Effects of in vivo exposure to UV filters (4-MBC, OMC, BP-3, 4-HB, OC, OD-PABA) on endocrine signaling genes in the insect Chironomus riparius.

Effects of in vivo exposure to UV filters (4-MBC, OMC, BP-3, 4-HB, OC, OD-PABA) on endocrine signaling genes in the insect Chironomus riparius.

Sci Total Environ. 2013 Apr 13;456-457C:120-126

Authors: Oz�ez I, Mart�nez-Guitarte JL, Morcillo G

Abstract
There is increasing evidence indicating that several UV filters might have endocrine disruptive effects. Numerous studies have evaluated hormonal effects in vertebrates, mainly reporting estrogenic and androgenic activities in mammals and fishes. There is only limited knowledge about potential endocrine activity in invertebrate hormonal systems. In this work, the effects on endocrine signaling genes of six frequently used UV filters were investigated in Chironomus riparius, a reference organism in aquatic toxicology. The UV filters studied were: octyl-p-methoxycinnamate (OMC) also called 2-ethylhexyl-4-methoxycinnamate (EHMC); 4-methylbenzylidene camphor (4-MBC); benzophenone-3 (BP-3); 4-hidroxybenzophenone (4-HB); octocrylene (OC); and octyldimethyl-p-aminobenzoate (OD-PABA). After in vivo exposure at different dosages, expression levels of the genes coding for the ecdysone receptor (EcR), the ultraspiracle (usp, ortholog of the RXR) and the estrogen-related receptor (ERR) were quantified by Real Time PCR. The EcR gene was significantly upregulated by 4-MBC, OMC/EHMC and OD-PABA, with a dose-related response following 24h exposure. In contrast, the benzophenones, BP-3 and 4-HB, as well as OC did not alter this gene at the same exposure conditions. The transcription profiles of the usp and ERR genes were not significantly affected, except for BP-3 that inhibited the usp gene at the highest concentration. To our knowledge, this is the first experimental evidence in invertebrates of a direct effect of UV filters on endocrine-related genes, and is consistent with the known effects on vertebrate hormonal receptor genes. The capability of 4-MBC, OMC/EHMC and OD-PABA to stimulate the expression of the ecdysone receptor, a key transcription factor for the ecdysone-genomic response in arthropods, suggests the possibility of a broad and long-term effect on this hormonal pathway. These findings strengthen the need for further research about the ecotoxicological implications of chronic exposure to these compounds in aquatic invertebrates.

PMID: 23591065 [PubMed - as supplied by publisher]

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Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

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Cytotoxic Activity of Tivantinib (ARQ 197) Is Not Due Solely to c-MET Inhibition.

Cytotoxic Activity of Tivantinib (ARQ 197) Is Not Due Solely to c-MET Inhibition.

Cancer Res. 2013 Apr 18;

Authors: Katayama R, Aoyama A, Yamori T, Qi J, Oh-Hara T, Song Y, Engelman JA, Fujita N

Abstract
The receptor tyrosine kinase c-MET is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-MET axis is often dysregulated in tumors. c-MET activation can be caused by MET gene amplification, activating mutations, and auto- or paracrine mechanisms. Thus, c-MET inhibitors are under development as anticancer drugs. Tivantinib (ARQ 197) was reported as a small-molecule c-MET inhibitor and early clinical studies suggest antitumor activity. To assess whether the antitumor activity of tivantinib was due to inhibition of c-MET, we compared the activity of tivantinib with other c-MET inhibitors in both c-MET-addicted and nonaddicted cancer cells. As expected, other c-MET inhibitors, crizotinib and PHA-665752, suppressed the growth of c-MET-addicted cancers, but not the growth of cancers that are not addicted to c-MET. In contrast, tivantinib inhibited cell viability with similar potency in both c-MET-addicted and nonaddicted cells. These results suggest that tivantinib exhibits its antitumor activity in a manner independent of c-MET status. Tivantinib treatment induced a G2-M cell-cycle arrest in EBC1 cells similarly to vincristine treatment, whereas PHA-665752 or crizotinib treatment markedly induced G0-G1 cell-cycle arrest. To identify the additional molecular target of tivantinib, we conducted COMPARE analysis, an in silico screening of a database of drug sensitivities across 39 cancer cell lines (JFCR39), and identified microtubule as a target of tivantinib. Tivantinib-treated cells showed typical microtubule disruption similar to vincristine and inhibited microtubule assembly in vitro. These results suggest that tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET. Cancer Res; 1-10. �2013 AACR.

PMID: 23598276 [PubMed - as supplied by publisher]

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Impaired acetylcholine-induced cutaneous vasodilation in young smokers: roles of nitric oxide and prostanoids.

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Impaired acetylcholine-induced cutaneous vasodilation in young smokers: roles of nitric oxide and prostanoids.

