u-0126 and Neoplasm-Metastasis

TGFβ-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased TGFβ-SMAD3 and a reduced BMP-SMAD1/5 targeted gene expression signature correlated with shortened distant metastasis free survival and overall survival of patients. At molecular levels, we discovered that TGFβ abolished BMP-induced SMAD1/5 activation in the highly-invasive breast cancer MDA-MB-231 cells, but to a less extent in the non-invasive cancer and normal breast cells. This suggests an inverse correlation between BMP signaling and invasiveness of tumor cells and TGFβ signaling acts in a double whammy fashion in driving cancer invasion and metastasis. Sustained ERK activation by TGFβ was specifically observed in MDA-MB-231 cells, and MEK inhibitor (MEKi) treatment restored BMP-SMAD1/5 signaling while not affecting SMAD2/3 activation. FK506 potently activated BMP, but not TGFβ signaling in breast cancer cells. MEKi or FK506 alone inhibited MDA-MB-231 extravasation in a zebrafish xenograft cancer model. Importantly, when administrated at suboptimal concentrations MEKi and FK506 strongly synergized in promoting BMP-SMAD1/5 signaling and inhibiting cancer cell extravasation. Furthermore, this combination of suboptimal concentrations treatment in a mouse tumor model resulted in real-time reduction of BMP-SMAD1/5 signaling in live tumors, and consequently potently inhibited tumor self-seeding, liver and bone metastasis, but not lung and brain metastasis. Mechanistically, it is the first time to identify BMP-SMAD1/5 signaling as an underlying molecular driver for organ-specific metastasis. Combining of MEKi and FK506, or their analogues, may be explored for clinical development of breast cancer.

Topics: Animals; Bone Morphogenetic Proteins; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice; Neoplasm Metastasis; NIH 3T3 Cells; Nitriles; Organ Specificity; Protein Kinase Inhibitors; Signal Transduction; Tacrolimus; Xenograft Model Antitumor Assays; Zebrafish

BACKGROUND Ginkgo biloba extract (EGb761), a standard extract of the Chinese traditional medicine Ginkgo biloba, plays an anti-tumor role in various cancers. However, whether EGb761 is involved in the invasion and metastasis of gastric cancer remains unclear. MATERIAL AND METHODS In the current study, cell viability assay, Western blotting, wound-healing assay, Transwell invasion assay, and orthotopic transplantation model were performed to explore the effects of EGb761 on gastric cancer. RESULTS In vitro, the results showed that EGb761 suppressed the proliferation of gastric cancer cells in a dose-dependent manner. Furthermore, the migration and invasiveness were weakened and the protein levels of p-ERK1/2, NF-kappaB P65, NF-kappaB p-P65, and MMP2 were decreased by EGb761 or U0126 (an inhibitor of ERK signaling pathway) exposure in gastric cancer cells. Moreover, the combined treatment with EGb761 and U0126 significantly inhibited ERK, NF-kappaB signaling pathway, and the expression of MMP2 than those of single drug. In vivo, EGb761 inhibited the tumor growth and hepatic metastasis of gastric cancer in the mouse model. Results of immunohistochemistry indicated that the expression of ERK1/2, NF-kappaB P65 and MMP2 were decreased by EGb761 in the tumor tissues. CONCLUSIONS EGb761 plays a vital role in the suppression of metastasis and ERK/NF-kappaB signaling pathway in gastric cancer.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Ginkgo biloba; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; NF-kappa B; Nitriles; Phosphorylation; Plant Extracts; Signal Transduction; Stomach Neoplasms

Purvalanol and roscovitine are specific cyclin-dependent kinase (CDK) inhibitors, which have antiproliferative and apoptotic effects on various types of cancer. Although, the apoptotic accomplishment of purvalanol and roscovitine was elucidated at the molecular level, the underlying exact of drug-induced apoptosis through mitogen-activated protein kinase (MAPK) signaling still speculative. In addition, the role of CDK inhibitors in the downregulation of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated epithelial-mesenchymal transition (EMT) remains unclear. Here, we investigated the potential effect of each CDK inhibitors on cell proliferation, migration, and generation of reactive oxygen species due to the inhibition of MAPKs in metastatic DU145 and PC3 prostate cancer cells. We reported that purvalanol and roscovitine induced mitochondria membrane potential loss-dependent apoptotic cell death, which was also characterized by activation of several caspases, cleavage of poly (ADP-ribose) polymerase-1 in DU145 and PC3 cells. Cotreatment of either purvalanol or roscovitine with ERK1/2 inhibitor, U0126, synergistically suppressed cell proliferation, and induced apoptotic action. Also, ERK1/2 inhibition potentiated the effect of each CDK inhibitor on the downregulation of EMT processes via increasing the epithelial marker and decreasing mesenchymal markers through reduction of Wnt signaling regulators in DU145 cells. This study provides biological evidence about purvalanol and roscovitine have apoptotic and antimetastatic effects via MAPK signaling on prostate cancer cell by activation of GSK3β signaling and inhibition of phosphoinositide-3-kinase/AKT (PI3K/AKT) pathways involved in the EMT process.

