Activation of the RAF/Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase/Extracellular Signal-Regulated Kinase Pathway Mediates Apoptosis Induced by Chelerythrine in Osteosarcorna

Department of Pediatrics and Molecular Pharmacology, The Albert Einstein College of Medicine, The Children's Hospital at Montefiore, Bronx, New York 10467, USA.
Clinical Cancer Research (Impact Factor: 8.19). 10/2008; 14(20):6396-404. DOI: 10.1158/1078-0432.CCR-07-5113
Source: PubMed

ABSTRACT 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.
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.
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.
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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of Rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including Rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells we identified a series of molecules antagonized by Rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine (NAC) or GSH co-treatment protected TSC2-null cells from chelerythrine's effects, indicating that chelerythrine-induced cell death is ROS-dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress-dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors. Implications: This study demonstrates that TSC2-deficient tumor cells are hypersensitive to oxidative stress-dependent cell death, and provide critical proof-of-concept that TSC2-deficient cells can be therapeutically targeted without the use of a Rapalog to induce a cell death response.
    Molecular Cancer Research 09/2014; 13(1). DOI:10.1158/1541-7786.MCR-14-0440 · 4.50 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this investigation was to assess the in-vitro interaction of two antifungal agents, econazole-nitrate and chelerythrine, against ten fluconazole-resistant clinical isolates and one ATCC type strain 10231 of Candida albicans. The checkerboard microdilution method was performed according to the recommendations of the National Committee for Clinical Laboratory Standards, and the results were determined by visual examination. The interaction intensity was tested in all isolates using the fractional inhibitory concentration index (FICI). These experiments showed synergism between econazole-nitrate and chelerythrine in antifungal activity against C. albicans, and no antagonistic activity was observed in any of the strains tested. Moreover, time-kill curves were performed with selected strains to confirm the positive interactions. The similarity between the results of the FICI values and the time-kill curves revealed that chelerythrine greatly enhances the antifungal effects of econazole-nitrate against isolates of C. albicans. This synergistic effect may markedly reduce the dose of econazole-nitrate required to treat candidiasis, thereby decreasing the econazole-nitrate toxic side effects. This novel synergism might provide a potential combination treatment against fungal infections.
    Iranian journal of pharmaceutical research (IJPR) 03/2014; 13(2):567-73. · 0.51 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Results of previous study showed promising but short-lived activity of sorafenib in the treatment of patients with unresectable advanced and metastatic osteosarcoma. This treatment failure has been attributed to the mTOR pathway and might therefore be overcome with the addition of mTOR inhibitors. We aimed to investigate the activity of sorafenib in combination with everolimus in patients with inoperable high-grade osteosarcoma progressing after standard treatment. We did this non-randomised phase 2 trial in three Italian Sarcoma Group centres. We enrolled adults (≥18 years) with relapsed or unresectable osteosarcoma progressing after standard treatment (methotrexate, cisplatin, and doxorubicin, with or without ifosfamide). Patients received 800 mg sorafenib plus 5 mg everolimus once a day until disease progression or unacceptable toxic effects. The primary endpoint was 6 month progression-free survival (PFS). All analyses were intention-to-treat. This trial is registered with, number NCT01804374. We enrolled 38 patients between June 16, 2011, and June 4, 2013. 17 (45%; 95% CI 28-61) of 38 patients were progression free at 6 months. Toxic effects led to dose reductions, or short interruptions, or both in 25 (66%) of 38 patients and permanent discontinuation for two (5%) patients. The most common grade 3-4 adverse events were lymphopenia and hypophosphataemia each in six (16%) patients, hand and foot syndrome in five (13%), thrombocytopenia in four (11%), and fatigue, oral mucositis, diarrhoea, and anaemia each in two (5%). One patient (3%) had a grade 3 pneumothorax that required trans-thoracic drainage, and that recurred at the time of disease progression. This was reported as a serious adverse event related to the study drugs in both instances. No other serious adverse events were reported during the trial. There were no treatment-related deaths. Although the combination of sorafenib and everolimus showed activity as a further-line treatment for patients with advanced or unresectable osteosarcoma, it did not attain the prespecified target of 6 month PFS of 50% or greater. Italian Sarcoma Group. Copyright © 2014 Elsevier Ltd. All rights reserved.
    The Lancet Oncology 12/2014; DOI:10.1016/S1470-2045(14)71136-2 · 24.73 Impact Factor