u-0126 and Carcinoma

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

Nasopharyngeal carcinoma (NPC) is a common malignancy in southern China and Southeast Asia. NPC frequently metastasizes to the bone in advanced patients resulting in high mortality. The molecular mechanisms for NPC development and cancer-induced bone lesions are unclear. In this study, we firstly determined chemokine receptor CCR2 and CXCR6 expressions in clinical specimens and CNE2, SUNE1, CNE1, and HK1 cell lines. Then, we measured chemokine CCL2 and CXCL16 production in these NPC cell lines by ELISA. Expression levels of these chemokines and their receptors were observed to positively correlate with tumor aggressiveness. Furthermore, U0126 (MEK inhibitor) was used to treat these NPC cell lines. CCL2 and CXCL16 expression levels and cell proliferation were significantly inhibited by U0126 in a dose- and time-dependent manner. Finally, we collected conditioned medium (CM) from NPC cell cultures in the presence of U0126 treatment. When mouse bone marrow non-adherent cells were treated with the CM, the numbers of multinucleated osteoclast formation were dramatically diminished. These results indicate that MEK inhibitor diminishes NPC cell proliferation and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expressions. This study provides novel therapeutic targets such as CCL2/CCR2 and CXCL16/CXCR6 for advanced NPC patients.

Topics: Animals; Bone Marrow Cells; Butadienes; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chemokine CCL2; Chemokine CXCL16; Chemokines, CXC; Culture Media, Conditioned; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinases; Mice; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Nitriles; Osteoclasts; Protein Kinase Inhibitors; Receptors, Scavenger

Nedaplatin, a cisplatin analog, was developed to reduce the toxicity of cisplatin, whereas it can be cross-resistant with cisplatin in some circumstances. This study aimed to investigate the role of autophagy in nedaplatin induced cell death in cisplatin-resistant nasopharyngeal carcinoma cells. Here, we showed that HNE1/DDP and CNE2/DDP cells were resistant to nedaplatin-induced cell death with reduced apoptotic activity. Nedaplatin treatment resulted in autophagosome accumulation and increased expression of LC3-II, indicating the induction of autophagy by nedaplatin in HNE1/DDP and CNE2/DDP cells. Inhibition of autophagy by Bafilomycin A1 (Baf A1) and 3-Methyladenine (3-MA) remarkably enhanced the antitumor efficacy of nedaplatin in HNE1/DDP and CNE2/DDP cells, suggesting that the resistance to nedaplatin-induced cell death was caused by enhanced autophagy in nedaplatin-resistant NPC cells. Additionally, Baf A1 enhanced reactive oxygen species (ROS) generation and apoptosis induced by nedaplatin in HNE1/DDP cells. Mechanistically, nedaplatin treatment caused activation of ERK1/2 and suppression of Akt/mTOR signaling pathways. While inhibition of ERK1/2 by MEK1/2 inhibitor, U0126, could reduce the expression of LC3-II in nedaplatin-resistant NPC cells. Furthermore, suppression of ROS could inhibit nedaplatin-induced ERK activation in HNE1/DDP cells, indicating that ROS and ERK were involved in nedaplatin-induced autophagy. Together, these findings suggested that autophagy played a cytoprotective role in nedaplatin-induced cytotoxicity of HNE1/DDP and CNE2/DDP cells. Furthermore, our results highlighted a potential approach to restore the sensitivity of cisplatin-resistant nasopharyngeal cancer cells to nedaplatin in combination with autophagy inhibitors.

Topics: Adenine; Antineoplastic Agents; Apoptosis; Autophagy; Butadienes; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Drug Resistance, Neoplasm; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Macrolides; Microtubule-Associated Proteins; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Nitriles; Organoplatinum Compounds; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; TOR Serine-Threonine Kinases

Tumor microenvironment influences targeted drug therapy. In this study we compared drug responses to RAF and MEK inhibitors on tumor cell migration in 2D and 3D culture of BRAF(V600E) mutant cell lines derived from human papillary (BCPAP) and anaplastic (SW1736) thyroid carcinomas. Scratch wounding was compared to a double-layered collagen gel model developed for analysis of directed tumor cell invasion during prolonged culture. In BCPAP both PLX4720 and U0126 inhibited growth and migration in 2D and decreased tumor cell survival in 3D. In SW1736 drugs had no effect on migration in 2D but decreased invasion in 3D, however this related to reduced growth. Dual inhibition of BRAF(V600E) and MEK reduced but did not prevent SW1736 invasion although rebound phosphorylation of ERK in response to PLX4720 was blocked by U0126. These findings indicate that anti-tumor drug effects in vitro differ depending on culture conditions (2D vs. 3D) and that the invasive features of anaplastic thyroid cancer depend on non-MEK mechanism(s).