Topics: Apoptosis; Butadienes; Cyclin-Dependent Kinases; Humans; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Metastasis; Neoplasm Proteins; Nitriles; PC-3 Cells; Prostatic Neoplasms

Lung cancer (LC) is a devastating malignancy with no effective treatments, due to its complex genomic profile. Using bioinformatics analysis and immunohistochemical of lung carcinoma tissues, we show that TRIM59 as a critical oncoprotein relating to LC proliferation and metastasis. In this study, high TRIM59 expression was significantly correlated with lymph node metastasis, distant metastasis, and tumour stage. Furthermore, up-regulation of TRIM59 expression correlated with poorer outcomes in LC patients. Mechanistically, TRIM59 play a key role in promoting LC growth and metastasis through regulation of extracellular-signal regulated protein kinase (ERK) signalling pathway and epithelial-to-mesenchymal transition (EMT)-markers, as validated by loss-of-function studies. In-depth bioinformatics analysis showed that there is preliminary evidence of co-expression of TRIM59 and cyclin dependent kinase 6 (CDK6) in LC. Notably, CDK6 expression significantly decreased when TRIM59 was knocked down in the LC cells. In contrast, exogenous up-regulation of TRIM59 expression also induced significant increases in the expression of CDK6. Moreover, the expression of CDK6 was also inhibited by the ERK signalling inhibitor, U0126. The results of both loss- and gain-of-function studies showed that TRIM59 could regulate the expression of CDK6. Collectively, these data provide evidence that TRIM59 is involved in lung carcinoma growth and progression possibly through the induction of CDK6 expression and EMT process by activation of ERK pathway.

Topics: Biomarkers, Tumor; Butadienes; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase 6; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; MAP Kinase Signaling System; Neoplasm Metastasis; Nitriles; Signal Transduction; Tripartite Motif Proteins

Mitogen‑activated protein kinase kinase (MEK) small molecule inhibitors have been investigated in preclinical or clinical trials for the treatment of cancer. In the present study the genetic test results of 120 patients with colorectal cancer (CRC) were screened and the mutation rate of MEK1 was identified to be 1.67%. MEK inhibition by U0126 significantly decreased the growth of SW48 cells that harbored the MEK1 Q56P mutation, although it did not evidently affect the growth of NCI‑H508 cells with MEK1 wild‑type. In addition, U0126 increased the sensitivity of SW48 cells to 5‑fluorouracil (5‑FU) and oxaliplatin by producing more γH2AX foci and decreasing the expression of excision repair cross‑complementation group 1 and thymidylate synthase. The results suggested that MEK inhibitors in combination with oxaliplatin/5‑FU may offer an improved therapeutic effect in patients with MEK‑mutant CRC.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Butadienes; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; DNA-Binding Proteins; Drug Synergism; Endonucleases; Female; Fluorouracil; Gene Expression Regulation, Neoplastic; Histones; Humans; Male; MAP Kinase Kinase 1; Middle Aged; Mutation; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Nitriles; Oxaliplatin; Signal Transduction; Thymidylate Synthase

Baicalein, a naturally occurring flavonoid isolated from the roots of Scutellaria baicalensis, is historically and widely used as anti-inflammatory and anticancer therapy. Nevertheless, the anti-metastatic effect and underlying molecular mechanisms of baicalein on colorectal carcinoma (CRC) remain unclear. The aim of the present study was, therefore, to invastigate the anti-metastatic activity of baicalein and related mechanism(s) on CRC cells. In this study, we observed that baicalein treatment inhibited proliferation, as well as migration and invasion of HT-29 and DLD1 cells. Baicalein decreased the expression of the matrix metalloproteinases-2 (MMP-2) and MMP-9 in a dose-dependent manner. Also, baicalein treatment significantly reduced phosphorylation of extracellular signal regulated kinases (ERK). Furthermore, in DLD1 cells, MEK1 overexpression partially blocked the anti-metastatic effects of baicalein. Combined treatment with an ERK inhibitor (U0126) and baicalein led to the synergistic reduction of MMP-2/9 expression; and the invasive capabilities of DLD1 cells were also inhibited markedy. Finally, intragastric administration of baicalein inhibited CRC xenograft growth in vivo and suppressed the phosphorylation of ERK and the expression of MMP-2/9 in tumor tissues. Consequently, baicalein suppresses CRC cell invasion via inhibition of the ERK signaling pathways, indicating that baicalein is a potential agent for CRC treatment.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Synergism; Flavanones; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Nitriles; Phosphorylation; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is one of the most common malignant human tumors worldwide, but no effective therapeutic options are currently available. The cancer stem cell (CSC) has proven to play a central role in the development, metastasis, and recurrence of HCC. In this study, we report a dual functional mitogen-activated protein kinase inhibitor (U0126)-based therapy for treating both bulk HCC and HCC CSCs, using poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles as the drug carrier. It is demonstrated that nanoparticle encapsulation enhanced the cell uptake of U0126 in HCC CSCs and that enhanced endocytosis lead to augmented cytotoxicity of U0126 in HCC CSCs. Moreover, the nanoparticle encapsulation increased the inhibition of self-renewal capability, prolonged the circulation time, and increased the tumor accumulation of U0126 when compared with the use of the free inhibitor. The systemic delivery of U0126 remarkably enhanced the suppression of tumor development with decreased CSCs in the HepG2 xenograft simultaneously with reduced systemic toxicity.

