Tumor necrosis factor-alpha induces insulin resistance in endothelial cells via a p38 mitogen-activated protein kinase-dependent pathway

Chronic inflammation contributes to vascular insulin resistance and endothelial dysfunction. Systemic infusion of TNF-alpha abrogates insulin's action to enhance skeletal muscle microvascular perfusion. In skeletal muscle TNF-alpha induces insulin resistance via the p38 MAPK pathway. To examine whether p38 MAPK also regulates TNF-alpha-induced vascular insulin resistance, bovine aortic endothelial cells (bAECs) were incubated+/-TNF-alpha (5 ng/ml) for 6 h in the presence or absence of SB203580 (p38 MAPK specific inhibitor, 10 microM) after serum starvation for 10 h. For the last 30 min, cells were treated+/-1 nM insulin, and insulin receptor substrate (IRS)-1, Akt, endothelial nitric oxide synthase (eNOS), p38 MAPK, ERK1/2, c-Jun N-terminal kinase, and AMP-activated protein kinase (AMPK) phosphorylation, and eNOS activity were measured. TNF-alpha increased p38 MAPK phosphorylation, potently stimulated IRS-1 serine phosphorylation, and blunted insulin-stimulated IRS-1 tyrosine and Akt phosphorylation and eNOS activity. TNF-alpha also potently stimulated the phosphorylation of ERK1/2 and AMPK. Treatment with SB203580 decreased p38 MAPK phosphorylation back to the baseline and restored insulin sensitivity of IRS-1 tyrosine and Akt phosphorylation and eNOS activity in TNF-alpha-treated bAECs without affecting TNF-alpha-induced ERK1/2 and AMPK phosphorylation. We conclude that in cultured bAECs, TNF-alpha induces insulin resistance in the phosphatidylinositol 3-kinase/Akt/eNOS pathway via a p38 MAPK-dependent mechanism and enhances ERK1/2 and AMPK phosphorylation independent of the p38 MAPK pathway. This differential modulation of TNF-alpha's actions by p38 MAPK suggests that p38 MAPK plays a key role in TNF-alpha-mediated vascular insulin resistance and may contribute to the generalized endothelial dysfunction seen in type 2 diabetes mellitus and the cardiometabolic syndrome.     

Lysophosphatidylcholine activates mitogen-activated protein kinases by a tyrosine kinase-dependent pathway in bovine aortic endothelial cells

Lysophosphatidylcholine (lyso-PC) is a major component of an atherogenic lipoprotein. In this study, to investigate the involvement of mitogen-activated protein kinases in the signaling pathway by lyso-PC in endothelial cells, we measured the activity of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in bovine aortic endothelial cells. Lyso-PC activated ERK and JNK in a dose-dependent manner. However, the time courses of activation of these kinases were different. ERK and JNK activation by lyso-PC was inhibited by a tyrosine kinase inhibitor, herbimycin A, but not by a protein kinase C (PKC) specific inhibitor. We conclude, therefore, that lyso-PC-mediated ERK and JNK activation is caused by a tyrosine kinase-dependent mechanism, but not conventional types of PKC-dependent mechanisms.     

Lysophosphatidylcholine induces apoptosis in AR42J cells

Phospholipase A2 (PLA2) has been suggested to play an important role in the pathogenesis of acute pancreatitis, in part through the PLA2-generated phospholipid by-products, most notably lysophosphatidylcholine (lyso-PC). The effects of lyso-PC on pancreatic acinar cells, other than the induction of necrosis, are poorly characterized. Here we examined the effects of lyso-PC on the induction of apoptosis in rat pancreatic AR42J cells. Lyso-PC induced apoptosis in a dose-dependent manner at 10 and 25 microM, but induced cell lysis at > or = 50 microM. Lyso-PC-induced (at 25 microM) apoptosis was not blocked by a protein kinase C inhibitor (staurosporine) or by inhibitors of caspases (acetyl-DEVD-aldehyde and benzoyloxycarbonyl-VAD-fluoromethylketone). Lyso-PC at 10 and 25 microM induced the expression of clusterin mRNA and wild-type p53. Apoptosis induction by lyso-PC (at 25 microM) was not inhibited by a specific antagonist of platelet-activating factor (PAF) receptor, suggesting that the action was independent of th     

Enediyne lidamycin induces apoptosis in human multiple myeloma cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase

In the present study, the effects of lidamycin (LDM), a member of the enediyne antibiotic family, on two human multiple myeloma (MM) cell lines, U266 and SKO-007, were evaluated. In MTS assay, LDM showed much more potent cytotoxicity than conventional anti-MM agents to both cell lines. The IC₅₀ values of LDM for the U266 and SKO-007 cells were 0.0575 ± 0.0015 and 0.1585 ± 0.0166 nM, respectively, much lower than those of adriamycin, dexamethasone, and vincristine. Mechanistically, LDM triggered MM cells apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in LDM-induced cell death. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed the LDM-induced apoptosis through decreasing the level of cleaved PARP and caspase-3/7. Interestingly, phosphorylation of extracellular signal-related kinase was increased by LDM; conversely, MEK inhibitor synergistically enhanced LDM-induced cytotoxicity and apoptosis in MM cells. The results demonstrated that LDM suppresses MM cell growth through the activation of p38 MAPK and JNK, with the potential to be developed as a chemotherapeutic agent for MM.     

The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism

Antibodies against CD20 can activate complement and induce antibody-dependent cellular cytotoxicity (ADCC) in B lymphocytes. In B-cell lines, such antibodies also induce apoptosis. In this study, the expression and function of CD20 on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed. Flow cytometric analysis demonstrated that B-CLL cells express CD20 with a fluorescence intensity that is significantly weaker than that of normal CD5(+) and CD5(-) B cells and that of malignant CD5(-) low-grade non-Hodgkin lymphoma cells. A small population of cells from healthy donors that have an expression pattern of CD5 and CD20 identical to that of B-CLL cells were identified, and this population was confirmed to be of T lineage, not B lineage. Culture of freshly isolated B-CLL cells in the presence of the chimeric anti-CD20 antibody rituximab and a cross-linking F(ab)(2) fragment, resulted in dose- and time-dependent induction of apoptosis. The induction of apoptosis occurred under conditions in which the influence of complement activation and ADCC was negligible. Cross-linking of rituximab induced strong and sustained phosphorylation of the 3 mitogen activated protein (MAP) kinases c-Jun NH2-terminal protein kinase, extracellular signal-regulated kinase, and p38. Introduction of the p38 inhibitor SB203580 into the system completely blocked signaling downstream of p38, as evidenced by the absence of MAPKAP K2 activity, and significantly reduced the degree of anti-CD20-induced apoptosis. These results demonstrate that cross-linking of rituximab bound to CD20 on freshly isolated B-CLL cells induces apoptosis through a signaling pathway that is dependent on p38 MAP-kinase activation.     

Iodide excess induces apoptosis in thyroid cells through a p53-independent mechanism involving oxidative stress

Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; in vitro iodide is cytotoxic, inhibits cell growth, and induces morphological changes in thyroid cells of some species. In this study, we investigated the effect of iodide excess in an immortalized thyroid cell line (TAD-2) in primary cultures of human thyroid cells and in cells of nonthyroid origin. Iodide displayed a dose-dependent cytotoxicity in both TAD-2 and primary thyroid cells, although at different concentrations, whereas it had no effect on cells of nonthyroid origin. Thyroid cells treated with iodide excess underwent apoptosis, as evidenced by morphological changes, plasma membrane phosphatidylserine exposure, and DNA fragmentation. Apoptosis was unaffected by protein synthesis inhibition, whereas inhibition of peroxidase enzymatic activity by propylthiouracil completely blocked iodide cytotoxicity. During KI treatment, reactive oxygen species were produced, and lipid peroxide levels increased markedly. Inhibition of endogenous p53 activity did not affect the sensitivity of TAD-2 cells to iodide, and Western blot analysis demonstrated that p53, Bcl-2, Bcl-XL, and Bax protein expression did not change when cells were treated with iodide. These data indicate that excess molecular iodide, generated by oxidation of ionic iodine by endogenous peroxidases, induces apoptosis in thyroid cells through a mechanism involving generation of free radicals. This type of apoptosis is p53 independent, does not require protein synthesis, and is not induced by modulation of Bcl-2, Bcl-XL, or Bax protein expression.     

Chlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers p38 mitogen-activated protein kinase-dependent apoptosis in chronic myelogenous leukemic cells

We report that chlorogenic acid (Chl) induces apoptosis of several Bcr-Abl-positive chronic myelogenous leukemia (CML) cell lines and primary cells from CML patients in vitro and destroys Bcr-Abl-positive K562 cells in vivo. In contrast, this compound has no effect on the growth and viability of Bcr-Abl-negative lymphocytic and myeloid cell lines and primary CML cells. Sodium chlorogenate (NaChl) exhibits 2-fold higher efficiency in killing K562 cells compared with Chl. NaChl also induces growth inhibition of squamous cell carcinoma (HSC-2) and salivary gland tumor cells (HSG), although at 50-fold higher concentration. NaChl inhibits autophosphorylation of p210(Bcr-Abl) fusion protein rapidly. We demonstrate that p38 phosphorylation is increased in Bcr-Abl-positive cells after treatment with NaChl and closely paralleled the inhibition of Bcr-Abl phosphorylation. NaChl did not increase phosphorylation of p38 in Bcr-Abl-negative cells including HSC-2 and HSG that are responsive to this compound, indicating that p38 activation by NaChl is dependent on Bcr-Abl kinase inhibition. Inhibition of p38 activity by SB203580 significantly reduced NaChl-induced apoptosis of K562 cells, whereas activation of p38 by anisomycin augmented the apoptosis. These findings indicate that inhibition of Bcr-Abl kinase leading to activation of p38 mitogen-activated protein (MAP) kinase may play an important role in the anti-CML activity of Chl.     

Bone morphogenic protein-4 induces endothelial cell apoptosis through oxidative stress-dependent p38MAPK and JNK pathway

The expression of bone morphogenic protein 4 (BMP4), a new pro-inflammatory marker, is increased by disturbed flow in endothelial cells (ECs). BMP4 stimulates production of reactive oxygen species (ROS) and causes endothelial cell dysfunction. The present study examined BMP4-induced apoptosis in ECs and isolated arteries from rat, mouse, and human, and the signaling pathways mediating BMP4-induced apoptosis. Apoptosis was assessed by flow cytometry to detect Annexin-V positive cells, and terminal deoxynucleotidyl transferase dUTP nick end (TUNEL) labeling. The superoxide production was measured by dihydroethidium fluorescence. BMP4 induced EC apoptosis in human mesenteric arteries, mouse aortic endothelium, rat primary ECs, and human ECs. BMP4-induced EC apoptosis was mediated through ROS production by activation of NADPH oxidase, which led to cleaved caspase-3 expression. BMP4 also induced sequential activation of p38 MAPK and JNK which was upstream of caspase 3 activation. Knockdown of BMP receptor 1A by lentiviral shRNA or NOX4 siRNA transfection inhibited BMP4-induced ROS production, p38 and JNK phosphorylation, and caspase-3 activation in ECs. JNK siRNA inhibited BMP4-induced JNK phosphorylation and caspase-3 activation. The present study delineates that BMP4 causes EC apoptosis through activation of caspase-3 in a ROS/p38MAPK/JNK-dependent signaling cascade.     

Asymmetric dimethylarginine induces apoptosis via p38 MAPK/caspase-3-dependent signaling pathway in endothelial cells

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is emerging as a key contributor for endothelial dysfunction and its effects on endothelium are not yet completely defined. The aim of this study was to investigate ADMA-induced apoptosis and its mechanisms in human umbilical vein endothelial cells (HUVECs). Apoptosis was evaluated by in situ terminal uridine nick end labeling (TUNEL) assay and DNA fragmentation analysis. Caspase-3 activity was measured using a colorimetric protease assay kit. Activations of mitogen-activated protein kinases (MAPKs) were characterized by Western blot and immunofluorescence. Intracellular oxidant production was measured using H(2)DCF-DA, an oxidant-sensitive fluorescent indicator. ADMA (3-30 microM) induced apoptosis of HUVECs in a dose- and time-dependent manner. Caspase-3 was activated during apoptosis and its specific inhibitor DEVD-CHO significantly attenuated ADMA-induced apoptosis. Phosphorylation of p38 MAPK was induced by ADMA, and p38 MAPK specific inhibitor SB203580 concentration-dependently prevented ADMA-induced caspase-3 activation and cell apoptosis. ADMA increased intracellular oxidant production, which was significantly suppressed by intracellular antioxidant PDTC, l-arginine or antisense endothelial NOS mRNA. They also markedly prevented ADMA-induced phosphorylation of p38 MAPK and cell apoptosis. In conclusion, our present results demonstrate that ADMA induces apoptosis of endothelial cell via elevation of intracellular oxidant production, which involves p38 MAPK/caspase-3-dependent signaling pathway.     