Am J Physiol Heart Circ Physiol. 2013 Mar 1;304(5):H667-73

Authors: Fujii N, Reinke MC, Brunt VE, Minson CT

Abstract
Cigarette smoking attenuates acetylcholine (ACh)-induced cutaneous vasodilation in humans, but the underlying mechanisms are unknown. We tested the hypothesis that smokers have impaired nitric oxide (NO)- and cyclooxygenase (COX)-dependent cutaneous vasodilation to ACh infusion. Twelve young smokers, who have smoked more than 5.2 � 0.7 yr with an average daily consumption of 11.4 � 1.2 cigarettes, and 12 nonsmokers were tested. Age, body mass index, and resting mean arterial pressure were similar between the groups. Cutaneous vascular conductance (CVC) was evaluated as laser-Doppler flux divided by mean arterial pressure, normalized to maximal CVC (local heating to 43.0�C plus sodium nitroprusside administration). We evaluated the increase in CVC from baseline to peak (CVC?peak) and area under the curve of CVC (CVCAUC) during a bolus infusion (1 min) of 137.5 ?M ACh at four intradermal microdialysis sites: 1) Ringer (control), 2) 10 mM N(G)-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor), 3) 10 mM ketorolac (COX inhibitor), and 4) combination of l-NAME + ketorolac. CVC?peak and CVCAUC at the Ringer site in nonsmokers were greater than in smokers (CVC?peak, 42.9 � 5.1 vs. 22.3 � 3.5%max, P < 0.05; and CVCAUC, 8,085 � 1,055 vs. 3,145 � 539%max�s, P < 0.05). In nonsmokers, CVC?peak and CVCAUC at the l-NAME site were lower than the Ringer site (CVC?peak, 29.5 � 6.2%max, P < 0.05; and CVCAUC, 5,377 � 1,109%max�s, P < 0.05), but in smokers, there were no differences between the Ringer and l-NAME sites (CVC?peak, 16.8 � 4.3%max, P = 0.11; and CVCAUC, 2,679 � 785%max�s, P = 0.30). CVC?peak and CVCAUC were reduced with ketorolac in nonsmokers (CVC?peak, 13.3 � 3.6%max, P < 0.05; and CVCAUC, 1,967 � 527%max�s, P < 0.05) and smokers (CVC?peak, 7.8 � 1.8%max, P < 0.05; and CVCAUC, 1,246 � 305%max�s, P < 0.05) and at the combination site in nonsmokers (CVC?peak, 15.9 � 3.1%max, P < 0.05; and CVCAUC, 2,660 � 512%max�s, P < 0.05) and smokers (CVC?peak, 11.5 � 2.6%max, P < 0.05; and CVCAUC, 1,693 � 409%max�s, P < 0.05), but the magnitudes were greater in nonsmokers (P < 0.05). These results suggest that impaired ACh-induced skin vasodilation in young smokers is related to diminished NO- and COX-dependent vasodilation.

PMID: 23316063 [PubMed - indexed for MEDLINE]

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Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

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Image-based high-throughput screening for inhibitors of angiogenesis.

Related Articles

Image-based high-throughput screening for inhibitors of angiogenesis.

Methods Mol Biol. 2013;931:139-51

Authors: Evensen L, Link W, Lorens JB

Abstract
Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling.

PMID: 23027002 [PubMed - indexed for MEDLINE]

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Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

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Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

Nucleic Acids Res. 2013 Feb 1;41(3):1669-83

Authors: Mu�oz-Galv�n S, L�pez-Saavedra A, Jackson SP, Huertas P, Cort�s-Ledesma F, Aguilera A

Abstract
While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome system for the repair of replication-born DSBs by SCR in yeast. We provide evidence that MRX, particularly its Mre11 nuclease activity, and Sae2 are required for SCR-mediated repair of DSBs. The phenotype of nuclease-deficient MRX mutants is suppressed by ablation of Yku70 or overexpression of Exo1, suggesting a competition between NHEJ and resection factors for DNA ends arising during replication. In addition, we observe partially redundant roles for Sgs1 and Exo1 in SCR, with a more prominent role for Sgs1. Using human U2OS cells, we also show that the competitive nature of these reactions is likely evolutionarily conserved. These results further our understanding of the role of DNA resection in repair of replication-born DSBs revealing unanticipated differences between these events and repair of enzymatically induced DSBs.

PMID: 23254329 [PubMed - in process]

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Effects of in vivo exposure to UV filters (4-MBC, OMC, BP-3, 4-HB, OC, OD-PABA) on endocrine signaling genes in the insect Chironomus riparius.

Effects of in vivo exposure to UV filters (4-MBC, OMC, BP-3, 4-HB, OC, OD-PABA) on endocrine signaling genes in the insect Chironomus riparius.

Sci Total Environ. 2013 Apr 13;456-457C:120-126

Authors: Oz�ez I, Mart�nez-Guitarte JL, Morcillo G

Abstract
There is increasing evidence indicating that several UV filters might have endocrine disruptive effects. Numerous studies have evaluated hormonal effects in vertebrates, mainly reporting estrogenic and androgenic activities in mammals and fishes. There is only limited knowledge about potential endocrine activity in invertebrate hormonal systems. In this work, the effects on endocrine signaling genes of six frequently used UV filters were investigated in Chironomus riparius, a reference organism in aquatic toxicology. The UV filters studied were: octyl-p-methoxycinnamate (OMC) also called 2-ethylhexyl-4-methoxycinnamate (EHMC); 4-methylbenzylidene camphor (4-MBC); benzophenone-3 (BP-3); 4-hidroxybenzophenone (4-HB); octocrylene (OC); and octyldimethyl-p-aminobenzoate (OD-PABA). After in vivo exposure at different dosages, expression levels of the genes coding for the ecdysone receptor (EcR), the ultraspiracle (usp, ortholog of the RXR) and the estrogen-related receptor (ERR) were quantified by Real Time PCR. The EcR gene was significantly upregulated by 4-MBC, OMC/EHMC and OD-PABA, with a dose-related response following 24h exposure. In contrast, the benzophenones, BP-3 and 4-HB, as well as OC did not alter this gene at the same exposure conditions. The transcription profiles of the usp and ERR genes were not significantly affected, except for BP-3 that inhibited the usp gene at the highest concentration. To our knowledge, this is the first experimental evidence in invertebrates of a direct effect of UV filters on endocrine-related genes, and is consistent with the known effects on vertebrate hormonal receptor genes. The capability of 4-MBC, OMC/EHMC and OD-PABA to stimulate the expression of the ecdysone receptor, a key transcription factor for the ecdysone-genomic response in arthropods, suggests the possibility of a broad and long-term effect on this hormonal pathway. These findings strengthen the need for further research about the ecotoxicological implications of chronic exposure to these compounds in aquatic invertebrates.

PMID: 23591065 [PubMed - as supplied by publisher]

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Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

Related Articles

Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

Nucleic Acids Res. 2013 Feb 1;41(3):1669-83

Authors: Mu�oz-Galv�n S, L�pez-Saavedra A, Jackson SP, Huertas P, Cort�s-Ledesma F, Aguilera A

Abstract
While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome system for the repair of replication-born DSBs by SCR in yeast. We provide evidence that MRX, particularly its Mre11 nuclease activity, and Sae2 are required for SCR-mediated repair of DSBs. The phenotype of nuclease-deficient MRX mutants is suppressed by ablation of Yku70 or overexpression of Exo1, suggesting a competition between NHEJ and resection factors for DNA ends arising during replication. In addition, we observe partially redundant roles for Sgs1 and Exo1 in SCR, with a more prominent role for Sgs1. Using human U2OS cells, we also show that the competitive nature of these reactions is likely evolutionarily conserved. These results further our understanding of the role of DNA resection in repair of replication-born DSBs revealing unanticipated differences between these events and repair of enzymatically induced DSBs.

