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Lars Edvinsson - One of the best experts on this subject based on the ideXlab platform.

  • mek1 2 inhibitor U0126 but not nimodipine reduces upregulation of cerebrovascular contractile receptors after subarachnoid haemorrhage in rats
    PLOS ONE, 2019
    Co-Authors: Simon Topp Christensen, Sara Ellinor Johansson, Karin Warfvinge, Kristian Agmund Haanes, Lars Edvinsson, Aneta Radziwonbalicka
    Abstract:

    Vascular pathophysiological changes after haemorrhagic stroke, such as phenotypic modulation of the cerebral arteries and cerebral vasospasms, are associated with delayed cerebral ischemia (DCI) and poor outcome. The only currently approved drug treatment shown to reduce the risk of DCI and improve neurologic outcome after aneurysmal subarachnoid haemorrhage (SAH) is nimodipine, a dihydropyridine L-type voltage-gated Ca2+ channel blocker. MEK1/2 mediated transcriptional upregulation of contractile receptors, including endothelin-1 (ET-1) receptors, has previously been shown to be a factor in the pathology of SAH. The aim of the study was to compare intrathecal and subcutaneous treatment regimens of nimodipine and intrathecal treatment regimens of U0126, a MEK1/2 inhibitor, in a single injection experimental rat SAH model with post 48 h endpoints consisting of wire myography of cerebral arteries, flow cytometry of cerebral arterial tissue and behavioural evaluation. Following ET-1 concentration-response curves, U0126 exposed arteries had a significantly lower ET-1max than vehicle arteries. Arteries from both the intrathecal- and subcutaneous nimodipine treated animals had significantly higher ET-1max contractions than the U0126 arteries. Furthermore, Ca2+ concentration response curves (precontracted with ET-1 and in the presence of nimodipine) showed that nimodipine treatment could result in larger nimodipine insensitive contractions compared to U0126. Flow cytometry showed decreased protein expression of the ETB receptor in U0126 treated cerebral vascular smooth muscle cells compared to vehicle. Only U0126 treatment lowered ET-1max contractions and ETB receptor levels, as well as decreased the contractions involving nimodipine-insensitive Ca2+ channels, when compared to both intrathecal and subcutaneous nimodipine treatment. This indicate that targeting gene expression might be a better strategy than blocking specific receptors or ion channels in future treatments of SAH.

  • Synergistic effects of a cremophor EL drug delivery system and its U0126 cargo in an ex vivo model.
    Drug Delivery, 2019
    Co-Authors: Simon Topp Christensen, Sara Ellinor Johansson, Anne-sofie Grell, C. M. Andersson, Lars Edvinsson, Kristian Agmund Haanes
    Abstract:

    Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ETB) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K+ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system.

  • Synergistic effects of a cremophor EL drug delivery system and its U0126 cargo in an ex vivo model.
    Drug Delivery, 2019
    Co-Authors: Simon Topp Christensen, Sara Ellinor Johansson, Anne-sofie Grell, C. M. Andersson, Lars Edvinsson, Kristian Agmund Haanes
    Abstract:

    Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ETB) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K+ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system.

  • The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries
    Toxicology and Applied Pharmacology, 2016
    Co-Authors: Na-na Ping, Karin Warfvinge, Wei Li, Lars Edvinsson
    Abstract:

    Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8 weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4 weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ETA receptor mRNA and protein levels as well as contractile responses mediated by ETA receptors. The immunoreactivity of increased ETA receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ETB receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries.

  • U0126 Attenuates Cerebral Vasoconstriction and Improves Long-Term Neurologic Outcome after Stroke in Female Rats:
    Journal of Cerebral Blood Flow & Metabolism, 2015
    Co-Authors: Hilda Ahnstedt, Karin Warfvinge, Maryam Mostajeran, Frank W Blixt, Saema Ansar, Diana N. Krause, Lars Edvinsson
    Abstract:

    Sex differences are well known in cerebral ischemia and may impact the effect of stroke treatments. In male rats, the MEK1/2 inhibitor U0126 reduces ischemia-induced endothelin type B (ETB) receptor upregulation, infarct size and improves acute neurologic function after experimental stroke. However, responses to this treatment in females and long-term effects on outcome are not known. Initial experiments used in vitro organ culture of cerebral arteries, confirming ERK1/2 activation and increased ETB receptor-mediated vasoconstriction in female cerebral arteries. Transient middle cerebral artery occlusion (tMCAO, 120 minutes) was induced in female Wistar rats, with U0126 (30 mg/kg intraperitoneally) or vehicle administered at 0 and 24 hours of reperfusion, or with no treatment. Infarct volumes were determined and neurologic function was assessed by 6-point and 28-point neuroscores. ETB receptor-mediated contraction was studied with myograph and protein expression with immunohistochemistry. In vitro organ culture and tMCAO resulted in vascular ETB receptor upregulation and activation of ERK1/2 that was prevented by U0126. Although no effect on infarct size, U0126 improved the long-term neurologic function after experimental stroke in female rats. In conclusion, early prevention of the ERK1/2 activation and ETB receptor-mediated vasoconstriction in the cerebral vasculature after ischemic stroke in female rats improves the long-term neurologic outcome.

Stephen Leong - One of the best experts on this subject based on the ideXlab platform.

  • Abstract A256: Rational combination of the IGF‐1R/IR tyrosine kinase inhibitor (TKI), OSI‐906, with the MEK inhibitor, U0126, results in synergistic and apoptotic effects in human colorectal cancer (CRC) cell lines
    Therapeutic Agents - Small Molecule Kinase Inhibitors, 2009
    Co-Authors: Sara A. Flanigan, Todd M. Pitts, Gillian N. Kulikowski, Aik Choon Tan, Sujatha Nallapareddy, John J. Tentler, S. Gail Eckhardt, Stephen Leong
    Abstract:

    Background: Signaling by the insulin‐like growth factor 1 receptor (IGF‐1R) promotes cell growth, migration and survival in several human tumor types. IGF‐1R is overexpressed in CRC and is associated with a poor prognosis and resistance to chemotherapy. Our prior transcriptional profiling analysis of CRC cell lines treated with the small molecule IGF‐1R/IR TKI, OSI‐906, indicated that overexpression of the mitogen‐activated protein kinase (MAPK) pathway conferred resistance to OSI‐906. The purpose of this study was to evaluate the rational combination of the MEK1/2 inhibitor, U0126, and OSI‐906 against human CRC cell lines. Methods and Results: The antiproliferative effects of OSI‐906 and U0126 were assessed as single agents and in combination using the Sulforhodamine B (SRB) cell viability assay. Twenty‐eight CRC cell lines were exposed to either OSI‐906 (0–5µM), or U0126 (0–20µM). In reference to OSI‐906, cell lines with IC50 ≤ 1µM were considered sensitive (S) and cell lines with IC50 ≥ 5µM were deemed resistant (R). Likewise, cell lines with IC50 10µM were considered R. We selected 12 cell lines for combination studies according to the following conditions: 1) S to both drugs, 2) R to both drugs, 3) S to OSI‐906 but R to U0126, and 4) R to OSI‐906 but S to U0126, and treated them with varying doses of the two drugs as single agents and in combination. Combination effects between OSI‐906 and U0126 were evaluated using the Chou and Talalay method. Robust synergy was observed in most CRC cell lines, including those that were resistant to OSI‐906 or U0126. Apoptosis was then analyzed using bioluminescent caspase 3/7 detection and validated through analysis of PARP cleavage by immunoblotting. Surprisingly, some of the cell lines demonstrated induction of apoptosis when exposed to the combination but not with either agent alone. The CRC cell lines with the greatest apoptotic induction were inherently sensitive to OSI‐906 in the SRB assay. Conclusion: Our prior transcriptional profiling data revealed elevated expression of genes in the MAPK pathway in cell lines that were less sensitive to OSI‐906, providing the basis for rational combination therapy with U0126. Interestingly, the combination of OSI‐906 and U0126 displayed synergy in nearly all cell lines tested, regardless of sensitivity to either compound. Similarly, the apoptosis exhibited by some CRC cell lines could not be predicted by the sensitivity profile, or RAS/RAF mutational status. However, CRC cell lines with the greatest synergistic induction of apoptosis were sensitive to OSI‐906. Further pathway and transcriptional profiling analysis is ongoing to reveal the underlying mechanism(s) of the observed synergy. These results, if further validated in vivo, will support the rational combination of OSI‐906 and a MEK inhibitor in patients with CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A256.

