u-0126 and Colonic-Neoplasms

Lipid rafts play a key role in the regulation of fundamentally important cellular processes, including cell proliferation, differentiation, and survival. The composition of such detergent-resistant microdomains (DRMs) is altered under pathologic conditions, including cancer. Although DRMs have been analyzed in colorectal carcinoma little information exists about their composition upon treatment with targeted drugs. Hence, a quantitative proteomic profiling approach was performed to define alterations within the DRM fraction of colorectal carcinoma cells upon treatment with the drug U0126, an inhibitor of the mitogen-activated protein kinase pathway. Comparative expression profilings resulted in the identification of 300 proteins, which could partially be linked to key oncogenic signaling pathways and tumor-related cellular features, such as cell proliferation, adhesion, motility, invasion, and apoptosis resistance. Most of these proteins were downregulated upon inhibitor treatment. In addition, quantitative proteomic profilings of cholesterol-depleted versus intact lipid rafts were performed to define, which U0126-regulated target structures represent bona fide raft proteins. Selected differentially abundant raft proteins were validated at the mRNA and/or protein level using U0126- or Trametinib-treated cells. The presented data provide insights into the molecular mechanisms associated with the response to the treatment with MEK inhibitors and might also lead to novel candidates for therapeutic interventions.

Topics: Butadienes; Cell Line, Tumor; Colon; Colonic Neoplasms; Detergents; Humans; Membrane Microdomains; Mitogen-Activated Protein Kinases; Nitriles; Protein Kinase Inhibitors; Proteome; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Our previous study has demonstrated that tissue factor-factor VIIa (TF/FVIIa) complex promotes the proliferation and migration of colon cancer cell line SW620 through the activation of protease-activated receptor 2 (PAR2). In the current study, the underlying molecular mechanisms of TF/FVIIa/PAR2 signaling in SW620 cells were further explored, with the focus on the role of activator protein-1 (AP-1) subunit c-Jun. The results revealed that PAR2-AP and FVIIa could upregulate c-Jun expression and c-Jun phosphorylation in SW620 cells in a time-dependent manner. The effect of FVIIa was significantly blocked by anti-TF and anti-PAR2 antibodies. Protein kinase Cα (PKCα) inhibitor safingol and extracellular signal-regulated kinase 1 and 2 (ERK1/2) inhibitor U0126 abrogated the activation of c-Jun. In contrast, Ca(2+) chelators EGTA and thapsigargin, and p38MAPK inhibitor SB203580 had no effect. Suppression of c-Jun/AP-1 activation using a natural inhibitor curcumin decreased the expression of caspase-3, MMP-9, and TF, as well as the proliferation and migration of SW620 cells induced by PAR2-AP or FVIIa. Collectively, our findings suggest that c-Jun/AP-1 activation is required for TF/FVIIa/PAR2-induced SW620 cell proliferation and migration. PKCα and ERK1/2 are located upstream of c-Jun/AP-1 in this signaling pathway. Pharmacological inhibition of this pathway might be a novel strategy for colon cancer therapy.

Topics: Antineoplastic Agents; Butadienes; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colonic Neoplasms; Curcumin; Extracellular Signal-Regulated MAP Kinases; Factor VIIa; Humans; MAP Kinase Signaling System; Nitriles; Protein Kinase C-alpha; Proto-Oncogene Proteins c-jun; Receptor, PAR-2; Sphingosine; Thromboplastin; Transcription Factor AP-1

To investigate mechanisms of discrepancy in response to a MEK/ERK inhibitor, U0126, in KRAS- and BRAF-mutant colorectal cancer cells.. Multiparametric flow cytometry was performed on two colon cancer cell lines, HCT116 and HT29. Cells were treated with U0126, and phospho-specific antibodies were used to monitor ERK signaling.. HCT116 and HT29 cells were treated with increasing amounts of U0126. The western blot analysis revealed that by increasing the amount of U0126 resulted in inhibition of phospho-ERK, in HCT116 and to a lesser degree in HT29 cells. Microarray profiling identified CYP1A1 and 1A2 overexpression in HT29 cells and that inhibition of CYP1A1 with α-naphthoflavone and furanfylline restored sensitivity to U0126 in HT29 cells.. Combination of a CYP inhibitor with MEK/ERK inhibitor enhances the inhibitory effect on ERK in BRAF-mutant colon cancer cells.

