Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway

Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72 h. Peak expression occurred at 12 h (3- to 4-fold, p < 0.05). p38 inhibitor (SB203580) blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs’ proliferation and migration. Over-expression of Bcl-2 increased HAECs’ tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation.Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.     

NF-kappaB activation mechanism of 4-hydroxyhexenal via NIK/IKK and p38 MAPK pathway

4-Hydroxyhexenal (HHE) is known to affect redox balance during aging, included are vascular dysfunctions. To better understand vascular abnormality through the molecular alterations resulting from HHE accumulation in aging processes, we set out to determine whether up-regulation of mitogen-activated protein kinase (MAPK) by HHE is mediated through nuclear factor kappa B (NF-kappaB) activation in endothelial cells. HHE induced NF-kappaB activation by inhibitor of kappaB (IkappaB) phosphorylation via the IkappaB kinase (IKK)/NF-kappaB inducing kinase (NIK) pathway. HHE increased the activity of p38 MAPK and extracellular signal regulated kinase (ERK), but not c-jun NH(2)-terminal kinase, indicating that p38 MAPK and ERK are closely involved in HHE-induced NF-kappaB transactivation. Pretreatment with ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, attenuated the induction of p65 translocation, IkappaB phosphorylation, and NF-kappaB luciferase activity. These findings strongly suggest that HHE induces NF-kappaB activation through IKK/NIK pathway and/or p38 MAPK and ERK activation associated with oxidative stress in endothelial cells.     

Pim-3 protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway

Although anoxic preconditioning (APC) in the myocardium has been investigated for many years, its physiological mechanism is still not completely understood. Increasing evidence indicates that transiently increased resistance to ischemic damage following APC is dependent on de novo proteins synthesis. However, the key effector pathway(s) associated with APC still remains unclear. The proto-oncogene Pim kinase belongs to a serine/threoine protein kinase family, consists of Pim-1, Pim-2 and Pim-3 and has been implicated in stimulating cell growth and inhibiting cell apoptosis. Therefore we assumed that Pim-3 expression might be aberrantly induced in cardiomyocytes that were subjected to anoxia/reoxygenation (A/R) injury and that Pim-3 might also contribute to cardio-protection after APC. To address this hypothesis, we cloned a Pim-3 expression vector, transfected it into rat cardiomyocytes, and examined Pim-3 expression in rat cardiomyocytes that were subjected to A/R injury. Moreover, we studied the role of three major MAPK pathways, e.g. p38 MAPK, JNK, and ERK1/2, in order to evaluate the molecular mechanism underlying Pim-3 up-regulation and A/R induced cardiomyocyte injury. Our experiments showed that APC induced an up-regulation of Pim-3 and the transfection of Pim-3 gene into the cardiomyocytes attenuated A/R injury. The inhibition of p38 MAPK by SB203580 abolished both the Pim-3 up-regulation and the cardio-protection provided by APC. Overall, these results suggest that APC could act to protect the heart from A/R injury with cooperation from the proto-oncogene Pim-3; in addition, it up-regulates Pim-3 expression through a p38 MAPK signaling pathway.     

MAPKAPK-2 modulates p38-MAPK localization and small heat shock protein phosphorylation but does not mediate the injury associated with p38-MAPK activation during myocardial ischemia