PMID: 23254329 [PubMed - in process]

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Activity of the Fibroblast Growth Factor Receptor Inhibitors Dovitinib (TKI258) and NVP-BGJ398 in Human Endometrial Cancer Cells.

Activity of the Fibroblast Growth Factor Receptor Inhibitors Dovitinib (TKI258) and NVP-BGJ398 in Human Endometrial Cancer Cells.

Mol Cancer Ther. 2013 Feb 26;

Authors: Konecny GE, Kolarova T, O'Brien NA, Winterhoff B, Yang G, Qi J, Qi Z, Venkatesan N, Ayala R, Luo T, Finn RS, Kristof J, Galderisi C, Graus Porta D, Anderson L, Shi MM, Yovine A, Slamon DJ

Abstract
The recent identification of activating fibroblast growth factor receptor 2 (FGFR2) mutations in endometrial cancer (EC) has generated an opportunity for a novel target-based therapy. Here we explore the therapeutic potential of two FGFR inhibitors, the multi-kinase inhibitor dovitinib (TKI258) and the more selective FGFR inhibitor NVP-BGJ398 for the treatment of EC. We examined the effects of both inhibitors on tumor cell growth, FGFR2 signaling, cell cycle and apoptosis using a panel of 20 molecularly characterized human EC cell lines. Anchorage independent growth was studied using soft agar assays. In vivo studies were conducted using EC xenograft models. Cell lines with activating FGFR2 mutations (S252W, N550K) were more sensitive to dovitinib or NVP-BGJ398 when compared to their FGFR2 wild-type counterparts (p=0.073 and p=0.021, respectively). Both agents inhibited FGFR2 signaling, induced cell cycle arrest and significantly increased apoptosis in FGFR2 mutant lines. In vitro, dovitinib and NVP-BGJ398 were both potent at inhibiting cell growth of FGFR2 mutant EC cells but the activity of dovitinib was less restricted to FGFR2 mutant lines when compared to NVP-BGJ398. In vivo, dovitinib and NVP-BGJ398 significantly inhibited the growth of FGFR2 mutated EC xenograft models. In addition, dovitinib showed significant antitumor activity in FGFR2 wild-type EC xenograft models including complete tumor regressions in a long term in vivo study. Dovitinib and NVP-BGJ398 warrant further clinical evaluation in patients with FGFR2 mutated EC. Dovitinib may have antitumor activity in EC beyond FGFR2 mutated cases and may permit greater flexibility in patient selection.

PMID: 23443805 [PubMed - as supplied by publisher]

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A preliminary study on the radiation-resistance mechanism in ovarian cancer.

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A preliminary study on the radiation-resistance mechanism in ovarian cancer.

J Cancer Res Ther. 2013 Jan-Mar;9(1):22-4

Authors: Liao Q, Zhang HM, Li HH, Zhou R, Mao HL, Chen YB, Cui MH

Abstract
Aim: The present study was designed to explore the radiation-resistance mechanism by interfering in checkpoints kinase 1 (CHK1) and DNA-activated protein kinase (DNA-PK) genes with short hairpin RNA (shRNA) transfection into Skov3 cells derived from ovarian cancer and HeLa cells derived from cervical cancer. Materials and Methods: The cultured Skov3 and HeLa cells were transfected with plasmid vectors containing CHK1 shRNA and DNA-PK shRNA, respectively, through Lipofectimine? 2000 mediation, and cultured for 20 hours before exposure to 2 Gy X-radiation. The cells were harvested 4 and 28 after X-irradiation respectively then washed 3 times with PBS. These cells were stained with Annexin V/PI and applied by flow cytometer to analyze alteration of apoptosis with software CellQuest. Results: The apoptotic response in Skov3 cells to X-radiation was significantly lower than that in HeLa cells at 4 hour (t = 15.22, P < 0.001) and 28 hours (t = 15.78, P < 0.001) of post-irradiation. The shRNA might not affect the apoptosis of Skov3 and HeLa cells, while shRNA-transfection significantly enhanced the apoptotic response in Skov3 cells to X-radiation as compared with that in HeLa cells. Conclusions: The present work suggests that the CHK1 and DNA-PK genes are very likely to play a role in developing a radiation resistance in ovarian cancer.

PMID: 23575069 [PubMed - in process]

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Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

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Activity of the Fibroblast Growth Factor Receptor Inhibitors Dovitinib (TKI258) and NVP-BGJ398 in Human Endometrial Cancer Cells.

Activity of the Fibroblast Growth Factor Receptor Inhibitors Dovitinib (TKI258) and NVP-BGJ398 in Human Endometrial Cancer Cells.

Mol Cancer Ther. 2013 Feb 26;

Authors: Konecny GE, Kolarova T, O'Brien NA, Winterhoff B, Yang G, Qi J, Qi Z, Venkatesan N, Ayala R, Luo T, Finn RS, Kristof J, Galderisi C, Graus Porta D, Anderson L, Shi MM, Yovine A, Slamon DJ

Abstract
The recent identification of activating fibroblast growth factor receptor 2 (FGFR2) mutations in endometrial cancer (EC) has generated an opportunity for a novel target-based therapy. Here we explore the therapeutic potential of two FGFR inhibitors, the multi-kinase inhibitor dovitinib (TKI258) and the more selective FGFR inhibitor NVP-BGJ398 for the treatment of EC. We examined the effects of both inhibitors on tumor cell growth, FGFR2 signaling, cell cycle and apoptosis using a panel of 20 molecularly characterized human EC cell lines. Anchorage independent growth was studied using soft agar assays. In vivo studies were conducted using EC xenograft models. Cell lines with activating FGFR2 mutations (S252W, N550K) were more sensitive to dovitinib or NVP-BGJ398 when compared to their FGFR2 wild-type counterparts (p=0.073 and p=0.021, respectively). Both agents inhibited FGFR2 signaling, induced cell cycle arrest and significantly increased apoptosis in FGFR2 mutant lines. In vitro, dovitinib and NVP-BGJ398 were both potent at inhibiting cell growth of FGFR2 mutant EC cells but the activity of dovitinib was less restricted to FGFR2 mutant lines when compared to NVP-BGJ398. In vivo, dovitinib and NVP-BGJ398 significantly inhibited the growth of FGFR2 mutated EC xenograft models. In addition, dovitinib showed significant antitumor activity in FGFR2 wild-type EC xenograft models including complete tumor regressions in a long term in vivo study. Dovitinib and NVP-BGJ398 warrant further clinical evaluation in patients with FGFR2 mutated EC. Dovitinib may have antitumor activity in EC beyond FGFR2 mutated cases and may permit greater flexibility in patient selection.

