Low concentration of arsenic could induce caspase-3 mediated head kidney macrophage apoptosis with JNK-p38 activation in Clarias batrachus

We had earlier demonstrated that chronic exposure (30 days) to micro-molar concentration (0.50 μM) of arsenic induced head kidney macrophage (HKM) death in Clarias batrachus. The purpose of the present study is to characterize the nature of HKM death induced by arsenic and elucidate the signal transduction pathways involved in the process. Arsenic-induced HKM death was apoptotic in nature as evident from DNA gel, Annexin V-propidium iodide, Hoechst 33342 staining and TdT-mediated dUTP nick end labeling (TUNEL) assays. Inhibitor studies and immunoblot analyses further demonstrated that arsenic-induced HKM apoptosis involved activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, a well-characterized caspase-3 substrate. Preincubation with antioxidants N-acetyl-cysteine or dimethyl sulfoxide significantly lowered reactive oxygen species (ROS) levels in arsenic-treated HKM and prevented caspase activation, malondialdehyde formation and HKM apoptosis. Arsenic induced membrane translocation of the NADPH oxidase subunit p47^phox. Preincubation with apocynin and diphenyleneiodonium chloride, both selective inhibitors of NADPH oxidases, prevented p47^phox translocation, ROS production and HKM death. Exposure of HKM to arsenic induced the activation of mitogen-activated protein kinase family (MAPK) proteins including c-Jun NH₂-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38). Preincubation of HKM with p38 inhibitor SB203580 and JNK inhibitor SP600125 protected the HKM against arsenic-induced apoptosis. We conclude that exposure to micro-molar concentration of arsenic induces ROS generation through the activation of NADPH oxidases, which in turn causes caspase-3 mediated HKM apoptosis. In addition, the study also indicates a role of p38-JNK pathway in arsenic-induced HKM apoptosis in C. batrachus.     

Capsaicin-induced apoptosis is regulated by endoplasmic reticulum stress- and calpain-mediated mitochondrial cell death pathways

Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca²⁺, calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.     

Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O₂⁻ generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67^phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91^phox knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47^phox, p67^phox and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67^phox siRNA. Exposure of MPMVEC obtained from gp91^phox knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to activation of MAPKs through induced phosphorylation of p38 and ERK1/2 MAPKs that may further result in endothelial dysfunction through production of cytokines such as IL-6. Our results suggest that endothelial oxidative stress may be an important mechanism for PM-induced cardiovascular effects.     

Mechanism of apoptotosis induced by ortho-topolin riboside in human hepatoma cell line SMMC-7721

The naturally occurring cytokinin, ortho-topolin riboside (oTR), has been recently reported to have a strong anticancer effect. However, the molecular mechanism has not been elucidated. From our research we found that oTR strongly inhibited the proliferation of SMMC-7721 cells inducing apoptosis. After oTR treatment, up-regulation of the protein levels of pro-apoptotic Bax and the down-regulation of the anti-apoptotic proteins, Bcl-2 and Bcl-xL was observed, leading to the loss of mitochondrial membrane potential, the release of cytochrome c from the mitochondria into the cytosol, the downstream activation of caspase-9 and caspase-3, as well as the cleavage of poly ADP-ribose-polymerase (PARP), the effect of apoptosis could be blocked by the pan-specific caspase inhibitor z-VAD-fmk and caspase-9-specific inhibitor z-LEHD-fmk. Moreover, oTR was shown to inhibit the activation of the extracellular signal-regulated kinase-1/2 (ERK_1/2) as well as the Akt pathway. These results suggest that oTR interferes with the mitogen-activated protein kinase (MAPK) and Akt pathways and induces the apoptosis of human SMMC-7721 cells through the activation of intrinsic mitochondria-mediated pathways. However, the apoptosis was completely prevented when cells were treated with A-134974, an inhibitor of adenosine kinase, it indicated that the intracellular phosphorylation of oTR is necessary for its cytotoxic effects to SMMC-7721 cells.     

Arachidonic acid release mediated by OX1 orexin receptors

Background and purpose:

We have previously shown that lipid mediators, produced by phospholipase D and C, are generated in OX₁ orexin receptor signalling with high potency, and presumably mediate some of the physiological responses to orexin. In this study, we investigated whether the ubiquitous phospholipase A₂ (PLA₂) signalling system is also involved in orexin receptor signalling.

