Requires P38 MAPK Activation Research Articles

EGFR-dependent signalling reduced and p38 dependent apoptosis required by Gallic acid in Malignant Mesothelioma cells.

The unrestrained EGFR signalling contributes to malignant phenotype in a number of cancers including Malignant Mesotheliomas. Present study was designed to evaluate EGFR-dependent anti-proliferative and apoptotic effects of Gallic acid in transformed Mesothelial (MeT-5A) and Malignant Mesothelioma (SPC212) cells. Gallic acid reduced the viability of Malignant Mesothelioma cells in a concentration and time-dependent manner. However, viability of mesothelial cells reduced only at high concentration and longer time periods. Gallic acid restrained the activation of EGFR, ERK1/2 and AKT proteins and down regulated expression of Cyclin D and Bcl-2 genes, but upregulated the expression of p21 gene in EGF-induced SPC212 cells. GA-induced transitory G1 arrest and triggered mitochondrial and death receptor mediated apoptosis, which requires p38MAPK activation. The data provided here indicate that GA is able to inhibit EGFR dependent proliferation and survival signals and induces p38 pathway dependent apoptosis in Malignant Mesothelioma cells. On the basis of these experimental findings it is worthwhile to investigate further the biological activity of Gallic acid on other Mesothelioma cell lines harbouring aberrant EGFR signals.

Tanshinone IIA enhances the effects of TRAIL by downregulating survivin in human ovarian carcinoma cells

BackgroundTanshinone IIA (TIIA), a diterpene quinone from the medicinal plant Salvia miltiorrhiza Bunge (Lamiaceae) was shown to possess apoptotic and TRAIL-sensitizing effects. Still, the molecular mechanisms whereby TIIA induces apoptosis remain largely unknown. PurposeThe role of survivin, an inhibitor of apoptosis protein, in TIIA-induced apoptosis has never been addressed before and hence was the primary goal of this study. MethodsIn this study, we explored the anticancer effect of TIIA in TOV-21G, SKOV3, and OVCAR3 ovarian carcinoma cells. Cytotoxicity was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of related signaling proteins. ResultsOur results illustrated that TIIA's cytotoxic effect was caused by apoptosis with the involvement of caspases activity. Moreover, TIIA downregulated survivin in a concentration-dependent manner without affecting the expression of Bcl-2, Bcl-xL, and Bax. TIIA-induced survivin downregulation is regulated by both transcriptional processes and proteasomal degradation. Using TOV-21G cells as our cellular model, we demonstrated that TIIA-induced survivin downregulation requires p38 MAPK activation. Importantly, genetic overexpression of survivin rendered cells more resistant to TIIA, indicating an essential role of survivin downregulation in TIIA-induced apoptosis. This TRAIL sensitization effect of TIIA is ascribed to survivin downregulation because the effect was abrogated in cells that overexpressed survivin. ConclusionOur findings provide new insights into the action modes of TIIA-mediated anticancer effects and further implicate a rational design for cancer therapeutic regimens by combining TIIA-sensitized TRAIL via downregulating survivin to elicit ovarian cancer cell death.

The formation of proximal and distal definitive endoderm populations in culture requires p38 MAPK activity

Endoderm formation in the mammal is a complex process with two lineages forming during the first weeks of development, the primitive (or extraembryonic) endoderm, which is specified in the blastocyst, and the definitive endoderm that forms later, at gastrulation, as one of the germ layers of the embryo proper. Fate mapping evidence suggests that the definitive endoderm arises as two waves, which potentially reflect two distinct cell populations. Early primitive ectoderm-like (EPL) cell differentiation has been used successfully to identify and characterise mechanisms regulating molecular gastrulation and lineage choice during differentiation. The roles of the p38 MAPK family in the formation of definitive endoderm were investigated using EPL cells and chemical inhibitors of p38 MAPK activity. These approaches define a role for p38 MAPK activity in the formation of the primitive streak and a second role in the formation of the definitive endoderm. Characterisation of the definitive endoderm populations formed from EPL cells demonstrates the formation of two distinct populations, defined by gene expression and ontogeny, that were analogous to the proximal and distal definitive endoderm populations of the embryo. Formation of the proximal definitive endoderm was found to require p38 MAPK activity and is correlated with molecular gastrulation, defined by the expression of brachyury (T). Distal definitive endoderm formation also requires p38 MAPK activity but can form when T expression is inhibited. Understanding lineage complexity will be a prerequisite for the generation of endoderm derivatives for commercial and clinical use.

