Protein kinase G activates the JNK1 pathway via phosphorylation of MEKK1.
ABSTRACT We recently obtained evidence that treatment of human colon cancer cells with exisulind (sulindac sulfone) and related compounds induces apoptosis by activation of protein kinase G (PKG) and c-Jun kinase (JNK1). The present study further explores this mechanism. We demonstrate that in NIH3T3 cells a constitutively active mutant of PKG causes a dose-dependent activation of JNK1 and thereby transactivates c-Jun and stimulates transcription from the AP-1 enhancer element. The activation of JNK1 and the transactivation of c-Jun by this mutant of PKG were inhibited by a dominant negative MEKK1. In vitro assays showed that a purified PKG directly phosphorylated the N-terminal domain of MEKK1. PKG also directly phosphorylated a full-length MEKK1, and this was associated with enhanced MEKK1 phosphorylation. Thus, it appears that PKG activates JNK1 through a novel PKG-MEKK1-SEK1-JNK1 pathway, by directly phosphorylating and activating MEKK1.
Article: Suppression of gastric cancer cell growth by targeting the beta-catenin/T-cell factor pathway.[show abstract] [hide abstract]
ABSTRACT: Functional activation of beta-catenin/T-cell factor (Tcf) signaling plays an important role in the early events of carcinogenesis. Recently, it was demonstrated that adenomatous polyposis coli or beta-catenin genes are mutated frequently in gastric cancer cells. The objective of the current study was to use a gene-targeting approach to kill human gastric cancer cells selectively with activated beta-catenin/Tcf signaling. A recombinant adenovirus that carries a lethal gene (p53 up-regulated modulator of apoptosis [PUMA]) under the control of a beta-catenin/Tcf-responsive promoter (AdTOP-PUMA) was used selectively to target gastric cancer cells (AGS) that posses an active beta-catenin/Tcf pathway. The combined effect of AdTOP-PUMA and several chemotherapeutic agents (5-florouracil, doxorubicin, paclitaxel) also was evaluated. Cell viability was measured by methylene blue assay, protein expression was measured by Western blot analysis, and cell cycle and apoptosis were evaluated by fluorescent-activated cell sorter analysis. RESULTS.: The TOP-PUMA adenovirus inhibited AGS cell growth in a dose- and time-dependent fashion. Growth inhibition was associated with the up-regulation of PUMA expression and the induction of apoptosis. Chemotherapy synergistically enhanced the killing effect of AdTOP-PUMA. Selective targeting of gastric cancer cells with the activated beta-catenin pathway may be a novel and effective therapy in gastric cancer. Combination of this gene-therapy approach with standard therapy may improve efficacy and reduce toxicity.Cancer 02/2007; 109(2):188-97. · 4.77 Impact Factor
Article: Alpha2(I) collagen gene regulation by protein kinase C signaling in human dermal fibroblasts.[show abstract] [hide abstract]
ABSTRACT: We investigated the mechanisms by which protein kinase C (PKC) regulates the expression of the alpha2(I) collagen gene in normal dermal fibroblasts. Reduction of PKC-alpha activity by treatment with Gö697-6 or by overexpression of a dominant negative (DN) mutant form decreased alpha2(I) collagen gene expression. This decrease required a sequence element in the collagen promoter that contains Sp1/Sp3 binding sites. Reduction of PKC-delta activity by rottlerin or overexpression of DN PKC-delta also decreased alpha2(I) collagen gene expression. This effect required a separate sequence element containing Sp1/Sp3-binding sites and an Ets-binding site. In both cases, point mutations within the response elements abrogated the response to PKC inhibition. Forced overexpression of Sp1 rescued the PKC inhibitor-mediated reduction in collagen protein expression. A DNA affinity precipitation assay revealed that inhibition of PKC-delta by rottlerin increased the binding activity of endogenous Fli1 and decreased that of Ets1. On the other hand, TGF-beta1, which increased the expression of PKC-delta, had the opposite effect, increasing the binding activity of Ets1 and decreasing that of Fli1. Our results suggest that PKC-delta is involved in the regulation of the alpha2(I) collagen gene in the presence or absence of TGF-beta. Alteration of the balance of Ets1 and Fli1 may be a novel mechanism regulating alpha2(I) collagen expression.Nucleic Acids Research 02/2005; 33(4):1337-51. · 8.03 Impact Factor
Article: Sulindac derivatives inhibit cell growth and induce apoptosis in primary cells from malignant peripheral nerve sheath tumors of NF1-patients.[show abstract] [hide abstract]
ABSTRACT: BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are neoplasms leading to death in most cases. Patients with Neurofibromatosis type 1 have an increased risk of developing this malignancy. The metabolites of the inactive prodrug Sulindac, Sulindac Sulfide and Sulindac Sulfone (Exisulind) are new chemopreventive agents that show promising results in the treatment of different cancer types. In this study we examined the antineoplastic effect of these compounds on primary cells derived from two MPNSTs of Neurofibromatosis type 1 patients. RESULTS: Exisulind and Sulindac Sulfide showed a dramatic time- and dose-dependent growth inhibitory effect with IC50-values of 120 microM and 63 microM, respectively. The decrease in viability of the tested cells correlated with induction of apoptosis. Treatment with 500 microM Exisulind and 125 microM Sulindac Sulfide for a period of 2 days increased the rate of apoptosis 21-27-fold compared to untreated cells. Reduced expression of RAS-GTP and phosphorylated ERK1/2 was detected in treated MPNST cells. Moreover, elevated levels of phosphorylated SAPK/JNK were found after drug treatment, and low activation of cleaved caspase-3 was seen. CONCLUSIONS: Our results suggest that this class of compounds may be of therapeutic benefit for Neurofibromatosis type 1 patients with MPNST.Cancer Cell International 06/2004; 4(1):4. · 1.97 Impact Factor