Experimental approach:

Recombinant Chinese hamster ovary-K1 cells, expressing human OX₁ receptors, were used as a model system. Arachidonic acid (AA) release was measured from ³H-AA-labelled cells. Ca²⁺ signalling was assessed using single-cell imaging.

Key results:

Orexins strongly stimulated [³H]-AA release (maximally 4.4-fold). Orexin-A was somewhat more potent than orexin-B (pEC₅₀= 8.90 and 8.38 respectively). The concentration–response curves appeared biphasic. The release was fully inhibited by the potent cPLA₂ and iPLA₂ inhibitor, methyl arachidonyl fluorophosphonate, whereas the iPLA₂ inhibitors, R- and S-bromoenol lactone, caused only a partial inhibition. The response was also fully dependent on Ca²⁺ influx, and the inhibitor studies suggested involvement of the receptor-operated influx pathway. The receptor-operated pathway, on the other hand, was partially dependent on PLA₂ activity. The extracellular signal-regulated kinase, but not protein kinase C, were involved in the PLA₂ activation at low orexin concentrations.

Conclusions and implications:

Activation of OX₁ orexin receptors induced a strong, high-potency AA release, possibly via multiple PLA₂ species, and this response may be important for the receptor-operated Ca²⁺ influx. The response coincided with other high-potency lipid messenger responses, and may interact with these signals.     

Ketoconazole-induced JNK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells

This study examined the effect of ketoconazole on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca²⁺ levels in MG63 osteosarcoma cells. Ketoconazole at 20–200 μM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that ketoconazole induced phosphorylation of ERK and JNK, but not p38, MAPKs. Ketoconazole-induced cell death and apoptosis were partially reversed by the selective JNK inhibitor SP600125, but not by the selective ERK inhibitor PD98059, suggesting that ketoconazole’s cytotoxic action was via JNK, but not via ERK and p38 MAPKs. Ketoconazole at a concentration of 100 μM induced [Ca²⁺]_i increases. Chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) totally inhibited ketoconazole-induced [Ca²⁺]_i increases without reversing ketoconazole-induced cell death. Collectively, in MG63 cells, ketoconazole induced cell death and apoptosis via evoking JNK phosphorylation in a Ca²⁺-independent manner.     

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.     

Antitumor effects of emodin on LS1034 human colon cancer cells in vitro and in vivo: Roles of apoptotic cell death and LS1034 tumor xenografts model

Emodin, an active natural anthraquinone derivative, is found in the roots and rhizomes of numerous Chinese medicinal herbs and exhibits anticancer effects on many types of human cancer cell lines. The aim of this study investigated that emodin induced apoptosis of human colon cancer cells (LS1034) in vitro and inhibited tumor nude mice xenografts bearing LS1034 in vivo. In in vitro study, emodin induced cell morphological changes, decreased the percentage of viability, induced G2/M phase arrest and increased ROS and Ca²⁺ productions as well as loss of mitochondrial membrane potential (ΔΨ_m) in LS1034 cells. Emodin-triggered apoptosis was also confirmed by DAPI staining and these effects are concentration-dependent. Western blot analysis indicated that the protein levels of cytochrome c, caspase-9 and the ratio of Bax/Bcl-2 were increased in LS1034 cells after emodin exposure. Emodin induced the productions of ROS and Ca²⁺ release, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. In in vivo study, emodin effectively suppressed tumor growth in tumor nude mice xenografts bearing LS1034. Overall, the potent in vitro and in vivo antitumor activities of emodin suggest that it might be developed for treatment of colon cancer in the future.     

Euphol from Euphorbia tirucalli selectively inhibits human gastric cancer cell growth through the induction of ERK1/2-mediated apoptosis

Gastric cancer is one of the most common malignancies worldwide, and the main cause of cancer-related death in Asia. The present study assessed the anticancer effects of euphol, a triterpene alcohol with anti-inflammatory and antiviral activities on human gastric cancer cells. Euphol showed higher cytotoxicity activity against human gastric CS12 cancer cells than against noncancer CSN cells. In addition, it up-regulated the pro-apoptotic protein BAX and down-regulated the prosurvival protein Bcl-2, causing mitochondrial dysfunction, possibly by caspase-3 activation. The anti-proliferative effects of euphol were associated with the increased p27^kip1 levels and decreased cyclin B1 levels. Inhibition of ERK1/2 activation by PD98059 reversed euphol-induced pro-apoptotic protein expression and cell death. Taken together, these findings suggest that euphol selectively induced gastric cancer cells apoptosis by modulation of ERK signaling, and could thus be of value for cancer therapy.     