Apigenin in Combination with Akt Inhibition Significantly Enhances Thyrotropin-Stimulated Radioiodide Accumulation in Thyroid Cells

Selectively increased radioiodine accumulation in thyroid cells by thyrotropin (TSH) allows targeted treatment of thyroid cancer. However, the extent of TSH-stimulated radioiodine accumulation in some thyroid tumors is not sufficient to confer therapeutic efficacy. Hence, it is of clinical importance to identify novel strategies to selectively further enhance TSH-stimulated thyroidal radioiodine accumulation. PCCl3 rat thyroid cells, PCCl3 cells overexpressing BRAF(V600E), or primary cultured tumor cells from a thyroid cancer mouse model, under TSH stimulation were treated with various reagents for 24 hours. Cells were then subjected to radioactive iodide uptake, kinetics, efflux assays, and protein extraction followed by Western blotting against selected antibodies. We previously reported that Akt inhibition increased radioiodine accumulation in thyroid cells under chronic TSH stimulation. Here, we identified Apigenin, a plant-derived flavonoid, as a reagent to further enhance the iodide influx rate increased by Akt inhibition in thyroid cells under acute TSH stimulation. Akt inhibition is permissive for Apigenin's action, as Apigenin alone had little effect. This action of Apigenin requires p38 MAPK activity but not PKC-δ. The increase in radioiodide accumulation by Apigenin with Akt inhibition was also observed in thyroid cells expressing BRAF(V600E) and in primary cultured thyroid tumor cells from TRβ(PV/PV) mice. Taken together, Apigenin may serve as a dietary supplement in combination with Akt inhibitors to enhance therapeutic efficacy of radioiodine for thyroid cancer.

The formation of proximal and distal definitive endoderm populations in culture requires p38 MAPK activity

Endoderm formation in the mammal is a complex process with two lineages forming during the first weeks of development, the primitive (or extraembryonic) endoderm, which is specified in the blastocyst, and the definitive endoderm that forms later, at gastrulation, as one of the germ layers of the embryo proper. Fate mapping evidence suggests that the definitive endoderm arises as two waves, which potentially reflect two distinct cell populations. Early primitive ectoderm-like (EPL) cell differentiation has been used successfully to identify and characterise mechanisms regulating molecular gastrulation and lineage choice during differentiation. The roles of the p38 MAPK family in the formation of definitive endoderm were investigated using EPL cells and chemical inhibitors of p38 MAPK activity. These approaches define a role for p38 MAPK activity in the formation of the primitive streak and a second role in the formation of the definitive endoderm. Characterisation of the definitive endoderm populations formed from EPL cells demonstrates the formation of two distinct populations, defined by gene expression and ontogeny, that were analogous to the proximal and distal definitive endoderm populations of the embryo. Formation of the proximal definitive endoderm was found to require p38 MAPK activity and is correlated with molecular gastrulation, defined by the expression of brachyury (T). Distal definitive endoderm formation also requires p38 MAPK activity but can form when T expression is inhibited. Understanding lineage complexity will be a prerequisite for the generation of endoderm derivatives for commercial and clinical use.

MMPs 2 and 9 are essential for coronary collateral growth and are prominently regulated by p38 MAPK

Transient, repetitive ischemia (RI) stimulates coronary collateral growth (CCG) in normal, healthy (SD) rats, which requires p38 MAPK activation. In contrast, RI does not induce CCG in the metabolic syndrome (JCR) rats, which is associated with lack of p38 MAPK activation. The functional consequences of p38 MAPK activation in CCG remain unknown. Theoretically, effective collateral growth would require extracellular matrix remodeling; however, direct assessment as well as identification of proteases responsible for this degradation are lacking. In this study, we investigated the role of p38 MAPK in the regulation of matrix metalloproteinases 2 and 9 (MMPs 2 and 9) and their requirement for CCG in SD vs. JCR rats. The rats underwent the RI protocol (8 LAD occlusions, 40s each, every 20min, in 8h cycles for 0, 3, 6, or 9days). MMP expression was measured in the ischemic, collateral-dependent zone (CZ) and the normal zone (NZ) by Western blot, and MMP activity by zymography. Expression and activation of MMP 2 and 9 were significantly increased (~3.5 fold) on day 3 of RI in the CZ of SD rats. In vivo p38 MAPK inhibition completely blocked RI-induced MMP 2 and 9 expression and activation. MMP activation correlated with increased degradation of components of the basement membrane and the vascular elastic laminae: elastin (~3 fold), laminin (~3 fold) and type IV collagen (~2 fold). This was blocked by MMP 2 and 9 inhibition, which also abolished RI-induced CCG. In contrast, in JCR rats, RI did not induce expression or activation of MMP 2 or 9 and there was no associated degradation of elastin, laminin or type IV collagen. In conclusion, MMP 2 and 9 activation is essential for CCG and is mediated, in part, by p38 MAPK. Furthermore, compromised CCG in the metabolic syndrome may be partially due to the lack of p38 MAPK-dependent activation of MMP 2 and 9 and resultant decreased extracellular matrix degradation.