  • abstract a256 rational combination of the igf 1r ir tyrosine kinase inhibitor tki osi 906 with the mek inhibitor U0126 results in synergistic and apoptotic effects in human colorectal cancer crc cell lines
    Molecular Cancer Therapeutics, 2009
    Co-Authors: Sara A. Flanigan, Todd M. Pitts, Gillian N. Kulikowski, Aik Choon Tan, Sujatha Nallapareddy, John J. Tentler, Gail S Eckhardt, Stephen Leong
    Abstract:

    Background: Signaling by the insulin‐like growth factor 1 receptor (IGF‐1R) promotes cell growth, migration and survival in several human tumor types. IGF‐1R is overexpressed in CRC and is associated with a poor prognosis and resistance to chemotherapy. Our prior transcriptional profiling analysis of CRC cell lines treated with the small molecule IGF‐1R/IR TKI, OSI‐906, indicated that overexpression of the mitogen‐activated protein kinase (MAPK) pathway conferred resistance to OSI‐906. The purpose of this study was to evaluate the rational combination of the MEK1/2 inhibitor, U0126, and OSI‐906 against human CRC cell lines. Methods and Results: The antiproliferative effects of OSI‐906 and U0126 were assessed as single agents and in combination using the Sulforhodamine B (SRB) cell viability assay. Twenty‐eight CRC cell lines were exposed to either OSI‐906 (0–5µM), or U0126 (0–20µM). In reference to OSI‐906, cell lines with IC50 ≤ 1µM were considered sensitive (S) and cell lines with IC50 ≥ 5µM were deemed resistant (R). Likewise, cell lines with IC50 10µM were considered R. We selected 12 cell lines for combination studies according to the following conditions: 1) S to both drugs, 2) R to both drugs, 3) S to OSI‐906 but R to U0126, and 4) R to OSI‐906 but S to U0126, and treated them with varying doses of the two drugs as single agents and in combination. Combination effects between OSI‐906 and U0126 were evaluated using the Chou and Talalay method. Robust synergy was observed in most CRC cell lines, including those that were resistant to OSI‐906 or U0126. Apoptosis was then analyzed using bioluminescent caspase 3/7 detection and validated through analysis of PARP cleavage by immunoblotting. Surprisingly, some of the cell lines demonstrated induction of apoptosis when exposed to the combination but not with either agent alone. The CRC cell lines with the greatest apoptotic induction were inherently sensitive to OSI‐906 in the SRB assay. Conclusion: Our prior transcriptional profiling data revealed elevated expression of genes in the MAPK pathway in cell lines that were less sensitive to OSI‐906, providing the basis for rational combination therapy with U0126. Interestingly, the combination of OSI‐906 and U0126 displayed synergy in nearly all cell lines tested, regardless of sensitivity to either compound. Similarly, the apoptosis exhibited by some CRC cell lines could not be predicted by the sensitivity profile, or RAS/RAF mutational status. However, CRC cell lines with the greatest synergistic induction of apoptosis were sensitive to OSI‐906. Further pathway and transcriptional profiling analysis is ongoing to reveal the underlying mechanism(s) of the observed synergy. These results, if further validated in vivo, will support the rational combination of OSI‐906 and a MEK inhibitor in patients with CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A256.

Bianca M. Zani - One of the best experts on this subject based on the ideXlab platform.

  • MEK/ERK inhibitor U0126 affects in vitro and in vivo growth of embryonal rhabdomyosarcoma
    Molecular Cancer Therapeutics, 2009
    Co-Authors: Francesco Marampon, Gianluca Bossi, Carmela Ciccarelli, Agnese Di Rocco, Ada Sacchi, Richard G. Pestell, Bianca M. Zani
    Abstract:

    We reported previously that the disruption of c-Myc through mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibition blocks the expression of the transformed phenotype in the embryonal rhabdomyosarcoma (ERMS) cell line (RD), thereby inducing myogenic differentiation in vitro. In this article, we investigate whether MEK/ERK inhibition, by the MEK/ERK inhibitor U0126, affects c-Myc protein level and growth of RMS tumor in an in vivo xenograft model. U0126 significantly reduced RMS tumor growth in RD cell line-xenotransplanted mice. Immunobiochemical and immunohistochemical analysis showed (a) phospho-active ERK levels were reduced by U0126 therapy and unaltered in normal tissues, (b) phospho-Myc and c-Myc was reduced commensurate with phospho-ERK inhibition, and (c) reduction in Ki-67 and endothelial (CD31) marker expression. These results indicate that MEK/ERK inhibition affects growth and angiogenic signals in tumor. The RD-M1 cultured xenograft tumor-derived cell line and the ERMS cell line TE671 responded to U0126 by arresting growth, down-regulating c-Myc, and initiating myogenesis. All these results suggest a tight correlation of MEK/ERK inhibition with c-Myc down-regulation and arrest of tumor growth. Thus, MEK inhibitors may be investigated for a signal transduction-based targeting of the c-Myc as a therapeutic strategy in ERMS.

  • mek erk inhibitor U0126 affects in vitro and in vivo growth of embryonal rhabdomyosarcoma
    Molecular Cancer Therapeutics, 2009
    Co-Authors: Francesco Marampon, Gianluca Bossi, Carmela Ciccarelli, Agnese Di Rocco, Ada Sacchi, Richard G. Pestell, Bianca M. Zani
    Abstract:

    We reported previously that the disruption of c-Myc through mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibition blocks the expression of the transformed phenotype in the embryonal rhabdomyosarcoma (ERMS) cell line (RD), thereby inducing myogenic differentiation in vitro. In this article, we investigate whether MEK/ERK inhibition, by the MEK/ERK inhibitor U0126, affects c-Myc protein level and growth of RMS tumor in an in vivo xenograft model. U0126 significantly reduced RMS tumor growth in RD cell line-xenotransplanted mice. Immunobiochemical and immunohistochemical analysis showed (a) phospho-active ERK levels were reduced by U0126 therapy and unaltered in normal tissues, (b) phospho-Myc and c-Myc was reduced commensurate with phospho-ERK inhibition, and (c) reduction in Ki-67 and endothelial (CD31) marker expression. These results indicate that MEK/ERK inhibition affects growth and angiogenic signals in tumor. The RD-M1 cultured xenograft tumor-derived cell line and the ERMS cell line TE671 responded to U0126 by arresting growth, down-regulating c-Myc, and initiating myogenesis. All these results suggest a tight correlation of MEK/ERK inhibition with c-Myc down-regulation and arrest of tumor growth. Thus, MEK inhibitors may be investigated for a signal transduction-based targeting of the c-Myc as a therapeutic strategy in ERMS.

  • mek erk inhibitor U0126 affects in vitro and in vivo growth of embryonal rhabdomyosarcoma
    Molecular Cancer Therapeutics, 2009
    Co-Authors: Francesco Marampon, Gianluca Bossi, Carmela Ciccarelli, Agnese Di Rocco, Ada Sacchi, Richard G. Pestell, Bianca M. Zani
    Abstract:

    We reported previously that the disruption of c-Myc through mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibition blocks the expression of the transformed phenotype in the embryonal rhabdomyosarcoma (ERMS) cell line (RD), thereby inducing myogenic differentiation in vitro . In this article, we investigate whether MEK/ERK inhibition, by the MEK/ERK inhibitor U0126, affects c-Myc protein level and growth of RMS tumor in an in vivo xenograft model. U0126 significantly reduced RMS tumor growth in RD cell line-xenotransplanted mice. Immunobiochemical and immunohistochemical analysis showed ( a ) phospho-active ERK levels were reduced by U0126 therapy and unaltered in normal tissues, ( b ) phospho-Myc and c-Myc was reduced commensurate with phospho-ERK inhibition, and ( c ) reduction in Ki-67 and endothelial (CD31) marker expression. These results indicate that MEK/ERK inhibition affects growth and angiogenic signals in tumor. The RD-M1 cultured xenograft tumor-derived cell line and the ERMS cell line TE671 responded to U0126 by arresting growth, down-regulating c-Myc, and initiating myogenesis. All these results suggest a tight correlation of MEK/ERK inhibition with c-Myc down-regulation and arrest of tumor growth. Thus, MEK inhibitors may be investigated for a signal transduction-based targeting of the c-Myc as a therapeutic strategy in ERMS. [Mol Cancer Ther 2009;8(3):543–51]

Sara A. Flanigan - One of the best experts on this subject based on the ideXlab platform.