Activation of Lysophosphatidic Acid Receptor Is Coupled to Enhancement of Ca2+-Activated Potassium Channel Currents

The calcium-activated K⁺ (BK_Ca) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. Ca²⁺ is the main regulator of BK_Ca channel activation. The BK_Ca channel contains two high affinity Ca²⁺ binding sites, namely, regulators of K⁺ conductance, RCK1 and the Ca²⁺ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular Ca²⁺ levels through diverse G proteins such as Gα_q/11, Gα_i, Gα_12/13, and Gαs and the related signal transduction pathway. In the present study, we examined LPA effects on BK_Ca channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated BK_Ca channel activation was also attenuated by the PLC inhibitor U-73122, IP₃ inhibitor 2-APB, Ca²⁺ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated BK_Ca channel activation. The present study indicates that LPA-mediated activation of the BK_Ca channel is achieved through the PLC, IP₃, Ca²⁺, and PKC pathway and that LPA-mediated activation of the BK_Ca channel could be one of the biological effects of LPA in the nervous and vascular systems.     

Cadmium induces N-cadherin cleavage via ERK-mediated γ-secretase activation in C6 astroglia cells

N-cadherin has known to be involved in tumor progression and metastasis. However, it is still obscure about the signaling pathway involving in the processing of N-cadherin. Thus, we examined which signaling pathway plays a major role in the processing of N-cadherin in C6 glioma cells following treatment of cadmium (Cd), a highly ubiquitous heavy metal. A cleavage product of N-cadherin, N-cad/CTF2 was observed by the treatment of Cd to C6 cells in a time and concentration-dependent manner. The production of N-cad/CTF2 was inhibited by pretreatment of γ-secretase inhibitors or siRNA transfection of nicastrin, indicating that γ-secretase is involved in the cleavage. Interestingly, Cd could activate both ERK and JNK signaling pathways in C6 cells; however, γ-secretase-mediated N-cad/CTF2 production by Cd was completely blocked by MEK1/2 inhibitors PD184352 and U0126, but not by a JNK inhibitor SP600125, demonstrating that the ERK signaling pathway plays a major role in the cleavage. In addition, pretreatment of an antioxidant or Ca²⁺ blocker blocked the production of N-cad/CTF2 by Cd together with the inhibition of ERK1/2 phosphorylation. Collectively, these results suggest that Cd increases intracellular Ca²⁺ or ROS, which induces γ-secretase-dependent N-cad/CTF2 production via the activation of the ERK signaling pathway in C6 glial cells.     

Involvement of oxidative stress-induced ERK/JNK activation in the Cu(2+)/pyrrolidine dithiocarbamate complex-triggered mitochondria-regulated apoptosis in pancreatic β-cells

Oxidative stress was demonstrated to promote the progression of diabetes mellitus (DM). It has been suggested that copper may play a specific role in the progression and pathogenesis of DM. Pyrrolidine dithiocarbamate (PDTC), a widely apply to the medicine, was known to be capable of enhancing copper accumulation. In this study, we investigated the effect of submicromolar-concentration Cu²⁺/PDTC complex on pancreatic β-cell damage and evaluated the role of oxidative stress in this effect. CuCl₂ (0.01-300 μM) did not affect the cell viability in β-cell line RIN-m5F cells. However, combination of CuCl₂ (0.5 μM) and PDTC (0.3 μM) markedly reduced RIN-m5F cell viability. Cu²⁺/PDTC complex could also increase the LPO and decrease the intracellular reduced GSH levels, and display several features of apoptosis signals including: increase in sub-G1 cell population, annexin-V binding, and caspase-3 activity, mitochondrial dysfunctions, and the activation of PARP and caspase cascades, which accompanied with the marked increase the intracellular Cu²⁺ levels. These apoptotic-related responses of Cu²⁺/PDTC complex-induced could be effectively prevented by antioxidant N-acetylcysteine (NAC). Furthermore, Cu²⁺/PDTC complex was capable of increasing the phosphorylations of ERK1/2 and JNK, and its upstream kinase MEK1/2 and MKK4, which could be reversed by NAC. Transfection with ERK2- and JNK-specific si-RNA and specific inhibitors SP600125 and PD98059 could inhibit ERK1/2 and JNK activation and attenuate MMP loss and caspase-3 activity induced by the Cu²⁺/PDTC complex. Taken together, these results are the first report to demonstrate that the Cu²⁺/PDTC complex triggers a mitochondria-regulated apoptosis via an oxidative stress-induced ERK/JNK activation-related pathway in pancreatic β-cells.     