PMID: 23443805 [PubMed - as supplied by publisher]

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Effects of preoperative single dose Etoricoxib on postoperative pain and sleep after lumbar diskectomy: prospective randomized double blind controlled study.

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Effects of preoperative single dose Etoricoxib on postoperative pain and sleep after lumbar diskectomy: prospective randomized double blind controlled study.

Middle East J Anesthesiol. 2012 Jun;21(5):725-30

Authors: Srivastava S, Gupta D, Naz A, Rizvi MM, Singh PK

Abstract
BACKGROUND: Etoricoxib, a selective Cox-2 inhibitor has been found to be effective in the management of acute pain. This study evaluates the effect of preoperative use of oral Etoricoxib on post operative pain relief and sleep in patients undergoing single level diskectomy.
METHODS: In this prospective, randomized, controlled study, forty four patient (ASA 1 & 2, age 18-60 years) scheduled to undergo single level lumber diskectomy were given either placebo (control group) or Etoricoxib 120 mg orally one hour before surgery. Post operatively fentanyl intravenous (IV) PCA pump was started. Visual analog score (VAS) was assessed at 0, 6, 12, 18 and 24 hours at rest and movement. Primary end point was total pain relief over 24 hours. Sleep overnight, total fentanyl consumption, incidence of nausea and vomiting, intra-operative blood loss and patient satisfaction were noted.
RESULTS: Forty three patients completed the study. Reductions in VAS at rest and on movement were observed in the Etoricoxib group when compared with the Control group at all the intervals till 24 hours postoperatively, except on movement at 24 hours postoperative (P < 0.05). Total fentanyl consumption (microg/kg/hr) was higher in Control group (P = 0.007). More patients in Etoricoxib group had a contented facial expression (p = 0.003), relaxed body language (p = 0.00) and better sleep at night than control group (p = 0.0004).
CONCLUSION: Single preoperative oral dose (120 mg) of Etoricoxib, given one hour before surgery, has significantly reduced the post operative pain at rest and movement and improved sleep in patients undergoing single level diskectomy without any side effects and with good patient satisfaction.

PMID: 23265037 [PubMed - indexed for MEDLINE]

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Image-based high-throughput screening for inhibitors of angiogenesis.

Related Articles

Image-based high-throughput screening for inhibitors of angiogenesis.

Methods Mol Biol. 2013;931:139-51

Authors: Evensen L, Link W, Lorens JB

Abstract
Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling.

PMID: 23027002 [PubMed - indexed for MEDLINE]

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Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

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Image-based high-throughput screening for inhibitors of angiogenesis.

Related Articles

Image-based high-throughput screening for inhibitors of angiogenesis.

Methods Mol Biol. 2013;931:139-51

Authors: Evensen L, Link W, Lorens JB

Abstract
Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling.

PMID: 23027002 [PubMed - indexed for MEDLINE]

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Upregulated glial cell line-derived neurotrophic factor through cyclooxygenase-2 activation in the muscle is required for mechanical hyperalgesia after exercise in rats.

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Upregulated glial cell line-derived neurotrophic factor through cyclooxygenase-2 activation in the muscle is required for mechanical hyperalgesia after exercise in rats.

J Physiol. 2013 Apr 15;

Authors: Murase S, Terazawa E, Hirate K, Yamanaka H, Kanda H, Noguchi K, Ota H, Queme F, Taguchi T, Mizumura K

Abstract
Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes delayed onset muscle soreness (DOMS), a kind of muscular mechanical hyperalgesia. Previously we reported that bradykinin-like substance released from the muscle during exercise plays a pivotal role in triggering the process of muscular mechanical hyperalgesia by upregulating nerve growth factor (NGF) in exercised muscle of rats. We show here that cyclooxygenase (COX)-2 and glial cell line-derived neurotrophic factor (GDNF) are also involved in DOMS. COX-2 inhibitors but not COX-1 inhibitors given orally before LC completely suppressed the development of DOMS, but when given 2 days after LC they failed to reverse the mechanical hyperalgesia. COX-2 mRNA and protein in exercised muscle increased 6-13 fold in mRNA and 1.7-2 fold in protein 0-12 hours after LC. COX-2 inhibitors did not suppress NGF upregulation after LC. Instead, we found GDNF mRNA was upregulated 7-8 fold in the exercised muscle 12 hours to 1 day after LC and blocked by pretreatment of COX-2 inhibitors. In situ hybridization studies revealed that both COX-2 and GDNF mRNA signals increased at the periphery of skeletal muscle cells 12 hours after LC. The accumulation of COX-2 mRNA signals was also observed in small blood vessels. Intramuscular injection of anti-GDNF antibody 2 days after LC partly reversed DOMS. Based on these findings, we conclude that GDNF upregulation through COX-2 activation is essential to mechanical hyperalgesia after exercise.

PMID: 23587883 [PubMed - as supplied by publisher]

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Activity of the Fibroblast Growth Factor Receptor Inhibitors Dovitinib (TKI258) and NVP-BGJ398 in Human Endometrial Cancer Cells.

Activity of the Fibroblast Growth Factor Receptor Inhibitors Dovitinib (TKI258) and NVP-BGJ398 in Human Endometrial Cancer Cells.