UCN-01 induces cytotoxicity toward human CLL cells through a p53-independent mechanism

OBJECTIVES: UCN-01, a novel protein kinase C inhibitor, is currently being tested in phase I clinical trials after being noted to induce apoptosis in lymphoid cell lines. We sought to study the in vitro activity of UCN-01 against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing this cytotoxicity. METHODS: Detailed in vitro studies were performed from tumor cells derived from patients with CLL cells following attainment of written informed consent. RESULTS: The 50% loss of viability (LC(50)) in mononuclear cells from CLL patients (n = 10) following exposure to UCN-01 for 4 days was 0.4 microM (95% CI +/- 0.21; range 0.09-1.16). Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.4 and 5.0 microM of UCN-01 resulted in decreased expression of p53 protein. We therefore investigated the dependence of UCN-01 on intact p53 by exposing splenocytes from wild-type (p53(+/+)) and p53 null (p53(-/-)) mice, which demonstrated no preferential cytotoxicity when compared to the marked differential induced by F-Ara-A and radiation. CONCLUSIONS: UCN-01 has significant in vitro activity against human CLL cells that appears to occur independent of p53 status. While demonstration of in vitro cytotoxicity does not establish in vivo efficacy, the findings described support the early introduction of UCN-01 into clinical trials for patients with B-CLL.     

p38 mitogen-activated protein kinase mediates palmitate-induced apoptosis but not inhibitor of nuclear factor-kappaB degradation in human coronary artery endothelial cells

Plasma free fatty acids are elevated in patients with type 2 diabetes and contribute to the pathogenesis of insulin resistance and endothelial dysfunction. The p38 MAPK mediates stress, inflammation, and apoptosis. Whether free fatty acids induce apoptosis and/or activate nuclear factor-kappaB inflammatory pathway in human coronary artery endothelial cells (hCAECs) and, if so, whether this involves the p38 MAPK pathway is unknown. hCAECs (passages 4-6) were grown to 70% confluence and then incubated with palmitate at concentrations of 0-300 microm for 6-48 h. Palmitate at 100, 200, or 300 microm markedly increased apoptosis after 12 h of incubation. This apoptotic effect was time (P=0.008) and dose (P=0.006) dependent. Palmitate (100 microm for 24 h) induced a greater than 2-fold increase in apoptosis, which was accompanied with a 4-fold increase in p38 MAPK activity (P<0.001). Palmitate did not affect the phosphorylation of Akt1 or ERK1/2. SB203580 (a specific inhibitor of p38 MAPK) alone did not affect cellular apoptosis; however, it abolished palmitate-induced apoptosis and p38 MAPK activation. Palmitate significantly reduced the level of inhibitor of nuclear factor-kappaB (IkappaB). However, treatment of cells with SB203580 did not restore IkappaB to baseline. We conclude that palmitate induces hCAEC apoptosis via a p38 MAPK-dependent mechanism and may participate in coronary endothelial injury in diabetes. However, palmitate-mediated IkappaB degradation in hCAECs is independent of p38 MAPK activity.     

MCP-1 induces activation of MAP-kinases ERK,JNK and p38 MAPK in human endothelial cells

Activation of vascular endothelial cells (ECs) plays an important pathogenic role in the development of atherosclerosis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant of monocytes. Besides induction of monocyte recruitment, it has been suggested that MCP-1 can also affect the cellular responses of ECs. We investigated whether MCP-1 activated the three major mitogen activated protein (MAP)-kinases extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38 MAPK. Stimulation of ECs with MCP-1 induced a time- and concentration-dependent activation of all three MAP-kinases, concentrations as low as 0.1 ng/ml were sufficient for this mechanism. MCP-1 also induced secretion of matrix metalloproteinase (MMP)-2 which along with ERK activation was inhibited by PD098059. The results demonstrate that MCP-1 can lead to direct activation of MAP kinases together with induction of MMP2 in ECs. Our data thus propose a new mechanism for the proatherogenic effect of MCP-1.     

