fenretinide and safingol

The unfolded protein response (UPR) is induced by proteotoxic stress of the endoplasmic reticulum (ER). Here we report that ATF6, a major mammalian UPR sensor, is also activated by specific sphingolipids, dihydrosphingosine (DHS) and dihydroceramide (DHC). Single mutations in a previously undefined transmembrane domain motif that we identify in ATF6 incapacitate DHS/DHC activation while still allowing proteotoxic stress activation via the luminal domain. ATF6 thus possesses two activation mechanisms: DHS/DHC activation and proteotoxic stress activation. Reporters constructed to monitor each mechanism show that phenobarbital-induced ER membrane expansion depends on transmembrane domain-induced ATF6. DHS/DHC addition preferentially induces transcription of ATF6 target lipid biosynthetic and metabolic genes over target ER chaperone genes. Importantly, ATF6 containing a luminal achromatopsia eye disease mutation, unresponsive to proteotoxic stress, can be activated by fenretinide, a drug that upregulates DHC, suggesting a potential therapy for this and other ATF6-related diseases including heart disease and stroke.

Topics: Activating Transcription Factor 6; Cell Line; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Fenretinide; Humans; Protein Serine-Threonine Kinases; Sphingosine; Transcription, Genetic; Unfolded Protein Response

N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model.. CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling.. No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells.. In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

Topics: Analysis of Variance; Antineoplastic Agents; Apoptosis; Cell Proliferation; Cell Survival; Fenretinide; Humans; Leukemia; Oxidoreductases; Sphingolipids; Sphingosine; Tumor Cells, Cultured

Cytotoxicity assays in 96-well tissue culture plates allow rapid sample handling for multicondition experiments but have a limited dynamic range. Using DIMSCAN, a fluorescence digital image system for quantifying relative cell numbers in tissue culture plates, we have developed a 96-well cytotoxicity assay with a >4-log dynamic range.. To overcome background fluorescence that limits detection of viable cells with fluorescein diacetate, we used 2'4'5'6'-tetrabromofluorescein (eosin Y) to quench background fluorescence in the medium and in nonviable cells to enhance the reduction of background fluorescence achieved with digital image thresholding. The sensitivity and linearity of the new assay were tested with serial dilutions of neuroblastoma and leukemia cell lines. DIMSCAN was compared with other in vitro cytotoxicity assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and trypan blue dye exclusion.. Without background fluorescence reduction, scans produced a nearly flat curve across various cell concentrations from 100 to 10(6) cells per well. Either digital image thresholding or eosin Y dramatically reduced background fluorescence, and combining them achieved a linear correlation (r > 0.9) of relative fluorescence to viable cell number over >4 logs of dynamic range, even in the presence of 4 x 10(4) nonviable cells per well. Cytotoxicity of deferoxamine for neuroblastoma cell lines measured by the DIMSCAN assay achieved dose-response curves similar to data obtained by manual trypan blue counts or colony formation in soft agar but with a wider dynamic range. Long-term cultures documented the clonogenic ability of viable cells detected by DIMSCAN over the entire dynamic range. The cytotoxicity of two drug combinations (buthionine sulfoximine + melphalan or fenretinide + safingol) was tested using both DIMSCAN and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the wider dynamic range of DIMSCAN facilitated detection of synergistic interactions.. DIMSCAN offers the ability to rapidly and efficiently conduct cytotoxicity assays in 96-well plates with a dynamic range of >4 logs. This assay enables rapid testing of anticancer drug combinations in microplates.

Topics: Antineoplastic Combined Chemotherapy Protocols; Buthionine Sulfoximine; Cell Proliferation; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Inhibitors; Eosine Yellowish-(YS); Fenretinide; Fluorescent Dyes; Humans; Leukemia; Melphalan; Microscopy, Fluorescence; Neuroblastoma; Protein Kinase C; Sphingosine; Tumor Cells, Cultured; Tumor Stem Cell Assay