Topics: Antineoplastic Agents; Butadienes; Carcinoma; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Survival; Humans; Indoles; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Invasiveness; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides; Thyroid Carcinoma, Anaplastic

The aim of the present study is to investigate the relationship between urinary-type plasminogen activator (uPA) expression and clinicopathological features in papillary thyroid carcinoma (PTC) and to determine the signal transduction of PTC cells in vitro. PTC tissues from 42 patients were analyzed for the expression of uPA and the BRAF(V600E) mutation. BCPAP, a PTC cell line harboring the BRAF(V600E) mutation, was used to study MAPK signaling. PCR and direct sequencing were applied to analyze BRAF(V600E) mutation status. uPA mRNA expression was measured using a quantitative RT-PCR method, and uPA protein was localized using an immunohistochemical method. The ERK protein status was detected by Western blot analysis. uPA gene expression was significantly increased in PTC tissues as compared to the corresponding non-tumor tissues. Furthermore, the up-regulation of uPA mRNAs was correlated with high-risk clinicopathological features, including extrathyroid invasion, loss of cellular polarity/cohesiveness, and the BRAF(V600E) mutation. Marked dephosphorylation of ERK1/2 and down-regulation of uPA expression were detected when BCPAP was treated with a MEK inhibitor, U0126. MEK inhibitors might be a potential treatment strategy for aggressive PTC with BRAF(V600E) through inhibition of uPA expression.

Topics: Adult; Aged; Butadienes; Carcinoma; Carcinoma, Papillary; Down-Regulation; Enzyme Inhibitors; Female; Humans; Male; MAP Kinase Signaling System; Middle Aged; Mutation; Nitriles; Phosphorylation; Proto-Oncogene Proteins B-raf; Thyroid Cancer, Papillary; Thyroid Neoplasms; Up-Regulation; Urokinase-Type Plasminogen Activator

BRAF is a main oncogene in human thyroid cancer. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with BRAF inhibitor PLX4720 decreases migration and invasion in thyroid cancer cells expressing oncogenic (V600E)BRAF through a MEK/ERK-dependent mechanism, since treatment with the MEK inhibitor U0126 exerts the same effect. Moreover, over-expression of (V600E)BRAF increases migration and invasion of wild-type BRAF thyroid cells. Using the same strategies, we demonstrate that these effects are mediated by upregulation of the transcriptional repressor Snail with a concomitant decrease of its target E-cadherin, both hallmarks of EMT. These results reveal a novel (V600E)BRAF-induced mechanism in thyroid tumours progression and provides a rationale for using the PLX4720 inhibitor to target (V600E)BRAF signalling to effectively control progression of thyroid cancer.

Topics: Antigens, CD; Butadienes; Cadherins; Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; Cell Movement; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Indoles; MAP Kinase Kinase Kinases; Mutation, Missense; Neoplasm Invasiveness; Nitriles; Proto-Oncogene Proteins B-raf; RNA, Small Interfering; Snail Family Transcription Factors; Sulfonamides; Thyroid Cancer, Papillary; Thyroid Neoplasms; Transcription Factors

Phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells and thus has been considered as a promising drug target. To ascertain a therapeutical approach of nasopharyngeal carcinoma (NPC), we hypothesized NVP-BEZ235, a novel and potent imidazo[4,5-c] quinolone derivative, that dually inhibits both PI3K and mTOR kinases activities, had antitumor activity in NPC. Expectedly, we found that NVP-BEZ235 selectively inhibited proliferation of NPC cells rather than normal nasopharyngeal cells using MTT assay. In NPC cell lines, with the extended exposure, NVP-BEZ235 selectively inhibited proliferation of NPC cells harboring PIK3CA mutation, compared to cells with wild-type PIK3CA. Furthermore, exposure of NPC cells to NVP-BEZ235 resulted in G1 growth arrest by Propidium iodide uptake assay, reduction of cyclin D1and CDK4, and increased levels of P27 and P21 by Western blotting, but negligible apoptosis. Moreover, we found that cisplatin (CDDP) activated PI3K/AKT and mTORC1 pathways and NVP-BEZ235 alleviated the activation by CDDP through dually targeting PI3K and mTOR kinases. Also, NVP-BEZ235 combining with CDDP synergistically inhibited proliferation and induced apoptosis in NPC cells. In CNE2 and HONE1 nude mice xenograft models, orally NVP-BEZ235 efficiently attenuated tumor growth with no obvious toxicity. In combination with NVP-BEZ235 and CDDP, there was dramatic synergy in shrinking tumor volumes and inducing apoptosis through increasing Noxa, Bax and decreasing Mcl-1, Bcl-2. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential as a monotherapy or in combination with CDDP for NPC treatment.

Topics: Animals; Apoptosis; Blotting, Western; Butadienes; Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cisplatin; Enzyme Inhibitors; Female; Humans; Imidazoles; Male; Mice; Mice, Inbred BALB C; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

More than 25% of patients diagnosed with endometrial carcinoma have an invasive primary cancer accompanied by metastases. Gonadotropin-releasing hormone (GnRH) plays an important role in reproduction. In mammals, expression of GnRH-II is higher than GnRH-I in reproductive tissues. Here, we examined the effect of a GnRH-II agonist on the motility of endometrial cancer cells and its mechanism of action in endometrial cancer therapy.. Immunoblotting and immunohistochemistry (IHC) were used to determine the expression of the GnRH-I receptor protein in human endometrial cancer. The activity of MMP-2 in the conditioned medium was determined by gelatin zymography. Cell motility was assessed by invasion and migration assay. GnRH-I receptor si-RNA was applied to knockdown GnRH-I receptor.. The GnRH-I receptor was expressed in the endometrial cancer cells. The GnRH-II agonist promoted cell motility in a dose-dependent manner. The GnRH-II agonist induced the phosphorylation of ERK1/2 and JNK, and the phosphorylation was abolished by ERK1/2 inhibitor (U0126) and the JNK inhibitor (SP600125). Cell motility promoted by GnRH-II agonist was suppressed in cells that were pretreated with U0126 and SP600125. Moreover, U0126 and SP600125 abolished the GnRH-II agonist-induced activation of MMP-2. The inhibition of MMP-2 with MMP-2 inhibitor (OA-Hy) suppressed the increase in cell motility in response to the GnRH-II agonist. Enhanced cell motility mediated by GnRH-II agonist was also suppressed by the knockdown of the endogenous GnRH-I receptor using siRNA.. Our study indicates that GnRH-II agonist promoted cell motility of endometrial cancer cells through the GnRH-I receptor via the phosphorylation of ERK1/2 and JNK, and the subsequent, MAPK-dependent activation of MMP-2. Our findings represent a new concept regarding the mechanism of GnRH-II-induced cell motility in endometrial cancer cells and suggest the possibility of exploring GnRH-II as a potential therapeutic target for the treatment of human endometrial cancer.

Topics: Anthracenes; Butadienes; Carcinoma; Cell Line, Tumor; Cell Movement; Endometrial Neoplasms; Enzyme Activation; Female; Gene Knockdown Techniques; Gonadotropin-Releasing Hormone; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Nitriles; Phosphorylation; Receptors, LHRH

Aquaporins (AQPs) are a family of small, integral membrane proteins that have been shown to play an important role in tumor development and metastasis. Several studies have demonstrated that expression of AQP3 contributes to the enhanced migration of epithelial cells and is related to differentiation, metastasis and vascular invasion in lung and gastric cancer. Therefore, we investigated whether AQP3 could enhance human colorectal carcinoma cell migration and we examined the role of AQP3 in the prognosis of colorectal carcinoma. Our results showed that human epidermal growth factor (hEGF) increased the expression of AQP3 and, subsequently, the migration ability of human colorectal carcinoma cells HCT116 in a dose- and time-dependent manner. The enhanced migration ability of HCT116 cells was blocked by the AQP3 inhibitor, CuSO(4). Overexpression of AQP3 induced by hEGF was inhibited by a PI3K/AKT inhibitor, LY294002, but the ERK inhibitor U0126 had a minor effect on the hEGF-induced AQP3 upregulation. Immunohistochemical staining of the cancer tissues and corresponding normal tissues showed that AQP3 expression in cancer tissue was higher compared to that in normal tissue. The expression intensity of AQP3 was associated with the differentiation, lymph node and distant metastasis of colorectal carcinoma patients. Our results suggest that AQP3 overexpression could facilitate colorectal carcinoma cell migration and AQP3 may be considered a potential indicator and therapeutic target for colon tumor metastasis and prognosis.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Aquaporin 3; Butadienes; Carcinoma; Cell Movement; Chi-Square Distribution; Chromones; Colorectal Neoplasms; Copper Sulfate; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; HCT116 Cells; Humans; Lymphatic Metastasis; Male; MAP Kinase Signaling System; Middle Aged; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; Up-Regulation

