Involvement of endoplasmic reticulum stress and activation of MAP kinases in beta-lapachone-induced human prostate cancer cell apoptosis

Beta-lapachone, an o-naphthoquinone, induces various carcinoma cells to undergo apoptosis, but the mechanism is poorly understood. In the present study, we found that the beta-lapachone-induced apoptosis of DU145 human prostate carcinoma cells was associated with endoplasmic reticulum (ER) stress, as shown by increased intracellular calcium levels and induction of GRP-78 and GADD-153 proteins, suggesting that the endoplasmic reticulum is a target of beta-lapachone. Beta-Lapachone-induced DU145 cell apoptosis was dose-dependent and accompanied by cleavage of procaspase-12 and phosphorylation of p38, ERK, and JNK, followed by activation of the executioner caspases, caspase-7 and calpain. However, pretreatment with the general caspase inhibitor, z-VAD-FMK, or calpain inhibitors, including ALLM or ALLN, failed to prevent beta-lapachone-induced apoptotic cell death. Blocking the enzyme activity of NQO1 with dicoumarol, a known NQO1 inhibitor, or preventing an increase in intracellular calcium levels using BAPTA-AM, an intracellular calcium chelator, substantially inhibited MAPK phosphorylation, abolished the activation of calpain, caspase-12 and caspase-7, and provided significant protection of beta-lapachone-treated cells. These findings show that beta-lapachone-induced ER stress and MAP kinase phosphorylation is a novel signaling pathway underlying the molecular mechanism of the anticancer effect of beta-lapachone.     

Calpain activation after mitochondrial permeability transition in microcystin-induced cell death in rat hepatocytes

Previous studies have shown that microcystin-LR (MLR), a specific hepatotoxin, induces onset of mitochondrial permeability transition (MPT) and apoptosis in cultured rat hepatocytes. Here we attempted to investigate the downstream events after the onset of MPT in MLR-treated hepatocytes. Various mitochondrial electron transport chain (ETC) inhibitors effectively prevented the onset of MPT, suggesting that the mitochondrial ETC plays an important role in MLR-induced MPT. MLR also induced mitochondrial cytochrome c release, which can be prevented by a specific MPT inhibitor (cyclosporin A, CsA), and by various ETC inhibitors. Interestingly, the release of cytochrome c did not activate caspase-9 and -3, the main caspases involved in apoptosis. Instead, MLR activated calpain in rat hepatocytes, probably through the increase of intracellular Ca(2+) released from mitochondria. Both ALLN and ALLM, two calpain inhibitors, significantly blocked MLR-induced calpain activation and subsequent cell death. CsA also prevented MLR-induced calpain activation and cell death, suggesting that the activation of calpain may be a post-mitochondrial event. These data demonstrate for the first time that calpain rather than caspases plays an important role in MLR-induced apoptosis.     

Calpain inhibitors stimulate phagocyte functions via activation of human formyl peptide receptors

Calpain inhibitors induce pertussis toxin (PTx)-sensitive chemotaxis in human neutrophils and monocytes. Here, we show that various calpain inhibitors (PD150606, PD151746, N-acetyl-Leu-Leu-Nle-CHO [ALLN], N-acetyl-Leu-Leu-Met-CHO [ALLM], and calpeptin) and γ-secretase inhibitor I induced PTx-sensitive increase in cytoplasmic free Ca²⁺ ([Ca²⁺]_i) in human neutrophils and neutrophil migration. HEK-293 cells stably expressing human formyl peptide receptor (hFPR) or hFPR-like 1 (hFPRL1) displayed stimulus-specific increase in [Ca²⁺]_i in response to calpain inhibitors (PD150606, PD151746, ALLN, ALLM, MG-132, and calpeptin), γ-secretase inhibitor I, and N-formyl-Met-Leu-Phe. Parent HEK-293 cells also displayed PTx-sensitive increase in [Ca²⁺]_i in response to calpeptin and γ-secretase inhibitor I, whereas they displayed PTx-resistant increase in [Ca²⁺]_i in response to MG-132. MDL-28170 induced neither an increase in [Ca²⁺]_i in neutrophils and HEK-293 cells nor neutrophil migration. Ionomycin-induced cleavage of talin (a substrate of calpain) in neutrophils was inhibited by all inhibitors used here. These findings suggest that potent calpain inhibitors could stimulate phagocyte functions via activation of hFPR, hFPRL1 and/or other G-protein coupled receptors depending on the inhibitors used.     