Patients with disseminated Ewing's family of tumors (ESFT) often experience drug-resistant relapse. We hypothesize that targeting minimal residual disease with the cytotoxic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR; fenretinide) may decrease relapse. We determined the following: (a) 4-HPR cytotoxicity against 12 ESFT cell lines in vitro; (b) whether 4-HPR increased ceramide species (saturated and desaturated ceramides); (c) whether physiological hypoxia (2% O(2)) affected cytotoxicity, mitochondrial membrane potential (DeltaPsi(m)) change, or ceramide species or reactive oxygen species levels; (d) whether cytotoxicity was enhanced by l-threo-dihydrosphingosine (safingol); (e) whether physiological hypoxia increased acid ceramidase (AC) expression; and (f) the effect of the AC inhibitor N-oleoyl-ethanolamine (NOE) on cytotoxicity and ceramide species. Ceramide species were quantified by thin-layer chromatography and scintillography. Cytotoxicity was measured by a fluorescence-based assay using digital imaging microscopy (DIMSCAN). Gene expression profiling was performed by oligonucleotide array analysis. We observed, in 12 cell lines tested in normoxia (20% O(2)), that the mean 4-HPR LC(99) (the drug concentration lethal to 99% of cells) = 6.1 +/- 5.4 microm (range, 1.7-21.8 microm); safingol (1-3 microm) synergistically increased 4-HPR cytotoxicity and reduced the mean 4-HPR LC(99) to 3.2 +/- 1.7 microm (range, 2.0-8.0 microm; combination index < 1). 4-HPR increased ceramide species in the three cell lines tested (up to 9-fold; P < 0.05). Hypoxia (2% O(2)) reduced ceramide species increase, DeltaPsi(m) loss, reactive oxygen species increase (P < 0.05), and 4-HPR cytotoxicity (P = 0.05; 4-HPR LC(99), 19.7 +/- 23.9 microm; range, 2.3-91.4). However, hypoxia affected 4-HPR + safingol cytotoxicity to a lesser extent (P = 0.04; 4-HPR LC(99), 4.9 +/- 2.3 microm; range, 2.0-8.2). Hypoxia increased AC RNA expression; the AC inhibitor NOE enhanced 4-HPR-induced ceramide species increase and cytotoxicity. The antioxidant N-acetyl-l-cysteine somewhat reduced 4-HPR cytotoxicity but did not affect ceramide species increase. We conclude the following: (a) 4-HPR was active against ESFT cell lines in vitro at concentrations achievable clinically, but activity was decreased in hypoxia; and (b) combining 4-HPR with ceramide modulators synergized 4-HPR cytotoxicity in normoxia and hypoxia.

Topics: Acetylcysteine; Antineoplastic Agents; Antioxidants; Apoptosis; Cell Hypoxia; Ceramides; Drug Synergism; Enzyme Inhibitors; Fenretinide; Galactosylgalactosylglucosylceramidase; Gene Expression Profiling; Humans; Membrane Potentials; Mitochondria; Neoplasm, Residual; Neuroectodermal Tumors, Primitive; Oligonucleotide Array Sequence Analysis; Protein Kinase C; Reactive Oxygen Species; Sarcoma, Ewing; Sphingosine; Tumor Cells, Cultured

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR; fenretinide) is cytotoxic to a variety of cancer cell lines, and we previously showed an association between ceramide generation and 4-HPR cytotoxicity for neuroblastoma cell lines (B. J. Maurer et al., J. Natl. Cancer Inst. (Bethesda), 91: 1138-1146, 1999). Here we determine whether the increased ceramide mediated by 4-HPR in the CHLA-90 human neuroblastoma cell line results from de novo ceramide synthesis. Treatment of CHLA-90 with 4-HPR for 2 h, in the presence of [(3)H]palmitic acid, caused sequential formation of [(3)H]sphinganine (220% over control) and [(3)H]ceramide (160% over control), with sphinganine returning to baseline at 4 h, and ceramide continuing to increase (215% over control). 4-HPR treatment did not accelerate cellular decay of sphingomyelin. Preincubation of cells with either L-cycloserine, an inhibitor of serine palmitoyltransferase (SPT), or fumonisin B(1), an inhibitor of ceramide synthase, retarded ceramide formation in response to 4-HPR treatment, although sphinganine was still generated when 4-HPR and FB(1) were present. Data from in vitro enzyme assays using microsomes showed that preexposure of intact cells to 4-HPR resulted in a time (175% over control; 6 h)- and dose-dependent increase (173% over control; 10 microM) in SPT activity as well as a time (265% over control)- and dose-dependent increase (215% above control; 10 microM) in ceramide synthase activity. Our results show that 4-HPR-mediated ceramide generation is derived from the de novo synthetic pathway by coordinate activation of SPT and ceramide synthase. Knowledge of these biochemical events is of utility when downstream modulators of ceramide metabolism are used to heighten the cytotoxic response to chemotherapy.

Topics: Acyltransferases; Antineoplastic Agents; Ceramides; Enzyme Activation; Enzyme Induction; Fenretinide; Humans; Neuroblastoma; Oxidoreductases; Serine C-Palmitoyltransferase; Sphingosine; Tumor Cells, Cultured

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.. 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.. 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.. 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.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Apoptosis; Ceramides; Drug Synergism; Enzyme Inhibitors; Estrogen Receptor Modulators; Fenretinide; Glucosyltransferases; Humans; Morpholines; Necrosis; Neoplasms; Protein Kinase C; Sphingosine; Tamoxifen; Tumor Cells, Cultured; Tumor Suppressor Protein p53