Thyroid hormone regulates a wide range of cellular activities, including the balance between cell proliferation and differentiation. The thyroid-hormone-inactivating type 3 deiodinase (DIO3, D3) has been shown to be reactivated in human neoplasias. Here, we evaluated DIO3 expression in human papillary thyroid carcinoma (PTC).. Tumor and surrounding normal thyroid tissue were collected from 26 unselected patients with PTC. Clinical data were retrospectively reviewed in medical records. DIO3 mRNA levels were measured by real-time polymerase chain reaction and D3 activity by paper-descendent chromatography. Studies of DIO3 gene regulation were performed in a human PTC-derived cell line (K1 cells). BRAF(V600E) mutation was identified in DNA from paraffin-embedded tissues by direct sequencing. Immunohistochemistry analyses were performed using a specific human D3 antibody.. Increased D3 activity was detected in all 26 PTC samples analyzed as compared with adjacent thyroid tissue. The augmentations in D3 activity were paralleled by increased DIO3 mRNA levels (approximately fivefold). In PTC-derived cells, DIO3 transcripts were further upregulated by the transforming growth factor β1 (TGFβ1). Interestingly, preincubation with mitogen-activated protein kinase (MAPK) cascade inhibitors U0126 (ERK pathway) and SB203580 (p38 pathway) decreased DIO3 mRNA levels and blocked the TGFβ1-induced increase in DIO3 transcripts, suggesting that D3 induction might be mediated through the MAPK signaling pathway. Accordingly, DIO3 mRNA and activity levels were significantly higher in BRAF(V600E)-mutated samples (p=0.001). Increased D3 activity was correlated with tumor size (r=0.68, p=0.003), and associated with lymph node (p=0.03) or distant metastasis (p=0.006) at diagnosis. Conversely, decreased levels of the thyroid-hormone-activating type 2 deiodinase (DIO2) gene were observed in PTC, which might contribute to further decreases in intracellular thyroid hormone levels. Increased D3 expression was also observed in follicular thyroid carcinoma but not in medullary or anaplastic thyroid carcinoma samples.. These results indicate that the malignant transformation of thyroid follicular cell toward PTC promotes opposite changes in DIO3 and DIO2 expression by pretranscriptional mechanisms. The association between increased levels of D3 activity and advanced disease further supports a role for intracellular triiodothyronine concentration on the thyroid tumor cell proliferation or/and dedifferentiation.

Topics: Adult; Butadienes; Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; Child; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Immunohistochemistry; Iodide Peroxidase; Male; MAP Kinase Signaling System; Middle Aged; Mutation; Nitriles; Proto-Oncogene Proteins B-raf; Pyridines; Retrospective Studies; Thyroid Cancer, Papillary; Thyroid Neoplasms; Transforming Growth Factor beta1; Young Adult