Flagellin is required for salmonella-induced expression of heat shock protein Hsp25 in intestinal epithelium

Flagellin is a bacterial protein responsible for activation of Toll-like receptor 5 (TLR5), which we hypothesize is involved in Salmonella's induction of cytoprotective heat shock proteins in intestinal epithelial cells. Flagellin induces the cytoprotective heat shock protein Hsp25 in different intestinal epithelial cell lines and in mouse intestine. Flagellin induces Hsp25 expression in a time-dependent manner in vitro. This effect is transcriptional, as confirmed by luciferase reporter assays and actinomycin D treatment. In addition, Hsp25 induction requires p38 MAPK activation and is only observed when flagellin is added to the basolateral side of polarized intestinal epithelial cells, consistent with the known location of TLR5. Flagellin-mediated Hsp25 induction is associated with increased protective effects against oxidant stress, an effect that is at least partially mediated by p38 MAPK. Use of small interfering RNA against Hsp25 demonstrates that flagellin-mediated protection against oxidant stress is to some degree mediated through Hsp25 induction. This suggests that, by protecting against oxidant injury, the induction of Hsp25 expression by flagellin may contribute to intestinal homeostasis. In a coculture cell model and in a mouse model of Salmonella enterica Serovar Typhimurium infection, not only does infection with wild-type and a flagellin-deletion mutant strain of Salmonella show that flagellin induces Hsp25 in vivo, but it also demonstrates that in the case of live Salmonella infection, flagellin serves as a major stimulus for the induction of Hsp25 expression. These data provide evidence that flagellin is required for Salmonella-mediated induction of Hsp25 expression in intestinal epithelium.

EGFR Activation Results in Enhanced Cyclooxygenase-2 Expression through p38 Mitogen-Activated Protein Kinase–Dependent Activation of the Sp1/Sp3 Transcription Factors in Human Gliomas

Expression of cyclooxygenase-2 (COX-2) has been linked to many cancers and may contribute to malignant phenotypes, including enhanced proliferation, angiogenesis, and resistance to cytotoxic therapies. Malignant gliomas are highly aggressive brain tumors that display many of these characteristics. One prominent molecular abnormality discovered in these astrocytic brain tumors is alteration of epidermal growth factor (EGF) receptor (EGFR) through gene amplification and/or mutation resulting in excessive signaling from this receptor. We found that EGF-mediated stimulation of EGFR tyrosine kinase in human glioma cell lines induces expression of both COX-2 mRNA and protein. The p38 mitogen-activated protein kinase (p38-MAPK) pathway was a strong downstream factor in this activation with inhibition of this pathway leading to strong suppression of COX-2 induction. The p38-MAPK pathway can activate the Sp1/Sp3 transcription factors and this seems necessary for EGFR-dependent transactivation of the COX-2 promoter. Analysis of COX-2 promoter/luciferase constructs revealed that transcriptional activation of the COX-2 promoter by EGFR requires the Sp1 binding site located at -245/-240. Furthermore, Sp1/Sp3 binding to this site in the promoter is enhanced by EGFR activation both in vitro and in vivo. Enhanced DNA binding by Sp1/Sp3 requires p38-MAPK activity and correlates with increased phosphorylation of the Sp1 transcription factor. Thus, EGFR activation in malignant gliomas can transcriptionally activate COX-2 expression in a process that requires p38-MAPK and Sp1/Sp3. Finally, treatment of glioma cell lines with prostaglandin E2, the predominant product of COX-2 activity, results in increased vascular endothelial growth factor expression, thus potentially linking elevations in COX-2 expression with tumor angiogenesis in malignant gliomas.

Capsaicin‐induced apoptosis of glioma cells is mediated by TRPV1 vanilloid receptor and requires p38 MAPK activation

We provide evidence on the expression of the transient receptor potential vanilloid type-1 (TRPV1) by glioma cells, and its involvement in capsaicin (CPS)-induced apoptosis. TRPV1 mRNA was identified by quantitative RT-PCR in U373, U87, FC1 and FLS glioma cells, with U373 cells showing higher, and U87, FC1 and FLS cells lower TRPV1 expression as compared with normal human astrocytes. By flow cytometry we found that a substantial portion of both normal human astrocytes, and U87 and U373 glioma cells express TRPV1 protein. Moreover, we analyzed the expression of TRPV1 at mRNA and protein levels of glioma tissues with different grades. We found that TRPV1 gene and protein expression inversely correlated with glioma grading, with marked loss of TRPV1 expression in the majority of grade IV glioblastoma multiforme. We also described that CPS trigger apoptosis of U373, but not U87 cells. CPS-induced apoptosis involved Ca(2+) influx, p38 but not extracellular signal-regulated mitogen-activated protein kinase activation, phosphatidylserine exposure, mitochondrial permeability transmembrane pore opening and mitochondrial transmembrane potential dissipation, caspase 3 activation and oligonucleosomal DNA fragmentation. TRPV1 was functionally implicated in these events as they were markedly inhibited by the TRPV1 antagonist, capsazepine. Finally, p38 but not extracellular signal-regulated protein kinase activation was required for TRPV1-mediated CPS-induced apoptosis of glioma cells.

