Abstract
Non-small-cell lung cancer (NSCLC) is characterized by severe resistance to chemotherapy. Here, we demonstrate that A549 adenocarcinoma cells permanently differentiate with the antimetabolites methotrexate (MTX) and gemcitabine (GE) when blocking the resistance mechanism that normally counteracts this process. MTX (1-10 μM) and GE (1 μM) induced growth arrest accompanied by sustained extracellular signal-regulated kinase (ERK1/2) phosphorylation and moderate reduction of c-Myc levels after 96 h, whereas only a low percentage of the cells differentiated. Combination with the mitogen-activated protein kinase kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis-(methylthio)butadiene (U0126) reduced MTX- or GE-induced ERK1/2 over-phosphorylation, nearly abolished c-Myc expression, and provoked radical morphological changes in all cells. Besides the appearance of multilamellar bodies and intracellular cytokeratin reorganization, modulation of molecular markers occurred in a manner consistent with differentiation (gelsolin, +300%; surfactant protein A and C, -70%). Similar to U0126, c-Myc inactivation with specific small interfering RNA initiated differentiation only in the presence of MTX, demonstrating that inhibition of the mitogen-activated protein kinase/ERK pathway alone or down-regulation of c-Myc is not sufficient to induce this process. It is noteworthy that withdrawal of antitumoral drugs and U0126 neither reversed differentiation nor reactivated proliferation. Our results reveal that maintenance of a certain threshold of c-Myc expression through sustained ERK1/2 activation represents a molecular mechanism that confers resistance to antimetabolite-induced differentiation in A549 cells, and provide a novel molecular basis for therapeutic strategies based on irreversible differentiation of cancer cells using conventional chemotherapeutic antimetabolites in combination with inhibitors of the MEK/ERK pathway or c-Myc.
Footnotes
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This work was supported by Grants “Nous estudis immunogenotipics en la classificació i tractament de la leucèmia mieloide aguda i síndrome mielodisplàsica”, ISCII number G03/008 and PROGECIB-8A (Conselleria d'Economia, Hisenda i Innovació, Govern Balear). The Dorothy and Jim Bird Foundation also provided funds for this study. R.A. and O.V. are supported by the Spanish I3 Programme (“Programa de Incentivación de la Incorporación e Intensificación de la Actividad Investigadora”).
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J.M.S., A.G., and R.A. contributed equally to this work.
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ABBREVIATIONS: NSCLC, non-small-cell lung cancer; MTX, methotrexate; GE, gemcitabine; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; siRNA, small interfering RNA; PARP, poly(ADP-ribose) polymerase; SP-A, surfactant protein A; SP-C, surfactant protein C; TS, thymidylate synthase.
- Received November 9, 2007.
- Accepted March 19, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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