Protection of glioblastoma cells from cisplatin cytotoxicity via protein kinase Ciota-mediated attenuation of p38 MAP kinase signaling

Glioblastoma multiforme is an aggressive form of brain cancer that responds poorly to chemotherapy and is generally incurable. The basis for the poor response of this cancer to chemotherapy is not well understood. The atypical protein kinases C (PKCiota and PKCzeta) have previously been implicated in leukaemia cell chemoresistance. To assess the role of atypical PKC in glioblastoma cell chemoresistance, RNA interference was used to deplete human glioblastoma cells of PKCiota. Transfection of cells with either of two different RNA duplexes specific for PKCiota caused a partial sensitisation to cell death induced by the chemotherapy agent cisplatin. To screen for possible mechanisms for PKCiota-mediated chemoresistance, microarray analysis of gene expression was performed on RNA from glioblastoma cells that were either untreated or depleted of PKCiota. This identified sets of genes that were regulated either positively or negatively by PKCiota. Within the set of genes that were negatively regulated by PKCiota, the function of the gene coding for GMFbeta, an enhancer of p38 mitogen-activated protein kinase (MAP kinase) signaling, was investigated further, as the p38 MAP kinase pathway has been previously identified as a key mediator of cisplatin cytotoxicity. The expression of both GMFbeta mRNA and protein increased upon PKCiota depletion, and this was accompanied by an increase in cisplatin-activated p38 MAP kinase signaling. Transient overexpression of GMFbeta increased cisplatin-activated p38 MAP kinase signaling and also sensitised cells to cisplatin cytotoxicity. The increase in cisplatin cytotoxicity seen with PKCiota depletion was blocked by the p38 MAP kinase inhibitor SKF86002. These data show that PKCiota can confer partial resistance to cisplatin in glioblastoma cells by suppressing GMFbeta-mediated enhancement of p38 MAP kinase signaling.     

Activation of p38 mitogen-activated protein kinase is necessary for gemcitabine-induced cytotoxicity in human pancreatic cancer cells

BACKGROUND: Gemcitabine is a pyrimidine nucleoside analog that is clinically active against pancreatic cancer. We have recently demonstrated that p38 MAPK is specifically activated by gemcitabine and that pharmacological blockade of p38 MAPK signaling prevented gemcitabine-induced apoptosis in human pancreatic cancer cells. In this study, we further investigated the implication of p38 MAPK in the cytotoxic action of gemcitabine. MATERIALS AND METHODS: Cells expressing a dominant-negative mutant of p38 MAPK were generated. Clonogenic assays were used to assess the long-term effect on cancer cell viability in the human pancreatic cancer cells, PK1 and PCI43. The p38 MAPK activation level was assessed using an antibody specific to the phosphorylated form. RESULTS: Gemcitabine increased the activation level of p38 MAPK in a dose-dependent manner and induced apoptosis in the two tested human pancreatic cancer cell lines. The selective p38 MAPK inhibitors, SB203580 and SB202190, reduced gemcitabine-induced activation of p38 MAPK, prevented the gemcitabine-induced apoptosis and increased long-term clonogenic survival. Overexpression of a dominant-negative p38 mutant in cells resulted in the reduction of gemcitabine-induced p38 MAPK activation and apoptosis, and increases in clonogenic survival. CONCLUSION: These results strongly suggest that the activation of p38 MAPK signaling is necessary for gemcitabine-induced cell death in human pancreatic cancer cells. Based upon these results, we suggest that molecules of p38 MAPK signaling pathways should be listed as novel targets for gemcitabine-based therapy.     