  • Abstract A256: Rational combination of the IGF‐1R/IR tyrosine kinase inhibitor (TKI), OSI‐906, with the MEK inhibitor, U0126, results in synergistic and apoptotic effects in human colorectal cancer (CRC) cell lines
    Therapeutic Agents - Small Molecule Kinase Inhibitors, 2009
    Co-Authors: Sara A. Flanigan, Todd M. Pitts, Gillian N. Kulikowski, Aik Choon Tan, Sujatha Nallapareddy, John J. Tentler, S. Gail Eckhardt, Stephen Leong
    Abstract:

    Background: Signaling by the insulin‐like growth factor 1 receptor (IGF‐1R) promotes cell growth, migration and survival in several human tumor types. IGF‐1R is overexpressed in CRC and is associated with a poor prognosis and resistance to chemotherapy. Our prior transcriptional profiling analysis of CRC cell lines treated with the small molecule IGF‐1R/IR TKI, OSI‐906, indicated that overexpression of the mitogen‐activated protein kinase (MAPK) pathway conferred resistance to OSI‐906. The purpose of this study was to evaluate the rational combination of the MEK1/2 inhibitor, U0126, and OSI‐906 against human CRC cell lines. Methods and Results: The antiproliferative effects of OSI‐906 and U0126 were assessed as single agents and in combination using the Sulforhodamine B (SRB) cell viability assay. Twenty‐eight CRC cell lines were exposed to either OSI‐906 (0–5µM), or U0126 (0–20µM). In reference to OSI‐906, cell lines with IC50 ≤ 1µM were considered sensitive (S) and cell lines with IC50 ≥ 5µM were deemed resistant (R). Likewise, cell lines with IC50 10µM were considered R. We selected 12 cell lines for combination studies according to the following conditions: 1) S to both drugs, 2) R to both drugs, 3) S to OSI‐906 but R to U0126, and 4) R to OSI‐906 but S to U0126, and treated them with varying doses of the two drugs as single agents and in combination. Combination effects between OSI‐906 and U0126 were evaluated using the Chou and Talalay method. Robust synergy was observed in most CRC cell lines, including those that were resistant to OSI‐906 or U0126. Apoptosis was then analyzed using bioluminescent caspase 3/7 detection and validated through analysis of PARP cleavage by immunoblotting. Surprisingly, some of the cell lines demonstrated induction of apoptosis when exposed to the combination but not with either agent alone. The CRC cell lines with the greatest apoptotic induction were inherently sensitive to OSI‐906 in the SRB assay. Conclusion: Our prior transcriptional profiling data revealed elevated expression of genes in the MAPK pathway in cell lines that were less sensitive to OSI‐906, providing the basis for rational combination therapy with U0126. Interestingly, the combination of OSI‐906 and U0126 displayed synergy in nearly all cell lines tested, regardless of sensitivity to either compound. Similarly, the apoptosis exhibited by some CRC cell lines could not be predicted by the sensitivity profile, or RAS/RAF mutational status. However, CRC cell lines with the greatest synergistic induction of apoptosis were sensitive to OSI‐906. Further pathway and transcriptional profiling analysis is ongoing to reveal the underlying mechanism(s) of the observed synergy. These results, if further validated in vivo, will support the rational combination of OSI‐906 and a MEK inhibitor in patients with CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A256.