Topics: Animals; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Carriers; Endocytosis; Enzyme Inhibitors; Female; Hep G2 Cells; Humans; Immunohistochemistry; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Transplantation; Nitriles; Polyethylene Glycols; Stem Cell Transplantation

The pro-apoptotic protein Bnip3 is induced by hypoxia and is present in the core regions of most solid tumors. Bnip3 induces programmed necrosis by an intrinsic caspase independent mitochondrial pathway. Many tumor cells have evolved pathways to evade Bnip3-mediated death attesting to the physiological relevance of the survival threat imposed by Bnip3. We have reported that acidosis can trigger the Bnip3 death pathway in hypoxic cells therefore we hypothesized that manipulation of intracellular pH by pharmacological inhibition of the vacuolar (v)ATPase proton pump, a significant pH control pathway, may activate Bnip3 and promote death of hypoxic cells within the tumor. Here we confirm that bafilomycin A1 (BafA1), a selective vATPase inhibitor, significantly increased death of breast cancer cells in a hypoxia and Bnip3-dependent manner and significantly reduced tumor growth in MCF7 and MDA-MB-231 mouse xenografts. Combined treatment of cells with BafA1 and the ERK1/2 inhibitor U0126 further augmented cell death. Combined treatment of mice containing MDA-MB-231 xenografts with BafA1 and the ERK1/2 inhibitor sorafenib was superior to either treatment alone and supported tumor regression. BafA1 and sorafenib treatments alone reduced MDA-MB-231 cell metastasis and again the combination was significantly more effective than either treatment alone and was without apparent side effects. These results present a novel mechanism to destroy hypoxic tumor cells that may help reverse the resistance of hypoxic tumors to radiation and chemotherapy and perhaps target tumor stem cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Butadienes; Cell Hypoxia; Enzyme Inhibitors; Gene Knockdown Techniques; Heterografts; Humans; Hydrogen-Ion Concentration; Macrolides; MAP Kinase Signaling System; MCF-7 Cells; Membrane Proteins; Mice; Neoplasm Metastasis; Niacinamide; Nitriles; Phenylurea Compounds; Proto-Oncogene Proteins; Sorafenib; Tumor Burden; Vacuolar Proton-Translocating ATPases

The expression and function of ribosomal s6 protein kinase 4 (RSK4) in renal cell carcinoma (RCC) are unknown.. Immunohistochemistry was used to detect the expression of RSK4 in RCC, and the relationship between RSK4 expression and clinicopathological features as well as prognosis of RCC patients was statistically analysed. Ectopic RSK4 expression in RCC cell lines was performed to determine its effect on cell cycle regulation, tumour invasiveness, and metastatic capability.. RSK4 was overexpressed in RCCs (P=0.003), compared with normal tissues, and the expression varied in different RCC subtypes (P=0.021), especially in two subtypes of papillary RCCs (P=0.001). RSK4 expression was positively correlated with high pT stage (P<0.001), high Fuhrman grade (P<0.001), lymph node involvement (P<0.001), and presence of distant metastasis (P=0.039), and could predict poor outcome in RCC patients. Molecular studies showed that overexpression of RSK4 could promote cell cycle progression and enhance the invasive and metastatic capability of RCC cell lines and vice versa.. The expression pattern and molecular mechanisms of RSK4 in RCCs indicate that it could be a potential independent prognostic factor and serve as a new potential therapeutic target for RCC patients.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Butadienes; Carcinoma, Renal Cell; Cell Cycle; Cell Line, Tumor; Child; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Hyaluronan Receptors; Kidney Neoplasms; Male; Matrix Metalloproteinase 9; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Nitriles; Prognosis; Pteridines; Ribosomal Protein S6 Kinases, 90-kDa; RNA Interference; RNA, Small Interfering; Young Adult