Lack of p38 MAP kinase activation in TRAIL-resistant cells is not related to the resistance to TRAIL-mediated cell death

Activation of MAP kinases is involved in various cellular processes, including immunoregulation, inflammation, cell growth, cell differentiation, and cell death. To investigate the role of p38 MAP kinase activation in the signaling pathway of TRAIL-mediated apoptosis, we compared TRAIL-mediated MAP kinase activation in TRAIL-susceptible human colon cancer cell line DLD1 and TRAIL-resistant DLD1/TRAIL-R cells. TRAIL-mediated activation of ERK occurred in both cell lines. In contrast, both DLD1 and DLD1/TRAIL-R cells showed no obvious JNK activation after treatment with TRAIL. Interestingly, TRAIL-mediated activation of p38 MAP kinases was observed in DLD1 cells but not in DLD1/TRAIL-R cells. However, activation of p38 MAP kinases was observed in both DLD1 and DLD1/TRAIL-R cells after treatment with anisomycin. Furthermore, inhibiting activated p38 MAP kinases with known inhibitors or with an adenovector expressing dominant negative p38alpha did not block TRAIL-mediated cell death in DLD1 cells. Moreover, activation of p38 MAP kinases by adenovectors expressing constitutive MKK3 or MKK6 (Ad/MKK3bE or Ad/MKK6bE) did not induce cell death in either DLD1 or DLD1/TRAIL-R cell lines. Our results suggest that activation of p38 MAP kinases does not play a major role in TRAIL-mediated apoptosis in DLD1 cells and that lack of TRAIL-mediated p38 MAP kinase activation may not be the mechanism of TRAIL-resistance in DLD1/TRAIL-R cells.     

p38 kinase is a key signaling molecule for H-Ras-induced cell motility and invasive phenotype in human breast epithelial cells

Ras expression has been suggested as a marker for tumor aggressiveness of breast cancer,including the degrees of invasion and tumor recurrence.We showed previously that H-ras, but not N-ras, up-regulates matrix metalloproteinase 2 expression and induces invasive phenotype in MCF10A human breast epithelial cells (A. Moon, et al. Int. J. Cancer, 85: 176-181, 2000). In this study, we show that H-ras also promotes cell motility more effectively than N-ras in MCF10A cells. We have investigated H-ras-specific signaling pathway(s) critical for H-ras-mediated cell motility and invasive phenotype. Whereas neither H-ras nor N-ras activated c-Jun NH(2)-terminal kinase 1, both H-ras and N-ras effectively activated extracellular signal-regulated protein kinase (ERK) -1,2. Importantly, prominent activation of p38 mitogen-activated protein kinase was shown only in H-ras-activated cells but not in N-ras-activated MCF10A cells. Functional significance of H-ras-activated p38 in invasiveness and cell motility was evidenced by studies using SB203580, a chemical inhibitor of p38, and a dominant-negative construct of p38. Whereas inhibition of c-Jun NH(2)-terminal kinase 1 activity had no effect on H-ras-induced MCF10A cell invasion and motility, the inhibition of the ERK pathway using a chemical inhibitor PD98059 or dominant-negative mutant of mitogen-activated protein/ERK kinase 1, an activator of ERKs, significantly reduced H-ras-induced invasion and migration. We also provide evidence that p38 and, to a lesser degree, ERKs, are critical for H-ras-mediated up-regulation of matrix metalloproteinase 2. Taken together, the present study shows that H-ras activation of both p38 and ERKs induces cell invasion and motility, whereas N-ras activation of ERKs alone is not sufficient. This study reveals the p38 kinase as a key signaling molecule differentially regulated by H-ras and N-ras, leading to H-ras-specific cell invasive and migrative phenotypes in human breast epithelial cells.     

Phorbol esters inhibit fibroblast growth factor-2-stimulated fibroblast proliferation by a p38 MAP kinase dependent pathway

Treatment of fibroblasts with the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), specifically inhibits fibroblast growth factor-2 (FGF-2) induced proliferation. TPA treatment has little or no effect on FGF receptor activation but specifically inhibits the activation of p38 MAPK but not other downstream signaling pathways implicated in cell proliferation. p38 MAPK was recently shown to be required for the FGF-2-stimulated proliferation of fibroblasts. The effect of TPA on both p38 MAPK activation and cell proliferation can be reversed by treatment with the PKC inhibitor Go6983. The TPA-mediated inhibition of p38 MAPK activation requires phosphatase activity and is at least partially mediated by ERKs since it is reduced by treatment with the MEK inhibitor PD98059. In contrast, the FGF-2-stimulated differentiation of PC12 cells, which express the same FGF receptor as Swiss 3T3 fibroblasts, is not affected by TPA treatment, consistent with a lack of involvement of p38 MAPK activity in this process. These data indicate that the effects of TPA treatment on cellular function are not only cell type but also stimulus specific and are dependent upon the distinct pathways activated downstream of each stimulus.     

Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53

The anti-cancer agent paclitaxel (Taxol) stabilizes microtubules leading to G2/M cell cycle arrest and apoptotic cell death. In order to analyse the molecular mechanisms of Taxol-induced cytotoxicity, we studied the involvement of mitogen-activated protein kinases (MAPK) ERK and p38 as well as the p53 pathways in Taxol-induced apoptosis. The human breast carcinoma cell line MCF7 and its derivatives, MCF7/HER-2 and MDD2, were used in the study. We found that Taxol treatment strongly activated ERK, p38 MAP kinase and p53 in MAP kinase MCF7 cells prior to apoptosis. PD98059 or SB203580, specific inhibitors of ERK and p38 kinase activities, significantly decreased apoptosis, leaving the surviving cells arrested in G2/M. These inhibitors did not significantly affect Taxol-induced alterations in the cell cycle regulatory proteins Rb, p53, p21/Waf1 and Cdk-2. In addition, inactivation of p53 did not affect cellular sensitivity to Taxol killing. However, cells with inactivated p53, unlike cells harboring wild type p53, failed to arrest in G2/M after treatment with Taxol and continued to divide or go into apoptosis. Our data show that both ERK and p38 MAP kinase cascades are essential for apoptotic response to Taxol-induced cellular killing and are independent of p53 activity. However, p53 may serve as a survival factor in breast carcinoma cells treated with Taxol by blocking cells in G2/M phase of the cell cycle.     

Differential regulation of Jun N-terminal kinase and p38MAP kinase by Galpha12

Based on the findings that the overexpression of the wild-type Galpha(12) (Galpha(12)WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Galpha(12) can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Galpha(12)WT or the expression of an activated mutant of Galpha(12) (Galpha(12)QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Galpha(12)WT or the expression of Galpha(12)QL results in a potent inhibition of p38MAPK, and that the mechanism by which Galpha(12) inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Galpha(12) attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Galpha(12) activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Galpha(12) differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Galpha(12) stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK- and JNK-mediated signaling pathways.     

Differentiation of human melanoma cells through p38 MAP kinase is associated with decreased retinoblastoma protein phosphorylation and cell cycle arrest

The retinoblastoma protein (pRB), the product of the retinoblastoma gene, is a key regulator of the cell cycle, affecting apoptosis, proliferation and differentiation. Dysregulation of pRB is implicated in the pathogenesis of many cancers, including malignant melanoma. Recently we demonstrated that alpha-melanocyte-stimulating hormone (alpha-MSH)-induced activation of p38 mitogen-activated protein (MAP) kinase leads to differentiation of B16 murine melanoma cells. The current study assesses the ability of alpha-MSH to activate p38 MAP kinase in COLO 853 human melanoma cells and determines whether this is linked to modulation of pRB activity. Treatment of COLO 853 cells with alpha-MSH induced time- and concentration-dependent increases in the phosphorylation of p38 MAP kinase, which corresponded with its ability to induce melanogenesis and inhibit cell growth. SB 203580, a selective inhibitor of p38 MAP kinase, blocked both the alpha-MSH-induced melanogenic response and inhibition of cell growth. Cell cycle analysis using flow cytometry revealed that treatment of COLO 853 cells with alpha-MSH for 72 h led to an increase in the proportion of cells in the G(1) phase and a marked reduction in the amount of phosphorylated pRB. Both of these effects were reversed by pre-treatment of cells with SB 203580. In summary, we have demonstrated for the first time that the alpha-MSH-induced differentiation of COLO 853 human melanoma cells proceeds via a p38 MAP kinase-mediated pathway and is associated with decreased pRB phosphorylation and accumulation of cells in the G(1) phase.     