Topics: Benzoflavones; Butadienes; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Signaling System; Nitriles; Proto-Oncogene Proteins B-raf

Diets high in fish and curcumin are associated with a decreased risk of CRC. Insulin resistance and obesity are associated with increased CRC risk and higher reoccurrence rates. We utilized cell culture to determine if dietary compounds could reduce insulin-induced cell proliferation comparing the response in normal and metastatic colon epithelial cells. We treated model normal murine colon epithelial cells (YAMC) and adenocarcinoma cells (MC38) with docosahexaenoic acid (DHA) or curcumin alone and then co-treatments of the diet-derived compound and insulin were combined. Cell proliferation was stimulated with insulin (1 ug/mL) to model insulin resistance in obesity. Despite the presence of insulin, proliferation was reduced in the MC38 cells treated with 10 μM curcumin (p<0.001) and 50 μM DHA (p<0.001). Insulin stimulated MAPK and MEK phosphorylation was inhibited by DHA and curcumin in MC38 cancer cells. Here we show that curcumin and DHA can block insulin-induced colon cancer cell proliferation in vitro via a MEK mediated mechanism.

Topics: Animals; Antineoplastic Agents; Butadienes; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Curcumin; Docosahexaenoic Acids; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Insulin; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Nitriles

To evaluate the activity of U0126, a MEK1/2 inhibitor, in downregulating the phosphorylation of ERK in colon cancer cell lines and to explore the correlation of phospho-flow cytometry with standardized methods to validate its use in clinical settings. Phospho-specific flow cytometry provides an optimal platform for the analysis of signaling abnormalities in cancer. In this study, we used phospho-specific flow cytometry to monitor intracellular signaling in cells stimulated with phorbol 12-myristate 13-acetate (PMA).. Multiparametric flow cytometry was performed on two colon cancer cell lines, HCT116 and HT29. PMA-stimulated cells were treated with U0126, and phospho-specific antibodies were used to monitor ERK signaling. The resulting data were compared to western blotting and immunofluorescence staining.. HCT116 and HT29 cells were treated with increasing amounts of U0126 after PMA stimulation. The western blot analysis revealed that increasing the amount of U0126 resulted in inhibition of phospho-ERK (p-ERK). Fluorescence-activated cell sorting plots of phosphorylation of ERK demonstrated that the levels of p-ERK decreased with increasing concentrations of U0126. Results of immunofluorescence staining indicated that the staining density of the immunofluorescent dye decreased as the concentration of U0126 increased from 0.1 microM to 100 microM.. Quantitative and correlated expression profiles for ERK signaling suggest that phospho-specific flow cytometry will provide new insights into mechanisms underlying defective signaling in cancer and enable us to predict drug responses in cancer cell lines.

Topics: Blotting, Western; Butadienes; Carcinogens; Colonic Neoplasms; Enzyme Inhibitors; Flow Cytometry; Fluorescent Antibody Technique; HCT116 Cells; HT29 Cells; Humans; MAP Kinase Signaling System; Nitriles; Phosphorylation; Predictive Value of Tests; Reproducibility of Results; Tetradecanoylphorbol Acetate

An aberrant WNT signaling contributes to the development and progression of multiple cancers. WNT5a is one of the WNT signaling molecules. This study was designed to test the hypothesis that amino acid deprivation induces changes in the WNT signaling pathway in colon cancer cells. Results showed that targets of the amino acid response pathway, ATF3 and p21, were induced in the human colon cancer cell line SW480 during amino acid limitation. There was a significant decrease in the WNT5a mRNA level following amino acid deprivation. The down-regulation of WNT5a mRNA by amino acid deprivation is not due to mRNA destabilization. There is a reduction of nuclear beta-catenin protein level by amino acid limitation. Under amino acid limitation, phosphorylation of ERK1/2 was increased and the blockage of ERK1/2 by the inhibitor U0126 partially restored WNT5a mRNA level. In conclusion, amino acid limitation in colon cancer cells induces phosphorylation of ERK1/2, which then down-regulates WNT5a expression.