The rate of morbidity and mortality of hepatocellular carcinoma (HCC) in Taiwan has not lessened because of difficulty in treating tumor metastasis. Norcantharidin (NCTD) is currently used as an anticancer drug for hepatoma, breast cancer, and colorectal adenocarcinoma. NCTD possesses various biological anticancer activities, including apoptosis. However, detailed effects and molecular mechanisms of NCTD on metastasis are unclear. Thus, HCC cells were subjected to treatment with NCTD and then analyzed to determine the effects of NCTD on cell metastasis.. Modified Boyden chamber assays revealed that NCTD treatment inhibited cell migration and invasion capacities of HCC cells substantially. Results of zymography and western blotting showed that activities and protein levels of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (u-PA) were inhibited by NCTD. Western blot analysis showed that NCTD inhibits phosphorylation of ERK1/2. Testing of mRNA level, quantitative real-time PCR, and promoter assays evaluated the inhibitory effects of NCTD on MMP-9 and u-PA expression in HCC cells. The chromatin immunoprecipitation (ChIP) assay for analyzing the genomic DNA sequences bound to these proteins was reactive to the transcription protein nuclear factor (NF)-kappaB, which was inhibited by NCTD. The expression of NF-kappa B was measured by western blot analysis, which revealed decreased nuclear-factor DNA-binding activity after NCTD treatment.. NCTD inhibited MMP-9 and u-PA expression through the phosphorylation of ERK1/2 and NF-kappaB signaling pathway which serves as a powerful chemopreventive agent in HCC cell metastasis.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Survival; Hepatocytes; Humans; Liver Neoplasms; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Neoplasm Invasiveness; Neoplasm Metastasis; NF-kappa B; Nitriles; Phosphatidylinositol 3-Kinases; Plasminogen Activator Inhibitor 1; Proto-Oncogene Proteins c-akt; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Transcription, Genetic; Urokinase-Type Plasminogen Activator; Wound Healing

Epidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined.. We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration.. Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Analysis of Variance; Butadienes; Carcinoma, Hepatocellular; Cell Movement; Epidermal Growth Factor; Guanine Nucleotide Exchange Factors; Hep G2 Cells; Humans; Immunoblotting; In Vitro Techniques; Microscopy, Fluorescence; Mutation, Missense; Neoplasm Metastasis; Nitriles; rac1 GTP-Binding Protein; RNA, Small Interfering; Signal Transduction

ErbB2+ human breast cancer is a major clinical problem. Prior results have suggested that tetraspanin CD151 might contribute to ErbB2-driven breast cancer growth, survival, and metastasis. In other cancer types, CD151 sometimes supports tumor growth and metastasis. However, a definitive test of CD151 effects on de novo breast cancer initiation, growth, and metastasis has not previously been done. We used CD151 gene-deleted mice expressing the MMTV-ErbB2 transgene to show that CD151 strongly supports ErbB2+ mammary tumor initiation and metastasis. Delayed tumor onset (by 70-100 days) in the absence of CD151 was accompanied by reduced survival of mammary epithelial cells and impaired activation of FAK- and MAPK-dependent pathways. Both primary tumors and metastatic nodules showed smooth, regular borders, consistent with a less invasive phenotype. Furthermore, consistent with impaired oncogenesis and decreased metastasis, CD151-targeted MCF-10A/ErbB2 cells showed substantial decreases in three-dimensional colony formation, EGF-stimulated tumor cell motility, invasion, and transendothelial migration. These CD151-dependent functions were largely mediated through α6β4 integrin. Moreover, CD151 ablation substantially prevented PKC- and EGFR/ERK-dependent α6β4 integrin phosphorylation, consistent with retention of epithelial cell polarity and intermediate filament cytoskeletal connections, which helps to explain diminished metastasis. Finally, clinical data analyses revealed a strong correlation between CD151 and ErbB2 expression and metastasis-free survival of breast cancer patients. In conclusion, we provide strong evidence that CD151 collaborates with LB integrins (particularly α6β4 and ErbB2 (and EGFR) receptors to regulate multiple signaling pathways, thereby driving mammary tumor onset, survival, and metastasis. Consequently, CD151 is a useful therapeutic target in malignant ErbB2+ breast cancer.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Female; Focal Adhesion Kinase 1; Humans; Integrin alpha6beta4; Lapatinib; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Neoplasm Metastasis; Nitriles; Phosphorylation; Quinazolines; Receptor, ErbB-2; Tetraspanin 24; Transendothelial and Transepithelial Migration

MUC1 is overexpressed and aberrantly glycosylated in more than 60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In this study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared with both KC and KCM. Cell lines derived from KCKO tumors have significantly less tumorigenic capacity compared with cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared with mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor, platelet-derived growth factor, or matrix metalloproteinase 9. Further, significantly less KCKO cells entered the G(2)-M phase of the cell cycle compared with the KCM cells. Proteomics and Western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of mitogen-activated protein kinase (MAPK), as well as a significant decrease in nestin and tubulin-α2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro.

