan-induced increase of paracellular permeability in T84 cells by RhoA and basolateral potassium channels

Mastoparan, a polypeptide known to activate heterotrimeric GTP-binding proteins, enhances the transport of Ca2+ and K+ across membranes. In the present study we investigated the influence of mastoparan on transepithelial resistance (TER) and on short circuit current (SCC) of the intestinal cell line T84. Mastoparan decreased the TER by 80% of baseline and induced a SCC of 8.34+/-1.38 microAcm(-2). The changes in paracellular conductance were estimated using the nystatin technique and showed that mastoparan increased the paracellular conductance 4-fold. Basolateral Cl(-)-free medium, or blockade of the basolateral Cl(-) uptake via the Na+/K+/2Cl(-) co-transporter with bumetanide, reduced SCC of T84 cells, but did not abolish the effect of mastoparan on the TER. Luminal addition of the Cl(-)-channel blocker DIDS or NPPB had no effect on the increase in SCC. In contrast, blocking the basolateral K(+)-channels by 2mM Ba2+ inhibited both the resistance decrease and elevation of the SCC, and further inhibited the mastoparan-induced increase in intracellular free Ca2. This indicates that mastoparan acts primarily via activating K+ channels with a secondary Cl(-) secretion and Ca2+ influx. Reduction of intracellular free Ca2+ did not alter the effect of mastoparan on TER. Stimulation with mastoparan led to a biphasic rearrangement of actin filaments and increased globular actin content in T84 cells. Depolymerization of actin filaments also correlated with inactivation of Rho-proteins, which are known regulators of the cytoskeleton. Mastoparan induced a 2-fold increase in GDI-complexed Rho.We conclude that mastoparan-induced changes in paracellular permeability are mediated via enhanced basolateral K+ conductance and Rho-protein inactivation. A secondary increase in intracellular Ca2+ or direct interaction of small GTPases with the cytoskeleton are likely mediators of the remodeling of the cytoskeleton with subsequent changes in paracellular permeability.     

Mitochondria as an important target in heavy metal toxicity in rat hepatoma AS-30D cells

The mechanisms of toxic effects of divalent cations of three heavy metals Hg, Cd and Cu in rat ascites hepatoma AS-30D cells cultivated in vitro were compared. It was found that the toxicity of these ions, applied in the micromolar range (10-500 microM), decreased from Hg(2+) (most toxic) to Cu(2+) (least toxic). Hg(2+) and Cd(2+) produced a high percentage of cell death by both necrosis and apoptosis, whereas Cu(2+) at concentrations up to 500 microM was weakly effective. Hg(2+) at concentration of 10 microM appeared slightly uncoupling (i.e., stimulated resting state respiration and decreased the mitochondrial transmembrane potential), whereas it exerted a strong inhibitory effect on the respiratory chain and rapid dissipation of the membrane potential at higher concentrations. Cu(2+) had inhibitory effect on cell respiration only at 500 microM concentration and after incubation of 48 h but produced a significant uncoupling effect at lower concentrations. Cu(2+) induced an early and sharp increase of intracellular production of reactive oxygen species (ROS). The action of Hg(2+) and Cd(2+) on ROS generation was biphasic. They stimulated ROS generation within the cells at low concentrations and at short incubation times but decreased ROS generation at higher concentrations and at longer incubation. It is concluded that mitochondria are an important target for toxic effects of Hg(2+), Cd(2+) and Cu(2+) in AS-30D rat hepatoma cells.     

A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation

Although reactive oxygen species (ROS) have been implicated in cadmium (Cd)-induced hepatotoxicity, the role of ROS in this pathway remains unclear. Therefore, we attempted to determine the molecular mechanisms relevant to Cd-induced cell death in HepG2 cells. Cd was found to induce apoptosis in the HepG2 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis and TUNEL staining. In the early stages, both rapid and transient ROS generation triggered apoptosis via Fas activation and subsequent caspase-8-dependent Bid cleavage, as well as by calpain-mediated mitochondrial Bax cleavage. The timing of Bid activation was coincided with the timing at which the mitochondrial transmembrane potential (MMP) collapsed as well as the cytochrome c (Cyt c) released into the cytosol. Furthermore, mitochondrial permeability transition (MPT) pore inhibitors, such as cyclosporin A (CsA) and bongkrekic acid (BA), did not block Cd-induced ROS generation, MMP collapse and Cyt c release. N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of the Cd-induced apoptosis via catalase upregulation and subsequent Fas downregulation. NAC treatment also completely blocked the Cd-induced intracellular ROS generation, MMP collapse and Cyt c release, indicating that Cd-induced mitochondrial dysfunction may be regulated indirectly by ROS-mediated signaling pathway. Taken together, a rapid and transient ROS generation by Cd triggers apoptosis via caspase-dependent pathway and subsequent mitochondrial pathway. NAC inhibits Cd-induced apoptosis through the blocking of ROS generation as well as the catalase upregulation.     

The environmental carcinogen 3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells

The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca(2+) and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2',7'-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.