ICAM-1 and AMPK regulate cell detachment and apoptosis by N-methyl-N'-nitro-N-nitrosoguanidine, a widely spread environmental chemical, in human hormone-refractory prostate cancers

Poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, plays a crucial role in the regulation of DNA repair. PARP-1 hyperactivation causes DNA damage and cell death. The underlying mechanism is complicated and is through diverse pathways. The understanding of responsible signaling pathways may offer implications for effective therapies. After concentration-response determination of N-Methyl-N′-Nitro-N-Nitrosoguanidine (MNNG, a PARP-1 activating agent and an environmental mutagen) in human hormone-refractory prostate cancers, the data showed that concentrations below 5 μM did not change cell survival but cause a time-dependent up-regulation of intracellular adhesion molecule-1 (ICAM-1) in mRNA, total protein and cell surface levels. Detection of phosphorylation and degradation of IκB-α and nuclear translocation of NF-κB showed that MNNG induced the activation of NF-κB that was responsible for the ICAM-1 up-regulation since PDTC (a NF-κB inhibitor) significantly abolished this effect. However, higher concentrations (e.g., 10 μM) of MNNG induced a 61% detachment of the cells which were apoptosis associated with the activation of AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Further identification showed that both AMPK and JNK other than p38 MAPK functionally contributed to cell death. The remaining 39% attached cells were survival associated with high ICAM-1 expression. In conclusion, the data suggest that NF-κB-dependent up-regulation of ICAM-1 plays a key role on cell attachment and survival; whereas, activation of AMPK and JNK participates in cytotoxic signaling pathways in detached cells caused by PARP-1 activation.     

Protein tyrosine nitration and poly(ADP-ribose) polymerase activation in N-methyl-N-nitro-N-nitrosoguanidine-treated thymocytes: implication for cytotoxicity

1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) is a DNA alkylating agent. DNA alkylation by MNNG is known to trigger accelerated poly(ADP-ribose) metabolism. Various nitroso compounds release nitric oxide (NO). Therefore, we set out to investigate whether MNNG functions as NO donor and whether MNNG-derived NO or secondary NO metabolites such as peroxynitrite contribute to MNNG-induced cytotoxicity. MNNG in aqueous solutions resulted in time- and concentration-dependent NO release and nitrite/nitrate formation. Moreover, various proteins in MNNG-treated thymocytes were found to be nitrated, indicating that MNNG-derived NO may combine with cellular superoxide to form peroxynitrite, a nitrating agent. MNNG also caused DNA breakage and increased poly(ADP-ribose) polymerase activity and cytotoxicity in thymocytes. MNNG-induced DNA damage (measured by the comet assay) and thymocyte death (measured by propidium iodide uptake) was prevented by the PARP inhibitor PJ-34 and by glutathione (GSH) or N-acetylcysteine (NAC). The cytoprotection provided by PJ-34 against necrotic parameters was paralleled by increased outputs in apoptotic parameters (caspase activity, DNA laddering) indicating that PARP activation diverts apoptotic death toward necrosis. As MNNG-induced cytotoxicity showed many similarities to peroxynitrite-induced cell death, we tested whether peroxynitrite was responsible for at least part of the cytotoxicity induced by MNNG. Cell-permeable enzymic antioxidants (superoxide dismutase and catalase), the NO scavenger cPTIO or the peroxynitrite decomposition catalyst FP15 failed to inhibit MNNG-induced DNA breakage and cytotoxicity. In conclusion, MNNG induces tyrosine nitration in thymocytes. Furthermore, MNNG damages DNA by a radical mechanism that does not involve NO or peroxynitrite.     