Various stressors rapidly activate the p38-MAPK signaling pathway in Mytilus galloprovincialis (Lam.)

The stimulation of p38-MAPK signal transduction pathway by various stressful stimuli was investigated in the marine bivalve M. galloprovincialis. Oxidative stress (5 M H₂O₂) induced a biphasic pattern of p38-MAPK phosphorylation with maximal values attained at 15 min (8.1-fold) and 1 h (8.0-fold) of treatment respectively. Furthermore, 1 M SB203580 abolished the p38-MAPK phosphorylation induced by oxidative stress. Aerial exposure also induced a biphasic pattern of p38-MAPK phosphorylation, with maximal values attained at 1 h (6.8-fold) and 8 h (4.9-fold) respectively. Re-oxygenation following a 15 min of aerial exposure resulted in the progressive dephosphorylation of the kinase. Treatment with 0.5 M sorbitol (in normal seawater) induced the rapid kinase phosphorylation (9.2-fold) and this effect was reversible. Seawater salinities varying between 100–60% had no effect, whereas a salinity of 50% induced a significant p38-MAPK phosphorylation. Furthermore, hypertonicity (120% seawater) resulted in a moderate kinase phosphorylation. All the above results demonstrate for the first time in a marine invertebrate imposed to environmental and other forms of stress as an intact, living organism, that the p38-MAPK pathway is specifically activated by various stressful stimuli which this animal can often face and sustain in vivo.     

Involvement of NO/cGMP signaling in the apoptotic and anti-angiogenic effects of beta-lapachone on endothelial cells in vitro

Neovascularization is an essential process in tumor development, it is conceivable that anti-angiogenic treatment may block tumor growth. In angiogenesis, nitric oxide (NO) is an important factor which mediates vascular endothelial cell growth and migration. beta-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho-[1,2-b]pyran-5,6-dione), a natural product extracted from the lapacho tree (Tabebuia avellanedae), has been demonstrated to possess anti-cancer and anti-viral effects. Whether beta-lapachone can induce endothelial cell death or has an anti-angiogenic effect is still an enigma. We investigated the in vitro effect of beta-lapachone on endothelial cells, including human vascular endothelial cell line, EAhy926, and human umbilical vascular endothelial cells (HUVEC). Our results revealed that (1) the intracellular cGMP levels and the mitochondria membrane potential (MMP) decreased, and calpain and caspases were activated, during beta-lapachone-induced endothelial cell death; (2) co-treatment with calpain inhibitors (ALLM or ALLN) or the intracellular calcium chelator, BAPTA, but not the general caspase inhibitor, zVAD-fmk, provided significant protection against apoptosis by preventing the beta-lapachone-induced MMP decrease and cytoplasmic calcium increase; (3) addition of NO downregulated the beta-lapachone-induced cGMP depletion and protected the cells from apoptosis by blocking the MMP decrease and the calcium increase; and (4) exogenous NO protects endothelial cells against the cell death induced by beta-lapachone, but not the anti-angiogenic effect. From all the data above, we demonstrated that NO can attenuate the apoptotic effect of beta-lapachone on human endothelial cells and suggest that beta-lapachone may have potential as an anti-angiogenic drug.     

Cyclosporin A induces apoptosis in H9c2 cardiomyoblast cells through calcium-sensing receptor-mediated activation of the ERK MAPK and p38 MAPK pathways

The cardiotoxicity of cyclosporine A (CsA) limits its clinical application in extensive and long-term therapies. Our group has shown that CsA induces myocardium cell apoptosis in vivo and increases calcium-sensing receptor (CaSR) expression. However, its molecular mechanism remains unknown. The purpose of this study was to determine whether CaSR plays an essential role in CsA-induced apoptosis in H9c2 cells and to investigate the role of the mitogen-activated protein kinase (MAPK) signaling cascade in this process. H9c2 cells were treated with CsA in a dose-dependent manner, and decreased Bcl-2 expression, increased Bax expression, and caspase-3 activation were observed. In a time-dependent manner, CsA increased CaSR expression, activated the extracellularly regulated kinase (ERK) and p38 MAPK pathways, and inactivated the c-Jun N-terminal kinase (JNK) MAPK signaling pathway. When H9c2 cardiomyoblast cells pretreated with gadolinium chloride (GdCl(3)), a CaSR activator, were treated with CsA, decreased phosphorylation of ERK1/2, increased phosphorylation of p38, decreased Bcl-2 expression, increased Bax expression, and activated caspase-3 were observed. Cells pretreated with the CaSR inhibitor NPS2390 inhibited this process. Furthermore, the MEK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580 markedly blocked the effect of CsA on cell apoptosis, apoptotic-related protein expression, and caspase-3 activation. These findings showed that CsA induced apoptosis in H9c2 cells in vitro, and CaSR mediated the degradation of ERK MAPK and the upregulation of the p38 MAPK pathway involved in CsA-induced H9c2 cardiomyoblast cell apoptosis.     