Topics: Animals; Butadienes; Carcinoma, Pancreatic Ductal; Cell Cycle; Cell Growth Processes; Epidermal Growth Factor; Humans; Intermediate Filament Proteins; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Mucin-1; Neoplasm Metastasis; Nerve Tissue Proteins; Nestin; Nitriles; Pancreatic Neoplasms; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Tubulin

Metastasis accounts for the poor outcome of patients with pancreatic cancer. We recently discovered PRSS3 to be over-expressed in metastatic human pancreatic cancer cells. This study aimed to elucidate the role of PRSS3 in the growth and metastasis of human pancreatic cancer.. PRSS3 expression in human pancreatic cancer cell lines was detected by qPCR and immunoblotting. The effect of PRSS3 on cancer cell proliferation, migration and invasion in vitro, tumour growth and metastasis in vivo were investigated by manipulation of PRSS3 expression in human pancreatic cancer cell lines. VEGF expression was detected by ELISA, and the pathway through which PRSS3 regulates VEGF expression was investigated. The therapeutic effect of targeting this pathway on metastasis was assessed in vivo. Immunohistochemistry was employed to detect PRSS3 expression in human pancreatic cancer tissues.. PRSS3 was over-expressed in the metastatic PaTu8988s cell line, but not in the non-metastatic PaTu8988t cell line. Over-expression of PRSS3 promoted pancreatic cancer cell proliferation as well as invasion in vitro, and tumour progression and metastasis in vivo. Stepwise investigations demonstrated that PRSS3 upregulates VEGF expression via the PAR1-mediated ERK pathway. ERK inhibitor significantly delayed the progression of metastases of pancreatic cancer and prolonged the survival of animals bearing metastatic pancreatic cancer (p<0.05). 40.54% of human pancreatic cancers (n=74) were positive for PRSS3 protein. A significant correlation was observed between PRSS3 expression and metastasis (p<0.01). Multivariate Cox regression analysis indicated that patients with PRSS3 expression in their tumours had a shorter survival time compared to those without PRSS3 expression (p<0.05).. PRSS3 plays an important role in the progression, metastasis and prognosis of human pancreatic cancer. Targeting the PRSS3 signalling pathway may be an effective and feasible approach for treatment of this lethal cancer.

Topics: Adult; Aged; Animals; Butadienes; Cell Proliferation; Disease Progression; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Transplantation; Nitriles; Pancreatic Neoplasms; Prognosis; Survival Analysis; Trypsin; Tumor Cells, Cultured; Up-Regulation; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

To explore the signal pathway mediating the regulatory effect of Hepatitis B virus X protein (HBX) on c-met gene promoter in HepG2 cells.. The expression of c-met in HBX-transfected HepG2 cells treated with different signal pathway inhibitors was detected by western blot, the invasion capability of cells was determined by Matrigel invasion assay.. ERK inhibitor U0126 inhibited the expression of the c-Met in HBx-transfected HepG2 cells. However, both p38MAPK inhibitor SB203580 and PI-3K inhibitor wortmanin had no effect on expression of the c-Met in HBx-transfected HepG2 cells. Furthermore, the ERK inhibitor U0126 also inhibited the invasiveness of HBX-transfected HepG2 cells.. HBx induces invasion of HCC via activation of ERK pathway.

Topics: Blotting, Western; Butadienes; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Genetic Vectors; Hep G2 Cells; Hepatitis B virus; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Nitriles; Plasmids; Promoter Regions, Genetic; Proto-Oncogene Proteins c-met; Signal Transduction; Trans-Activators; Transfection; Viral Regulatory and Accessory Proteins