Long-term Activation of c-Jun N-terminal Kinase through Receptor Interacting Protein is Associated with DNA Damage-induced Cell Death

Activation of c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is an important cellular response that modulates the outcome of the cells which are exposed to the tumor necrosis factor (TNF) or the genotoxic stress including DNA damaging agents. Although it is known that JNK is activated in response to genotoxic stress, neither the pathways to transduce signals to activate JNK nor the primary sensors of the cells that trigger the stress response have been identified. Here, we report that the receptor interacting protein (RIP), a key adaptor protein of TNF signaling, was required to activate JNK in the cells treated with certain DNA damaging agents such as adriamycin (Adr) and 1-β-D-arabinofuranosylcytosine (Ara-C) that cause slow and sustained activation, but it was not required when treated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and short wavelength UV, which causes quick and transient activation. Our findings revealed that this sustained JNK activation was not mediated by the TNF (tumor necrosis factor) receptor signaling, but it required a functional ATM (ataxia telangiectasia) activity. In addition, JNK inhibitor SP-600125 significantly blocked the Adr-induced cell death, but it did not affect the cell death induced by MNNG. These findings suggest that the sustained activation of JNK mediated by RIP plays an important role in the DNA damage-induced cell death, and that the duration of JNK activation relays a different stress response to determine the cell fate.     

Role of oxidative stress and MAPK signaling in reference moist smokeless tobacco-induced HOK-16B cell death

The use of smokeless tobacco products is often associated with an oral injury at the site of repeated use. To further our understanding of this injury process, the effect of reference moist smokeless tobacco extract (STE) on cell death, oxidative stress, and MAPK signaling in a human oral keratinocyte cell line, HOK-16B, was investigated. STE caused dose-dependent cell death and reactive oxygen species (ROS) production within 30 min to 3 h of exposure. This same insult enhanced the activity of ERK1/2, JNK1/2, p38 MAPK and ASK1, an upstream activator of JNK1/2 and p38 MAPK. Inhibition of JNK1/2 and to a lesser extent p38 MAPK, but not ERK1/2, suppressed STE-induced cell death. Pretreatment with antioxidants and an iron chelator, deferoxamine suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Interestingly, extracellular free iron levels in STE (29.4 ± 0.5 μM) were significantly elevated as compared with cell culture medium (4.9 ± 0.6 μM) and the addition of extracellular free iron (14, 30 or 70 μM) to HOK-16B cultures (without STE) caused dose-dependent cell death after 3 h. Thus, acute exposure to STE leads to HOK-16B cell death in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways.     

Arachidonic acid-induced apoptosis of human neuroblastoma SK-N-SH cells is mediated through mitochondrial alteration elicited by ROS and Ca-evoked activation of p38α MAPK and JNK1

Arachidonic acid (AA)-induced apoptosis of human neuroblastoma SK-N-SH cells was characteristic of elevation of intracellular Ca²⁺ concentration ([Ca²⁺]i), ROS generation, activation of 38 MAPK and JNK and loss of mitochondrial membrane potential (ΔΨm). Subsequent modulation of Bcl-2 family members and cytochrome c release accompanied with activation of caspase-9 and -3 were involved in the death of SK-N-SH cells. BAPTA-AM (Ca²⁺ chelator) pretreatment rescued viability of AA-treated cells through abolishing phosphorylation of p38 MAPK and JNK, ΔΨm loss and ROS generation. N-Acetylcysteine (ROS scavenger) pretreatment reduced the dissipation of ΔΨm, but insignificantly affected AA-induced p38 MAPK and JNK activation. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) attenuated mitochondrial depolarization, degradation of Bcl-2/Bcl-xL, and mitochondrial translocation of Bax. Transfection of specific siRNA proved that p38α MAPK and JNK1 were involved in modulating Bcl-2 family proteins. Taken together, our data suggest that the cytotoxicity of AA toward SK-N-SH cells is mediated through mitochondria-dependent death pathway, eliciting by AA-induced ROS generation and Ca²⁺-evoked activation of p38α MAPK and JNK1.     

Importance of protein thiols during N-methyl-N'-nitro-N-nitrosoguanidine toxicity in primary rat hepatocytes