The problem of pyridinyl imidazole class inhibitors of MAPK14/p38α and MAPK11/p38β in autophagy research

In addition to its established role in inflammation, the stress-activated p38 MAP kinase pathway plays major roles in the regulation of cell cycle, senescence, and autophagy. Robust studies could establish mechanistic links between MAPK11-MAPK14/p38 signaling and macroautophagy converging at ATG9-trafficking and BECN1 phosphorylation. However, several reports seem to monitor MAPK11-MAPK14/p38-dependence of autophagy exclusively by the use of the SB203580/SB202190 class of MAPK14/MAPK11/p38α/β inhibitors. In this “Letter to the editor” we present data to support our claim that these inhibitors interfere with autophagic flux in a MAPK11-MAPK14/p38-independent manner and hence should no longer be used as pharmacological tools in the analysis of MAPK11-MAPK14/p38-dependence of autophagy. We propose a general guideline from Autophagy with regard to this issue to avoid such misinterpretations in the future.     

Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts

We have recently shown that insulin induced myogenesis in the mouse C2C12 skeletal muscle cell line by activation of phosphatidylinositol (PI) 3-kinase/p70S6-kinase and p38-mitogen-activated protein kinase (MAPK) and downregulation of p42/p44-MAPK. This study investigated the insulin-signaling pathways involved in mitogenesis, survival, and membrane ruffling in C2C12 myoblasts, a cellular system that besides IGF-I receptors, expressed a high number of functional insulin receptors. Insulin (10 nM) rapidly stimulated beta-chain insulin receptor and IRS-1 tyrosine phosphorylation, IRS-2 being poorly and SHC not phosphorylated at all. However, an association of SHC with IRS-1 was found under insulin stimulation. Insulin stimulated IRS-1 association with p85alpha leading to the activation of PI3-kinase, and, subsequently AKT and p70S6-kinases. Moreover, both p42/p44- and p38-MAPKs resulted in phosphorylation after insulin stimulation. Insulin treatment for 24 h produced mitogenesis, as demonstrated by the increase in ((3)H)-thymidine incorporation, DNA content, the expression of PCNA and cyclin D1 proteins, and the proportion of cells in S + G2/M phases of the cell cycle. This mitogenic effect of insulin was precluded by inhibition of p70S6-kinase (either by rapamycin or by the PI3-kinase inhibitor LY294002) as well as by inhibition of p44/p42-MAPK with PD098059, but was not affected by inhibition of p38-MAPK. Serum deprivation of C2C12 myoblasts resulted in growth arrest at the GO/G1 phases of the cell cycle and apoptosis, as detected either by DNA laddering or by increase in the percentage of hypodiploid cells. Insulin rescued serum-deprived cells from apoptosis in an AKT-dependent manner, as demonstrated by the inhibition of AKT-activity by the use of LY294002 and ML-9, meanwhile neither inhibition of p70S6-kinase, nor MAPK affected insulin-induced survival. Finally, we evaluated the capacity of insulin to modulate actin cytoskeleton rearrangement. Insulin stimulation of myoblasts produced membrane ruffling and decreased actin stress fibers; this biological response being dependent of p38-MAPK, as demonstrated by the use of the p38-MAPK inhibitors SB203580 or PD169316, but independent of PI3-kinase and p42/p44-MAPK.     

Heat stress prevents lipopolysaccharide-induced apoptosis in pulmonary microvascular endothelial cells by blocking calpain/p38 MAPK signalling