CXCL12 and CXCR4 signaling plays critical roles in development, homeostasis, and tumor metastasis. Previously, we have shown that epigenetic silencing of CXCL12 in colorectal and mammary carcinomas promotes metastasis. Anoikis is an essential process of colonic epithelial turnover and limits the metastatic progression of carcinoma. We sought to determine the role for anoikis in limiting tumor metastasis following reexpression of CXCL12 in human colorectal carcinoma cells.. Tumor formation and metastasis of colonic carcinoma cells was monitored using in vivo bioluminescence imaging. Anoikis was defined by using caspase-3/7, focal adhesion kinase (FAK) and p130Cas cleavage, DNA fragmentation, and cell survival assays. Phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) was monitored by immunoblot and immunohistochemistry, and activity was inhibited by using U0126.. Constitutive expression of CXCL12 in human colorectal carcinoma cells reduced orthotopic tumor formation and inhibited metastasis in severe combined immunodeficient mice. Further, CXCL12 expression induced apoptosis specifically in nonadherent colorectal carcinoma cells. Apoptotic cell death was preceded by hypophosphorylation and cleavage of FAK and p130Cas, leading to increased cellular detachment in culture, and depended on alterations in the extracellular matrix. Similar to in vivo colonic epithelium, CXCL12-induced anoikis of carcinoma cells depended on basal ERK1/2 activation.. These data significantly expand the current paradigm of chemokine signaling in carcinogenesis by showing that endogenous CXCL12, in marked contrast to exogenous ligand, inhibits tumor metastasis through increased anoikis. Altered ERK1/2 signaling provides a mechanism for the dichotomy between the physiologic and pathophysiologic roles of CXCL12-CXCR4 signaling in the intestinal epithelium.

Topics: Animals; Anoikis; Autocrine Communication; Butadienes; Caspase 3; Caspase 7; Cell Adhesion; Cell Survival; Chemokine CXCL12; Colorectal Neoplasms; Crk-Associated Substrate Protein; DNA Fragmentation; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; HCT116 Cells; HT29 Cells; Humans; Immunoblotting; Immunohistochemistry; Luminescent Measurements; Mice; Mice, SCID; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Metastasis; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; Time Factors; Transfection

Cell-cell contacts play important roles in the homeostasis of normal epithelium and in the steps of metastasis of tumor cells, although signaling mechanisms to regulate cell-cell contacts are unclear. In this study, we observed that phenotype of no cell-cell contacts in rat intestinal epithelial cell subline (RIE1-Sca) correlated with increased Erk1/2 signaling activity, compared to that of parental RIE1 cells growing in colonies. Furthermore, cell-cell contacts between RIE1-Sca cells were reformed by treatment with a specific MEK inhibitor (U0126), with translocation of ZO1 and beta-catenin to cell-cell contacts, without changes of their expression levels. U0126 treatment also increased EGFR phosphorylation in a ligand-independent manner. Pretreatment with EGFR kinase inhibitor abolished U0126 treatment-mediated EGFR phosphorylation, and expression of dominant negative H-Ras N17 allowed EGFR phosphorylation and cell-cell contacts even without U0126 treatment. Furthermore, the expression of a nonphosphorylatable EGFR Y5F mutant abolished U0126-mediated cell-cell contacts. U0126 treatment also caused less efficient wound healing by keeping monolayer integrity intact, compared to control untreated cells. This U0126-mediated reduction in wound healing was further altered either by pretreatment of EGFR kinase inhibitor or expression of H-Ras N17 or EGFR Y5F. Taken together, this study supports a unique mechanism of cell-cell contact formation through MEK/Erks inhibition-mediated EGFR phosphorylation.

Topics: Amino Acid Substitution; Animals; beta Catenin; Butadienes; Cell Communication; Cell Line, Tumor; Enzyme Inhibitors; ErbB Receptors; Genes, Dominant; Humans; MAP Kinase Signaling System; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation, Missense; Neoplasm Metastasis; Neoplasms; Nitriles; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); Rats; Wound Healing; Zonula Occludens-1 Protein

Although >66% of melanomas harbor activating mutations in BRAF and exhibit constitutive activity in the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase signaling pathway, it is unclear how effective MEK inhibition will be as a sole therapeutic strategy for melanoma. We investigated the anticancer activity of MEK inhibition in a panel of cell lines derived from radial growth phase (WM35) and vertical growth phase (WM793) of primary melanomas and metastatic melanomas (1205Lu, 451Lu, WM164, and C8161) in a three-dimensional spheroid model and found that the metastatic lines were completely resistant to MEK inhibition (U0126 and PD98059) but the earlier stage cell lines were not. Similarly, these same metastatic melanoma lines were also resistant to inhibitors of the phosphatidylinositol 3-kinase/Akt pathway (LY294002 and wortmannin). Under adherent culture conditions, the MEK inhibitors blocked growth through the induction of cell cycle arrest and up-regulation of p27, but this was readily reversible following inhibitor washout. However, when the phosphatidylinositol 3-kinase and MEK inhibitors were combined, the growth and invasion of the metastatic melanoma three-dimensional spheroids were blocked. Taken together, these results suggest that the most aggressive melanomas are resistant to strategies targeting one signaling pathway and that multiple signaling pathways may need to be targeted for maximal therapeutic efficacy. It is further suggested that BRAF mutational status is not predictive of response to MEK inhibition under three-dimensional culture conditions.