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent toxicant in isolated rat hepatocytes, was evaluated for its mechanism of cytotoxicity. This direct acting toxicant generates an alkylating carbonium ion that covalently binds to cell macromolecules, depletes nonprotein thiols (NPT), and subsequently kills cells. In this study MNNG depleted protein thiols (PT) in a two-phase process. The first phase (about 30% depletion) occurred rapidly, in parallel with the depletion of NPT. After a plateau, a second phase of PT depletion occurred 5-8 min prior to cell death. Indole-3-carbinol (I-3-C), added prior to MNNG, did not alter the depletion of NPT nor the first phase of PT depletion. However, cell killing was substantially retarded and was still immediately preceded by the second phase of PT depletion. The addition of o-phenanthroline or 5,10-dihydroindeno[1,2-b]indole (DHII) prior to MNNG did not alter the first phase of PT depletion, but partially protected (about 30%) against the depletion of NPT. However, o-phenanthroline or DHII completely protected against the MNNG-induced loss of cell viability and the second-phase depletion of PT. When DHII was added after MNNG and prior to the expected second phase of PT depletion, that depletion was markedly depressed, as was the subsequent loss of cell viability. We conclude that MNNG covalent binding, depletion of NPT, and first-phase depletion of PT may be necessary, but insufficient to kill cells. We propose that rapid depletion of cellular antioxidants predisposes the cell to oxidative stress and that oxygen toxicity is responsible for the second-phase depletion of PT and the final cytotoxic events. The fact that the second-phase depletion of PT is required for and immediately precedes cell death suggests the importance of critical but as yet unidentified target thiol proteins in MNNG hepatotoxicity.     

Phorbol 12-myristate 13-acetate protects against tumor necrosis factor (TNF)-induced necrotic cell death by modulating the recruitment of TNF receptor 1-associated death domain and receptor-interacting protein into the TNF receptor 1 signaling complex: Implication for the regulatory role of protein kinase C

Protein kinase C (PKC) triggers cellular signals that regulate proliferation or death in a cell- and stimulus-specific manner. Although previous studies have demonstrated that activation of PKC with phorbol 12-myristate 13-acetate (PMA) protects cells from apoptosis induced by a number of mechanisms, including death receptor ligation, little is known about the effect or mechanism of PMA in the necrotic cell death. Here, we demonstrate that PMA-mediated activation of PKC protects against tumor necrosis factor (TNF)-induced necrosis by disrupting formation of the TNF receptor (TNFR)1 signaling complex. Pretreatment with PMA protected L929 cells from TNF-induced necrotic cell death in a PKC-dependent manner, but it did not protect against DNA-damaging agents, including doxorubicin (Adriamycin) and camptothecin. Analysis of the upstream signaling events affected by PMA revealed that it markedly inhibited the TNF-induced recruitment of TNFR1-associated death domain protein (TRADD) and receptor-interacting protein (RIP) to TNFR1, subsequently inhibiting TNF-induced activation of nuclear factor-kappaB and c-Jun NH2-terminal kinase (JNK). However, JNK inhibitors do not significantly affect TNF-induced necrosis, suggesting that the inhibition of JNK activation by PMA is not part of the antinecrotic mechanism. In addition, PMA acted as an antagonist of TNF-induced reactive oxygen species (ROS) production, thereby suppressing activation of ROS-mediated poly(ADP-ribose)polymerase (PARP), and thus inhibiting necrotic cell death. Furthermore, during TNF-induced necrosis, PARP was significantly activated in wild-type mouse embryonic fibroblast (MEF) cells but not in RIP-/- or TNFR-associated factor 2-/-MEF cells. Taken together, these results suggest that PKC activation ensures effective shutdown of the death receptor-mediated necrotic cell death pathway by modulating formation of the death receptor signaling complex.     

JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells

N-Benzoyl-O-(N′-(1-benzyloxycarbonyl-4-piperidiylcarbonyl) -D-phenylalanyl)-D-phenylalaninol (BBP), a novel synthesized asperphenamate derivative with the increased solubility, showed growth inhibitory effect on human breast carcinoma MCF-7 cells in a time- and concentration-dependent manner. The growth inhibitory effect of BBP was associated with induction of autophagy, which was demonstrated by the development of acidic vesicular organelles, cleavage of LC3 and upregulation of Atg4 in BBP-treated MCF-7 cells. Since the application of Atg4 siRNA totally blocked the cleavage of LC3, we demonstrated a central role of Atg4 in BBP-induced autophagy. The further studies showed that BBP increased the levels of reactive oxygen species (ROS), and pretreatment with NAC effectively blocked the accumulation of ROS, autophagy and growth inhibition triggered by BBP. Moreover, BBP induced the activation of JNK, and JNK inhibitor SP600125 reversed autophagy, the increase of Atg4 levels, conversion of LC3 and growth inhibition induced by BBP. Knockdown of JNK by siRNA efficiently inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by BBP, indicating that JNK activation may be a upstream signaling of ROS and should be a core component in BBP-induced autophagic signaling pathway. These results suggest that BBP produces its growth inhibitory effect through induction of the autophagic cell death in MCF-7 cells, which is modulated by a JNK-dependent Atg4 upregulation involving ROS production.     