Topics: Amino Acids; beta Catenin; Butadienes; Cell Line, Tumor; Cell Nucleus; Colonic Neoplasms; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins; RNA Stability; RNA, Messenger; Transcription, Genetic; Wnt Proteins; Wnt-5a Protein

Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA-A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT-deficient and MEK-inhibited cells. Increased HLA-A2 surface expression was correlated with enhanced recognition and lysis by A2-specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells.

Topics: Antineoplastic Agents; Benzenesulfonates; Butadienes; Colonic Neoplasms; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3B; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; HCT116 Cells; HLA-A2 Antigen; Humans; Intercellular Signaling Peptides and Proteins; Killer Cells, Natural; Mitogen-Activated Protein Kinase Kinases; Mutation; Niacinamide; Nitriles; Phenylurea Compounds; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Pyridines; ras Proteins; Sorafenib

Elevated expression and aberrant activation of the src oncogene are strongly associated with cancer initiation and progression, thereby making Src a promising molecular target for anti-cancer therapy. Through drug screening using a temperature-inducible v-Src-transformed epithelial cell line, we found that andrographolide could suppress v-Src-induced transformation and down-regulate v-Src protein expression. In addition, actin cable dissolution and E-cadherin down-regulation, features of transformed phenotype, are perturbed by andrographolide. Moreover, andrographolide promoted v-Src degradation via a ubiquitin-dependent manner. Although andrographolide treatment altered the tyrosine phosphorylation pattern in v-Src-expressing cells, it did not directly affect the kinase activity of v-Src. Both the Erk and phosphatidylinositol 3-kinase signaling pathways were strongly inhibited in andrographolide-treated v-Src cells. However, only MKK inhibitors (PD98059 and U0126) were able to cause a non-transformed morphology similar to that of andrographolide-treated v-Src cells. Moreover, overexpression of constitutively active MKK1 in v-Src cells blocked andrographolide-mediated morphological inhibition. Interestingly, andrographolide treatment could also reduce the protein level of the c-Src truncation mutant (Src531), an Src mutant originally identified from human colon cancer cells. In summary, we demonstrated that andrographolide antagonized v-Src action through promotion of v-Src protein degradation. Furthermore, attenuation of the Erk1/2 signaling pathway is essential for andrographolide-mediated inhibition of v-Src transformation. Our results demonstrate that andrographolide can act as a v-Src inhibitor and reveal a novel action mechanism of andrographolide.

Topics: Antiviral Agents; Butadienes; Cell Line, Tumor; Cell Transformation, Neoplastic; Colonic Neoplasms; Diterpenes; Enzyme Inhibitors; Epithelial Cells; Flavonoids; Gene Expression Regulation, Neoplastic; Hot Temperature; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Nitriles; Oncogene Protein pp60(v-src); Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Tyrosine; Ubiquitin; Ubiquitination

The short fatty acid, butyrate, which is produced by intestinal anaerobic bacteria in the colon, has inhibitory activity on histone deacetylases (HDACs). Treatment of the human colon cancer cell line, LS174T, with 1-2 mM sodium butyrate stimulated MUC2 mucin production, as determined by histological PAS staining of carbohydrate chains of mucin, and confirmed at the protein and mRNA levels by immunoblotting with anti-MUC2 antibody and real-time RT-PCR, respectively. Increases in acetylated histone H3 in the LS174T cells treated with butyrate suggest inhibition of HDACs in these cells. Butyrate-stimulated MUC2 production in the LS174T cells was inhibited by the MEK inhibitor, U0126, implicating the involvement of extracellular signal-regulated kinase (ERK) cascades in this process. Proliferation of the LS174T cells was inhibited by butyrate treatment. Although apoptotic nuclear DNA fragmentation could not be detected, cell-cycle arrest at the G0/G1 phase in the butyrate-treated cells was demonstrated by flow cytometry. Thus butyrate, an HDAC inhibitor, inhibits proliferation of LS174T cells but stimulates MUC2 production in individual cells.

