Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas.
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)/C(18)-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 C(18)-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 C(18)-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/C(18)-ceramide in gemcitabine/doxorubicin-induced cell death via the activation of caspase-9/3 in HNSCC.
Senkal
Can E
CE
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA.
Ponnusamy
Suriyan
S
Rossi
Michael J
MJ
Bialewski
Jacek
J
Sinha
Debijyati
D
Jiang
James C
JC
Jazwinski
S Michal
SM
Hannun
Yusuf A
YA
Ogretmen
Besim
B
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy*; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D004317:Doxorubicin / Q000008:administration & dosage; D005455:Fluorescent Antibody Technique; D006258:Head and Neck Neoplasms / Q000188:drug therapy*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051049:Sphingosine N-Acyltransferase; D015996:Survival Rate; D000093542:Gemcitabine
D002518:Ceramides; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D003841:Deoxycytidine; D004317:Doxorubicin; D010088:Oxidoreductases; C473786:dihydroceramide synthase; C493790:CERS1 protein, human; C505722:CERS5 protein, human; D051049:Sphingosine N-Acyltransferase; D020169:Caspases; D000093542:Gemcitabine