Pulmonary microvascular endothelial cells (PMECs) injury including apoptosis plays an important role in the pathogenesis of acute lung injury during sepsis. Our recent study has demonstrated that calpain activation contributes to apoptosis in PMECs under septic conditions. This study investigated how calpain activation mediated apoptosis and whether heat stress regulated calpain activation in lipopolysaccharides (LPS)-stimulated PMECs. In cultured mouse primary PMECs, incubation with LPS (1 μg/ml, 24 h) increased active caspase-3 fragments and DNA fragmentation, indicative of apoptosis. These effects of LPS were abrogated by pre-treatment with heat stress (43 °C for 2 h). LPS also induced calpain activation and increased phosphorylation of p38 MAPK. Inhibition of calpain and p38 MAPK prevented apoptosis induced by LPS. Furthermore, inhibition of calpain blocked p38 MAPK phosphorylation in LPS-stimulated PMECs. Notably, heat stress decreased the protein levels of calpain-1/2 and calpain activities, and blocked p38 MAPK phosphorylation in response to LPS. Additionally, forced up-regulation of calpain-1 or calpain-2 sufficiently induced p38 MAPK phosphorylation and apoptosis in PMECs, both of which were inhibited by heat stress. In conclusion, heat stress prevents LPS-induced apoptosis in PMECs. This effect of heat stress is associated with down-regulation of calpain expression and activation, and subsequent blockage of p38 MAPK activation in response to LPS. Thus, blocking calpain/p38 MAPK pathway may be a novel mechanism underlying heat stress-mediated inhibition of apoptosis in LPS-stimulated endothelial cells.     

Bigendothelin-1 via p38-MAPK-dependent mechanism regulates adult rat ventricular myocyte contractility in sepsis

We tested the hypothesis that exogenous administration of the ET-1 precursor, bigET-1, would regulate adult rat ventricular myocyte (ARVM) contractility in a p38-mitogen activated protein kinase (p38-MAPK)-dependent mechanism during sepsis. Ventricular myocytes from adult rat hearts (both sham and septic) were stimulated to contract at 0.5 Hz and mechanical properties were evaluated using an IonOptix Myocam system. Immunoblot analysis was used to determine the phosphorylation of p38-MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2). ARVMs were treated with vehicle, bigET-1 and inhibitors for 24 h and then subjected to functional and biochemical estimations. Septic ARVM displayed a distorted cell membrane and irregular network within the cells along with increased cell contractility as evidenced by elevated peak shortening (PS), maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt) in comparison to sham ARVM. BigET-1 treatment caused ARVM enlargement in both sham and sepsis groups. BigET-1 (100 nM) produced an increase in ARVM contractility in sham group as compared to vehicle treatment. However, septic ARVM treated with bigET-1 exhibited unaltered ARVM contractility, and upregulated ET(B) receptors as compared to respective sham group. BigET-1 increased the concentration of ET-1 and upregulated phosphorylation of p38-MAPK but not of ERK1/2 in sham and septic ARVM. Furthermore, inhibition of p38-MAPK by SB203580 (10 microM) increased ARVM contractility in sham but not in sepsis group. BigET-1 reversed SB203580-induced increase in PS in sham group but accentuated it in sepsis group. BigET-1 also reversed SB203580-induced inhibition of p38-MAPK phosphorylation in sham but not in septic ARVM. SB203580 pretreatment followed by bigET-1 administration significantly decreased p38-MAPK phosphorylation and downregulated ET(B) receptor expression as compared to bigET-1 treatment per se in sepsis group but not in sham. We concluded that a bigET-1-induced non-responsive effect on septic ARVM contractile function could be due to upregulation of p38-MAPK phosphorylation and ET(B) receptor expression.     

Gastrin-induced DNA synthesis requires p38-MAPK activation via PKC/Ca(2+) and Src-dependent mechanisms

We present evidence that gastrin, binding to a G protein-coupled receptor, activates the p38-mitogen-activated protein kinase (MAPK) pathway. Blockage of protein kinase C (PKC) by GF109203X, depletion of intracellular calcium by thapsigargin or inhibition of Src family kinases by PP2 prevented p38-MAPK activation and the Src kinase activity stimulated by gastrin. Inhibition of the PI 3-kinase by wortmannin or LY294002 did not affect these responses. In addition, the p38-MAPK inhibitor, SB203580, repressed gastrin-induced [(3)H]thymidine incorporation, indicating a major role of p38-MAPK in the growth-promoting effect of gastrin. Our results demonstrate that gastrin-induced DNA synthesis requires p38-MAPK activation through mechanisms that involve calcium mobilization, PKC and Src family kinases.     

Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27

Evidence is presented that calpain, a calcium-activated protease, degrades the cyclin-dependent kinase inhibitor, p27, during the mitotic clonal expansion phase of 3T3-L1 preadipocyte differentiation. Calpain activity is required during an early stage of the adipocyte differentiation program. Thus, inhibition of calpain with N-acetyl-Leu-Leu-norleucinal (ALLN) blocks clonal expansion and acquisition of the adipocyte phenotype only when added between 12 and 24 h after the induction of differentiation. Likewise, inhibition of calpain by overexpression of calpastatin, the specific endogenous inhibitor of calpain, prevents 2-day post-confluent preadipocytes from reentering the cell cycle triggered by the differentiation inducers. Inhibition of calpain with ALLN causes preadipocytes to arrest just prior to S phase and prevents phosphorylation of the retinoblastoma gene product, DNA replication, clonal expansion, and subsequent adipocyte differentiation but does not affect the expression of immediate early genes (i.e. fos, jun, C/EBPbeta, and C/EBPdelta). Inhibition of calpain by either ALLN or by overexpression of calpastatin blocks the degradation of p27. p27 is degraded in vitro by cell-free extracts from clonally expanding preadipocytes that contain "active" calpain but not by extracts from pre-mitotic preadipocytes that do not. This action is inhibited by calpastatin or ALLN. Likewise, p27 in preadipocyte extracts is a substrate for purified calpain; this proteolytic action was inhibited by heat inactivation, EGTA, or ALLN. Thus, extracellular signals from the differentiation inducers appear to activate calpain, which degrades p27 allowing density-dependent inhibited preadipocytes to reenter the cell cycle and undergo mitotic clonal expansion.     

Alterations of load-induced p38 MAP kinase activation in failing rat hearts.

Hemodynamic load-induced cardiac p38 mitogen-activated protein kinase (MAPK) activation was studied in normotensive control Dahl rats (n = 10) and hypertensive Dahl rats with heart failure (n = 16). The isolated heart from each animal was stretched on a Langendorff apparatus at an equivalent diastolic wall stress, and the p38-MAPK activity of the left ventricular (LV) myocardium was analyzed by immunoprecipitation-kinase assay. Compared to the control hearts, the stretch-induced p38-MAPK activities were significantly decreased, and inversely correlated with the LV diameter (r = -0.73, P < 0.01). Chronic treatment with an angiotensin II AT1-receptor antagonist, valsartan (10 mg/kg/day), ameliorated cardiac function and remodeling process in the failing hearts, which was associated with an improvement of the p38-MAPK activities. Thus, the mechano-signal transduction of p38-MAPK pathway is downregulated in the failing hearts, along with progressive ventricular remodeling. The data also suggest that the beneficial effects of the AT1-receptor antagonists are potentially mediated by the restoration of cardiac growth-related signal transduction.     

Alterations in STriatal‐Enriched protein tyrosine Phosphatase expression,activation, and downstream signaling in early and late stages of the YAC128 Huntington's disease mouse model

Striatal neurodegeneration and synaptic dysfunction in Huntington's disease are mediated by the mutant huntingtin (mHtt) protein. MHtt disrupts calcium homeostasis and facilitates excitotoxicity, in part by altering NMDA receptor (NMDAR) trafficking and function. Pre‐symptomatic (excitotoxin‐sensitive) transgenic mice expressing full‐length human mHtt with 128 polyglutamine repeats (YAC128 Huntington's disease mice) show increased calpain activity and extrasynaptic NMDAR (Ex‐NMDAR) localization and signaling. Furthermore, Ex‐NMDAR stimulation facilitates excitotoxicity in wild‐type cortical neurons via calpain‐mediated cleavage of STriatal‐Enriched protein tyrosine Phosphatase 61 (STEP61). The cleavage product, STEP33, cannot dephosphorylate p38 mitogen‐activated protein kinase (MAPK), thereby augmenting apoptotic signaling. Here, we show elevated extrasynaptic calpain‐mediated cleavage of STEP61 and p38 phosphorylation, as well as STEP61 inactivation and reduced extracellular signal‐regulated protein kinase 1/2 phosphorylation (ERK1/2) in the striatum of 6‐week‐old, excitotoxin‐sensitive YAC128 mice. Calpain inhibition reduced basal and NMDA‐induced STEP61 cleavage. However, basal p38 phosphorylation was normalized by a peptide disrupting NMDAR‐post‐synaptic density protein‐95 (PSD‐95) binding but not by calpain inhibition. In 1‐year‐old excitotoxin‐resistant YAC128 mice, STEP33 levels were not elevated, but STEP61 inactivation and p38 and ERK 1/2 phosphorylation levels were increased. These results show that in YAC128 striatal tissue, enhanced NMDAR–PSD‐95 interactions contributes to elevated p38 signaling in early, excitotoxin‐sensitive stages, and suggest that STEP61 inactivation enhances MAPK signaling at late, excitotoxin‐resistant stages.