The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine triggers apoptosis through p53-dependent and -independent pathways

One of the cellular responses to DNA damaging events is the activation of programmed cell death, also known as apoptosis. Apoptosis is an important process in limiting tumorigenesis by eliminating cells with damaged DNA. This view is reinforced by the finding that many genes with pro-apoptotic function are absent or altered in cancer cells. The tumor suppressor p53 performs a significant role in apoptotic signaling by controlling expression of a host of genes that have pro-apoptotic or pro-survival function. The S(N)1 DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggers apoptosis and the upregulation/phosphorylation of p53; however, the mechanism(s) governing MNNG-induced cell death remain unresolved. We observed that the human lymphoblastoid cell line WTK-1, which expresses mutant p53, shows far less sensitivity to the cytotoxic effects of MNNG than the closely related, p53-normal line TK-6. Exposure to 15 muM MNNG (LD50 at 24 h in TK-6) leads to a kinetically slower rate of apoptotic onset in WTK-1 cells compared to TK-6 as judged by viability assays and approaches that directly examine apoptotic onset. Similar results were obtained using an unrelated human lymphoblastoid line B310 expressing reduced levels of p53 due to E6 oncoprotein expression, indicating that MNNG activates both p53-dependent and -independent apoptotic mechanisms and that these two mechanisms are discernable by the rates which they trigger apoptotic onset. We document, during time points corresponding to peak apoptotic response in TK6, WTK-1, B310, and B310-E6, that these cell lines show marked decreases in mitochondrial transmembrane potential and increases in cytochrome c within the cytosolic fraction of MNNG-treated cells. Consistent with these events, we observed that both caspase-9 and -3 are activated in our panel of lymphoblastoid cells after MNNG exposure. We also found, using both broad spectrum and specific inhibitors, that blocking caspase activity in TK-6 and B310 cells had a significant effect on apoptotic advance, but that this treatment had no effect on entry of WTK-1 or B310-E6 cells into apoptosis. Finally, the PARP inhibitors benzamide and 6(5H)-phenanthridinone exerted notable inhibition of PARP activity and the nuclear translocation of the mitochondrial protein AIF (apoptosis-inducing factor) in MNNG-treated cells; however, these compounds exhibited no detectable inhibitory effects on MNNG-induced death in human lymphoblastoid cells. These observations suggest that PARP activity is not required during MNNG-triggered apoptosis in this cell type. Taken together, our observations support the conclusion that MNNG activates multiple apoptogenic pathways that contain both common and unique mechanisms.     

Inhibition of poly(ADP-ribose) polymerase (PARP) influences the mode of sulfur mustard (SM)-induced cell death in HaCaT cells

Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, beta-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 microM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (> or =1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms.     

低浓度甲基硝基亚硝胍激活p38MAPK信号转导通路

在低浓度甲基硝基亚硝胍（MNNG）诱发猴肾vero细胞遗传不稳定的实验模型中,曾经证明受试细胞中酪氨酸磷酸化蛋白谱的改变和JNK/SAPK信号通路的激活. 同样条件下,现又发现p38MAPK及其上游激酶MKK3/MKK6,以及JNK/SAPK的上游激酶SEK1/MKK4的磷酸化程度增高,提示低浓度MNNG可通过激活MAPK家族的两条应激信号转导通路诱导细胞的应激反应,且不同通路之间可能存在交互作用。     

Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH2-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.     

