Synergistic cytotoxicity in solid tumor cell lines between N-(4-hydroxyphenyl)retinamide and modulators of ceramide metabolism.
BACKGROUND
We previously reported that N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) treatment caused large increases of ceramide levels in neuroblastoma cell lines and induced cell death by a combination of apoptosis and necrosis through p53 (also known as TP53)-independent and caspase-independent pathways. Our goal was to determine if several molecules that inhibit enzymes involved in ceramide metabolism-L-threo-dihydrosphingosine (safingol), d, l-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), and tamoxifen-enhanced 4-HPR-mediated cytotoxicity and/or affected ceramide levels.


METHODS
Cellular lipids were quantified by radiolabeling and thin-layer chromatography. Cytotoxicity and cytotoxic synergy (expressed as combination index, where combination index <1 indicates synergy and >1 indicates antagonism) were measured in cultured cancer cell lines with the use of a fluorescence-based assay of cell viability employing digital imaging microscopy. Statistical tests were two-sided.


RESULTS
4-HPR increased ceramide levels by de novo synthesis. Safingol (1-4 microM) was incorporated into a stereochemical variant of ceramide and synergized with a 3:1 molar ratio of 4-HPR (3-12 microM), to produce a 100-fold to 10 000-fold (2 to 4 logs) increase in cytotoxicity relative to 4-HPR alone in neuroblastoma (combination index <0.1), lung (combination index <0.1-0.2), melanoma (combination index <0.1-0.2), prostate (combination index <0.1-1.0), colon (combination index 0.1-0.3), breast (combination index = 0.1-0.5), and pancreas (combination index = 0.2) cell lines, including p53 mutant and alkylator-resistant cell lines. The 4-HPR and safingol combination was cytotoxic in low-oxygen conditions and was minimally toxic to normal fibroblasts and bone marrow myeloid progenitor cells. Addition of agents that retard ceramide glucosylation and/or acylation, such as PPMP or tamoxifen, to 4-HPR or to the combination of 4-HPR and safingol further increased cytotoxicity to tumor cells.


CONCLUSIONS
Combinations of 4-HPR and modulators of ceramide metabolism may form the basis for a novel chemotherapy that is functional under hypoxic conditions (e.g., such as those within tumors) and is p53 independent and caspase independent.
Maurer
B J
BJ
Division of Hematology-Oncology, Childrens Hospital Los Angeles and Department of Pediatrics, University of Southern California Keck School of Medicine, Los Angeles, CA, USA.
Melton
L
L
Billups
C
C
Cabot
M C
MC
Reynolds
C P
CP
D000970:Antineoplastic Agents / Q000494:pharmacology*; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism*; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020847:Estrogen Receptor Modulators / Q000494:pharmacology; D017313:Fenretinide / Q000494:pharmacology*; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors*; D006801:Humans; D009025:Morpholines / Q000494:pharmacology*; D009336:Necrosis; D009369:Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013629:Tamoxifen / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000970:Antineoplastic Agents; D018931:Antineoplastic Agents, Hormonal; D002518:Ceramides; D004791:Enzyme Inhibitors; D020847:Estrogen Receptor Modulators; D009025:Morpholines; D016159:Tumor Suppressor Protein p53; D013629:Tamoxifen; D017313:Fenretinide; C033110:RV 538; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D011493:Protein Kinase C; D013110:Sphingosine; C005682:safingol