Topics: Butadienes; Cell Line, Tumor; Chromones; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Humans; MAP Kinase Kinase Kinases; Melanoma; Morpholines; Neoplasm Metastasis; Nitriles; Phosphatidylinositol 3-Kinases; Protein Subunits; Signal Transduction; Spheroids, Cellular

Malignant gliomas are highly invasive tumors which are lethal despite aggressive therapy. The motility behavior of two human glioma cell lines i.e. T98G and U87-MG cells was analysed. The glioma cells showed a high degree of basal motility (especially U87-MG cells) that may be related to the considerable local invasiveness of such tumours even in the absence of exogenous factors. The two cell lines responded equally well to platelet-derived growth factor (PDGF) as chemoattractant factor. The phosphatidylinositol 3-kinase (PI3-K) signaling, but not the extracellular signal-related kinase (ERK) signaling, was strongly involved in the PDGF-stimulated glioma cell motility. Somatostatin was capable of inhibiting the migration in both glioma cell lines without affecting crucial targets for motility control like PI3-K and Rac activity. These data suggest that somatostatin, by interfering with a target further downstream to Rac, negatively affects glioma cell motility, and may thus offer a pharmacological approach to controlling the deregulated motility of these aggressive tumoral cells.

Topics: Butadienes; Cell Line, Tumor; Cell Movement; Chemotactic Factors; Chromones; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Glioma; Hormones; Humans; Morpholines; Neoplasm Metastasis; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-akt; rac GTP-Binding Proteins; Somatostatin

Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by (V600E)B-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)-mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant (V600E)B-Raf signaling cascade to inhibit melanoma metastases.

Topics: Amino Acid Substitution; Animals; Butadienes; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Metastasis; Nitriles; Proto-Oncogene Proteins B-raf; RNA, Neoplasm; RNA, Small Interfering; Signal Transduction

Mitogen-activated protein kinase kinase 2 (MEK2) was detected as an invasion-metastasis related factor between highly invasive (PC-1.0) and weakly invasive (PC-1) pancreatic cancer cell lines in our previous study. On the other hand, tight junction (TJ) was found to be correlated with carcino-genesis and tumor development. In this study, the expressions and correlation of TJ transmembrane protein occludin and MEK/extracellular signal-regulated kinase (ERK) signaling pathway were analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and human (AsPC-1 and CAPAN-2) pancreatic cancer cell lines were analyzed immunocytochemically with anti-occludin, phosphorylated MEK1/2 (p-MEK1/2), phosphorylated ERK1/2 (p-ERK1/2) antibodies. MEK1/2 inhibitor U0126 significantly induced the expression of occludin at the cell-cell junction and substantially suppressed the p-MEK1/2 and p-ERK1/2 expressions in PC-1.0 and AsPC-1 cells. In contrast, dissociation factor (DF) treatment obviously disrupted the occludin expressions at the sites of cell-cell junction and markedly induced the p-MEK1/2 and p-ERK1/2 expressions in PC-1 and CAPAN-2 cells. In addition, occludin expressions at cell-cell junction were restored and p-MEK1/2 and p-ERK1/2 expressions were suppressed by subsequent U0126-treatment in DF treated PC-1 and CAPAN-2 cells. Correspondingly, light microscopic images showed that DF induced the dissociation of cell island-like colonies in PC-1 and CAPAN-2 cells, and U0126-treatment induced cell aggregation in these pancreatic cancer cells. Occludin is involved in the cell dissociation in pancreatic cancer cells. Moreover, MEK/ERK signaling pathway probably regulates the cell dissociation status of pancreatic cancer through influencing the intracellular localization and expression of occludin.

Topics: Animals; Butadienes; Cell Line, Tumor; Cricetinae; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Neoplasm Metastasis; Nitriles; Occludin; Pancreatic Neoplasms; Tight Junctions

Primary cancer of the gallbladder is not unusual. Most cases of gallbladder cancer are found at an advanced stage, accompanied by the invasion to the liver, metastases to the lymph nodes and distant organs, and peritoneal dissemination. In this study, we first examined the effect of mitogen-activated protein kinase kinase (MEK) inhibitors on the production of matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA), and tissue inhibitors of metalloproteinases (TIMPs) in a human gallbladder cancer cell line, NOZ cells in vitro. MEK inhibitors (PD98059 and U0126) inhibited the production of MMP-2, MMP-9 and high MW uPA, and upregulated TIMPs (TIMP-1, TIMP-2 and TIMP-3). Subsequently, we examined the effect of U0126 on invasion and metastasis of orthotopically inoculated NOZ cells in nude mice. Direct liver invasion by cancer cells was detected in all of the mice in the control group, but in only one mouse in the U0126-treated group. Most of the primary tumors in the U0126-treated group expanded to the liver, but did not invade into the liver. Vessel invasion in the liver was evident in 4 out of 5 mice in the control group, but in only one mouse in the U0126-treated group. Lymph node metastases and peritoneal dissemination were recognized in all of the mice in both groups. All 5 mice in the U0126-treated group, and 4 out of 5 mice in the vehicle control group, had metastases in the lungs. The present results suggest that a MEK inhibitor, U0126, prolonged the survival of the mice with NOZ tumor by inhibiting direct liver invasion and vessel invasion of the cancer cells via down-regulation of the matrix degrading ability of the cancer cells.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Flavonoids; Gallbladder Neoplasms; Humans; Liver; Liver Neoplasms; Lymphatic Metastasis; MAP Kinase Kinase 1; Mice; Mice, Nude; Models, Anatomic; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Nitriles; Urokinase-Type Plasminogen Activator