  • abstract a256 rational combination of the igf 1r ir tyrosine kinase inhibitor tki osi 906 with the mek inhibitor U0126 results in synergistic and apoptotic effects in human colorectal cancer crc cell lines
    Molecular Cancer Therapeutics, 2009
    Co-Authors: Sara A. Flanigan, Todd M. Pitts, Gillian N. Kulikowski, Aik Choon Tan, Sujatha Nallapareddy, John J. Tentler, Gail S Eckhardt, Stephen Leong
    Abstract:

    Background: Signaling by the insulin‐like growth factor 1 receptor (IGF‐1R) promotes cell growth, migration and survival in several human tumor types. IGF‐1R is overexpressed in CRC and is associated with a poor prognosis and resistance to chemotherapy. Our prior transcriptional profiling analysis of CRC cell lines treated with the small molecule IGF‐1R/IR TKI, OSI‐906, indicated that overexpression of the mitogen‐activated protein kinase (MAPK) pathway conferred resistance to OSI‐906. The purpose of this study was to evaluate the rational combination of the MEK1/2 inhibitor, U0126, and OSI‐906 against human CRC cell lines. Methods and Results: The antiproliferative effects of OSI‐906 and U0126 were assessed as single agents and in combination using the Sulforhodamine B (SRB) cell viability assay. Twenty‐eight CRC cell lines were exposed to either OSI‐906 (0–5µM), or U0126 (0–20µM). In reference to OSI‐906, cell lines with IC50 ≤ 1µM were considered sensitive (S) and cell lines with IC50 ≥ 5µM were deemed resistant (R). Likewise, cell lines with IC50 10µM were considered R. We selected 12 cell lines for combination studies according to the following conditions: 1) S to both drugs, 2) R to both drugs, 3) S to OSI‐906 but R to U0126, and 4) R to OSI‐906 but S to U0126, and treated them with varying doses of the two drugs as single agents and in combination. Combination effects between OSI‐906 and U0126 were evaluated using the Chou and Talalay method. Robust synergy was observed in most CRC cell lines, including those that were resistant to OSI‐906 or U0126. Apoptosis was then analyzed using bioluminescent caspase 3/7 detection and validated through analysis of PARP cleavage by immunoblotting. Surprisingly, some of the cell lines demonstrated induction of apoptosis when exposed to the combination but not with either agent alone. The CRC cell lines with the greatest apoptotic induction were inherently sensitive to OSI‐906 in the SRB assay. Conclusion: Our prior transcriptional profiling data revealed elevated expression of genes in the MAPK pathway in cell lines that were less sensitive to OSI‐906, providing the basis for rational combination therapy with U0126. Interestingly, the combination of OSI‐906 and U0126 displayed synergy in nearly all cell lines tested, regardless of sensitivity to either compound. Similarly, the apoptosis exhibited by some CRC cell lines could not be predicted by the sensitivity profile, or RAS/RAF mutational status. However, CRC cell lines with the greatest synergistic induction of apoptosis were sensitive to OSI‐906. Further pathway and transcriptional profiling analysis is ongoing to reveal the underlying mechanism(s) of the observed synergy. These results, if further validated in vivo, will support the rational combination of OSI‐906 and a MEK inhibitor in patients with CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A256.

Francesco Marampon - One of the best experts on this subject based on the ideXlab platform.

  • key role of mek erk pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem like cell population
    Molecular Cancer, 2016
    Co-Authors: Carmela Ciccarelli, Francesco Marampon, Francesca Vulcano, Luisa Milazzo, Giovanni Luca Gravina, Giampiero Macioce, Adele Giampaolo, Vincenzo Tombolini, Virginia Di Paolo, H J Hassan
    Abstract:

    The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD). Rhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium. MEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells. Our results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance. Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma.

  • MEK/ERK inhibitor U0126 affects in vitro and in vivo growth of embryonal rhabdomyosarcoma
    Molecular Cancer Therapeutics, 2009
    Co-Authors: Francesco Marampon, Gianluca Bossi, Carmela Ciccarelli, Agnese Di Rocco, Ada Sacchi, Richard G. Pestell, Bianca M. Zani
    Abstract:

    We reported previously that the disruption of c-Myc through mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibition blocks the expression of the transformed phenotype in the embryonal rhabdomyosarcoma (ERMS) cell line (RD), thereby inducing myogenic differentiation in vitro. In this article, we investigate whether MEK/ERK inhibition, by the MEK/ERK inhibitor U0126, affects c-Myc protein level and growth of RMS tumor in an in vivo xenograft model. U0126 significantly reduced RMS tumor growth in RD cell line-xenotransplanted mice. Immunobiochemical and immunohistochemical analysis showed (a) phospho-active ERK levels were reduced by U0126 therapy and unaltered in normal tissues, (b) phospho-Myc and c-Myc was reduced commensurate with phospho-ERK inhibition, and (c) reduction in Ki-67 and endothelial (CD31) marker expression. These results indicate that MEK/ERK inhibition affects growth and angiogenic signals in tumor. The RD-M1 cultured xenograft tumor-derived cell line and the ERMS cell line TE671 responded to U0126 by arresting growth, down-regulating c-Myc, and initiating myogenesis. All these results suggest a tight correlation of MEK/ERK inhibition with c-Myc down-regulation and arrest of tumor growth. Thus, MEK inhibitors may be investigated for a signal transduction-based targeting of the c-Myc as a therapeutic strategy in ERMS.

  • mek erk inhibitor U0126 affects in vitro and in vivo growth of embryonal rhabdomyosarcoma
    Molecular Cancer Therapeutics, 2009
    Co-Authors: Francesco Marampon, Gianluca Bossi, Carmela Ciccarelli, Agnese Di Rocco, Ada Sacchi, Richard G. Pestell, Bianca M. Zani
    Abstract:

    We reported previously that the disruption of c-Myc through mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibition blocks the expression of the transformed phenotype in the embryonal rhabdomyosarcoma (ERMS) cell line (RD), thereby inducing myogenic differentiation in vitro. In this article, we investigate whether MEK/ERK inhibition, by the MEK/ERK inhibitor U0126, affects c-Myc protein level and growth of RMS tumor in an in vivo xenograft model. U0126 significantly reduced RMS tumor growth in RD cell line-xenotransplanted mice. Immunobiochemical and immunohistochemical analysis showed (a) phospho-active ERK levels were reduced by U0126 therapy and unaltered in normal tissues, (b) phospho-Myc and c-Myc was reduced commensurate with phospho-ERK inhibition, and (c) reduction in Ki-67 and endothelial (CD31) marker expression. These results indicate that MEK/ERK inhibition affects growth and angiogenic signals in tumor. The RD-M1 cultured xenograft tumor-derived cell line and the ERMS cell line TE671 responded to U0126 by arresting growth, down-regulating c-Myc, and initiating myogenesis. All these results suggest a tight correlation of MEK/ERK inhibition with c-Myc down-regulation and arrest of tumor growth. Thus, MEK inhibitors may be investigated for a signal transduction-based targeting of the c-Myc as a therapeutic strategy in ERMS.

  • mek erk inhibitor U0126 affects in vitro and in vivo growth of embryonal rhabdomyosarcoma
    Molecular Cancer Therapeutics, 2009
    Co-Authors: Francesco Marampon, Gianluca Bossi, Carmela Ciccarelli, Agnese Di Rocco, Ada Sacchi, Richard G. Pestell, Bianca M. Zani
    Abstract:

    We reported previously that the disruption of c-Myc through mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibition blocks the expression of the transformed phenotype in the embryonal rhabdomyosarcoma (ERMS) cell line (RD), thereby inducing myogenic differentiation in vitro . In this article, we investigate whether MEK/ERK inhibition, by the MEK/ERK inhibitor U0126, affects c-Myc protein level and growth of RMS tumor in an in vivo xenograft model. U0126 significantly reduced RMS tumor growth in RD cell line-xenotransplanted mice. Immunobiochemical and immunohistochemical analysis showed ( a ) phospho-active ERK levels were reduced by U0126 therapy and unaltered in normal tissues, ( b ) phospho-Myc and c-Myc was reduced commensurate with phospho-ERK inhibition, and ( c ) reduction in Ki-67 and endothelial (CD31) marker expression. These results indicate that MEK/ERK inhibition affects growth and angiogenic signals in tumor. The RD-M1 cultured xenograft tumor-derived cell line and the ERMS cell line TE671 responded to U0126 by arresting growth, down-regulating c-Myc, and initiating myogenesis. All these results suggest a tight correlation of MEK/ERK inhibition with c-Myc down-regulation and arrest of tumor growth. Thus, MEK inhibitors may be investigated for a signal transduction-based targeting of the c-Myc as a therapeutic strategy in ERMS. [Mol Cancer Ther 2009;8(3):543–51]