Activation of the RAF/mitogen-activated protein/extracellular signal-regulated kinase Kinase/extracellular signal-regulated kinase pathway mediates apoptosis induced by chelerythrine in osteosarcoma

Rui Yang, Sajida Piperdi, Richard Gorlick

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Abstract

Purpose: Chelerythrine, a widely used broad-range protein kinase C inhibitor, induces apoptosis in many cell types. In this study, the mechanism of chelerythrine-induced apoptosis in osteosarcoma was investigated. Experimental Design: Signaling pathways activated by chelerythrine in osteosarcoma were detected by Western blots. Impacts of RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK on apoptosis and cell survival were studied using genetic approaches and pharmacologic pathway-specific inhibitors. Results: Osteosarcoma cells underwent apoptosis rapidly after treatment with chelerythrine. Three parallel MAPKs pathways, including the ERKs, c-Jun NH 2 kinases, and p38, were activated by chelerythrine in a dose-dependent and time-dependent fashion. For the ERKs, the activation was evident at the earliest time point tested (2 minutes) and sustained for >4 hours. Introduction of a dominant-negative H-RAS mutant (17N) partially attenuated ERK activation and delayed the onset of apoptosis induced by chelerythrine. The ERK activation and apoptotic effects of chelerythrine were greatly abrogated by the pharmaceutical inhibitors of MEK, but not by those of c-Jun NH 2 kinase or p38. Moreover, osteosarcoma cells were sensitized to chelerythrine by transient transfection with wild-type MEK1 or constitutively active MEK1 and became resistant with dominant-negative MEK1. Other protein kinase C inhibitors, including GF109203X or Gö6976, did not cause ERK activation or apoptosis in the same timeframe tested. Conclusion: In osteosarcoma, chelerythrine-induced apoptosis is mediated through activation of the RAF/MEK/ERK pathway. These findings suggest that activating the ERK MAPK, as opposed to inhibiting it, may be a therapeutic strategy in osteosarcoma.

Original languageEnglish (US)
Pages (from-to)6396-6404
Number of pages9
JournalClinical Cancer Research
Volume14
Issue number20
DOIs
StatePublished - Oct 15 2008

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Extracellular Signal-Regulated MAP Kinases
Osteosarcoma
Mitogens
Phosphotransferases
Apoptosis
Proteins
Mitogen-Activated Protein Kinase 9
Mitogen-Activated Protein Kinases
Protein C Inhibitor
Mitogen-Activated Protein Kinase Kinases
Protein Kinase Inhibitors
Protein Kinase C
MAP Kinase Kinase Kinases
chelerythrine
MAP Kinase Signaling System
Transfection
Cell Survival
Research Design
Western Blotting

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

@article{e85ea49e08064c8d83c2484de6b7ddf3,
title = "Activation of the RAF/mitogen-activated protein/extracellular signal-regulated kinase Kinase/extracellular signal-regulated kinase pathway mediates apoptosis induced by chelerythrine in osteosarcoma",
abstract = "Purpose: Chelerythrine, a widely used broad-range protein kinase C inhibitor, induces apoptosis in many cell types. In this study, the mechanism of chelerythrine-induced apoptosis in osteosarcoma was investigated. Experimental Design: Signaling pathways activated by chelerythrine in osteosarcoma were detected by Western blots. Impacts of RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK on apoptosis and cell survival were studied using genetic approaches and pharmacologic pathway-specific inhibitors. Results: Osteosarcoma cells underwent apoptosis rapidly after treatment with chelerythrine. Three parallel MAPKs pathways, including the ERKs, c-Jun NH 2 kinases, and p38, were activated by chelerythrine in a dose-dependent and time-dependent fashion. For the ERKs, the activation was evident at the earliest time point tested (2 minutes) and sustained for >4 hours. Introduction of a dominant-negative H-RAS mutant (17N) partially attenuated ERK activation and delayed the onset of apoptosis induced by chelerythrine. The ERK activation and apoptotic effects of chelerythrine were greatly abrogated by the pharmaceutical inhibitors of MEK, but not by those of c-Jun NH 2 kinase or p38. Moreover, osteosarcoma cells were sensitized to chelerythrine by transient transfection with wild-type MEK1 or constitutively active MEK1 and became resistant with dominant-negative MEK1. Other protein kinase C inhibitors, including GF109203X or G{\"o}6976, did not cause ERK activation or apoptosis in the same timeframe tested. Conclusion: In osteosarcoma, chelerythrine-induced apoptosis is mediated through activation of the RAF/MEK/ERK pathway. These findings suggest that activating the ERK MAPK, as opposed to inhibiting it, may be a therapeutic strategy in osteosarcoma.",
author = "Rui Yang and Sajida Piperdi and Richard Gorlick",
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T1 - Activation of the RAF/mitogen-activated protein/extracellular signal-regulated kinase Kinase/extracellular signal-regulated kinase pathway mediates apoptosis induced by chelerythrine in osteosarcoma