Mol Cancer Ther. 2013 Feb 26;

Authors: Konecny GE, Kolarova T, O'Brien NA, Winterhoff B, Yang G, Qi J, Qi Z, Venkatesan N, Ayala R, Luo T, Finn RS, Kristof J, Galderisi C, Graus Porta D, Anderson L, Shi MM, Yovine A, Slamon DJ

Abstract
The recent identification of activating fibroblast growth factor receptor 2 (FGFR2) mutations in endometrial cancer (EC) has generated an opportunity for a novel target-based therapy. Here we explore the therapeutic potential of two FGFR inhibitors, the multi-kinase inhibitor dovitinib (TKI258) and the more selective FGFR inhibitor NVP-BGJ398 for the treatment of EC. We examined the effects of both inhibitors on tumor cell growth, FGFR2 signaling, cell cycle and apoptosis using a panel of 20 molecularly characterized human EC cell lines. Anchorage independent growth was studied using soft agar assays. In vivo studies were conducted using EC xenograft models. Cell lines with activating FGFR2 mutations (S252W, N550K) were more sensitive to dovitinib or NVP-BGJ398 when compared to their FGFR2 wild-type counterparts (p=0.073 and p=0.021, respectively). Both agents inhibited FGFR2 signaling, induced cell cycle arrest and significantly increased apoptosis in FGFR2 mutant lines. In vitro, dovitinib and NVP-BGJ398 were both potent at inhibiting cell growth of FGFR2 mutant EC cells but the activity of dovitinib was less restricted to FGFR2 mutant lines when compared to NVP-BGJ398. In vivo, dovitinib and NVP-BGJ398 significantly inhibited the growth of FGFR2 mutated EC xenograft models. In addition, dovitinib showed significant antitumor activity in FGFR2 wild-type EC xenograft models including complete tumor regressions in a long term in vivo study. Dovitinib and NVP-BGJ398 warrant further clinical evaluation in patients with FGFR2 mutated EC. Dovitinib may have antitumor activity in EC beyond FGFR2 mutated cases and may permit greater flexibility in patient selection.

PMID: 23443805 [PubMed - as supplied by publisher]

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A preliminary study on the radiation-resistance mechanism in ovarian cancer.

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A preliminary study on the radiation-resistance mechanism in ovarian cancer.

J Cancer Res Ther. 2013 Jan-Mar;9(1):22-4

Authors: Liao Q, Zhang HM, Li HH, Zhou R, Mao HL, Chen YB, Cui MH

Abstract
Aim: The present study was designed to explore the radiation-resistance mechanism by interfering in checkpoints kinase 1 (CHK1) and DNA-activated protein kinase (DNA-PK) genes with short hairpin RNA (shRNA) transfection into Skov3 cells derived from ovarian cancer and HeLa cells derived from cervical cancer. Materials and Methods: The cultured Skov3 and HeLa cells were transfected with plasmid vectors containing CHK1 shRNA and DNA-PK shRNA, respectively, through Lipofectimine? 2000 mediation, and cultured for 20 hours before exposure to 2 Gy X-radiation. The cells were harvested 4 and 28 after X-irradiation respectively then washed 3 times with PBS. These cells were stained with Annexin V/PI and applied by flow cytometer to analyze alteration of apoptosis with software CellQuest. Results: The apoptotic response in Skov3 cells to X-radiation was significantly lower than that in HeLa cells at 4 hour (t = 15.22, P < 0.001) and 28 hours (t = 15.78, P < 0.001) of post-irradiation. The shRNA might not affect the apoptosis of Skov3 and HeLa cells, while shRNA-transfection significantly enhanced the apoptotic response in Skov3 cells to X-radiation as compared with that in HeLa cells. Conclusions: The present work suggests that the CHK1 and DNA-PK genes are very likely to play a role in developing a radiation resistance in ovarian cancer.

PMID: 23575069 [PubMed - in process]

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Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

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Ionizing radiation in glioblastoma initiating cells.

Ionizing radiation in glioblastoma initiating cells.

Front Oncol. 2013;3:74

Authors: Rivera M, Sukhdeo K, Yu J

Abstract
Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with a median survival of 12-15?months with treatment consisting of surgical resection followed by ionizing radiation (IR) and chemotherapy. Even aggressive treatment is often palliative due to near universal recurrence. Therapeutic resistance has been linked to a subpopulation of GBM cells with stem cell-like properties termed GBM initiating cells (GICs). Recent efforts have focused on elucidating resistance mechanisms activated in GICs in response to IR. Among these, GICs preferentially activate the DNA damage response (DDR) to result in a faster rate of double-strand break (DSB) repair induced by IR as compared to the bulk tumor cells. IR also activates NOTCH and the hepatic growth factor (HGF) receptor, c-MET, signaling cascades that play critical roles in promoting proliferation, invasion, and resistance to apoptosis. These pathways are preferentially activated in GICs and represent targets for pharmacologic intervention. While IR provides the benefit of improved survival, it paradoxically promotes selection of more malignant cellular phenotypes of GBM. As reviewed here, finding effective combinations of radiation and molecular inhibitors to target GICs and non-GICs is essential for the development of more effective therapies.

PMID: 23579692 [PubMed - in process]

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Immunosuppression in HCV-positive liver-transplant recipients.

Related Articles

Immunosuppression in HCV-positive liver-transplant recipients.

Curr Opin Organ Transplant. 2012 Dec;17(6):648-54

Authors: Chan AJ, Lake JR

Abstract
PURPOSE OF REVIEW: The purpose of this review is to evaluate the historical and recent literature as it pertains to current immunosuppression regimens in hepatitis C virus (HCV)-positive (+) liver-transplant recipients.
RECENT FINDINGS: Recent findings suggest that there are unique differences between HCV transplant recipients and non-HCV transplant recipients, not only in the graft's inflammatory response, but also to the treatments used to prevent and combat rejection.
SUMMARY: HCV (+) transplant recipients present unique challenges. Over the years, there has been progress but there is clearly no consensus regarding the optimal immunosuppressive medications or drug regimens; however, there continues to be advancements in the management of patients with HCV. Though current studies do not provide clear evidence as to optimal immunosuppression, they do identify questions ideally addressed by large, randomized controlled trials.

PMID: 23128793 [PubMed - indexed for MEDLINE]

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Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer.

Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer.