Homocysteine induces cardiomyocyte dysfunction and apoptosis through p38 MAPK-mediated increase in oxidant stress

Elevated plasma homocysteine (Hcy) is a risk factor for cardiovascular disease. While Hcy has been shown to promote endothelial dysfunction by decreasing the bioavailability of nitric oxide and increasing oxidative stress in the vasculature, the effects of Hcy on cardiomyocytes remain less understood. In this study we explored the effects of hyperhomocysteinemia (HHcy) on myocardial function ex vivo and examined the direct effects of Hcy on cardiomyocyte function and survival in vitro. Studies with isolated hearts from wild type and HHcy mice (heterozygous cystathionine-beta synthase deficient mice) demonstrated that HHcy mouse hearts had more severely impaired cardiac relaxation and contractile function and increased cell death following ischemia reperfusion (I/R). In isolated cultured adult rat ventricular myocytes, exposure to Hcy for 24 h impaired cardiomyocyte contractility in a concentration-dependent manner, and promoted apoptosis as revealed by terminal dUTP nick-end labeling and cleaved caspase-3 immunoblotting. These effects were associated with activation of p38 MAPK, decreased expression of thioredoxin (TRX) protein, and increased production of reactive oxygen species (ROS). Inhibition of p38 MAPK by the selective inhibitor SB203580 (5 μM) prevented all of these Hcy-induced changes. Furthermore, adenovirus-mediated overexpression of TRX in cardiomyocytes significantly attenuated Hcy-induced ROS generation, apoptosis, and impairment of myocyte contractility. Thus, Hcy may increase the risk for CVD not only by causing endothelial dysfunction, but also by directly exerting detrimental effects on cardiomyocytes.     

All-trans retinoic acid induces apoptosis in human mesangial cells: involvement of stress activated p38 kinase

All-trans retinoic acid (AR-t) is used for treating acute promyelocytic leukemia and renal cell carcinoma and it also has therapeutic value in several animal models of renal disease. Among its renal targets, mesangial cells have been widely studied: they have both retinoic acid receptors (RAR) and retinoid X receptors (RXR) and the cell growth is inhibited when human mesangial cells are incubated with 1-10 microM AR-t. Although his effect has been related with the antiproliferative action of AR-t, there are no studies on the involvement of apoptosis in AR-t induced cell growth when higher concentrations of retinoid are used. Our studies show that 25 microM AR-t triggers mesangial cell apoptosis assessed by light and fluorescence microscopy (Giemsa stain and acridine orange stain, respectively), DNA electrophoresis, flow cytometry (annexin-V) and immunocytochemistry (TUNEL). AR-t induced apoptosis was not inhibited by preincubation with the RXR pan-antagonist HX531 nor with the RAR pan-antagonist AGN 193109, this suggesting RAR and RXIR are not involved in AR-t induced cell death. Previous results of our group showed that ERK (extracellular regulated kinase) and INK (c-Jun kinase), two members of the MAP (mitogen activated protein) kinase family, are involved in non apoptotic effects of AR-t on mesangial cells. Therefore we focussed on the stress activated p38 kinase, the third member of the MAPK family, to investigate its involvement in AR-t induced apoptosis. The results confirmed a role of p38 since: 1) preincubation with B5203589, a p38 inhibitor, inhibited ARA induced apoptosis; 2) incubation with AR-t induced p38 phosphorilation after few minutes and p38 remained phosphorilated for at least 8 hours and 3) AR-t induced p38 phosphorilation was inhibited by SB203589. These data suggest that AR-t might have toxic side effects on the kidney but also suggest that AR-t could be an useful inhibitor of pathological mesangial cell expansion.     

Metformin induces apoptosis of lung cancer cells through activating JNK/p38 MAPK pathway and GADD153

There are epidemiological and experimental evidences that metformin, an insulin-sensitizer agent widely used for diabetes treatment, has inhibitory effects on the growth of various human cancers. However, the underlying molecular mechanisms for its anti-neoplastic activity has not been yet clarified and the effect of metformin on human lung cancer remains unknown. In this study we revealed for the first time that metformin treatment led to increased apoptosis in human lung cancer cell lines A549 and NCI-H1299 and significantly inhibited the cells proliferation in a?dose- and time-dependent manner, which was further demonstrated by the data obtained from A549 tumor xenografts in nude mice. We also found that metformin treatment can activate AMP-activated protein kinase, JNK/p38 MAPK signaling pathway and caspases, as well as upregulate the expression of growth arrest and DNA damage inducible gene 153 (GADD153). Either blockade of JNK/p38 MAPK pathway or knockdown of GADD153 gene abrogated the apoptosis-inducing effect of metformin. Taken together, our data suggest that metformin inhibits the growth of lung cancer cells and induces apoptosis through activating JNK/p38 MAPK pathway and GADD153.