低浓度甲基硝基亚硝胍激活p38 MAPK信号转导通路(英文)

在低浓度甲基硝基亚硝胍 (MNNG)诱发猴肾vero细胞遗传不稳定的实验模型中 ,曾经证明受试细胞中酪氨酸磷酸化蛋白谱的改变和JNK/SAPK信号通路的激活 .同样条件下 ,现又发现p38MAPK及其上游激酶MKK3/MKK6,以及JNK/SAPK的上游激酶SEK 1/MKK 4的磷酸化程度增高 ,提示低浓度MNNG可通过激活MAPK家族的两条应激信号转导通路诱导细胞的应激反应 ,且不同通路之间可能存在交互作用     

Calcium-mediated activation of c-Jun NH2-terminal kinase (JNK) and apoptosis in response to cadmium in murine macrophages.

Cadmium is a well-known carcinogenic and immunotoxic metal commonly found in cigarette smoke and industrial effluent. An altered intracellular calcium ([Ca(2+)](i)) level has been implicated in the pathophysiology of immune dysfunction. The present study was designed to determine the possible involvement of calcium (Ca(2+)) and mitogen-activated protein kinases (MAPKs) signaling pathways on cadmium-induced cell death in J774A.1 murine macrophage cells. Cadmium caused a low-amplitude [Ca(2+)](i) elevation at 20 microM and rapid and high-amplitude [Ca(2+)](i) elevation at 500 microM. Exposure to cadmium dose-dependently induced phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and deactivated p38 MAPK. Use of the selective JNK inhibitor SP600125 suggested that activation of JNK is pro-apoptotic and pro-necrotic. Buffering of the calcium response with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA-AM) and ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) completely blocked cadmium-induced apoptotic response. The pretreatment of cells with BAPTA-AM and EGTA suppressed the cadmium-induced cell injury, including growth arrest, mitochondrial activity impairment, and necrosis, and it also recovered the cadmium-altered JNK and p38 MAPK activity. Chelating [Ca(2+)](i) also reversed cadmium-induced hydrogen peroxide generation, suggesting that production of reactive oxygen species (ROS) is related to [Ca(2+)](i). The present study showed that cadmium induces a [Ca(2+)](i)-ROS-JNK-caspase-3 signaling pathway leading to apoptosis. Furthermore, cadmium-induced [Ca(2+)](i) regulates phosphorylation/dephosphorylation of JNK and p38, and it modulates signal transduction pathways to proliferation, mitochondrial activity, and necrosis.     

RIP1-mediated mitochondrial dysfunction and ROS production contributed to tumor necrosis factor alpha-induced L929 cell necroptosis and autophagy

Tumor necrosis factor alpha (TNFα) induces necroptosis and autophagy; however, the detailed molecular mechanism is not fully understood. In this study, we found that TNFα administration caused mitochondrial dysfunction and reactive oxygen species (ROS) production, which led to necroptosis and autophagy in murine fibrosarcoma L929 cells. Notably, the RIP1 (serine–threonine kinase receptor-interacting protein 1, a main adaptor protein of necroptosis) specific inhibitor necrostatin-1 (Nec-1) recovered mitochondrial dysfunction and ROS production due to TNFα administration. Moreover, pan-caspase inhibitor z-VAD-fmk (zVAD) increased RIP1 expression and exacerbated TNFα-induced mitochondrial dysfunction and ROS production, indicating that RIP1 led to mitochondrial dysfunction and ROS production. In addition, cytochrome c release from mitochondria was accompanied with TNFα administration, and Nec-1 blocked the release of cytochrome c upon TNFα administration, while zVAD enhanced the release. These further suggested that RIP1 induced mitochondrial dysfunction accompanied with cytochrome c release. Furthermore, autophagy inhibitor 3-methyladenine (3MA) did not affect RIP1 expression as well as mitochondrial dysfunction and ROS production. Together with our previous publication that autophagy was a downstream consequence of necroptosis, we concluded that TNFα induced mitochondrial dysfunction accompanied with ROS production and cytochrome c release via RIP1, leading to necroptosis and resulting autophagic cell death.     