Topics: Adenocarcinoma; Butadienes; Butyrates; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Humans; Mitogen-Activated Protein Kinase Kinases; Mucin-2; Mucins; Nitriles

XIAP-associated factor 1 (XAF1) negatively regulates the function of the X-linked inhibitor of apoptosis protein (XIAP), a member of the IAP family that exerts antiapoptotic effects. The extracellular signal-regulated kinase (ERK) pathway is thought to increase cell proliferation and to protect cells from apoptosis. The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.. Four human colon cancer cell lines, HCT1116 and Lovo (wildtype p53), DLD1 and SW1116 (mutant p53), were used. Lovo stable transfectants with XAF1 sense and antisense were established. The effects of dominant-negative MEK1 (DN-MEK1) and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined. The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay. Cell proliferation was measured by MTT assay. Apoptosis was determined by Hoechst 33258 staining.. U0126 increased the expression of XAF1 in a time- and dose-dependent manner. A similar result was obtained in cells transfected with DN-MEK1 treatment. Conversely, the expression of XIAP was down-regulated. Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection. rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression. Overexpression of XAF1 was more sensitive to U0126-induced apoptosis, whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.. XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation, which required de novo protein synthesis. The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Apoptosis Regulatory Proteins; Butadienes; Cell Line, Tumor; Colonic Neoplasms; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Humans; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Neoplasm Proteins; Nitriles; Phosphorylation; Promoter Regions, Genetic; Transcriptional Activation; Transfection; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein

The purpose of this study was to investigate whether proteasome inhibition acts as a thermal sensitizing agent to induce tumor cell death in a colon cancer cell line.. HT-29 colon cancer cells were exposed to hyperthermia (43 degrees C) in the presence of proteasome inhibition for 1 h. Viable cell mass and apoptosis were measured by MTT and annexin V staining, respectively. Protein levels were determined by Western blot analysis.. A significant synergistic effect on cell viability with proteasome inhibition was noted under hyperthermic conditions compared to hyperthermia alone (p < 0.05). Increases in phosphorylated ERK and decreases in HSP27 levels were observed in the cells exposed to proteasome inhibition at 43 degrees C. Pretreatment with an inhibitor of ERK yielded an additional increase in apoptosis when used in combination with proteasome inhibition and hyperthermia. Decreased expression of HSP27 by siRNA also resulted in increased thermally induced apoptotic cell death.. Thermal sensitization through proteasome inhibition may represent a novel approach to increase the efficacy of hyperthermia as an anticancer modality.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Butadienes; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Synergism; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Heat-Shock Proteins; HT29 Cells; Humans; Hyperthermia, Induced; Leupeptins; Nitriles; Protease Inhibitors; Proteasome Inhibitors; RNA, Small Interfering

Higher levels of focal adhesion kinase (FAK) are expressed in colon metastatic carcinomas. However, the signaling pathways and their mechanisms that control cell adhesion and motility, important components of cancer metastasis, are not well understood. We sought to identify the integrin-mediated mechanism of FAK cleavage and downstream signaling as well as its role in motility in human colon cancer GEO cells. Our results demonstrate that phosphorylated FAK (tyrosine 397) is cleaved at distinct sites by integrin signaling when cells attach to collagen IV. Specific blocking antibodies (clone P1E6) to integrin alpha2 inhibited FAK activation and cell motility (micromotion). Ectopic expression of the FAK C-terminal domain FRNK attenuated FAK and ERK phosphorylation and micromotion. Calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal blocked FAK cleavage, cell adhesion, and micromotion. Antisense approaches established an important role for mu-calpain in cell motility. Expression of wild type mu-calpain increased cell micromotion, whereas its point mutant reversed the effect. Further, cytochalasin D inhibited FAK phosphorylation and cleavage, cell adhesion, locomotion, and ERK phosphorylation, thus showing FAK activation downstream of actin assembly. We also found a pivotal role for FAK Tyr(861) phosphorylation in cell motility and ERK activation. Our results reveal a novel functional connection between integrin alpha2 engagement, FAK, ERK, and mu-calpain activation in cell motility and a direct link between FAK cleavage and enhanced cell motility. The data suggest that blocking the integrin alpha2/FAK/ERK/mu-calpain pathway may be an important strategy to reduce cancer progression.