PLoS One. 2013;8(4):e60359

Authors: Lehner A, Magdolen V, Schuster T, Kotzsch M, Kiechle M, Meindl A, Sweep FC, Span PN, Gross E

Abstract
HTRA1 is a highly conserved serine protease which has been implicated in suppression of epithelial-to-mesenchymal-transition (EMT) and cell motility in breast cancer. Its prognostic relevance for breast cancer is unclear so far. Therefore, we evaluated the impact of HTRA1 mRNA expression on patient outcome using a cohort of 131 breast cancer patients as well as a validation cohort including 2809 publically available data sets. Additionally, we aimed at investigating for the presence of promoter hypermethylation as a mechanism for silencing the HTRA1 gene in breast tumors. HTRA1 downregulation was detected in more than 50% of the breast cancer specimens and was associated with higher tumor stage (p?=?0.025). By applying Cox proportional hazard models, we observed favorable overall (OS) and disease-free survival (DFS) related to high HTRA1 expression (HR?=?0.45 [CI 0.23-0.90], p?=?0.023; HR?=?0.55 [CI 0.32-0.94], p?=?0.028, respectively), with even more pronounced impact in node-positive patients (HR?=?0.21 [CI 0.07-0.63], p?=?0.006; HR?=?0.29 [CI 0.13-0.65], p?=?0.002, respectively). Moreover, HTRA1 remained a statistically significant factor predicting DFS among established clinical parameters in the multivariable analysis. Its impact on patient outcome was independently confirmed in the validation set (for relapse-free survival (n?=?2809): HR?=?0.79 [CI 0.7-0.9], log-rank p?=?0.0003; for OS (n?=?971): HR?=?0.63 [CI 0.48-0.83], log-rank p?=?0.0009). In promoter analyses, we in fact detected methylation of HTRA1 in a small subset of breast cancer specimens (two out of a series of 12), and in MCF-7 breast cancer cells which exhibited 22-fold lower HTRA1 mRNA expression levels compared to unmethylated MDA-MB-231 cells. In conclusion, we show that downregulation of HTRA1 is associated with shorter patient survival, particularly in node-positive breast cancer. Since HTRA1 loss was demonstrated to induce EMT and cancer cell invasion, these patients might benefit from demethylating agents or histone deacetylase inhibitors previously reported to lead to HTRA1 upregulation, or from novel small-molecule inhibitors targeting EMT-related processes.

PMID: 23580433 [PubMed - in process]

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Activity of the Fibroblast Growth Factor Receptor Inhibitors Dovitinib (TKI258) and NVP-BGJ398 in Human Endometrial Cancer Cells.

Activity of the Fibroblast Growth Factor Receptor Inhibitors Dovitinib (TKI258) and NVP-BGJ398 in Human Endometrial Cancer Cells.

Mol Cancer Ther. 2013 Feb 26;

Authors: Konecny GE, Kolarova T, O'Brien NA, Winterhoff B, Yang G, Qi J, Qi Z, Venkatesan N, Ayala R, Luo T, Finn RS, Kristof J, Galderisi C, Graus Porta D, Anderson L, Shi MM, Yovine A, Slamon DJ

Abstract
The recent identification of activating fibroblast growth factor receptor 2 (FGFR2) mutations in endometrial cancer (EC) has generated an opportunity for a novel target-based therapy. Here we explore the therapeutic potential of two FGFR inhibitors, the multi-kinase inhibitor dovitinib (TKI258) and the more selective FGFR inhibitor NVP-BGJ398 for the treatment of EC. We examined the effects of both inhibitors on tumor cell growth, FGFR2 signaling, cell cycle and apoptosis using a panel of 20 molecularly characterized human EC cell lines. Anchorage independent growth was studied using soft agar assays. In vivo studies were conducted using EC xenograft models. Cell lines with activating FGFR2 mutations (S252W, N550K) were more sensitive to dovitinib or NVP-BGJ398 when compared to their FGFR2 wild-type counterparts (p=0.073 and p=0.021, respectively). Both agents inhibited FGFR2 signaling, induced cell cycle arrest and significantly increased apoptosis in FGFR2 mutant lines. In vitro, dovitinib and NVP-BGJ398 were both potent at inhibiting cell growth of FGFR2 mutant EC cells but the activity of dovitinib was less restricted to FGFR2 mutant lines when compared to NVP-BGJ398. In vivo, dovitinib and NVP-BGJ398 significantly inhibited the growth of FGFR2 mutated EC xenograft models. In addition, dovitinib showed significant antitumor activity in FGFR2 wild-type EC xenograft models including complete tumor regressions in a long term in vivo study. Dovitinib and NVP-BGJ398 warrant further clinical evaluation in patients with FGFR2 mutated EC. Dovitinib may have antitumor activity in EC beyond FGFR2 mutated cases and may permit greater flexibility in patient selection.

PMID: 23443805 [PubMed - as supplied by publisher]

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Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer.

Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer.

PLoS One. 2013;8(4):e60359

Authors: Lehner A, Magdolen V, Schuster T, Kotzsch M, Kiechle M, Meindl A, Sweep FC, Span PN, Gross E

Abstract
HTRA1 is a highly conserved serine protease which has been implicated in suppression of epithelial-to-mesenchymal-transition (EMT) and cell motility in breast cancer. Its prognostic relevance for breast cancer is unclear so far. Therefore, we evaluated the impact of HTRA1 mRNA expression on patient outcome using a cohort of 131 breast cancer patients as well as a validation cohort including 2809 publically available data sets. Additionally, we aimed at investigating for the presence of promoter hypermethylation as a mechanism for silencing the HTRA1 gene in breast tumors. HTRA1 downregulation was detected in more than 50% of the breast cancer specimens and was associated with higher tumor stage (p?=?0.025). By applying Cox proportional hazard models, we observed favorable overall (OS) and disease-free survival (DFS) related to high HTRA1 expression (HR?=?0.45 [CI 0.23-0.90], p?=?0.023; HR?=?0.55 [CI 0.32-0.94], p?=?0.028, respectively), with even more pronounced impact in node-positive patients (HR?=?0.21 [CI 0.07-0.63], p?=?0.006; HR?=?0.29 [CI 0.13-0.65], p?=?0.002, respectively). Moreover, HTRA1 remained a statistically significant factor predicting DFS among established clinical parameters in the multivariable analysis. Its impact on patient outcome was independently confirmed in the validation set (for relapse-free survival (n?=?2809): HR?=?0.79 [CI 0.7-0.9], log-rank p?=?0.0003; for OS (n?=?971): HR?=?0.63 [CI 0.48-0.83], log-rank p?=?0.0009). In promoter analyses, we in fact detected methylation of HTRA1 in a small subset of breast cancer specimens (two out of a series of 12), and in MCF-7 breast cancer cells which exhibited 22-fold lower HTRA1 mRNA expression levels compared to unmethylated MDA-MB-231 cells. In conclusion, we show that downregulation of HTRA1 is associated with shorter patient survival, particularly in node-positive breast cancer. Since HTRA1 loss was demonstrated to induce EMT and cancer cell invasion, these patients might benefit from demethylating agents or histone deacetylase inhibitors previously reported to lead to HTRA1 upregulation, or from novel small-molecule inhibitors targeting EMT-related processes.