Reevesioside F induces potent and efficient anti-proliferative and apoptotic activities through Na/K-ATPase α3 subunit-involved mitochondrial stress and amplification of caspase cascades

Reevesioside F, isolated from Reevesia formosana, induced anti-proliferative activity that was highly correlated with the expression of Na⁺/K⁺-ATPase α₃ subunit in several cell lines, including human leukemia HL-60 and Jurkat cells, and some other cell lines. Knockdown of α₃ subunit significantly inhibited cell apoptosis suggesting a crucial role of the α₃ subunit. Reevesioside F induced a rapid down-regulation of survivin protein, followed by release of cytochrome c from mitochondria and loss of mitochondrial membrane potential (ΔΨ_m). Further examination demonstrated the mitochondrial damage in leukemic cells through Mcl-1 down-regulation, Noxa up-regulation and an increase of the formation of truncated Bid, tBim and a 23-kDa cleaved Bcl-2 fragment. Furthermore, reevesioside F induced an increase of mitochondria-associated acetyl α-tubulin that may also contribute to apoptosis. The caspase cascade was profoundly activated by reevesioside F. Notably, the specific caspase-3 inhibitor z-DEVD-fmk significantly blunted reevesioside F-induced loss of ΔΨ_m and apoptosis, suggesting that caspase-3 activation may further amplify mitochondrial damage and apoptotic signaling cascade. In spite of being a cardiac glycoside, reevesioside F did not increase the intracellular Ca²⁺ levels. Moreover, CGP-37157 which blocked Na⁺/Ca²⁺ exchanger on plasma membrane and mitochondria did not modify reevesioside F-mediated effect. In summary, the data suggest that reevesioside F induces apoptosis through the down-regulation of survivin and Mcl-1, and the formation of pro-apoptotic fragments from Bcl-2 family members. The loss of ΔΨ_m and mitochondrial damage are responsible for the activation of caspases. Moreover, the amplification of caspase-3-mediated signaling pathway contributes largely to the execution of apoptosis in leukemic cells.     

Arsenic trioxide induces the apoptosis in bone marrow mesenchymal stem cells by intracellular calcium signal and caspase-3 pathways

It was previously reported that excessive arsenic trioxide would produce cardiovascular toxicity. Bone marrow mesenchymal stem cells (BMSCs) have been shown to play a supporting role in cardiovascular functions. The increasing apoptosis of BMSCs commonly would promote the development of cardiovascular diseases. Thus we hypothesize that arsenic trioxide caused apoptosis in BMSCs, which provided a better understanding of arsenic toxicity in hearts. The present study was designed to investigate the proapoptotic effects of arsenic trioxide on BMSCs and explore the mechanism underlying arsenic trioxide-induced BMSCs apoptosis. We demonstrate that arsenic trioxide significantly inhibited survival ratios of BMSCs in a concentration-dependent and time-dependent manner. The Annexin V/PI staining and terminal deoxynucleotidyl transferasemediated dUTP nick-end labelling (TUNEL) assay also showed that arsenic trioxide markedly induced the apoptosis of BMSCs. The caspase-3 activity was obviously enhanced in the presence of arsenic trioxide in a concentration-dependent manner in BMSCs. Additionally, arsenic trioxide caused the increase of intracellular free calcium ([Ca²⁺]_i) in rat BMSCs. BAPTA pretreatment may attenuate the apoptosis of BMSCs induced by arsenic trioxide. Taken together, arsenic trioxide could inhibit the proliferation and induce the apoptosis of BMSCs by modulating intracellular [Ca²⁺]_i, and activating the caspase-3 activity.     

Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells

Tetrandrine, which is isolated from Chinese herb Stephania tetrandrae, possesses anti-inflammatory, immunosuppressive, and cytoprotective properties. Though it was previously shown that tetrandrine causes a G1 blockade and apoptosis in various cell types, however, the mechanism by which tetrandrine initiates apoptosis remains poorly understood. In present study, we investigated the mechanisms of apoptosis induced by tetrandrine in U937 leukemia cells. Tetrandrine inhibited U937 cell growth by inducing apoptosis. After treatment of U937 cells with tetrandrine (10microM) for 24h, alteration of cell morphology, chromatin fragmentation, cytochrome c release, and caspase activation were observed. Tetrandrine also induced early oxidative stress, which resulted in activation of JNK, but not ERK and p38 MAPK. A broad-spectrum caspase inhibitor and antioxidants significantly blocked tetrandrine-induced caspase-3 activation. However, inhibition of the JNK activity with SP600125 did not block tetrandrine-induced apoptosis. Tetrandrine-induced apoptosis of U937 cells also required activity of PKC-delta, because pretreatment with a specific PKC-delta inhibitor greatly blocked tetrandrine-induced caspase-3 activation. In addition, the apoptotic response to tetrandrine was significantly attenuated in dominant-negative PKC-delta transfected MCF-7 cells, suggesting that PKC-delta plays an important role in tetrandrine-induced apoptosis and can induce caspase activation. These results suggest that tetrandrine induces oxidative stress, JNK activation, and caspase activation. However, JNK activation by ROS is not involved in the tetrandrine-induced apoptosis. In addition, tetrandrine induces caspase-dependent generation of a catalytically active fragment of PKC-delta, and this fragment also appears to play a role in the activation of caspases.     

Effect of 15-lipoxygenase metabolites, 15-(S)-HPETE and 15-(S)-HETE on chronic myelogenous leukemia cell line K-562: reactive oxygen species (ROS) mediate caspase-dependent apoptosis

Growth inhibitory effects of 15-lipoxygenase-1 [13-(S)-HPODE and 13-(S)-HODE] and 15-lipoxygenase-2 [15-(S)-HPETE and 15-(S)-HETE] (15-LOX-1 and LOX-2) metabolites and the underlying mechanisms were studied on chronic myeloid leukemia cell line (K-562). The hydroperoxy metabolites, 15-(S)-HPETE and 13-(S)-HPODE rapidly inhibited the growth of K-562 cells by 3h with IC(50) values, 10 and 15microM, respectively. In contrast, the hydroxy metabolite of 15-LOX-2, 15-(S)-HETE, showed 50% inhibition only at 40microM by 6h and 13-(S)-HODE, hydroxy metabolite of 15-LOX-1, showed no significant effect up to 160microM. The cells exposed to 10microM of 15-(S)-HPETE and 40microM of 15-(S)-HETE showed typical apoptotic features like release of cytochrome c, caspase-3 activation and PARP-1 (poly(ADP) ribose polymerase-1) cleavage. A flow cytometry based DCFH-DA analysis and inhibitory studies with DPI, a pharmacological inhibitor of NADPH oxidase, NAC (N-acetyl cysteine) and GSH revealed that NADPH oxidase-mediated generation of ROS is responsible for caspase-3 activation and subsequent induction of apoptosis in the K-562 cell line.     

The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: Intracellular calcium increase, calpain/caspase 3 activation, and functional upregulation

Previous work by our group demonstrated that homomeric α7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca²⁺ increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific α7 agonist PNU 282987 with IC₅₀ values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human α7 but not with α4β2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and α-bungarotoxin but not by dihydro-β-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on α7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca²⁺ release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca²⁺ levels and induced an increase in α-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and α7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca²⁺-dependent enzymes such as protein kinase C and nitric oxide synthase, which are involved in the generation of ROS and the blockade of the dopamine transporter. This, together with caspase 3 activation, must play a role in MDMA-induced cytotoxicity.     

Brief low [Mg2+]o-induced Ca2+ spikes inhibit subsequent prolonged exposure-induced excitotoxicity in cultured rat hippocampal neurons

Reducing [Mg²⁺]_o to 0.1 mM can evoke repetitive [Ca²⁺]_i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg²⁺]_o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca²⁺ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg²⁺]_o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca²⁺ channel antagonist nimodipine, which blocked 0.1 mM [Mg²⁺]_o-induced [Ca²⁺]_i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca²⁺]_i spikes. The intracellular Ca²⁺ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca²⁺]_i spikes. While Gö6976, a specific inhibitor of PKCα had no effect on the tolerance, both the PKCε translocation inhibitor and the PKCζ pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca²⁺]_i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg²⁺]_o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca²⁺]_i spike-induced activation of PKCε and PKCξ, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.     

