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Research Articles: Therapeutics, Targets, and Development

Role of human longevity assurance gene 1 and C₁₈-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas

¹ Departments of Biochemistry and Molecular Biology, ² Hollings Cancer Center, and ³ Biostatistics, Bioinformatics and Epidemiology, Medical University of South Carolina, Charleston, South Carolina and ⁴ Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana

Requests for reprints: Besim Ogretmen, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425. Phone: 843-792-0940; Fax: 843-792-8568. E-mail: ogretmen{at}musc.edu

Abstract

In this study, quantitative isobologram studies showed that treatment with gemcitabine and doxorubicin, known inducers of ceramide generation, in combination, supra-additively inhibited the growth of human UM-SCC-22A cells in situ. Then, possible involvement of the human homologue of yeast longevity assurance gene 1 (LASS1)/C₁₈-ceramide in chemotherapy-induced cell death in these cells was examined. Gemcitabine/doxorubicin combination treatment resulted in the elevation of mRNA and protein levels of LASS1 and not LASS2-6, which was consistent with a 3.5-fold increase in the endogenous (dihydro)ceramide synthase activity of LASS1 for the generation of C₁₈-ceramide. Importantly, the overexpression of LASS1 (both human and mouse homologues) enhanced the growth-inhibitory effects of gemcitabine/doxorubicin with a concomitant induction of caspase-3 activation. In reciprocal experiments, partial inhibition of human LASS1 expression using small interfering RNA (siRNA) prevented cell death by about 50% in response to gemcitabine/doxorubicin. In addition, LASS1, and not LASS5, siRNA modulated the activation of caspase-3 and caspase-9, but not caspase-8, in response to this combination. Treatment with gemcitabine/doxorubicin in combination also resulted in a significant suppression of the head and neck squamous cell carcinoma (HNSCC) tumor growth in severe combined immunodeficiency mice bearing the UM-SCC-22A xenografts. More interestingly, analysis of endogenous ceramide levels in these tumors by liquid chromatography/mass spectroscopy showed that only the levels of C₁₈-ceramide, the main product of LASS1, were elevated significantly (about 7-fold) in response to gemcitabine/doxorubicin when compared with controls. In conclusion, these data suggest an important role for LASS1/C₁₈-ceramide in gemcitabine/doxorubicin-induced cell death via the activation of caspase-9/3 in HNSCC. [Mol Cancer Ther 2007;6(2):712–22]

Grant support: NIH DE01657 (B. Ogretmen), AG006168 (S.M. Jazwinski), and CA097132 (Y.A. Hannun), Department of Defense, Phase VII Program Project grant through Hollings Cancer Center (B. Ogretmen), and the National Science Foundation/EPSCoR, EPS-0132573 (B. Ogretmen). The animal facility used in this study was supported by the NIH grant C06 RR015455 (Medical University of South Carolina) from the Extramural Research Facilities Program of the National Center for Research Resources. This work was conducted in a facility constructed with support from the NIH, grant CO6 RR018823 from the Extramural Research Facilities Program of the National Center for Research Resources.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/12/06; revised 12/ 1/06; accepted 12/21/06.

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