PMID: 23580433 [PubMed - in process]

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Image-based high-throughput screening for inhibitors of angiogenesis.

Related Articles

Image-based high-throughput screening for inhibitors of angiogenesis.

Methods Mol Biol. 2013;931:139-51

Authors: Evensen L, Link W, Lorens JB

Abstract
Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling.

PMID: 23027002 [PubMed - indexed for MEDLINE]

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[Celecoxib increased cellular ATRA sensitivity of human colon cancer cell lines through COX-2-independent mechanisms].

Related Articles

[Celecoxib increased cellular ATRA sensitivity of human colon cancer cell lines through COX-2-independent mechanisms].

Yao Xue Xue Bao. 2009 Dec;44(12):1353-8

Authors: Liu JP, Wei HB, Zheng ZH, Guo WP, Fang JF

Abstract
Retinoid resistance has limited clinical activity of retinoids as differentiation-inducing and apoptosis-inducing drugs. The present study was designed to investigate whether celecoxib (selective COX-2 inhibitor) has effects on cellular retinoid sensitivity of human colon cancer cell lines and its possible mechanism. Cell viability was measured by MTT assay. Apoptosis was detected by Annexin-V/PI staining and flow cytometry assay. PGE2 production was measured by ELISA assay. Expression of RARbeta was assayed by Western blotting. The results showed that celecoxib enhanced the inhibitory effect of ATRA in both COX-2 high-expressing HT-29 and COX-2 low-expressing SW480 cell lines. Further study showed the ATRA and celecoxib combination induced greater apoptosis, and the addition of PGE2 did not affect the number of apoptotic cells induced by the combination. Moreover, NS398 (another selective COX-2 inhibitor) did not affect the inhibitory effects of ATRA on both cell lines. Western blotting showed that the expression of RARbeta in HT-29 cell lines increased in celecoxib group and combination group. And the addition of PGE2 did not affect the expression of RARbeta induced by celecoxib either. In conclusion, celecoxib increased expression of RARbeta and cellular ATRA sensitivity through COX-2-independent mechanisms, which may provide a potential strategy for combination therapy.

PMID: 21351468 [PubMed - indexed for MEDLINE]

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Feasibility of adding everolimus to carboplatin and paclitaxel, with or without bevacizumab, for treatment-naive, advanced non-small cell lung cancer.

Feasibility of adding everolimus to carboplatin and paclitaxel, with or without bevacizumab, for treatment-naive, advanced non-small cell lung cancer.

Invest New Drugs. 2013 Apr 12;

Authors: Eberhardt WE, Mitchell P, Schiller JH, Brown MP, Thomas M, Mills G, Jehl V, Urva SR, De Leo JJ, Gogov S, Papadimitrakopoulou V

Abstract
Introduction One standard of care for advanced non-small cell lung cancer (NSCLC) is paclitaxel plus carboplatin?�?bevacizumab. This two-step phase I study evaluated the feasibility of adding everolimus to paclitaxel plus carboplatin?�?bevacizumab for advanced NSCLC. Methods Adults with advanced NSCLC naive to systemic therapy were enrolled. A Bayesian dose-escalation model was used to identify feasible daily or weekly everolimus doses given with paclitaxel (200�mg/m(2) q21 days) and carboplatin (AUC 6�mg/mL/min q21 days) (step 1) and paclitaxel (200�mg/m(2) q21 days), carboplatin (AUC 6�mg/mL/min q21 days), and bevacizumab (15�mg/kg q21 days) (step 2). Primary endpoint was end-of-cycle 1 dose-limiting toxicity (DLT) rate. Secondary endpoints included safety; relative dose intensities of paclitaxel, carboplatin, and bevacizumab; pharmacokinetics; and tumor response. Results Fifty-two patients were enrolled and received everolimus 5�mg/day plus carboplatin and paclitaxel (step 1 daily; n?=?13); everolimus 30�mg/week plus carboplatin and paclitaxel (step 1 weekly; n?=?13); everolimus 5�mg/day plus carboplatin, paclitaxel, and bevacizumab (step 2 daily; n?=?13); or everolimus 30�mg/week plus carboplatin, paclitaxel, and bevacizumab (step 2 weekly; n?=?13). End-of-cycle 1 DLT rate was 16.7�% (step 1 daily), 30.8�% (step 1 weekly), 30.0�% (step 2 daily), and 16.7�% (step 2 weekly). Cycle 1 DLTs were grade 3 neutropenia, anal abscess, diarrhea, and thrombocytopenia and grade 4 myalgia, cellulitis, neutropenia, febrile neutropenia, pulmonary embolism, and thrombocytopenia. The most common adverse events were neutropenia, fatigue, anemia, and thrombocytopenia. One patient (step 2 daily) experienced complete response, 10 patients partial response. Conclusions The feasible everolimus doses given with carboplatin and paclitaxel?�?bevacizumab were 5�mg/day and 30�mg/week. Neither schedule was very well tolerated in this unselected NSCLC population.

PMID: 23579358 [PubMed - as supplied by publisher]

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Ionizing radiation in glioblastoma initiating cells.

Ionizing radiation in glioblastoma initiating cells.

Front Oncol. 2013;3:74

Authors: Rivera M, Sukhdeo K, Yu J

Abstract
Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with a median survival of 12-15?months with treatment consisting of surgical resection followed by ionizing radiation (IR) and chemotherapy. Even aggressive treatment is often palliative due to near universal recurrence. Therapeutic resistance has been linked to a subpopulation of GBM cells with stem cell-like properties termed GBM initiating cells (GICs). Recent efforts have focused on elucidating resistance mechanisms activated in GICs in response to IR. Among these, GICs preferentially activate the DNA damage response (DDR) to result in a faster rate of double-strand break (DSB) repair induced by IR as compared to the bulk tumor cells. IR also activates NOTCH and the hepatic growth factor (HGF) receptor, c-MET, signaling cascades that play critical roles in promoting proliferation, invasion, and resistance to apoptosis. These pathways are preferentially activated in GICs and represent targets for pharmacologic intervention. While IR provides the benefit of improved survival, it paradoxically promotes selection of more malignant cellular phenotypes of GBM. As reviewed here, finding effective combinations of radiation and molecular inhibitors to target GICs and non-GICs is essential for the development of more effective therapies.