Most cases of gallbladder cancer are found at an advanced stage accompanied by invasion to the liver, metastases to the lymph nodes and distant organs, and peritoneal dissemination. Then, the treatment for advanced gallbladder cancer is often ineffective, and the prognosis of this disease is poor. The specific aim of this study was to establish a model system for developing new therapeutic strategies, such as molecular target therapy, for human advanced gallbladder cancer. We used a human gallbladder cancer cell line, NOZ with K-ras mutation in this experiment. Then, we established a novel orthotopic inoculation model for gallbladder cancer by using NOZ cells in nude mice. Mitogen-activated protein kinase (MAPK) in NOZ cells was constitutively activated, and the activation of MAPK provided autonomous proliferation of NOZ cells. All of the mice orthotopically inoculated by NOZ cells developed tumors at the gallbladder. Direct invasion into the liver, and bloody ascites were observed. Metastases to the mediastinal lymph nodes were also recognized in all of the mice examined. Moreover, most of the mice showed lung metastases. Survival duration was 29-50 days after inoculation. Nude mice with NOZ tumor at gallbladder were treated by an intraperitoneal injection of a MAPK kinase inhibitor U0126 (25 micro mole/kg) twice a week. U0126 (p=0.0110, one-way ANOVA) significantly prolonged the survival duration of the mice with NOZ gallbladder tumor. Our orthotopic inoculation model is useful for developing new therapeutic strategies, such as molecular target therapy for advanced gallbladder cancer.

Topics: Animals; Blotting, Western; Butadienes; Cell Division; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Gallbladder; Gallbladder Neoplasms; Genes, ras; Humans; Immunohistochemistry; Liver; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Metastasis; Neoplasm Transplantation; Nitriles; RNA; Transcription, Genetic; Tumor Suppressor Protein p53

Little is known about the genetic regulation of medulloblastoma dissemination, but metastatic medulloblastoma is highly associated with poor outcome. We obtained expression profiles of 23 primary medulloblastomas clinically designated as either metastatic (M+) or non-metastatic (M0) and identified 85 genes whose expression differed significantly between classes. Using a class prediction algorithm based on these genes and a leave-one-out approach, we assigned sample class to these tumors (M+ or M0) with 72% accuracy and to four additional independent tumors with 100% accuracy. We also assigned the metastatic medulloblastoma cell line Daoy to the metastatic class. Notably, platelet-derived growth factor receptor alpha (PDGFRA) and members of the downstream RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway are upregulated in M+ tumors. Immunohistochemical validation on an independent set of tumors shows significant overexpression of PDGFRA in M+ tumors compared to M0 tumors. Using in vitro assays, we show that platelet-derived growth factor alpha (PDGFA) enhances medulloblastoma migration and increases downstream MAP2K1 (MEK1), MAP2K2 (MEK2), MAPK1 (p42 MAPK) and MAPK3 (p44 MAPK) phosphorylation in a dose-dependent manner. Neutralizing antibodies to PDGFRA blocks MAP2K1, MAP2K2 and MAPK1/3 phosphorylation, whereas U0126, a highly specific inhibitor of MAP2K1 and MAP2K2, also blocks MAPK1/3. Both inhibit migration and prevent PDGFA-stimulated migration. These results provide the first insight into the genetic regulation of medulloblastoma metastasis and are the first to suggest a role for PDGFRA and the RAS/MAPK signaling pathway in medulloblastoma metastasis. Inhibitors of PDGFRA and RAS proteins should therefore be considered for investigation as possible novel therapeutic strategies against medulloblastoma.

Topics: Butadienes; Enzyme Activation; Enzyme Inhibitors; Gene Expression Profiling; Humans; Immunohistochemistry; MAP Kinase Signaling System; Medulloblastoma; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Nitriles; Phenotype; Receptor, Platelet-Derived Growth Factor alpha