Topics: Biotinylation; Butadienes; Calpain; Cell Adhesion; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Cytochalasin D; Electric Impedance; Enzyme Activation; Enzyme Inhibitors; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin alpha2; Mitogen-Activated Protein Kinases; Nitriles; Nucleic Acid Synthesis Inhibitors; Oligonucleotides, Antisense; Phosphorylation; Point Mutation; Precipitin Tests; Protein Structure, Tertiary; Signal Transduction; Substrate Specificity; Tyrosine

Colorectal cancer is the second leading cause of cancer mortality in the United States. Substantial human and animal data support the ability of nonsteroidal anti-inflammatory drugs to cause regression of existing colon tumors and prevent new tumor formation. The mechanism by which the nonsteroidal anti-inflammatory drug sulindac prevents tumor growth is poorly understood and seems complex as sulindac can modulate several growth-related signaling pathways. Sulindac metabolites simultaneously (a) increase cellular cyclic GMP and subsequently activate cyclic GMP-dependent protein kinase (PKG); (b) activate c-jun NH2-terminal kinase (JNK); (c) inhibit extracellular signal-regulated kinase 1/2 (ERK1/2); and (d) decrease beta-catenin protein expression at times and doses consistent with apoptosis. The purpose of this study was to determine if PKG, ERK1/2, JNK, and beta-catenin are independent targets for sulindac in vitro. Pharmacologic activation of PKG with YC-1 increases JNK phosphorylation and induces apoptosis in colon cancer cells without modulating ERK1/2 phosphorylation or beta-catenin protein expression. Inhibition of ERK1/2 with U0126 induces apoptosis but fails to activate JNK phosphorylation or down-regulate beta-catenin protein expression. Cotreatment with U0126 and YC-1 synergistically increases apoptosis in colorectal cancer cells and recapitulates the effects of sulindac treatment on ERK1/2, JNK, and beta-catenin. These results indicate that sulindac metabolites modulate ERK1/2 and PKG pathways independently in colon cancer cells and suggest that the full apoptotic effect of sulindac is mediated by more than one pathway. Using similar combinatorial approaches in vivo may provide more effective, less toxic chemopreventive and chemotherapeutic strategies. Such therapies could dramatically reduce the incidence and death rate from colorectal cancer.

Topics: Antineoplastic Agents; Apoptosis; beta Catenin; Butadienes; Colonic Neoplasms; Cyclic GMP-Dependent Protein Kinases; Down-Regulation; Enzyme Activation; Enzyme Activators; Humans; Indazoles; MAP Kinase Kinase 1; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Sulindac; Tumor Cells, Cultured

Recently, the synthesized octahedral Pt(IV) compound trans,cis-Pt(acetato)2Cl2(1,4-butanediamine), K101, showed potent anti-tumor activity in vitro and in vivo. For the further investigation of K101-induced anti-cancer activity, we tested cytotoxicity against various cancer cell lines and performed the histoculture drug response assay (HDRA) against human colorectal tumor tissues in vitro. We investigated the signaling pathway of K101-induced apoptosis via expression of p53 and ERK1/2 in the human colon cell line HCT116. The cytotoxicity and the three-dimensional HDRA of K101 were evaluated using the MTT assay. To study the K101-induced apoptosis pathway, we performed FACS analysis and immunoblotting of p53, p21, Bax, Fas and ERK1/2 in HCT116 cells treated with or without K101. The cytotoxic IC50 values of K101 ranged from 1.15 to 2.38 micromol/l, compared to cisplatin ranging from 2.13 to 13.1 micromol/l. Among several cancer cell lines, K101 showed greater potency than cisplatin in colon cancer cell lines. In the HDRA, K101 showed 80.0-91.4% efficacy rates compared with 48.6% for cisplatin against colorectal cancer patient tissues. In the signaling pathway, the expression of p53 and phospho-ERK1/2 was increased in a time-dependent manner by treatment with K101 in the HCT116 cells. When K101 was treated with MEK inhibitor U0126, the cell death rate was increased. The octahedral Pt(IV) complex K101 could be an attractive candidate as a chemotherapeutic agent against colon cancer. ERK1/2 activation and the p53 pathway may play significant functions in mediating K101-induced apoptosis in human colon cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Cell Line, Tumor; Colonic Neoplasms; Enzyme Activation; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Organoplatinum Compounds; Signal Transduction; Tumor Suppressor Protein p53

Protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. Expression of PKCbetaII in the colon of transgenic mice induces hyperproliferation and increased susceptibility to colon cancer. To determine molecular mechanisms by which PKCbetaII promotes colon cancer, we established rat intestinal epithelial (RIE) cells stably expressing PKCbetaII. Here we show that RIE/PKCbetaII cells acquire an invasive phenotype that is blocked by the PKCbeta inhibitor LY379196. Invasion is not observed in RIE cells expressing a kinase-deficient PKCbetaII, indicating that PKCbetaII activity is required for the invasive phenotype. PKCbetaII induces activation of K-Ras and the Ras effector, Rac1, in RIE/PKCbetaII cells. PKCbetaII-mediated invasion is blocked by the Mek inhibitor, U0126, and by expression of either dominant negative Rac1 or kinase-deficient atypical PKCiota. Expression of constitutively active Rac1 induces Mek activation and invasion in RIE cells, indicating that Rac1 is the critical downstream effector of PKCbetaII-mediated invasion. Taken together, our results define a novel PKCbetaII --> Ras --> PKCiota /Rac1 --> Mek signaling pathway that induces invasion in intestinal epithelial cells. This pathway provides a plausible mechanism by which PKCbetaII promotes colon carcinogenesis.

Topics: Animals; Apoptosis Regulatory Proteins; Butadienes; Carrier Proteins; Cell Line; Colonic Neoplasms; Cyclooxygenase 2; Enzyme Activation; Enzyme Inhibitors; Genes, Dominant; Genetic Predisposition to Disease; Glutathione Transferase; GTP-Binding Proteins; Humans; Intestinal Mucosa; Intracellular Signaling Peptides and Proteins; Isoenzymes; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinases; Membrane Proteins; Mesylates; Models, Biological; Mutation; Neoplasm Invasiveness; Nitriles; p21-Activated Kinases; Phenotype; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Protein Kinase C beta; Protein Serine-Threonine Kinases; Pyrroles; rac1 GTP-Binding Protein; ras Proteins; Rats; Retroviridae; Signal Transduction; Transfection

Colorectal cancer is the second leading cause of cancer death in the United States. Nonsteroidal anti-inflammatory drugs including sulindac are promising chemopreventive agents for colorectal cancer. Sulindac and selective cyclooxygenase (COX)-2 inhibitors cause regression of colonic polyps in familial polyposis patients. Sulindac induces apoptotic cell death in cancer cells in vitro and in vivo. In tumor cells, activation of extracellular-regulated kinase (ERK) 1/2 results in phosphorylation of several ERK1/2 effectors, including the proapoptotic protein Bad. Phosphorylation of Ser112 by ERK1/2 inactivates Bad and protects the tumor cell from apoptosis. Sulindac metabolites and other nonsteroidal anti-inflammatory drugs selectively inhibit ERK1/2 phosphorylation in human colon cancer cells. In this study we show that epidermal growth factor (EGF) strongly induces phosphorylation of ERK1/2 and Bad in HT29 colon cancer cells. EGF-stimulated phosphorylation of ERK and Bad is blocked by pretreatment with U0126, a selective MAP kinase kinase (MKK)1/2 inhibitor. Similarly, pretreatment with sulindac sulfide blocks the ability of EGF to induce ERK1/2 and Bad phosphorylation, but also down-regulates total Bad but not ERK1/2 protein levels. The ability of sulindac to block ERK1/2 signaling by the EGF receptor may account for at least part of its potent growth-inhibitory effects against cancer cells.