MAPKAPK-2 (MK2) is a protein kinase activated downstream of p38-MAPK which phosphorylates the small heat shock proteins HSP27 and αB crystallin and modulates p38-MAPK cellular distribution. p38-MAPK activation is thought to contribute to myocardial ischemic injury; therefore, we investigated MK2 effects on ischemic injury and p38 cellular localization using MK2-deficient mice (KO). Immunoblotting of extracts from Langendorff-perfused hearts subjected to aerobic perfusion or global ischemia or reperfusion showed that the total and phosphorylated p38 levels were significantly lower in MK2^−/− compared to MK2^+/+ hearts at baseline, but the ratio of phosphorylated/total p38 was similar. These results were confirmed by cellular fractionation and immunoblotting for both cytosolic and nuclear compartments. Furthermore, HSP27 and αB crsytallin phosphorylation were reduced to baseline in MK2^−/− hearts. On semiquantitative immunofluorescence laser confocal microscopy of hearts during aerobic perfusion, the mean total p38 fluorescence was significantly higher in the nuclear compared to extranuclear (cytoplasmic, sarcomeric, and sarcolemmal compartments) in MK2^+/+ hearts. However, although the increase in phosphorylated p38 fluorescence intensity in all compartments following ischemia in MK2^+/+ hearts was lost in MK2^−/− hearts, it was basally elevated in nuclei of MK2^−/− hearts and was similar to that seen during ischemia in MK2^+/+ hearts. Despite these differences, similar infarct volumes were recorded in wild-type MK2^+/+ and MK2^−/− hearts, which were decreased by the p38 inhibitor SB203580 (1 μM) in both genotypes. In conclusion, p38 MAPK-induced myocardial ischemic injury is not modulated by MK2. However, the absence of MK2 perturbs the cellular distribution of p38. The preserved nuclear distribution of active p38 MAPK in MK2^−/− hearts and the conserved response to SB203580 suggests that activation of p38 MAPK may contribute to injury independently of MK2. Diana A Gorog and Rita I Jabr made equal contributions to this work.     

Galangin induces B16F10 melanoma cell apoptosis via mitochondrial pathway and sustained activation of p38 MAPK

Galangin, an active flavonoid present at high concentration in Alpinia officinarum Hance and propolis, shows cytotoxicity towards several cancer cell lines, including melanoma. However, the specific cellular targets of galangin-induced cytotoxicity in melanoma are still unknown. Here, we investigated the effects of galangin in B16F10 melanoma cells and explored the possible molecular mechanisms. Galangin significantly decreased cell viability of B16F10 cells, and also induced cell apoptosis shown by Hoechst 33342 staining and Annexin V-PI double staining flow cytometric assay. Furthermore, upon galangin treatment, disruption of mitochondrial membrane potential was observed by JC-1 staining. Western blotting analysis indicated that galangin activated apoptosis signaling cascades by cleavage of procaspase-9, procaspase-3 and PARP in B16F10 cells. Moreover, galangin significantly induced activation of phosphor-p38 MAPK in a time and dose dependent manner. SB203580, an inhibitor of p38, partially attenuated galangin-induced apoptosis in B16F10 cells. Taken together, this work suggests that galangin has the potential to be a promising agent for melanoma treatment and may be further evaluated as a chemotherapeutic agent.     

6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.     

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.     

Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway

A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.     

Differential regulation of survivin by p53 contributes to cell cycle dependent apoptosis

Recent studies indicate that cell-cycle checkpoints are tightly correlated with the regulation of apoptosis, in which p53 plays an important role. Our present works show that the expression of E6/E7 oncogenes of human papillomavirus in HeLa cells is inhibited in the presence of anti-tumor reagent tripchlorolide (TC), which results in the up-regulation of p53 in HeLa cells. Interestingly, under the same TC-treatment, the cells at the early S-phase are more susceptible to apoptosis than those at the middle S-phase although p53 protein is stabilized to the same level in both situations. Significant difference is exhibited between the two specified expression profiles. Further analysis demonstrates that anti-apoptotic gene survivin is up-regulated by p53 in the TC-treated middle-S cells, whereas it is down-regulated by p53 in the TC-treated early-S cells. Taken together, the present study indicates that the differential p53-regulated expression of survivin at different stages of the cell cycle results in different cellular outputs under the same apoptosis-inducer.     