Tenascin-W is found in malignant mammary tumors, promotes alpha8 integrin-dependent motility and requires p38MAPK activity for BMP-2 and TNF-alpha induced expression in vitro

Tenascins represent a family of extracellular matrix glycoproteins with distinctive expression patterns. Here we have analyzed the most recently described member, tenascin-W, in breast cancer. Mammary tumors isolated from transgenic mice expressing hormone-induced oncogenes reveal tenascin-W in the stroma around lesions with a high likelihood of metastasis. The presence of tenascin-W was correlated with the expression of its putative receptor, alpha8 integrin. HC11 cells derived from normal mammary epithelium do not express alpha8 integrin and fail to cross tenascin-W-coated filters. However, 4T1 mammary carcinoma cells do express alpha8 integrin and their migration is stimulated by tenascin-W. The expression of tenascin-W is induced by BMP-2 but not by TGF-beta1, though the latter is a potent inducer of tenascin-C. The expression of tenascin-W is dependent on p38MAPK and JNK signaling pathways. Since preinflammatory cytokines also act through p38MAPK and JNK signaling pathways, the possible role of TNF-alpha in tenascin-W expression was also examined. TNF-alpha induced the expression of both tenascin-W and tenascin-C, and this induction was p38MAPK- and cyclooxygenase-dependent. Our results show that tenascin-W may be a useful diagnostic marker for breast malignancies, and that the induction of tenascin-W in the tumor stroma may contribute to the invasive behavior of tumor cells.

Memory formation requires p38MAPK activity in the rat hippocampus.

Using the specific inhibitor of p38MAPK, SB203580, we show a direct involvement of this protein kinase in short- and long-term memory. When given into the CA1 region of the rat dorsal hippocampus immediately, but not 30 or 120 min after training in a one-trial inhibitory avoidance task, SB203580 blocked short- and long-term memory formation. The SB203580 inactive analog, SB202474, had no effect whatsoever. Learning of the avoidance task was accompanied by an immediate and transient increase in hippocampal p38MAPK phosphorylation. No change in p38MAPK phosphorylation was detected in control animals that only received the electric foot-shock associated with the learning paradigm. Therefore, formation of short and long-term memory for inhibitory avoidance requires p38MAPK activation in the rat hippocampus.

P38 Mitogen-Activated Protein Kinase Activation Is Required for Thromboxane- Induced Contraction in Perfused and Pressurized Rat Mesenteric Resistance Arteries

Thromboxane A₂ (TxA2) is a potent proaggregating, vasoconstrictor agent produced in many physiological and pathological situations. Although mitogen-activated protein (MAP) kinases [MAPK (ERK1/2 and p38)] have been shown to be activated after endoperoxide/thromboxane receptor (TP) stimulation, no study has investigated their potential role in resistance arteries, especially in physiological conditions of pressure and flow in which the arteries can contract. Thus, responses to TP stimulation by the stable agonist U46619 were studied in isolated rat mesenteric resistance arteries (inner diameter 262 ± 5 µm) mounted in an arteriograph. Changes in diameter were recorded under physiological levels of flow (90 µl/min) and pressure (50 mm Hg). TP stimulation induced a concentration-dependent contraction (EC₅₀ value of 1.94 ± 0.22 × 10^–7M), without desensitization. U46619-induced contraction was inhibited by calcium entry blockade (nifedipine) and protein kinase C inhibition (GF109203X), but it was not affected by tyrosine kinase inhibition (tyrphostin A25). MAPKK (MEK) inhibition (PD98059) did not alter U46619-dependent contraction, although ERK1/2 MAPK were activated. By contrast, p38 MAPK inhibition (SB203580) dose-dependently inhibited the contraction, and Western blot analysis showed activation of p38 MAPK in arteries contracted with U46619. Activation of p38 MAPK by U46619 was inhibited by nifedipine and in the absence of extracellular calcium. This study brings new insights in the transduction pathway involved in the contractile response of resistance arteries to TxA2/endoperoxide receptor stimulation. This contraction requires p38 MAPK activation, but did not involve ERK1/2 MAPK activation although both were activated.

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 [ 3H]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.