Topics: Antineoplastic Agents; bcl-Associated Death Protein; Butadienes; Carrier Proteins; Caspase Inhibitors; Caspases; Colonic Neoplasms; Enzyme Activation; Epidermal Growth Factor; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Sulindac; Tumor Cells, Cultured

Recently, we have shown that autocrine transforming growth factor-alpha (TGF-alpha) controls the expression of integrin alpha2, cell adhesion to collagen IV and motility in highly progressed HCT116 colon cancer cells (Sawhney, R. S., Zhou, G-H. K., Humphrey, L. E., Ghosh, P., Kreisberg, J. I., and Brattain, M. G. (2002) J. Biol. Chem. 277, 75-86). We now report that expression of basal integrin alpha2 and its biological effects are controlled by constitutive activation of the extracellular signal-regulated/mitogen-activated protein kinase (ERK/MAPK) pathway. Treatment of cells with selective mitogen-activated protein kinase kinase (MEK) inhibitors PD098059 and U0126 showed that integrin alpha2 expression, cell adhesion, and activation of ERK are inhibited in a parallel concentration-dependent fashion. Moreover, autocrine TGF-alpha-mediated epidermal growth factor receptor activation was shown to control the constitutive activation of the ERK/MAPK pathway, since neutralizing antibody to the epidermal growth factor receptor was able to block basal ERK activity. TGF-alpha antisense-transfected cells also showed attenuated activation of ERK. Using a real time electric cell impedance sensing technique, it was shown that ERK-dependent integrin alpha2-mediated cell micromotion signaling is controlled by autocrine TGF-alpha. Thus, this study implicates ERK/MAPK signaling activated by endogenous TGF-alpha as one of the mechanistic features controlling metastatic spread.

Topics: Butadienes; Chromones; Colonic Neoplasms; Enzyme Inhibitors; Flavonoids; Humans; Integrin alpha2; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Transforming Growth Factor alpha; Tumor Cells, Cultured

We have investigated the contribution of CDK4 and CDK2 inhibition to G1 arrest in colon cancers following inhibition of the MEK/MAP kinase pathway. CDK4 inhibition is sufficient to cause arrest, but inhibition of CDK2 by p27 Kip1 redistribution or ectopic expression has no effect on proliferation. Likewise, inhibition of CDK2 through expression of dominant-negative (DN) CDK2 or antisense oligonucleotides did not prevent cell proliferation in these cells. We therefore tested whether CDK2 activity is dispensable in other cells. Surprisingly, osteosarcomas and Rb-negative cervical cancers continued to proliferate after depletion of CDK2 through antisense oligonucleotides or small interfering (si) RNA. Here we report of sustained cell proliferation in the absence of CDK2, and we suggest that CDK2 is not a suitable target for cancer therapy.

Topics: Benzamides; Butadienes; CDC2-CDC28 Kinases; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Ecdysterone; Enzyme Inhibitors; Female; G1 Phase; HeLa Cells; Humans; MAP Kinase Kinase Kinases; Models, Biological; Mutagenesis, Site-Directed; Mutation; Nitriles; Oligonucleotides, Antisense; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Retinoblastoma Protein; Tumor Cells, Cultured

Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and sulindac is associated with a decreased mortality from colorectal cancer. Sulindac causes regression of precancerous adenomatous polyps and inhibits the growth of cultured colon cell lines. Whereas induction of apoptotic cell death is thought to account for the growth inhibitory effect of sulindac, less is known about its biochemical mechanism(s) of action. Sulindac is metabolized in vivo to sulfide and sulfone derivatives. Both the sulfide and sulfone metabolites of sulindac as well as more potent cyclic GMP-dependent phosphodiesterase inhibitors were shown to cause inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation at doses (40-600 microM) and times (1-5 days) consistent with the induction of apoptosis by the drugs. Treatment of HCT116 human colon cancer cells with the specific mitogen-activated protein kinase kinase, U0126 (5-50 microM) resulted in a time- and dose-dependent inhibition of ERK1/2 phosphorylation, and induction of apoptosis. U0126 treatment (20 microM) increased basal apoptosis, and potentiated the apoptotic effect of sulindac sulfide and sulindac sulfone. These results suggest that the inhibition of ERK1/2 phosphorylation is responsible for at least part of the induction of programmed cell death by sulindac metabolites. Inhibition of ERK1/2 activity may, therefore, be a useful biochemical target for the development of chemopreventive and chemotherapeutic drugs for human colon cancer.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Butadienes; Caspase 3; Caspase 7; Caspases; Colonic Neoplasms; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Protein Serine-Threonine Kinases; Sulindac; Tumor Cells, Cultured