PMID: 23579692 [PubMed - in process]

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Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer.

Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer.

PLoS One. 2013;8(4):e60359

Authors: Lehner A, Magdolen V, Schuster T, Kotzsch M, Kiechle M, Meindl A, Sweep FC, Span PN, Gross E

Abstract
HTRA1 is a highly conserved serine protease which has been implicated in suppression of epithelial-to-mesenchymal-transition (EMT) and cell motility in breast cancer. Its prognostic relevance for breast cancer is unclear so far. Therefore, we evaluated the impact of HTRA1 mRNA expression on patient outcome using a cohort of 131 breast cancer patients as well as a validation cohort including 2809 publically available data sets. Additionally, we aimed at investigating for the presence of promoter hypermethylation as a mechanism for silencing the HTRA1 gene in breast tumors. HTRA1 downregulation was detected in more than 50% of the breast cancer specimens and was associated with higher tumor stage (p?=?0.025). By applying Cox proportional hazard models, we observed favorable overall (OS) and disease-free survival (DFS) related to high HTRA1 expression (HR?=?0.45 [CI 0.23-0.90], p?=?0.023; HR?=?0.55 [CI 0.32-0.94], p?=?0.028, respectively), with even more pronounced impact in node-positive patients (HR?=?0.21 [CI 0.07-0.63], p?=?0.006; HR?=?0.29 [CI 0.13-0.65], p?=?0.002, respectively). Moreover, HTRA1 remained a statistically significant factor predicting DFS among established clinical parameters in the multivariable analysis. Its impact on patient outcome was independently confirmed in the validation set (for relapse-free survival (n?=?2809): HR?=?0.79 [CI 0.7-0.9], log-rank p?=?0.0003; for OS (n?=?971): HR?=?0.63 [CI 0.48-0.83], log-rank p?=?0.0009). In promoter analyses, we in fact detected methylation of HTRA1 in a small subset of breast cancer specimens (two out of a series of 12), and in MCF-7 breast cancer cells which exhibited 22-fold lower HTRA1 mRNA expression levels compared to unmethylated MDA-MB-231 cells. In conclusion, we show that downregulation of HTRA1 is associated with shorter patient survival, particularly in node-positive breast cancer. Since HTRA1 loss was demonstrated to induce EMT and cancer cell invasion, these patients might benefit from demethylating agents or histone deacetylase inhibitors previously reported to lead to HTRA1 upregulation, or from novel small-molecule inhibitors targeting EMT-related processes.

PMID: 23580433 [PubMed - in process]

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COX-2 in cancer: Gordian knot or Achilles heel?

COX-2 in cancer: Gordian knot or Achilles heel?

Front Pharmacol. 2013;4:34

Authors: Stasinopoulos I, Shah T, Penet MF, Krishnamachary B, Bhujwalla ZM

Abstract
The networks of blood and lymphatic vessels and of the extracellular matrix and their cellular and structural components, that are collectively termed the tumor microenvironment, are frequently co-opted and shaped by cancer cells to survive, invade, and form distant metastasis. With an enviable capacity to adapt to continually changing environments, cancer represents the epitome of functional chaos, a stark contrast to the hierarchical and organized differentiation processes that dictate the development and life of biological organisms. The consequences of changing landscapes such as hypoxia and acidic extracellular pH in and around tumors create a cascade of changes in multiple pathways and networks that become apparent only several years later as recurrence and metastasis. These molecular and phenotypic changes, several of which are mediated by COX-2, approach the complexities of a "Gordian Knot." We review evidence from our studies and from literature suggesting that cyclooxygenase-2 (COX-2) biology presents a nodal point in cancer biology and an "Achilles heel" of COX-2-dependent tumors.

PMID: 23579438 [PubMed - in process]

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Drug-eluting stenting of saphenous vein graft versus native coronary artery supplying the same myocardial perfusion territory: a pilot retrospective 3-year follow-up.

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Drug-eluting stenting of saphenous vein graft versus native coronary artery supplying the same myocardial perfusion territory: a pilot retrospective 3-year follow-up.

J Invasive Cardiol. 2012 Oct;24(10):516-20

Authors: Ho PC, Lee AC, Fortuna R

Abstract
BACKGROUND: In post-coronary artery bypass graft (CABG) patients undergoing drug-eluting stent implantation of either the saphenous vein graft (SVG) versus the native coronary artery supplying the same myocardial perfusion territory, which option confers better clinical outcomes when both lesions are technically feasible?
METHODS: From 2005 to 2008 at a single medical center, a total of 178 post-CABG patients (with 241 lesions) underwent PCI due to progressive SVG disease. Of them, 23 patients (with 29 lesions) had amenable disease for PCI in both the SVG and native coronary artery matching the same myocardial perfusion territory; chronic total occlusions were excluded. All patients included in the study were treated with drug-eluting stents. Sixteen patients (19 lesions) underwent PCI of the SVG, and 9 patients (10 lesions) underwent PCI in the native vessels.
RESULTS: Primary endpoints were in-hospital and 3-year rates of death, myocardial infarction (MI), target lesion revascularization (TLR), and target vessel revascularization (TVR). There were 2 in-hospital MIs in the SVG-treated group and 0 for the native vessel-treated group. The 3-year clinical follow-up showed 3 MIs, 2 TLRs, 4 TVRs, and 6 deaths in the SVG-treated group; only 1 MI occurred in the native-vessel treated group (P=.02). More PCIs of the SVG were performed than in the native coronary artery (19 vs 10 lesions).
CONCLUSIONS: This small study suggests improved clinical outcomes with PCI of the native vessel, but a tendency of operators to choose PCI of the SVG instead. Large, prospective, multicenter, randomized clinical trials with long-term follow-up can validate the advantage of selecting PCI of the native vessel over the SVG when both options are available.

PMID: 23043035 [PubMed - indexed for MEDLINE]

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