15-Deoxy-delta 12,14-prostaglandin J2 induces apoptosis via JNK-mediated mitochondrial pathway in osteoblastic cells

The cyclopentenone prostaglandin 15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) induces apoptosis in various cell types. However, the underlying mechanism of 15d-PGJ2-induced apoptosis is not fully understood. The present study was undertaken to determine the molecular mechanism by which 15d-PGJ2 induces apoptosis in MC3T3-E1 mouse osteoblastic cells. 15d-PGJ2 caused a concentration- and time-dependent apoptotic cell death. 15d-PGJ2 induced a transient activation of ERK1/2 and sustained activation of JNK. 15d-PGJ2-induced cell death was prevented by the JNK inhibitor SP6001, but not by inhibitors of ERK1/2 and p38. JNK activation by 15d-PGJ2 was blocked by antioxidants N-acetylcysteine (NAC) and GSH. 15d-PGJ2 caused ROS generation and 15d-PGJ2-induced cell death was prevented by antioxidants, suggesting involvement of ROS generation in 15d-PGJ2-induced cell death. 15d-PGJ2 triggered the mitochondrial apoptotic pathway indicated by enhanced Bax expression, loss of mitochondrial membrane potential, cytochrome c release, and caspase-3 activation. The JNK inhibitor blocked these events induced by 15d-PGJ2. Taken together, these results suggest that the 15d-PGJ2 induces cell death through the mitochondrial apoptotic pathway dependent of ROS and JNK activation in osteoblastic cells.     

PARP-1 inhibition-induced activation of PI-3-kinase-Akt pathway promotes resistance to taxol

PARP inhibitors combined with DNA-damage inducing cytostatic agents can lead to effective tumor therapy. However, inhibition of poly(ADP-ribose) polymerase (PARP-1; EC 2.4.2.30) induces the activation of PI-3-kinase-Akt pathway, which can counteract the effectiveness of this therapy. To understand the role of Akt activation in the combined use of cytostatic agent and PARP inhibition, we used taxol (paclitaxel) as an antineoplastic agent, which targets microtubules and up-regulates mitochondrial ROS production, together with (i) pharmacological inhibition (PJ-34), (ii) siRNA knock-down and (iii) transdominant expression of the DNA binding domain of PARP-1. In all cases, PARP-1 inhibition leads to suppressed poly-ADP-ribosylation of nuclear proteins, prevention of NAD⁺ depletion and significant resistance against taxol induced caspase-3 activation and apoptotic cell death. Paclitaxel induced a moderate increase in Akt activation, which was significantly augmented by PARP inhibition, suggesting that PARP inhibition-induced Akt activation could be responsible for the cytostatic resistance. When activation of the PI-3-kinase-Akt pathway was prevented by LY-294002 or Akt Inhibitor IV, the cytoprotective effect of PARP inhibition was significantly diminished showing that the activation of PI-3-kinase-Akt cascade had significantly contributed to the cytostatic resistance. Our study demonstrates that drug-induced drug resistance can be responsible for the reduced efficacy of antitumor treatment. Although inhibition of PARP-1 can promote cell death in tumor cells by the inhibition of DNA repair, PARP-inhibition promoted activation of the PI-3-kinase-Akt pathway can counteract this facilitating effect, and can cause cytostatic resistance. We suggest augmenting PARP inhibition by the inhibition of the PI-3-kinase-Akt pathway for antitumor therapy.     

Benzo[a]pyrene-induced necrosis in the HepG(2) cells via PARP-1 activation and NAD(+) depletion

Benzo[a]pyrene (BaP), a member of polycyclic aromatic hydrocarbons (PAH), has been reported to induce cell death in various cell types. However, the underlying mechanisms are controversial. In the present study, we report that BaP induces necrotic cell death in human hepatoma (HepG(2)) cells. The process is dependent on the activation of poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme responsible for repairing DNA damage. Once activated, PARP-1 catalyzes the formation of ADP-ribose polymers on acceptor proteins at the expense of NAD(+). Incubation of cells with high extracellular concentration of NAD(+) (5mM) after BaP treatment caused an elevation in intracellular NAD(+) level and blocked cell death. Inhibitor of PARP-1 suppressed both overactivation of PARP-1 activity and NAD(+) depletion. Moreover, addition of pyruvate (5mM), but not glutamate (5mM) or glutamine (5mM), could restore ATP production and prevent cell death. These results elucidated a sequence of events linking cellular metabolism to the progression of cell death induced by this organic toxicant.