Increased apoptotic efficacy of lonidamine plus arsenic trioxide combination in human leukemia cells. Reactive oxygen species generation and defensive protein kinase (MEK/ERK, Akt/mTOR) modulation

Lonidamine is a safe, clinically useful anti-tumor drug, but its efficacy is generally low when used in monotherapy. We here demonstrate that lonidamine efficaciously cooperates with the anti-leukemic agent arsenic trioxide (ATO, Trisenox™) to induce apoptosis in HL-60 and other human leukemia cell lines, with low toxicity in non-tumor peripheral blood lymphocytes. Apoptosis induction by lonidamine/ATO involves mitochondrial dysfunction, as indicated by early mitochondrial permeability transition pore opening and late mitochondrial transmembrane potential dissipation, as well as activation of the intrinsic apoptotic pathway, as indicated by Bcl-X_L and Mcl-1 down-regulation, Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release to the cytosol, XIAP down-regulation, and caspase-9 and -3 cleavage/activation, with secondary (Bcl-2-inhibitable) activation of the caspase-8/Bid axis. Lonidamine stimulates reactive oxygen species production, and lonidamine/ATO toxicity is attenuated by antioxidants. Lonidamine/ATO stimulates JNK phosphorylation/activation, and apoptosis is attenuated by the JNK inhibitor SP600125. In addition, lonidamine elicits ERK and Akt/mTOR pathway activation, as indicated by increased ERK, Akt, p70S6K and rpS6 phosphorylation, and these effects are reduced by co-treatment with ATO. Importantly, co-treatment with MEK/ERK inhibitor (U0126) and PI3K/Akt (LY294002) or mTOR (rapamycin) inhibitors, instead of ATO, also potentiates lonidamine-provoked apoptosis. These results indicate that: (i) lonidamine efficacy is restrained by drug-provoked activation of MEK/ERK and Akt/mTOR defensive pathways, which therefore represent potential therapeutic targets. (ii) Co-treatment with ATO efficaciously potentiates lonidamine toxicity via defensive pathway inhibition and JNK activation. And (iii) conversely, the pro-oxidant action of lonidamine potentiates the apoptotic efficacy of ATO as an anti-leukemic agent.     

A nitric oxide/Ca(2+)/calmodulin/ERK1/2 mitogen-activated protein kinase pathway is involved in the mitogenic effect of IL-1beta in human astrocytoma cells

Background and purpose:

Evidence is accumulating to support a role for interleukin-1β (IL-1β) in astrocyte proliferation. However, the mechanism by which this cytokine modulates this process is not fully elucidated.

Experimental approach:

In this study we used human astrocytoma U-373MG cells to investigate the role of nitric oxide (NO), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and extracellular signal-regulated protein kinase (ERK) in the signalling pathway mediating IL-1β-induced astrocyte proliferation.

Key results:

Low IL-1β concentrations induced dose-dependent ERK activation which paralleled upregulation of cell division, whereas higher concentrations gradually reversed both these responses by promoting apoptosis. Pretreatment with the nonspecific NOS inhibitor, N-ω-nitro-l-arginine methyl ester (L-NAME) or the selective iNOS inhibitor, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), antagonized ERK activation and cell proliferation induced by IL-1β. Inhibition of cGMP formation by the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), partially inhibited ERK activation and cell division. Functionally blocking Ca²⁺ release from endoplasmic reticulum with ryanodine or 2-aminoethoxydiphenylborane (2-APB), inhibiting calmodulin (CaM) activity with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W7) or MAPK kinase activity with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]butadiene (U0126) downregulated IL-1β-induced ERK activation as well as cell proliferation. The cytokine induced a transient and time-dependent increase in intracellular NO levels which preceded elevation in [Ca²⁺]_i.

Conclusions and implications:

These data identified the NO/Ca²⁺/CaM/ERK signalling pathway as a novel mechanism mediating the mitogenic effect of IL-1β in human astrocytes. As astrocyte proliferation is a hallmark of reactive astrogliosis, our results reveal a new potential target for therapeutic intervention in neuroinflammatory disorders.