Inhibition of TRAIL-induced apoptosis by IL-8 is mediated by the p38-MAPK pathway in OVCAR3 cells

Introduction: TRAIL (TNF-Related Apoptosis Inducing Ligand) is a member of the TNF superfamily of cell death inducing ligands. Interestingly, while malignant cells are responsive to TRAIL-induced cell death when used alone or in combination with other agents, normal cells do not appear to be sensitive to this ligand, making it a desirable therapeutic compound against many cancers, including many ovarian carcinomas. Interleukin-8 (IL-8), a member of the C-X-C chemokine family, has been found to be at significantly higher level in the ascites from patients with ovarian cancer. We have previously demonstrated a role for IL-8 in blocking TRAIL's ability to induce apoptosis in the ovarian cancer cell line, OVCAR3, possibly by repressing the DR4 TRAIL receptor expression and blocking caspase-8 cleavage. In addition, we showed a member of the mitogen-activated protein kinase (MAPK) superfamily, p38γ, is among the genes regulated in OVCAR3 cells by TRAIL and IL-8. The present study further investigates involvement of the p38 MAPK pathway in IL-8's ability to block TRAIL-induced apoptosis in the ovarian surface epithelial cancer cell line, OVCAR3. Results: In this study we demonstrate that p38γ as well as p38α play a significant role in IL-8's ability to block TRAIL-induced apoptosis. Through array analysis, as well as confirmation with other methods, we detected regulation of p38γ and p38α following treatment of the cancer cell line with IL-8 or TRAIL. We also tested two other isoforms of p38 MAPK, p38β and p38δ, but did not find significant regulation by IL-8 or TRAIL. We also examined activation of the p38 MAPK pathway, up-stream as well as down-stream, and noticed activation of the pathway following treatment with TRAIL and decreased activity when IL-8 was introduced. With the use of specific inhibitors, we were able to further confirm the role of this pathway in TRAIL-induced apoptosis, and IL-8's ability to block this apoptosis, in ovarian cancer cell lines. Conclusion: Taken together, these results further solidify the role of IL-8 in blocking the TRAIL-induced apoptosis in these ovarian carcinoma cells and provide new molecular insight into this potentially important therapeutic target.     

Periplocin mediates TRAIL-induced apoptosis and cell cycle arrest in human myxofibrosarcoma cells via the ERK/p38/JNK pathway

BackgroundPeriploca sepium is traditionally used in Chinese medicine to treat particularly rheumatic disorders and as a tonic. Periplocin was found as the most cytotoxic compound of its root bark and induced death receptor mediated apoptosis in liposarcoma cells. Sarcomas are a rare type of cancer with only a few treatment options. The five-year survival rate of advanced tumors is low.PurposeIn this study, we investigated the effects of periplocin in two myxofibrosarcoma (MFS)cell lines, MUG-Myx2a and MUG-Myx2b, which are subclones of the same tumor and reflect the tumor´s heterogeneity, and in T60 primary myxofibrosarcoma cells.MethodsThe xCELLigence system and the CellTiter 96® AQ_ueous assay were used for studying cell viability. FACS and Western blot experiments were used to investigate the effects of periplocin on apoptosis induction, cell cycle distribution, and the expression of cleaved PARP, caspase 3, p53, phospho-histone γH2AX, ERK/phospho ERK, p38/phospho p38, and, finally, JNK/phospho JNK. Additionally, the expression of the apoptotic markers Bim, NOXA, Bak, Bcl-2, Bcl-xl, and the death receptors IGFR, FADD, TRADD, TNFR1A, TRAIL-R1, and TRAIL-R2 were evaluated using reversed real-time PCR.ResultsPeriplocin decreased dose-dependently the viability of all MFS cell lines and was more effective than the standard chemotherapeutic doxorubicin. It arrested the cells in the G2/M phase and led to caspase activation. Moreover, periplocin increased the mRNA expression of NOXA, Bak, Bcl-2, and death receptors such as TRAIL-R1 and TRAIL-R2 and the protein expression of ERK/phospho ERK, p38/phospho p38, and JNK/phospho JNK. In all cases, differences in the effects in the different subclones were observed.ConclusionPeriplocin showed promising effects in MFS cells. The higher effectiveness compared to doxorubicin is an important aspect for further research with regard as a treatment option. The different effects of periplocin in the two subclones showed the great importance of intratumoral heterogeneity in MFS therapy.     

Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumors including malignant glioblastoma. The mechanism of TMZ-induced glioblastoma cell death and apoptosis, however, is not fully understood. Here, we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found that methylating agents TMZ and N-methyl-N'-nitro-N-nitrosoguanidine induce AMPK activation in primary cultured human glioblastoma and glioblastoma cell lines. TMZ-induced O(6)-methylguanine production is involved in AMPK activation. O(6)-benzylguanine, an O(6)-methylguanine-DNA methyltransferase inhibitor, enhances TMZ-induced O(6)-methylguanine production, leading to enhanced reactive oxygen species production, which serves as an upstream signal for AMPK activation. Activation of AMPK is involved in TMZ-induced glioblastoma cell death and apoptosis. AMPK inhibitor (Compound C) or AMPKα siRNA knockdown inhibits TMZ-induced glioblastoma cell death and apoptosis, whereas AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside enhances it. In further studies, we found that activation of AMPK is involved in TMZ-induced p53 activation and subsequent p21, Noxa, and Bax up-regulation. Activation of AMPK by TMZ also inhibits mTOR complex 1 (mTORC1) signaling and promotes anti-apoptosis protein Bcl-2 down-regulation, which together mediate TMZ-induced pro-cell apoptosis effects. Our study suggests that activation of AMPK by TMZ contributes to glioblastoma cell apoptosis, probably by promoting p53 activation and inhibiting mTORC1 signaling.     

Microglial activation of p38 contributes to scorpion envenomation-induced hyperalgesia

Intraplantar (i.pl.) injection of BmK I, a receptor site 3-specific modulator of voltage-gated sodium channels (VGSCs) from the venom of scorpion Buthus martensi Karsch (BmK), was shown to induce long-lasting and spontaneous nociceptive responses as demonstrated through experiments utilizing primary thermal and mirror-imaged mechanical hypersensitivity with different time course of development in rats. In this study, microglia was activated on both sides of L4–L5 spinal cord by i.pl. injection of BmK I. Meanwhile, the activation of p38/MAPK in L4–L5 spinal cord was found to be co-expressed with OX-42, the cell marker of microglia. The unilateral thermal and bilateral mechanical pain hypersensitivity of rat induced by BmK I was suppressed in a dose-dependent manner following pretreatment with SB203580 (a specific inhibitor of p-p38). Interestingly, microglia activity was also reduced in the presence of SB203580, which suggests that BmK I-induced microglial activation is mediated by p38/MAPK pathway. Combined with previously published literature, the results of this study demonstrate that p38-dependent microglial activation plays a role in scorpion envenomation-induced pain-related behaviors.     

Cadmium induces vascular permeability via activation of the p38 MAPK pathway

The vasculature of various organs is a targeted by the environmental toxin, cadmium (Cd). However, mechanisms leading to pathological conditions are poorly understood. In the present study, we examined the effect of cadmium chloride (CdCl₂) on human umbilical vein endothelial cells (HUVECs). At 4 μM, CdCl₂ induced a hyper-permeability defect in HUVECs, but not the inhibition of cell growth up to 24 h. This effect of CdCl₂ was dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. The p38 MAPK inhibitor SB203850 suppressed the CdCl₂-induced alteration in trans-endothelial electrical resistance in HUVEC monolayers, a model measurement of vascular endothelial barrier integrity. SB203850 also inhibited the Cd-induced membrane dissociation of vascular endothelial (VE) cadherin and β-catenin, the important components of the adherens junctional complex. In addition, SB203850 reduces the Cd-induced expression and secretion of tumor necrosis factor α (TNF-α). Taken together, our findings suggest that Cd induces vascular hyper-permeability and disruption of endothelial barrier integrity through stimulation of p38 MAPK signaling.