AU - Yang, Rui

AU - Piperdi, Sajida

AU - Gorlick, Richard

PY - 2008/10/15

Y1 - 2008/10/15

N2 - Purpose: Chelerythrine, a widely used broad-range protein kinase C inhibitor, induces apoptosis in many cell types. In this study, the mechanism of chelerythrine-induced apoptosis in osteosarcoma was investigated. Experimental Design: Signaling pathways activated by chelerythrine in osteosarcoma were detected by Western blots. Impacts of RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK on apoptosis and cell survival were studied using genetic approaches and pharmacologic pathway-specific inhibitors. Results: Osteosarcoma cells underwent apoptosis rapidly after treatment with chelerythrine. Three parallel MAPKs pathways, including the ERKs, c-Jun NH 2 kinases, and p38, were activated by chelerythrine in a dose-dependent and time-dependent fashion. For the ERKs, the activation was evident at the earliest time point tested (2 minutes) and sustained for >4 hours. Introduction of a dominant-negative H-RAS mutant (17N) partially attenuated ERK activation and delayed the onset of apoptosis induced by chelerythrine. The ERK activation and apoptotic effects of chelerythrine were greatly abrogated by the pharmaceutical inhibitors of MEK, but not by those of c-Jun NH 2 kinase or p38. Moreover, osteosarcoma cells were sensitized to chelerythrine by transient transfection with wild-type MEK1 or constitutively active MEK1 and became resistant with dominant-negative MEK1. Other protein kinase C inhibitors, including GF109203X or Gö6976, did not cause ERK activation or apoptosis in the same timeframe tested. Conclusion: In osteosarcoma, chelerythrine-induced apoptosis is mediated through activation of the RAF/MEK/ERK pathway. These findings suggest that activating the ERK MAPK, as opposed to inhibiting it, may be a therapeutic strategy in osteosarcoma.

AB - Purpose: Chelerythrine, a widely used broad-range protein kinase C inhibitor, induces apoptosis in many cell types. In this study, the mechanism of chelerythrine-induced apoptosis in osteosarcoma was investigated. Experimental Design: Signaling pathways activated by chelerythrine in osteosarcoma were detected by Western blots. Impacts of RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK on apoptosis and cell survival were studied using genetic approaches and pharmacologic pathway-specific inhibitors. Results: Osteosarcoma cells underwent apoptosis rapidly after treatment with chelerythrine. Three parallel MAPKs pathways, including the ERKs, c-Jun NH 2 kinases, and p38, were activated by chelerythrine in a dose-dependent and time-dependent fashion. For the ERKs, the activation was evident at the earliest time point tested (2 minutes) and sustained for >4 hours. Introduction of a dominant-negative H-RAS mutant (17N) partially attenuated ERK activation and delayed the onset of apoptosis induced by chelerythrine. The ERK activation and apoptotic effects of chelerythrine were greatly abrogated by the pharmaceutical inhibitors of MEK, but not by those of c-Jun NH 2 kinase or p38. Moreover, osteosarcoma cells were sensitized to chelerythrine by transient transfection with wild-type MEK1 or constitutively active MEK1 and became resistant with dominant-negative MEK1. Other protein kinase C inhibitors, including GF109203X or Gö6976, did not cause ERK activation or apoptosis in the same timeframe tested. Conclusion: In osteosarcoma, chelerythrine-induced apoptosis is mediated through activation of the RAF/MEK/ERK pathway. These findings suggest that activating the ERK MAPK, as opposed to inhibiting it, may be a therapeutic strategy in osteosarcoma.

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