u-0126 and dactolisib

Cancer cell migration requires that cells respond and adapt to their surroundings. In the absence of extracellular matrix cues, cancer cells will undergo a mesenchymal to ameboid transition, whereas a highly confining space will trigger a switch to "leader bleb-based" migration. To identify oncogenic signaling pathways mediating these transitions, we undertook a targeted screen using clinically useful inhibitors. Elevated Src activity was found to change actin and focal adhesion dynamics, whereas inhibiting Src triggered focal adhesion disassembly and blebbing. On non-adherent substrates and in collagen matrices, amoeboid-like, blebbing cells having high Src activity formed protrusions of the plasma membrane. To evaluate the role of Src in confined cells, we use a novel approach that places cells under a slab of polydimethylsiloxane (PDMS), which is held at a defined height. Using this method, we find that leader bleb-based migration is resistant to Src inhibition. High Src activity was found to markedly change the architecture of cortical actomyosin, reduce cell mechanical properties, and the percentage of cells that undergo leader bleb-based migration. Thus, Src is a signal transducer that can potently influence transitions between migration modes with implications for the rational development of metastasis inhibitors.

Topics: Actins; Amino Acid Substitution; Butadienes; Cell Adhesion; Cell Movement; Dasatinib; Humans; Imidazoles; Mutant Proteins; Neoplasms; Nitriles; Phosphorylcholine; Protein Kinase Inhibitors; Proto-Oncogene Proteins pp60(c-src); Quinolines; Tumor Cells, Cultured

Radiation therapy is a key therapeutic strategy for endometrial carcinomas. However, biomarkers that predict radiosensitivity and drugs to enhance this sensitivity have not yet been established. We aimed to investigate the roles of TP53 and MAPK/PI3K pathways in endometrial carcinomas and to identify appropriate radiosensitizing therapeutics. D10 values (the irradiating dose required to reduce a cell population by 90%) were determined in eight endometrial cancer cell lines with known mutational statuses for TP53, PIK3CA, and KRAS. Cells were exposed to ionizing radiation (2-6Gy) and either a dual PI3K/mTOR inhibitor (NVP-BEZ235) or a MEK inhibitor (UO126), and their radiosensitizing effects were evaluated using clonogenic assays. The effects of silencing hypoxia-inducible factor-1 α (HIF-1α) expression with small interfering RNAs (siRNAs) were evaluated following exposure to ionizing radiation (2-3Gy). D10 values ranged from 2.0 to 3.1Gy in three cell lines expressing wild-type TP53 or from 3.3 to more than 6.0Gy in five cell lines expressing mutant TP53. NVP-BEZ235, but not UO126, significantly improved radiosensitivity through the suppression of HIF-1α/vascular endothelial growth factor-A expression. HIF-1α silencing significantly increased the induction of the sub-G1 population by ionizing radiation. Our study data suggest that TP53 mutation and PI3K pathway activation enhances radioresistance in endometrial carcinomas and that targeting the PI3K/mTOR or HIF-1α pathways could improve radiosensitivity.

Topics: Butadienes; Carcinoma, Endometrioid; Cell Line, Tumor; Endometrial Neoplasms; Enzyme Inhibitors; Female; Genes, p53; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Radiation Tolerance; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Epidermal growth factor (EGF) and their receptor (EGFR) play an important role in the development of cancer proliferation, and metastasis, although the mechanism remains unclear. The present study aimed at investigating the role of EGF-EGFR signalling pathway in the development of human hepatocellular carcinoma (HCC) inflammatory environment. Gene profiles of inflammatory cytokines from HCC were measured. Cell bio-behaviours of HCC with low or high metastasis were detected by the live cell monitoring system. Cell proliferation was measured by CCK8. The protein level of CXCL5 and CXCL8 was measured by ELISA. The phosphorylation of PI3K, ERK, MAPK was measured by western blot. EGF significantly induced cell proliferation in HepG2 cells, but not in HCCLM3 cells. EGF prompted the cell movement in both HepG2 and HCCLM3 and regulated the production of CXCL5 and CXCL8 from HCC, which were inhibited by EGFR inhibitor, Erk inhibitor (U0126), or PI3K inhibitors (BEZ-235 and SHBM1009). HCC proliferation, metastasis and production of inflammatory cytokines were regulated via EGF-EGFR signal pathways. CXCL5 could interact with CXCL8, possibly by CXCR2 or the cross-talk between CXCR2 and EGFR. EGF-EGFR signaling pathway can be the potential target of therapies for HCC.