Human potassium chloride cotransporter-1 (KCC1) gene is expressed in endometrial cancer and related to metastasis of endometrial cancer. However, whether KCC1 contributes to invasion and metastasis of endometrial cancer has not been thoroughly investigated. The purpose of this study is to research the alternation effect of insulin-like growth factor I (IGF-I) on the expression of KCC1 in endometrial cancer HEC-1B cells and to explore the mechanism of how KCC1 regulates the invasion ability of HEC-1B cells through the extracellular signal-regulated kinase (ERK) signaling pathway.. First, the inhibitive effect of RNA interference to KCC1 was detected by semiquantitative reverse transcriptase-polymerase chain reaction. Western blot was used to measure expression changes of KCC1 after exposure to IGF-I in the HEC-1B cells. The change in quantity of phosphorylated ERK1/2 (p-ERK1/2) and cell invasion ability also were measured. After RNA interference and treatment with U0126, the quantity of p-ERK1/2 and the cell invasion ability were measured again.. After the application of IGF-I on the HEC-1B cells, the expression of KCC1 and p-ERK1/2 increased dramatically, and the cell invasion ability advanced. RNA interference could inhibit the expression of KCC1, and the quantity of p-ERK1/2 and the cell invasion ability decreased even under the effect of IGF-I. Furthermore, after treatment with U0126, the cell invasion ability no longer advanced even under the effect of IGF-I either.. Insulin-like growth factors I can induce the upregulation of KCC1 gene, and KCC1 gene participates in the invasion ability of HEC-1B cells through the ERK signaling pathway.

Topics: Butadienes; Carcinoma; Cell Adhesion; Cell Line, Tumor; Cell Movement; Drug Evaluation, Preclinical; Endometrial Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; K Cl- Cotransporters; MAP Kinase Kinase 1; MAP Kinase Signaling System; Neoplasm Invasiveness; Nitriles; Protein Kinase Inhibitors; Symporters; Up-Regulation

Integrins play an important role in tumor metastasis induced by vascular endothelial growth factor (VEGF). However, in the case of gastric cancer, the precise role of VEGF in regulating integrin alphavbeta6 is unclear. In this study, we found that most of the alphavbeta6 integrin-positive gastric cancer tissues were also VEGF-positive. Furthermore, when gastric carcinoma cells were exposed to VEGF, expression of alphavbeta6 integrin was up-regulated and the extracellular signal-related kinase (ERK) pathway was activated. When integrin alphavbeta6 was blocked either with beta6 siRNA or anti-alphavbeta6 antibody, the migration of tumor cells normally induced by VEGF, as well as the activation of ERK, were markedly inhibited. Blocking the ERK signaling pathway significantly inhibited cell mobility. Taken together, the data suggest that VEGF is critical to the invasive process in human gastric cancer and that this occurs via up-regulation of integrin alphavbeta6 expression and activation of ERK.

Topics: Antibodies; Antigens, Neoplasm; Butadienes; Carcinoma; Cell Line, Tumor; Cell Movement; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Integrins; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nitriles; Protein Kinase Inhibitors; Recombinant Proteins; RNA Interference; RNA, Messenger; Stomach Neoplasms; Transfection; Vascular Endothelial Growth Factor A

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway plays an important role in papillary and anaplastic thyroid cancer (PTC and ATC) due to activating mutations in BRAF, RAS, or rearrangements in RET/PTC1. The objective of this study was to thoroughly test whether the BRAF V600E mutation predicts response to mitogen-activated protein kinase kinase 1/2 (MKK1/2) inhibition, as shown in other tumor types, using an authenticated panel of thyroid cancer cell lines.. PTC and ATC cells harboring distinct mutations in the MAPK pathway were treated with two different inhibitors selective for MKK1/2 (CI-1040 or U0126). The consequences of MKK1/2 inhibition on cell growth, survival, invasion, and MAPK signaling was determined.. Inhibition of MKK1/2 using CI-1040 or U0126 differentially inhibits the growth of a panel of PTC and ATC cell lines in two-dimensional culture, with those harboring the BRAF V600E mutation (SW1736) or BRAF-V600E/PI3K-E542K mutations (K1) being the most sensitive, the RET/PTC1 rearrangement (TPC1) and BRAF V600E mutant (BCPAP), intermediate, and the HRAS-G13R mutant (C643), the least sensitive. Growth of these cells is more sensitive to MKK1/2 inhibition when grown in 2% versus 10% serum. Baseline levels of phospho-ERK1/2 were similar in all of the cell lines, and inhibition phospho-ERK1/2 did not predict sensitivity to MKK1/2 inhibition. When cells are grown in three-dimensional culture, MKK1/2 inhibition of growth correlates with mutational status (BRAF > RET/PTC1 > RAS). Finally, PTC and ATC invasiveness is differentially inhibited by CI-1040, which is independent of tumor type or mutation present.. Different mutations in the MAPK pathway play distinct roles in the growth and invasion of thyroid cancer cells. These results indicate that MKK1/2 inhibitors have the potential to inhibit thyroid cancer growth and invasion, but that responses differ based on mutation status and growth conditions.