A dual function for p38 MAP kinase in hematopoietic cells: involvement in apoptosis and cell activation.

In the present study we examined in more detail the dual role of the c-JUN N-terminal kinase (JNK) and p38 stress-activated protein kinase pathways in mediating apoptosis or cellular activation in hematopoietic cells. Growth factor deprivation of the erythroleukemic cell line TF-1 led to apoptosis which was associated with an enhanced activity of JNK and p38 and immediate dephosphorylation of the extracellular signal-regulated kinases (ERKs). Enhanced activity of p38 and JNK was not only observed during apoptosis but also in TF-1 cells stimulated with IL-1. IL-1 rescued TF-1 cells from apoptosis. In this case, the upregulation of p38 and JNK was associated with an enhanced activity of ERK. By using SB203580, a specific inhibitor of the p38 signaling pathway, it was demonstrated that p38 plays a pivotal role in the apoptotic process. SB203580 repressed the apoptotic process to a large extent. In contrast, PD98059, a specific inhibitor of the ERK pathway, counteracted the suppressive effects of SB203580 and IL-1 on the apoptotic process indicating that the protective effect of SB203580 and IL-1 might be the result of a shift in the balance between the ERK1/2 and p38/JNK route. This was also supported by experiments with TF-1 cells overexpressing the Shc protein that demonstrated a significantly lower percentage of apoptotic cells, which coincided with higher ERK activity. Finally, the IL-1 and SB203580-mediated effects were associated with an enhanced nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activity, which could also be blocked by PD98059. These data demonstrate a dual function of the p38 pathway whereby other factors, such as ERK kinases, AP-1 and NF-kappaB, might determine the final cellular response.     

The type III TGF-beta receptor signals through both Smad3 and the p38 MAP kinase pathways to contribute to inhibition of cell proliferation

Transforming growth factor beta (TGFbeta) has an important role as a negative regulator of cellular proliferation. The type III transforming growth factor beta receptor (TbetaRIII) has an emerging role as both a TGFbeta superfamily co-receptor and in mediating signaling through its cytoplasmic domain. In L6 myoblasts, TbetaRIII expression enhanced TGFbeta1-mediated growth inhibition, with this effect mediated, in part, by the TbetaRIII cytoplasmic domain. The effects of TbetaRIII were not due to altered ligand presentation or to differences in Smad2 phosphorylation. Instead, TbetaRIII specifically increased Smad3 phosphorylation, both basal and TGFbeta-stimulated Smad3 nuclear localization and Smad3-dependent activation of reporter genes independent of its cytoplasmic domain. Conversely, SB431542, a type I transforming growth factor beta receptor (TbetaRI) inhibitor, as well as dominant-negative Smad3 specifically and significantly abrogated the effects of TbetaRIII on TGFbeta1-mediated inhibition of proliferation. TbetaRIII also specifically increased p38 phosphorylation, and SB203580, a p38 kinase inhibitor, specifically and significantly abrogated the effects of TbetaRIII/TGFbeta1-mediated inhibition of proliferation in L6 myoblasts and in primary human epithelial cells. Importantly, treatment with the TbetaRI and p38 inhibitors together had additive effects on abrogating TbetaRIII/TGFbeta1-mediated inhibition of proliferation. In a reciprocal manner, short hairpin RNA-mediated knockdown of endogenous TbetaRIII in various human epithelial cells attenuated TGFbeta1-mediated inhibition of proliferation. Taken together, these data demonstrate that TbetaRIII contributes to and enhances TGFbeta-mediated growth inhibition through both TbetaRI/Smad3-dependent and p38 mitogen-activated protein kinase pathways.     

Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway

Introduction  

The pineal gland hormone, melatonin, has been shown by numerous studies to inhibit the proliferation of estrogen receptor α (ERα)-positive breast cancer cell lines. Here, we investigated the role of melatonin in the regulation of breast cancer cell invasion.     

The PI 3-kinase-Rac-p38 MAP kinase pathway is involved in the formation of signet-ring cell carcinoma

Signet-ring cell carcinoma is classified in poorly differentiated adenocarcinoma with an aggressive nature and a poor prognosis. We have shown that the activation of PI 3-kinase in highly differentiated adenocarcinomas induces loss of cell-cell contact and formation of vacuoles, giving phenotypes similar to those of signet-ring cell lines. SB203580, a potent p38 MAP kinase inhibitor, blocked this transition, and expression of an active form of MKK6 (MKK6DA), an activator of p38 MAP kinase, gave effects similar to those induced by expression of the active form of PI 3-kinase (BD110), although formation of large vacuoles was not induced. Activation of MKK3, another activator of p38 MAP kinase, was activated in native signet-ring carcinoma cell lines. Anchorage-independent growth of signet-ring cell lines was inhibited by LY294002 or SB203580. These results suggest that p38 MAP kinase is functioning downstream of PI 3-kinase in signaling of the malignant phenotype. Secretion of mucins was enhanced in BD110-expressing cells, but not in MKK6DA-expressing cells, suggesting that secretion of mucins is independent of the MKK6-p38 MAP kinase cascade. Thus, there may be at least two pathways, p38 MAP kinase-dependent and -independent, which are involved in regulation of cell-cell contact and the protein secretion system, respectively.     

p38alpha MAP kinase as a sensor of reactive oxygen species in tumorigenesis

p38alpha is a stress-activated protein kinase that negatively regulates malignant transformation induced by oncogenic H-Ras, although the mechanisms involved are not fully understood. Here, we show that p38alpha is not a general inhibitor of oncogenic signaling, but that it specifically modulates transformation induced by oncogenes that produce reactive oxygen species (ROS). This inhibitory effect is due to the ROS-induced activation of p38alpha early in the process of transformation, which induces apoptosis and prevents the accumulation of ROS and their carcinogenic effects. Accordingly, highly tumorigenic cancer cell lines have developed a mechanism to uncouple p38alpha activation from ROS production. Our results indicate that oxidative stress sensing plays a key role in the inhibition of tumor initiation by p38alpha.     

High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL)-B-cells are quiescent differentiated cells that produce interleukin (IL)-10 and accumulate due to resistance to apoptosis. The mechanisms underlying such resistance are poorly understood. Herein we show that all CLL B-cells tested (30/30) display high mRNA and protein expression of the tumor suppressor Mda-7/IL-24, an IL-10 family member, in comparison to normal B cells. A downstream Mda-7 signaling target, p38 mitogen-activated protein kinase (MAPK) was highly phosphorylated in all CLL cells but not in normal B-cells. Mda-7 expression and p38 MAPK phosphorylation diminished in culture and the latter could be reinduced by recombinant (r)-IL-24 or LPS and Mda-7 transfection. Mda-7/IL-24 siRNA specifically inhibited p38 MAPK phosphorylation in CLL without affecting p38 MAPK, bcl2, or Lyn expression, further demonstrating the direct role of Mda-7/IL-24 in p38 MAPK activation. Both pharmacological inhibition of p38 MAPK and Mda-7 silencing augmented spontaneous apoptosis by three-fold in CLL cells cultured in autologous serum, which was reversed by LPS and r-IL-24. We established the role of p38 MAPK in CLL cell survival and demonstrated a paradoxical effect, whereby Mda-7 and IL-24, inducers of apoptosis in diverse cancer cells, promote the survival of CLL B-cells through p38 MAPK activation.     

TRIM44 is indispensable for glioma cell proliferation and cell cycle progression through AKT/p21/p27 signaling pathway

Journal of Neuro-Oncology - Incidental discovery accounts for 30% of newly-diagnosed intracranial meningiomas. There is no consensus on their optimal management. This review aimed to evaluate the...