Topics: Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CXCL5; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Inflammation; Interleukin-8; Liver Neoplasms; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Quinolines; Receptor Cross-Talk; Receptors, Interleukin-8B; Signal Transduction; Tumor Microenvironment

Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with those of asbestos-associated disease. We previously demonstrated co-activation of multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET, and AXL in mesothelioma cell lines, suggesting that these kinases could serve as novel therapeutic targets. Although clinical trials have not shown activity for EGFR inhibitors in mesothelioma, concurrent inhibition of various activated RTKs has pro-apoptotic and anti-proliferative effects in mesothelioma cell lines. Thus, we hypothesised that a coordinated network of multi-RTK activation contributes to mesothelioma tumorigenesis.. Activation of PI3K/AKT/mTOR, Raf/MAPK, and co-activation of RTKs were evaluated in mesotheliomas. Effects of RTK and downstream inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, apoptosis, activation of signalling intermediates, expression of cell-cycle checkpoints, and cell-cycle alterations.. We demonstrate activation of the PI3K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, but not in non-neoplastic mesothelial cells. The AKT activation, but not MAPK activation, was dependent on coordinated activation of RTKs EGFR, MET, and AXL. In addition, PI3K/AKT/mTOR pathway inhibition recapitulated the anti-proliferative effects of concurrent inhibition of EGFR, MET, and AXL. Dual targeting of PI3K/mTOR by BEZ235 or a combination of RAD001 and AKT knockdown had a greater effect on mesothelioma proliferation and viability than inhibition of individual activated RTKs or downstream signalling intermediates. Inhibition of PI3K/AKT was also associated with MDM2-p53 cell-cycle regulation.. These findings show that PI3K/AKT/mTOR is a crucial survival pathway downstream of multiple activated RTKs in mesothelioma, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention.

Topics: Antineoplastic Agents; Butadienes; Cell Cycle; Cell Line, Tumor; Chromones; Drug Screening Assays, Antitumor; Enzyme Activation; Everolimus; Humans; Imidazoles; Indazoles; MAP Kinase Signaling System; Mesothelioma; Molecular Targeted Therapy; Morpholines; Neoplasm Proteins; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; raf Kinases; Receptor Protein-Tyrosine Kinases; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

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

To study the inhibitory effect of the dual usage of BEZ235 and U0126, the inhibitor of phosphatidyl inositol-3-kinase/protein kinase B pathway and extracellular regulated proteinkinase/mitogen-activated protein kinase pathway, respectively, on cell proliferation.. Phosphatase and tensin homolog knockout mouse embryonic fibroblast (PTEN-/-MEF) cell lines were used as the cellular model for malignant tumors. BEZ235, the dual inhibitor of phosphatidyl inositol-3-kinase and mammalian target of rapamycin, and U0126, the inhibitor of mitogen-activated protein kinase were used to treat the cells individually and in a combination manner. The inhibitory effects to cell proliferation were monitored by MTT.. Both BEZ235 and U0126 suppressed PTEN knockout cell proliferation, and their half inhibitory concentrations were 6.257 nmol/L and 22.85 μmol/L, respectively. However, the combination treatment of the two drugs showed antagonistic rather than synergistic effect on cell proliferation.. BEZ235 and U0126 are not suitable for a combined target therapy regimen.

Topics: Animals; Butadienes; Cell Line; Cell Proliferation; Drug Antagonism; Fibroblasts; Imidazoles; Mice; Mice, Knockout; Nitriles; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Quinolines

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.

Topics: Angiogenesis Inhibitors; Animals; Butadienes; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Humans; Imidazoles; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Morpholines; Multiprotein Complexes; Neoplasms; Neovascularization, Pathologic; Nitriles; Proteins; Pyrimidines; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

The molecular signaling pathways orchestrating the biology of cancer stem-like cells (CSLCs), including glioblastoma, remain to be elucidated. We investigated in this study the role of the MEK/extracellular signal-regulated kinase (ERK) pathway in the control of self-renewal and tumorigenicity of glioblastoma CSLCs, particularly in relation to the PI3K/mTOR (mammalian target of rapamycin) pathway. Targeted inactivation of MEK alone using pharmacological inhibitors or siRNAs resulted in reduced sphere formation of both cell line- and patient-derived glioblastoma CSLCs, accompanied by their differentiation into neuronal and glial lineages. Interestingly, this effect of MEK inactivation was apparently augmented in the presence of NVP-BEZ235, a dual inhibitor of PI3K and mTOR. As a potential explanation for this observed synergy, we found that inactivation of either the MEK/ERK or PI3K/mTOR pathway triggered activation of the other, suggesting that there may be mutually inhibitory crosstalk between these two pathways. Significantly, inactivation of either pathway led to the reduced activation of p70S6K, and siRNA-mediated knockdown of p70S6K resulted in the activation of both pathways, which no longer maintained the cross-inhibitory relationship. Finally, combinational blockade of both pathways in glioblastoma CSLCs suppressed their tumorigenicity, whether transplanted subcutaneously or intracranially, more efficiently than blockade of either alone. Our findings suggest that there is p70S6K-mediated, cross-inhibitory regulation between the MEK/ERK and PI3K/mTOR pathways, in which each contribute to the maintenance of the self-renewal and tumorigenic capacity of glioblastoma CSLCs. Thus, combinational disruption of these pathways would be a rational and effective strategy in the treatment of glioblastoma.

Topics: Aminoacetonitrile; Animals; Butadienes; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; Imidazoles; Male; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Neoplastic Stem Cells; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Cells, Cultured