Topics: Benzamides; Butadienes; Carcinoma; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mutation; Nitriles; Signal Transduction; Thyroid Neoplasms

Statins, which are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, suppress cell proliferation and induce apoptosis in various cancer cell lines. However, the effects of statins in head and neck carcinoma have not been reported. In this study, we investigated the mechanism by which fluvastatin induces apoptosis in HSC-3 cells. An increase in caspase-3 activity was observed. The apoptosis induced by fluvastatin was inhibited by the addition of geranylgeranyl pyrophosphate (GGPP) to the cell culture. When we examined the survival signals at the time of apoptotic induction, we also found that fluvastatin had caused a remarkable decrease in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Moreover, we also found that U0126, a MEK1/2 inhibitor, induces apoptosis in HSC-3 cells. These results suggested that fluvastatin induces apoptosis by inhibiting GGPP biosynthesis and consequently decreasing the level of phosphorylated ERK1/2. The results of this study also indicate that fluvastatin may be used as an anticancer agent for tongue carcinoma.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Carcinoma; Cell Line, Tumor; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fatty Acids, Monounsaturated; Fluvastatin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Nitriles; Phosphorylation; Polyisoprenyl Phosphates; Tongue Neoplasms

One of the intriguing questions regarding cell motility concerns the mechanism that makes stationary cells move. Here, we provide the first physical evidence that the onset of breast cancer cell motility in response to insulin-like growth factor I (IGF-I) correlates with lowering of adhesion strength from 2.52 +/- 0.20 to 1.52 +/- 0.13 microdynes/microm2 in cells attached to fibronectin via alpha5beta1 integrin. The adhesion strength depends on the dose of IGF-I and time of IGF-I treatment. Weakening of cell-matrix adhesion is blocked significantly (p < 0.01) by the catalytically inactive IGF-I receptor (IGF-IR) and the phosphoinositide 3-kinase (PI-3 kinase) inhibitor LY-294002, but it is unaffected by mitogen-activated protein kinase kinase inhibitor UO-126 and Src kinase inhibitor PP2. Sustained blockade of Rho-associated kinase (ROCK) with Y-27632 down-regulates adhesion strength in stationary, but not in IGF-I-treated, cells. Jasplakinolide, a drug that prevents actin filament disassembly, counteracts the effect of IGF-I on integrin-mediated cell adhesion. In the absence of growth factor signaling, ROCK supports a strong adhesion via alpha5beta1 integrin, whereas activation of the IGF-IR kinase reduces cell-matrix adhesion through a PI-3K-dependent, but ROCK-independent, mechanism. We propose that disassembly of the actin filaments via PI-3 kinase pathway contributes to weakening of adhesion strength and induction of cell movement. Understanding how cell adhesion and migration are coordinated has an important application in cancer research, developmental biology, and tissue bioengineering.

Topics: Actins; Amides; Blotting, Western; Breast Neoplasms; Butadienes; Carcinoma; Catalysis; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chromones; CSK Tyrosine-Protein Kinase; Culture Media, Serum-Free; Depsipeptides; Down-Regulation; Fibronectins; Flow Cytometry; Humans; Immunoprecipitation; Insulin-Like Growth Factor I; Integrin alpha5beta1; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Microscopy, Phase-Contrast; Models, Biological; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Pyridines; Pyrimidines; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; src-Family Kinases; Time Factors

Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.

Topics: Bone Neoplasms; Butadienes; Carcinoma; Chondrosarcoma; Depression, Chemical; Enzyme Activation; Enzyme Inhibitors; Female; Flavonoids; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; Membrane Glycoproteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nitriles; Ovarian Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Recombinant Fusion Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Transfection; Trypsin Inhibitor, Kunitz Soybean; Tumor Cells, Cultured

Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.

Topics: Antineoplastic Agents; Butadienes; Carcinoma; Enzyme Inhibitors; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-met; Quinazolines; RNA, Messenger; Suramin; Thyroid Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth an

Topics: Antibodies; Breast Neoplasms; Butadienes; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Carcinoma, Squamous Cell; Cell Death; Cell Division; DNA, Antisense; Dose-Response Relationship, Radiation; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Protein Kinases; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine