Affiliations of authors: B. J. Maurer, L. S. Metelitsa, R. C. Seeger, Division of Hematology-Oncology, Childrens Hospital Los Angeles, and Department of Pediatrics, University of Southern California School of Medicine, Los Angeles; M. C. Cabot, The John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA; C. P. Reynolds, Division of Hematology-Oncology, Childrens Hospital Los Angeles, and Departments of Pediatrics and Pathology, University of Southern California School of Medicine.
Correspondence to: C. Patrick Reynolds, M.D., Ph.D., Division of Hematology-Oncology, MS #57, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (e-mail: cpreynol{at}hsc.usc.edu).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been reported that reactive oxygen species (ROS) contribute to the induction of apoptosis by 4-HPR in cervical carcinoma (34) and myeloid leukemia (38) cell lines. Therefore, we determined if 4-HPR induced ROS in neuroblastoma cells and whether ROS was the sole mechanism of 4-HPR cytotoxicity.
Ceramide is a sphingosine-based lipid second messenger involved in the regulation of
diverse cellular responses, including the generation of hydrogen peroxide in mitochondria (39,40) and apoptosis (41,42). Ceramide can be
generated de novo by ceramide synthase or from sphingomyelin breakdown through the
activation of neutral or acidic sphingomyelinase by drugs, such as daunorubicin and doxorubicin,
or the drug resistance modulator SDZ PSC 833; through activation of receptors such as tumor
necrosis factor- or CD95/Fas/APO-1; or by ionizing radiation, ultraviolet-C, heat shock, or
oxidative stress (41,43,44). Ceramide has been reported to initiate
apoptosis under hypoxic conditions in a p53-independent manner via caspase-3 activation (45), causing the activation of the pro-death JNK/SAPK cascade, which is
opposed by spingosine-1-phosphate and ERK1/2 activation (46-48). To
date, no chemotherapeutic agent has been reported whose cytotoxicity is ascribed principally to
the generation of intracellular ceramide.
Because neuroblastomas commonly relapse in bone marrow (49), a tissue with a low oxygen tension (50), and since many common chemotherapeutic agents are antagonized by hypoxia (51,52), we are seeking to identify agents that retain cytotoxicity in reduced-oxygen environments for use in the treatment of neuroblastoma. To this end, we have examined the cytotoxic properties of 4-HPR in human neuroblastoma cell lines. We have determined 1) if 4-HPR increased ROS, 2) the effects of physiologic hypoxia (PO2, approximately 15 mm Hg) and the antioxidant N-acetyl-L-cysteine (NAC) on 4-HPR cytotoxicity, 3) the extent to which ceramide is induced by 4-HPR, and 4) whether 4-HPR induced cell death by an apoptotic or necrotic mechanism(s).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
4-HPR was provided by R. W. Johnson Pharmaceuticals (Spring House, PA). Fluorescein diacetate (FDA) was from the Eastman Kodak Company, Rochester, NY. Eosin Y, N-acetyl-D-sphingosine (C2 ceramide), NAC, and thin-layer chromatography (TLC)-grade organic solvents were from Sigma Chemical Co., St. Louis, MO. 5-(and-6)-Carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA) was from Molecular Probes, Inc., Eugene, OR. Ecolume scintillation cocktail was from ICN Biomedicals, Inc., Costa Mesa, CA. [9,10-3H(N)]Palmitic acid (50 Ci/mmol) was from Dupont NEN® Research Products, Boston, MA. Iscove's modified Dulbecco's medium (IMDM) was from BioWhittaker, Inc., Walkersville, MD. RPMI-1640 medium, fetal bovine serum (FBS), and L-glutamine were from Gemini BioProducts, Calabasas, CA. ITSTM Premix culture supplement (insulin, transferrin, and selenious acid) was from Collaborative Biomedical Products, Bedford, MA. Glucosylceramide (purified from Gaucher's spleen) was from Matreya, Inc., Pleasant Gap, PA. Monooleoylglycerol was from Nu-Check-Prep, Elysian, MN. Other lipid standards were from Avanti Polar Lipids, Inc., Alabaster, AL. Uniplate Silica Gel G 250-µm TLC plates were from Analtech, Inc., Newark, DE. Anti-p53 (DO-1) murine monoclonal antibody was from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Anti-Fas (CH-11) murine monoclonal immunoglobulin M (IgM) antibody was from Beckman Coulter-Immunotech, Miami, FL. BCA Protein AssayTM was from Pierce Chemical Co., Rockford, IL. Ready GelTM precast polyacrylamide gels were from Bio-Rad Laboratories, Hercules, CA. The ECLTM immunoblotting detection reagent kit was from Amersham Life Science, Inc., Arlington Heights, IL. The Protran® nitrocellulose membrane was obtained from Schleicher & Schuell, Inc., Keene, NH. The pan-caspase enzyme inhibitor BOC-d-fmk was from Enzyme Systems Products (Livermore, CA). The Apoptosis DNA-Ladder Kit was from Boehringer Mannheim, Indianapolis, IN. Specialty gases and gas mixtures were from Praxair, North Hollywood, CA. Modular incubation chambers were from Billups-Rothenberg, Del Mar, CA. Falcon brand plasticware was from Becton Dickinson and Co., Franklin Lakes, NJ. 4-HPR and C2 ceramide were dissolved in ethanol and stored at -20 °C. FDA, carboxy-DCFDA, and caspase inhibitors were dissolved in dimethyl sulfoxide (DMSO). FDA was stored at -20 °C, and carboxy-DCFDA was stored over liquid nitrogen.
Cell Culture
Human neuroblastoma cell lines SMS-LHN (53) and SMS-KCNR (54) were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (whole medium). The human neuroblastoma cell line CHLA-90 (55) was established from a tumor relapse in bone marrow after myeloablative chemoradiotherapy supported by autologous bone marrow transplant and was maintained in IMDM supplemented with 0.7 mM L-glutamine, insulin, and transferrin (5 µg/mL each), selenium (5 ng/mL), and 20% heat-inactivated FBS (whole medium). Cell lines were studied during passages 15 through 40 and cultured at 37 °C in a humidified incubator containing 95% room air plus 5% CO2 atmosphere. Cells were cultured without antibiotics to facilitate the detection of Mycoplasma, for which all cell lines tested negative. Cells were detached without trypsin from culture plates with the use of a modified Puck's Solution A plus EDTA (Puck's EDTA), which contains 140 mM NaCl, 5 mM KCl, 5.5 mM glucose, 4 mM NaHCO3, 0.8 mM ethylenediamine tetraacetic acid (EDTA), 13 µM Phenol Red, and 9 mM HEPES buffer (pH 7.3) (54).
For cytotoxicity assays (described below) under reduced oxygen conditions, cells were seeded into 96-well plates and placed into a sealed, humidified, modular incubation chamber. The chamber was flushed for 90 seconds at 10 psi with a mixture of 2% oxygen, 5% CO2, and 93% nitrogen and then incubated at 37 °C for 24 hours. Under these conditions, medium in microplate wells attains a PO2 of approximately 15 mm Hg as determined after the method of Vaupel et al. (56) (Anderson C: personal communication). This level of oxygen is below the degree of hypoxia found in bone marrow (50) and in the range of hypoxia found in tumor tissue (56). 4-HPR (10 mM stock in ethanol) was diluted in whole medium that had been allowed to equilibrate overnight in a loosely capped flask in a modular chamber flushed with the 2% oxygen mixture as described above. Tubes used for making drug dilutions were frequently flushed with 2% oxygen during mixing. Other agitation was minimized in an attempt to reduce atmospheric oxygen contamination, and the drug was added to plates as rapidly as possible. The same drug mix tubes were used to make plates to be studied in both hypoxic and normoxic atmospheres. After drug addition, plates were returned to a modular chamber and flushed with 2% oxygen for 90 seconds, and the chamber was sealed at approximately 5 psi over pressure to reduce atmospheric leaks, incubated at 37 °C, and reflushed with 2% oxygen every other day until assayed.
Cytotoxicity Assay
Cytotoxicity of 4-HPR was determined by use of a fluorescence-based assay employing digital imaging microscopy (DIMSCAN) (57,58). DIMSCAN employs digital imaging microscopy to quantify viable cells, which selectively accumulate FDA and thus are brightly fluorescent. DIMSCAN is capable of measuring cytotoxicity over a 4- to 5-log dynamic range by quantifying total fluorescence per well (which is proportional to viable, clonogenic cells) after eliminating background fluorescence with the use of digital thresholding and eosin Y quenching. Cell lines were seeded into 96-well plates in 100 µL of complete medium per well. CHLA-90 and SMS-KCNR cells (fast growing) were plated at 5000 cells per well; SMS-LHN cells (slow growing) were plated at 15 000 cells per well. Cells were allowed to attach 1 day (CHLA-90 and SMS-KCNR cells) or 2 days (SMS-LHN cells) prior to addition of 4-HPR in 50-µL volumes of whole medium to various final drug concentrations in replicates of 12 wells per concentration. Control wells received ethanol (final concentration = 0.15%) in whole medium equivalent to the maximum final ethanol concentration of 4-HPR-treated wells. For some assays, an antioxidant, NAC, was added in 50-µL volumes of whole medium to a final concentration of 1 mM, 3 hours before the addition of 4-HPR, which was then added in 20-µL volumes of whole medium to various final drug concentrations as described above. Plates were assayed at 4-5 days (CHLA-90 and SMS-KCNR cells) and 7 days (SMS-LHN cells) after initiation of drug exposure, depending on the growth properties of each cell line, to allow for maximum cell death and outgrowth of surviving cells. CHLA-90 cells were visibly rounded and detaching by 24 hours after drug addition, whereas SMS-LHN cells were not observed to begin to round up and detach before 48-72 hours after drug addition. For the measurement of the cytotoxicity, FDA (stock solution of 1 mg/mL in DMSO) was added, in 50 µL of whole medium per well, to a final concentration of 10 µg/mL. The plates were incubated for an additional 15-30 minutes at 37 °C, and then 30 µL of eosin Y (0.5% in normal saline) was added per well. Total fluorescence of each well was then measured with the use of digital imaging microscopy as previously described (58). Results were analyzed and expressed as the surviving fractions of treated cells compared with those of control cells with the use of Excel software (Microsoft, Seattle, WA) and graphed with the use of SigmaPlot 4.0 (Jandell Scientific, San Rafael, CA). The stability of 4-HPR in our tissue culture system for 7 days is not known.
Apoptosis and Necrosis Detection
The Apoptosis DNA-Laddering Kit (Boehringer Mannheim GmbH, Mannheim, Germany) was used to distinguish between apoptotic and necrotic cell death by detecting alterations in the DNA-fragmentation pattern visualized by gel electrophoresis following the manufacturer's instructions. U937 human myeloid leukemia cells, which express CD95/Fas/APO-1, were treated for 16 hours at 37 °C with 250 ng/mL of the anti-Fas IgM antibody CH-11 to serve as a positive control for apoptotic, internucleosomal DNA fragmentation. The pan-caspase inhibitor BOC-d-fmk was used to block the apoptotic component of cell death in both U937 and CHLA-90 cells. Cells were preincubated with either 20 µM or 40 µM BOC-d-fmk, respectively, for 1 hour before the addition of CH-11 in serum-free medium (U937 cells) or 10 µM 4-HPR in whole medium (CHLA-90 cells). DNA-fragmentation patterns were then examined after 24 hours. As an additional control, to demonstrate that 4-HPR did not inhibit BOC-d-fmk, we also preincubated U937 cells with 20 µM BOC-d-fmk and then treated them with 250 ng/mL of CH-11 plus 5 µM 4-HPR. In addition, CHLA-90 cells were pretreated with or without 40 µM BOC-d-fmk for 1 hour prior to the addition of 3-10 µM 4-HPR (six wells per drug concentration) for 24 hours and assayed by DIMSCAN to assess the effect of caspase inhibition on viability. Control cells were treated with vehicle solvents of 0.1% ethanol (4-HPR) and/or 0.2% DMSO (BOC-d-fmk). For further examination of cells treated with 4-HPR in the presence or absence of BOC-d-fmk, CHLA-90 cells were plated into Lab Tek chamber slides (Nunc, Naperville, IL), allowed to attach for 24 hours, and then treated with drugs as described above. Morphologic features of apoptosis (DNA condensation and/or apoptotic bodies) were visualized with the use of the blue nuclear fluorescence induced by the supravital DNA stain Hoechst 33342 (10 µg/mL for 30 minutes at 37 °C), whereas necrotic cells and advanced apoptotic cells were recognized by red fluorescent staining with propidium iodide (0.5 µg/mL) (59). Cells were observed by sequential use of filters appropriate for each dye on an Olympus Vanox epifluorescence microscope. Assessment of apoptosis by flow cytometry used propidium iodide in a hypotonic lysis buffer (60) to identify cells with a sub-G0/G1 DNA content (61). The stained nuclei were analyzed on a Coulter Epics ELITE flow cytometer with a 488-nm Argon laser and a 20-nm band-pass filter centered on 610 nm.
Determination of ROS
Production of ROS was detected by use of carboxy-DCFDA (34,38,62). A stock solution of carboxy-DCFDA (15 mM) was prepared in DMSO and stored over nitrogen vapor. CHLA-90 cells (1 x 106 in 5 mL of whole medium per T25 flask) were cultured in 20% oxygen and exposed to 5 µM 4-HPR for either 4 or 6 hours at 37 °C. Medium was discarded and, under low light conditions, replaced with 50 µM carboxy-DCFDA in 2 mL of whole medium for 20 minutes at 37 °C. Medium was decanted, and the cells were harvested with Puck's EDTA. The cells were resuspended in Puck's EDTA in foil-wrapped tubes and analyzed immediately by use of a Becton Dickinson FACStar flow cytometer with an argon laser at 488 nm through a 20-nm band-pass filter centered on 530 nm; nonviable cells were excluded by forward versus narrow angle light scatter. Ethanol-treated cells, at the same final concentration as that for the 4-HPR-exposed cells, served as the negative control. As a positive control, cells were loaded with carboxy-DCFDA for 20 minutes as above, the medium was discarded, and 100 µM hydrogen peroxide in 3 mL of medium was added for 15 minutes before harvesting for flow cytometry. Results of both time points were similar.
Lipid Analysis
Methods were modified from Lavie et al. (63,64). SMS-LHN or CHLA-90 cells were seeded into six-well plates (5 x 105 cells per well in 2 mL of whole medium) and allowed to recover for 1 day. All drug-treated or control samples were done in triplicate. At zero time, [3H]palmitic acid (1 µCi/mL medium) and 4-HPR (drug-treated) or ethanol (control) were added. Cells (drug-treated and control) were harvested at +6 hours (for dose-response studies) or at the appropriate time (for time-response studies), and lipids were extracted. We determined the effect of 4-HPR on [3H]palmitic acid uptake by assaying and comparing the remaining label in 100 µL of medium from each well. For lipid extraction, medium was aspirated, and cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then harvested with Puck's EDTA. At later time points, to ensure recovery of cells that may have detached during washing, cells harvested with Puck's EDTA were combined with cells centrifuged from the medium and then washed twice with ice-cold PBS. Cells were then transferred to a 4-mL glass sample vial and centrifuged at 150g for 5 minutes at room temperature, and the buffer was aspirated. To each sample vial were then added 1.2 mL of methanol-2% acetic acid, 1.0 mL of distilled water, and 1.2 mL of chloroform. The vial was vigorously vortexed for 30 seconds and centrifuged at 150g for 5 minutes at room temperature, and the lower (organic) phase was transferred to a new vial. Volatile organic solvents were evaporated under a nitrogen stream, and samples were stored at -20 °C.
For analysis, 100 µL of chloroform-methanol (2 : 1) was added to each vial, and the vial was vortexed. A 10-µL aliquot was assayed for tritium; from this, the tritium in the total lipid sample was calculated. Commercial lipid standards (5 µg per lane) were co-spotted onto the TLC plates with cellular 3H-lipids (10-µL aliquots). Ceramide was resolved in a solvent system containing chloroform-acetic acid (90 : 10, vol/vol). The lipid standards were visualized by iodine vapor, and the co-migrating tritiated lipid sample was assayed by scraping the TLC plate in the area of interest, adding 0.5 mL of water and 4.5 mL of Ecolume scintillation cocktail, vortexing, and measuring cpm tritium by liquid scintillation counting. This value was then corrected for the amount of the original sample previously removed for other assays. Ceramide increases are expressed as the mean fold increase in three drug-treated samples as compared with that of three matched controls.
Immunoblotting
SMS-LHN cells cultured as above were exposed to 4-HPR (10 µM) or cisplatin (10 µg/mL) in whole medium for the indicated time, and whole-cell protein lysates were prepared by standard procedures. Protein lysates were quantified with the use of the BCA Protein AssayTM (Pierce Chemical Co.), and 12-µg aliquots were separated in precast 15% Tris-HCl polyacrylamide gels (Bio-Rad Laboratories), transferred to Protran® nitrocellulose membranes, and hybridized with anti-human p53 mouse monoclonal antibody by standard procedures. Immunoblotting results were detected by chemoluminescence using ECLTM detection reagents (Amersham Life Science, Inc.) with x-ray film and stored digitally by use of the ScanJet IICX/T scanner (Hewlett-Packard, Palo Alto, CA).
Statistical Analysis
All data on cytotoxicity and ceramide are presented as means ± 95%
confidence interval. The 95% confidence interval was calculated as 1.96/
n, where
= standard deviation of ungrouped data and n
=
number of trials. The linearity of ceramide increases with dose and time was determined by the
Spearman rank correlation test. The statistical significance of the differences in mean
cytotoxicity, apoptosis, and necrosis induced by 4-HPR, NAC, and BOC-d-fmk treatments was
evaluated by the unpaired, two-sided Student's t test with the use of
Microsoft© Excel 97 software. All P values are two-sided. All experiments were
performed at least twice.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
4-HPR has been reported to increase ROS levels in a time- and
dose-dependent manner in HL-60 myeloid leukemia (38) and C33A
cervical carcinoma cells (34). To determine if 4-HPR increased
ROS levels in neuroblastoma cells, we exposed CHLA-90 cells to 5
µM 4-HPR for 4 or 6 hours and we measured ROS by using
carboxy-DCFDA. The results at both time points were similar. Fig.
1 shows that 4-HPR exposure increased the mean
fluorescence signal approximately 2.5-fold compared with that of the
controls in CHLA-90 neuroblastoma cells. As a further control, 4-HPR (5
µM) was co-incubated with carboxy-DCFDA in whole,
cell-free medium for 90 minutes, without activation of carboxy-DCFDA
fluorescence (data not shown).
|
4-HPR has been previously reported to be cytotoxic to neuroblastoma cell lines (18-20) and to be antagonized by the antioxidants NAC and L-ascorbic acid in HL-60 myeloid leukemia cells (38,65) and by pyrrolidine dithiocarbamate in C33A cervical carcinoma cells (34). To ascertain whether 4-HPR cytotoxicity for neuroblastoma was antagonized by antioxidants and/or hypoxia equivalent to that found in bone marrow (50), we determined cytotoxicity with the use of the DIMSCAN assay in 20% oxygen (normoxic) and 2% oxygen (hypoxia, approximately 15 mm Hg of oxygen) and in the presence of the antioxidant NAC (1 mM). NAC at 1 mM was chosen because it was shown previously to be sufficient to abrogate the ROS-mediated cytotoxicty induced in neuroblastoma cells by buthionine sulfoximine treatment (66).
As shown in Fig. 2, 4-HPR-mediated cytotoxicity was statistically
significantly reduced
under hypoxic conditions compared with normoxic conditions for CHLA-90 cells (at 1 µM, 2 µM, and 5 µM 4-HPR, P<.001; at 10
µM 4-HPR, P = .007), SMS-KCNR cells (at 5 µM
4-HPR, P<.001; at 10 µM 4-HPR, P = .03; at 15
µM 4-HPR, P = .003), and in SMS-LHN cells (at 1 µM 4-HPR, P = .05; at 2 µM 4-HPR, P = .02; at
5 µM and 10 µM 4-HPR, P<.001). Although hypoxia
reduced the activity of 4-HPR, dose escalation still achieved a substantial cell kill in all cell lines.
As shown in Fig. 2,
A, the antioxidant NAC (1 mM) statistically
significantly reduced the cytotoxicity of some, but not all, 4-HPR concentrations tested under
both normoxic and hypoxic conditions in CHLA-90 cells (in 20% oxygen, at 2 µM 4-HPR, P<.001; at 5 µM 4-HPR, P = .27; at 10
µM 4-HPR, P = .02; and in 2% oxygen, at 2 µM 4-HPR, P = .13; at 5 µM 4-HPR, P = .07; at
10 µM 4-HPR, P = .008), but 4-HPR still achieved a statistically
significant cell kill despite the combined antagonism of hypoxia and NAC (P<.001).
Similarly, in Fig. 2
, B, NAC (1 mM) statistically significantly
reduced the cytotoxicity
of high 4-HPR concentrations, most but not all, under both normoxic and hypoxic conditions for
SMS-LHN cells (in 20% oxygen, at 1 µM 4-HPR, P = .50;
at 2 µM 4-HPR, P = .03; at 10 µM 4-HPR, P = .05; and in 2% oxygen, at 1 µM 4-HPR, P =
.68; at 2 µM 4-HPR, P = .02; at 10 µM 4-HPR, P<.001), yet significant cytotoxicity was retained in the presence of NAC and hypoxia at
10 µM 4-HPR (P<.001). NAC (1 mM) had no appreciable
effect on 4-HPR cytotoxicity in SMS-KCNR cells under either normoxic or hypoxic conditions
(Fig. 2
, C).
|
Effect of 4-HPR Dose on Ceramide Levels
Because hypoxia and NAC, conditions expected to substantially reduce
ROS, did not completely abolish 4-HPR cytotoxicity in neuroblastoma
cells, a second mechanism of cytotoxicity was sought. Intracellular
ceramide levels were directly assayed in SMS-LHN and CHLA-90 cells
after a 6-hour exposure to increasing concentrations of 4-HPR. As shown
in Fig. 3, A, 4-HPR induced a statistically
significant increase in ceramide in a dose-dependent manner at 6 hours
in both neuroblastoma cell lines (P<.001). 4-HPR did not
influence the cellular uptake of [3H]palmitic acid (data
not shown).
|
Ceramide levels were assayed in SMS-LHN and CHLA-90 cells after
varying times of exposure to 4-HPR (10 µM). As shown in
Fig. 3, B, the level of ceramide was found to increase in a
time-dependent manner up to at least +48 hours (to approximately
13-fold in CHLA-90 cells,
= .90, P = .037 [Spearman
test]; to approximately sevenfold in SMS-LHN cells,
= .70,
P = .19 [Spearman test]; however, P<.001 for
SMS-LHN cells compared with controls at 48 hours [Student's ttest]). 4-HPR did not influence the cellular uptake of
[3H]palmitic acid (data not shown). Both cell lines
exhibited a biphasic increase, with an early ceramide peak occurring at
+6 hours. CHLA-90 cells were rounded and detaching at +36 hours after
4-HPR exposure. At +48 hours, the cytotoxic effects of 4-HPR were
morphologically apparent in only a minority of the SMS-LHN cells, but
cells became predominantly rounded and detached by +96 hours (data not shown).
Effect of 4-HPR on p53 Protein Expression
4-HPR (10 µM) did not appreciably increase p53 protein
levels in SMS-LHN cells after up to 72 hours of exposure in 20% oxygen
(Fig. 3, C), a time at which SMS-LHN cells were visibly dying. This
result suggests that 4-HPR cytotoxicity is not primarily dependent on
p53 induction in neuroblastoma cells. In contrast, cisplatin (10
µg/mL) readily increased p53 protein levels in SMS-LHN cells,
confirming that this cell line can increase p53 protein in response to
another chemotherapeutic agent (Fig. 3
, C).
4-HPR and Induction of Death by Mixed Apoptosis and Necrosis
To determine whether 4-HPR caused death in neuroblastoma cells by
apoptosis or necrosis, we assessed morphologic evidence of apoptosis
and necrosis as well as internucleosomal DNA-fragmentation patterns for
CHLA-90 cells in the presence or absence of the neural cell-penetrant,
pan-caspase enzyme inhibitor BOC-d-fmk. As shown in Fig.
4, A, the cytotoxicity of 4-HPR was significantly
reduced by 40 µM BOC-d-fmk across all 4-HPR concentrations
(P<.001), but 4-HPR still induced significant cytotoxicity
in the presence of 40 µM BOC-d-fmk (at 3 µM
4-HPR, P = .002; at >3 µM 4-HPR,
P<.001). The addition of BOC-d-fmk (40 µM)
significantly reduced (P = .001) the morphologic nuclear
changes indicative of apoptosis (Fig. 4
, B and C), typified by
condensed nuclear chromatin and fragmentation of the nuclei into
apoptotic bodies in cells that had not lost membrane integrity. 4-HPR
alone induced significant apoptosis (P = .006), but apoptosis
in cells treated with 4-HPR plus BOC-d-fmk was not significantly
different from that of controls (P = .48). However, the
statistically significant morphologic evidence of necrosis (P
= .002) induced by 4-HPR (loss of membrane integrity demonstrated by
propidium iodide staining, cell rounding, and cell detachment) was
minimally changed by BOC-d-fmk and was still statistically significant
(P = .016) relative to that of controls (Fig. 4
, B and C).
BOC-d-fmk (40 µM) also abrogated the minimal DNA
fragmentation induced by 4-HPR in CHLA-90 cells as detected by flow
cytometry (Fig. 4
, D) and gel electrophoresis (Fig. 4
, E) but only
partially decreased the cytotoxicity of 4-HPR (Fig. 4
, A). This finding
suggests that 4-HPR-induced cell death in neuroblastoma cell lines
proceeds predominantly by necrosis at higher drug concentrations. A
similar result was observed in neuroblastoma cells with the use of
exogenous C2 ceramide (Metelitsa LS, Keshelava N, Reynolds
CP, Durden D, Seeger RC: manuscript in preparation).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The retinoid 4-HPR as a chemopreventative agent is minimally toxic in human adults in chronic daily dosing of approximately 12 mg/kg orally (serum levels, 1-3 µM) and minimally toxic in animal models up to chronic daily dosing of approximately 800 mg/kg (8). We speculate that, since 4-HPR has not been reported to induce its own metabolism (8), in vivo levels similar to or higher than those of 13-cis-retinoic acid (5-10 µM) (70) will be achievable in patients, especially by use of a pulse-dosing schedule. High levels of 4-HPR (3-10 µM) have been reported to have in vitro cytotoxic activity against neuroblastoma cell lines (18-20), including those resistant to 13-cis- and trans-retinoic acid (71). For these reasons, we are investigating its mechanism(s) of cytotoxicity and its potential activity under hypoxic conditions less than the oxygen tension found in bone marrow, a common site of neuroblastoma metastases.
4-HPR has been reported to increase ROS in myeloid leukemia and cervical carcinoma cell lines (38,34), and we now confirm ROS generation in neuroblastoma cells. However, our dose-response studies in hypoxia and in the presence of the antioxidant NAC suggested additional mechanisms of 4-HPR cytotoxicity.
Our observation that 4-HPR induced a sustained increase in intracellular ceramide in neuroblastoma cell lines of up to approximately 10-fold in a time- and dose-dependent manner is novel. Recently, 4-HPR (3 µM) was reported to cause only a transient increase in ceramide in HL-60 leukemia cells and a modest 1.5-fold increase in the MCF7 breast cancer cell line (72). Other chemotherapeutic agents, at clinically achievable levels, such as the cyclosporine analogue SDZ PSC 833 (44), daunorubicin (73,74), doxorubicin (43), and irinotecan (CPT-11) (75), have been shown to increase intracellular ceramide, but to levels much lower than those that we observed in response to 4-HPR. Furthermore, our observation that p53 protein was not increased by 4-HPR in SMS-LHN neuroblastoma cells (which possess a p53 protein that is readily increased by cisplatin) suggests that 4-HPR cytotoxicity is generally p53 independent, which is in agreement with observations of 4-HPR cytotoxicity in HL-60 myeloid leukemia (65), small-cell lung cancer (76), and non-small-cell lung cancer (77) cell lines.
The mixed apoptosis-necrosis induced by 4-HPR in neuroblastoma cells is also noteworthy.
There have been reports in numerous cell types of apoptosis induced by low-dose 4-HPR
(approximately 3 µM) (20,22,27,29,30,34,36,38,65). These
conclusions have been based mainly on evidence of internucleosomal fragmentation of DNA.
However, the effect of caspase inhibitors (which block apoptotic cell death) on 4-HPR
cytotoxicity has only been minimally investigated (72). Herein we report
that, while 4-HPR-mediated apoptosis was abrogated and cytotoxicity was significantly reduced
by the pan-caspase enzyme inhibitor BOC-d-fmk, significant dose-dependent cytotoxicity was
retained compared with findings in controls, even in the presence of 40 µM
BOC-d-fmk (Fig. 4). These observations suggest that 4-HPR induced a
nonapoptotic cytotoxicity
(necrosis) in neuroblastoma cell lines, especially at higher drug concentrations. Recently, 4-HPR
(3 µM) has been shown to induce apoptosis in lymphoblastoid cell lines, but
higher concentrations of 4-HPR (10-20 µM) induced necrosis (78), and 10 µM 4-HPR has been shown to induce both apoptosis and
necrosis in an embryonal carcinoma cell line (59). The prominence of
necrotic death observed with high-dose 4-HPR suggests that it might be able to induce cell death
not only in p53-defective tumors but also in those with defective caspase cascades. In addition,
CHLA-90 is a cell line derived after myeloablative therapy that shows considerable resistance to
alkylating agents and etoposide (55,79). The observation that 4-HPR
demonstrated cytotoxicity in such a cell line suggests an ability to circumvent resistance to
commonly used chemotherapeutic agents in at least one tumor type.
The exact mechanism by which 4-HPR increased intracellular ceramide is not yet clear. Our preliminary experiments showed that pretreatment with Fumonisin B1 (100 µM) inhibited the 4-HPR-mediated increase in ceramide seen in CHLA-90 cells (data not shown), suggesting that 4-HPR (10 µM) may target the de novo ceramide synthesis pathway (72,80). Experiments are in progress that directly assay the effect of 4-HPR on ceramide synthase and neutral and acidic sphingomyelinase.
It is also not clear if 4-HPR initially increased ROS or ceramide. It is possible, depending on their intracellular compartmentalization, that these second messengers reciprocally stimulate their production. Our results with hypoxia and the antioxidant NAC, which should minimize ROS-induced cytotoxicity, suggest that 4-HPR might initially induce an increase in ceramide.
The discovery that high-dose 4-HPR induced a sustained increase in ceramide in neuroblastoma cell lines suggests that a similar increase might be induced in tumors in vivo. We have found 4-HPR to be highly active against neuroblastoma cell lines resistant to trans- and 13-cis-retinoic acid in vitro(71). This observation suggests that 4-HPR might be employed sequentially after 13-cis-retinoic acid in neuroblastoma treatment (1) to further eliminate minimal residual disease induced by high-dose chemotherapy by targeting tumor cells resistant to 13-cis-retinoic acid. Since high-dose 4-HPR has in vitro activity against other tumors that relapse from states of minimal residual disease (24,26,36,76), clinical trials in these tumors, similar to the 13-cis-retinoic acid trial in advanced neuroblastoma (1), would seem to be warranted.
Therefore, should it be clinically tolerated, high-dose 4-HPR may form the basis for a new, ceramide-based chemotherapy, which would have the advantages of being p53 and caspase independent and functional under conditions of reduced oxygen.
![]() |
NOTES |
---|
We thank Dr. Clarke Anderson and Dr. Robert Lavey for measuring oxygen tension, Lisa Melton and Paul Alfaro for technical assistance, and Dr. Susan Groshen for help in statistical analysis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Reynolds CP, Villablanca JG, Stram DO, Harris R, Seeger RC, Matthay KK. 13-cis-retinoic acid after intensive consolidation therapy for neuroblastoma improves event-free survival: a randomized Children's Cancer Group (CCG) study [abstract]. Proc ASCO 1998;17:2a.
2 Abou-Issa H, Moeschberger M, el-Masry W, Tejwani S, Curley RW Jr, Webb TE. Relative efficacy of glucarate on the initiation and promotion phases of rat mammary carcinogenesis. Anticancer Res 1995;15:805-10.[Medline]
3 Decensi A, Bruno S, Torrisi R, Parodi S, Polizzi A. Pilot study of high dose fenretinide and vitamin A supplementation in bladder cancer [letter]. Eur J Cancer 1994;30A:1909-10.
4 McCormick DL, Johnson WD, Rao KV, Bowman-Gram T, Steele VE, Lubet RA, et al. Comparative activity of N-(4-hydroxyphenyl)-all-trans-retinamide and alpha-difluoromethylornithine as inhibitors of lymphoma induction in PIM transgenic mice. Carcinogenesis 1996;17:2513-7.[Abstract]
5 Slawin K, Kadmon D, Park SH, Scardino PT, Anzano M, Sporn MB, et al. Dietary fenretinide, a synthetic retinoid, decreases the tumor incidence and the tumor mass of ras+myc-induced carcinomas in the mouse prostate reconstitution model system. Cancer Res 1993;53:4461-5.[Abstract]
6 Pienta KJ, Nguyen NM, Lehr JE. Treatment of prostate cancer in the rat with the synthetic retinoid fenretinide. Cancer Res 1993;53:224-6.[Abstract]
7 Pollard M, Luckert PH, Sporn MB. Prevention of primary prostate cancer in Lobund-Wistar rats by N-(4-hydroxyphenyl)retinamide. Cancer Res 1991;51:3610-1.[Abstract]
8 Kelloff GJ, Crowell JA, Boone CW, Steele VE, Lubet RA, Greenwald P, et al. Clinical development plan: N-(4-hydroxyphenyl)retinamide. J Cell Biochem Suppl 1994;20:176-96.[Medline]
9 Moon RC. Comparative aspects of carotenoids and retinoids as chemopreventive agents for cancer. J Nutr 1989;119:127-34.[Medline]
10 Abou-Issa H, Curley RW Jr, Panigot MJ, Wilcox KA, Webb TE. In vivo use of N-(4-hydroxyphenyl retinamide)-O-glucuronide as a breast cancer chemopreventive agent. Anticancer Res 1993;13:1431-6.[Medline]
11 Cobleigh MA, Dowlatshahi K, Deutsch TA, Mehta RG, Moon RC, Minn F, et al. Phase I/II trial of tamoxifen with or without fenretinide, an analog of vitamin A, in women with metastatic breast cancer. J Clin Oncol 1993;11:474-7.[Abstract]
12 Decensi A, Bruno S, Costantini M, Torrisi R, Curotto A, Gatteschi B, et al. Phase IIa study of fenretinide in superficial bladder cancer, using DNA flow cytometry as an intermediate end point. J Natl Cancer Inst 1994;86:138-40.[Medline]
13 Tradati N, Chiesa F, Rossi N, Grigolato R, Formelli F, Costa A, et al. Successful topical treatment of oral lichen planus and leukoplakias with fenretinide (4-HPR). Cancer Lett 1994;76:109-11.[Medline]
14 Costa A, Formelli F, Chiesa F, Decensi A, De Palo G, Veronesi U. Prospects of chemoprevention of human cancers with the synthetic retinoid fenretinide. Cancer Res 1994;54:2032s-2037s.[Abstract]
15 Chiesa F, Tradati N, Marazza M, Rossi N, Boracchi P, Mariani L, et al. Fenretinide (4-HPR) in chemoprevention of oral leukoplakia. J Cell Biochem Suppl 1993;17F:255-61.
16 Formelli F, Clerici M, Campa T, Di Mauro MG, Magni A, Mascotti G, et al. Five-year administration of fenretinide: pharmacokinetics and effects on plasma retinol concentrations. J Clin Oncol 1993;11:2036-42.[Abstract]
17 Costa A, Malone W, Perloff M, Buranelli F, Campa T, Dossena G, et al. Tolerability of the synthetic retinoid Fenretinide (HPR). Eur J Cancer Clin Oncol 1989;25:805-8.[Medline]
18 Ponzoni M, Bocca P, Chiesa V, Decensi A, Pistoia V, Raffaghello L, et al. Differential effects of N-(4-hydroxyphenyl)retinamide and retinoic acid on neuroblastoma cells: apoptosis versus differentiation. Cancer Res 1995;55:853-61.[Abstract]
19 Di Vinci A, Geido E, Infusini E, Giaretti W. Neuroblastoma cell apoptosis induced by the synthetic retinoid N-(4-hydroxyphenyl)retinamide. Int J Cancer 1994;59:422-6.[Medline]
20 Mariotti A, Marcora E, Bunone G, Costa A, Veronesi U, Pierotti MA, et al. N-(4-Hydroxyphenyl)retinamide: a potent inducer of apoptosis in human neuroblastoma cells. J Natl Cancer Inst 1994;86:1245-7.[Medline]
21 Ziv Y, Gupta MK, Milsom JW, Vladisavljevic A, Brand M, Fazio VW. The effect of tamoxifen and fenretinide on human colorectal cancer cell lines in vitro. Anticancer Res 1994;14:2005-9.[Medline]
22 Scher RL, Saito W, Dodge RK, Richtsmeier WJ, Fine RL. Fenretinide-induced apoptosis of human head and neck squamous carcinoma cell lines. Otolaryngol Head Neck Surg 1998;118:464-71.[Medline]
23 Kazmi SM, Plante RK, Visconti V, Lau CY. Comparison of N-(4-hydroxyphenyl)retinamide and all-trans-retinoic acid in the regulation of retinoid receptor-mediated gene expression in human breast cancer cell lines. Cancer Res 1996;56:1056-62.[Abstract]
24 Coradini D, Biffi A, Pellizzaro C, Pirronello E, Di Fronzo G. Combined effect of tamoxifen or interferon-ß and 4-hydroxyphenylretinamide on the growth of breast cancer cell lines. Tumour Biol 1997;18:22-9.[Medline]
25 Hsieh TC, Ng C, Wu JM. The synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) exerts antiproliferative and apoptosis-inducing effects in the androgen-independent human prostatic JCA-1 cells. Biochem Mol Biol Int 1995;37:499-506.[Medline]
26 Igawa M, Tanabe T, Chodak GW, Rukstalis DB. N-(4-Hydroxyphenyl) retinamide induces cell cycle specific growth inhibition in PC3 cells. Prostate 1994;24:299-305.[Medline]
27 Roberson KM, Penland SN, Padilla GM, Selvan RS, Kim CS, Fine RL, et al. Fenretinide: induction of apoptosis and endogenous transforming growth factor ß in PC-3 prostate cancer cells. Cell Growth Differ 1997;8:101-11.[Abstract]
28 Hsieh TC, Wu JM. Effects of fenretinide (4-HPR) on prostate LNCaP cell growth, apoptosis, and prostate-specific gene expression. Prostate 1997;33:97-104.[Medline]
29 Kalemkerian GP, Slusher R, Ramalingam S, Gadgeel S, Mabry M. Growth inhibition and induction of apoptosis by fenretinide in small-cell lung cancer cell lines. J Natl Cancer Inst 1995;87:1674-80.[Abstract]
30 Supino R, Crosti M, Clerici M, Warlters A, Cleris L, Zunino F, et al. Induction of apoptosis by fenretinide (4HPR) in human ovarian carcinoma cells and its association with retinoic acid receptor expression. Int J Cancer 1996;65:491-7.[Medline]
31
Sabichi AL, Hendricks DT, Bober MA, Birrer MJ. Retinoic acid
receptor beta expression and growth inhibition of gynecologic cancer cells by the synthetic
retinoid N-(4-hydroxyphenyl) retinamide. J Natl Cancer Inst 1998;90:597-605.
32 Formelli F, Cleris L. Synthetic retinoid fenretinide is effective against a human ovarian carcinoma xenograft and potentiates cisplatin activity. Cancer Res 1993;53:5374-6.[Abstract]
33 Oridate N, Lotan D, Mitchell MF, Hong WK, Lotan R. Inhibition of proliferation and induction of apoptosis in cervical carcinoma cells by retinoids: implications for chemoprevention. J Cell Biochem Suppl 1995;23:80-6.[Medline]
34
Oridate N, Suzuki S, Higuchi M, Mitchell MF, Hong WK,
Lotan R. Involvement of reactive oxygen species in N-(4-hydroxyphenyl)retinamide-induced apoptosis in cervical carcinoma cells. J Natl
Cancer Inst 1997;89:1191-8.
35
Benedetti L, Grignani F, Scicchitano BM, Jetten AM, Diverio
D, Lo Coco F, et al. Retinoid-induced differentiation of acute promyelocytic leukemia involves
PML-RARalpha-mediated increase of type II transglutaminase. Blood 1996;87:1939-50.
36 Delia D, Aiello A, Lombardi L, Pelicci PG, Grignani F, Formelli F, et al. N-(4-Hydroxyphenyl)retinamide induces apoptosis of malignant hemopoietic cell lines including those unresponsive to retinoic acid. Cancer Res 1993;53:6036-41.[Abstract]
37 Bagniewski PG, Reid JM, Villablanca JG, Reynolds CP, Ames MM. A phase I pharmacokinetic study of fenretinide (HPR) in children with high-risk tumors.Proc Am Assoc Cancer Res 1999;40:92.
38 Delia D, Aiello A, Meroni L, Nicolini M, Reed JC, Pierotti MA. Role of antioxidants and intracellular free radicals in retinamide-induced cell death. Carcinogenesis 1997;18:943-8.[Abstract]
39
Garcia-Ruiz C, Colell A, Mari M, Morales A, Fernandez-Checa
JC. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of
reactive oxygen species. Role of mitochondrial glutathione. J Biol Chem 1997;272:11369-77.
40
Quillet-Mary A, Jaffrezou JP, Mansat V, Bordier C, Naval J,
Laurent G. Implication of mitochondrial hydrogen peroxide generation in ceramide-induced
apoptosis. J Biol Chem 1997;272:21388-95.
41 Kolesnick RN, Kronke M. Regulation of ceramide production and apoptosis. Annu Rev Physiol 1998;60:643-65.[Medline]
42
Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG, Geley
S, et al. The central executioner of apoptosis: multiple connections between protease activation
and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J Exp Med 1997;186:25-37.
43 Cabot MC, Giuliano AE. Apoptosisa cell mechanism important for cytotoxic response to adriamycin and a lipid metabolic pathway that facilitates escape [abstract]. Breast Cancer Res Treat 1997;46:71.
44 Cabot MC, Han TY, Giuliano AE. The multidrug resistance modulator SDZ PSC 833 is a potent activator of cellular ceramide formation. FEBS Lett 1998;431:185-8.[Medline]
45
Yoshimura S, Banno Y, Nakashima S, Takenaka K, Sakai H,
Nishimura Y, et al. Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell
death. Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation. J Biol
Chem 1998;273:6921-7.
46
Cuvillier O, Rosenthal DS, Smulson ME, Spiegel S.
Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose)
polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes. J Biol Chem 1998;273:2910-6.
47
Jarvis WD, Fornari FA, Auer KL, Freemerman AJ, Szabo E,
Birrer MJ, et al. Coordinate regulation of stress- and mitogen-activated protein kinases in the
apoptotic actions of ceramide and sphingosine. Mol Pharmacol 1997;52:935-47.
48 Cuvillier O, Pirianov G, Kleuser B, Vanek PG, Coso OA, Gutkind S, et al. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 1996;381:800-3.[Medline]
49 Matthay KK, Atkinson JB, Stram DO, Selch M, Reynolds CP, Seeger RC. Patterns of relapse after autologous purged bone marrow transplantation for neuroblastoma: a Childrens Cancer Group pilot study. J Clin Oncol 1993;11:2226-33.[Abstract]
50 Grant JL, Smith B. Bone marrow gas tensions, bone marrow blood flow, and erythropoiesis in man. Ann Intern Med 1963;58:801-9.
51 Brown JM, Koong A. Therapeutic advantage of hypoxic cells in tumors: a theoretical study. J Natl Cancer Inst 1991;83:178-85.[Abstract]
52 Workman P, Stratford IJ. The experimental development of bioreductive drugs and their role in cancer therapy. Cancer Metastasis Rev 1993;12:73-82.[Medline]
53 Wada RK, Seeger RC, Brodeur GM, Einhorn PA, Rayner SA, Tomayko MM, et al. Human neuroblastoma cell lines that express N-myc without gene amplification. Cancer 1993;72:3346-54.[Medline]
54 Reynolds CP, Biedler JL, Spengler BA, Reynolds DA, Ross RA, Frenkel EP, et al. Characterization of human neuroblastoma cell lines established before and after therapy. J Natl Cancer Inst 1986;76:375-87.[Medline]
55 Keshelava N, Seeger RC, Groshen S, Reynolds CP. Drug resistance patterns of human neuroblastoma cell lines derived from patients at different phases of therapy. Cancer Res 1998;58:5396-404.[Abstract]
56 Vaupel P, Schlenger K, Knoop C, Hockel M. Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements. Cancer Res 1991;51:3316-22.[Abstract]
57 Fragala T, Proffitt RT, Reynolds CP. A novel 96-well plate cytotoxicity assay based on fluorescence digital imaging microscopy [abstract]. Proc Am Assoc Cancer Res 1995;36:303.
58 Proffitt RT, Tran JV, Reynolds CP. A fluorescence digital image microscopy system for quantifying relative cell numbers in tissue culture plates. Cytometry 1996;24:204-13.[Medline]
59
Clifford JL, Menter DG, Wang M, Lotan R, Lippman SM.
Retinoid receptor-dependent and -independent effects of N-(4-hydroxyphenyl)retinamide in F9 embryonal carcinoma cells. Cancer Res 1999;59:14-8.
60 Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biology 1975;66:188-93.[Abstract]
61 Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz MA, Lassota P, et al. Features of apoptotic cells measured by flow cytometry. Cytometry1992 ;13:795-808.[Medline]
62 Royall JA, Ischiropoulos H. Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 1993;302:348-55.[Medline]
63
Lavie Y, Cao HT, Bursten SL, Giuliano AE, Cabot MC.
Accumulation of glucosylceramide in multidug-resistant cancer cells. J Biol Chem 1996;271:19530-6.
64
Lavie Y, Cao HT, Volner A, Lucci A, Han TY, Geffen V, et al.
Agents that reverse multidrug resistance, tamoxifen, verapamil, and cyclosporin A, block
glycosphingolipid metabolism by inhibiting ceramide glycosylation in human cancer cells. J Biol Chem 1997;272:1682-7.
65
Delia D, Aiello A, Formelli F, Fontanella E, Costa A, Miyashita
T, et al. Regulation of apoptosis induced by the retinoid N-(4-hydroxyphenyl)
retinamide and effect of deregulated bcl-2. Blood 1995;85:359-67.
66 Anderson CP, Tsai JM, Meek WE, Liu RM, Tang Y, Forman HJ, et al. Depletion of glutathione by buthionine sulfoxine is cytotoxic for human neuroblastoma cell lines via apoptosis. Exp Cell Res 1999;246:183-92.[Medline]
67 Moulder JE, Rockwell S. Tumor hypoxia: its impact on cancer therapy. Cancer Metastasis Rev 1987;5:313-41.[Medline]
68 Teicher BA. Physiologic mechanisms of therapeutic resistance. Blood flow and hypoxia. Hematol Oncol Clin North Am 1995;9:475-506.[Medline]
69 Vaupel P, Schlenger K, Knoop C, Hockel M. Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements. Cancer Res 1991;51:3316-22.[Abstract]
70 Villablanca JG, Khan AA, Avramis VI, Seeger RC, Matthay KK, Ramsay NK, et al. Phase I trial of 13-cis-retinoic acid in children with neuroblastoma following bone marrow transplantation. J Clin Oncol 1995;13:894-901.[Abstract]
71 Reynolds CP, Melton LJ, Wang YL. N-(4-Hydroxyphenyl)retinamide is highly active against retinoic acid resistant neuroblastoma cell lines [abstract]. Proc Am Assoc Cancer Res 1997;38:25.
72 Dipietrantonio A, Hsieh TC, Olson SC, Wu JM. Regulation of G1/S transition and induction of apoptosis in HL-60 leukemia cells by fenretinide (4HPR). Int J Cancer 1998;78:53-61.[Medline]
73 Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks Z, Kolesnick R. Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 1995;82:405-14.[Medline]
74 Jaffrezou JP, Levade T, Bettaieb A, Andrieu N, Bezombes C, Maestre N, et al. Daunorubicin-induced apoptosis: triggering of ceramide generation through sphingomyelin hydrolysis. EMBO J 1996;15:2417-24.[Abstract]
75 Suzuki A, Iwasaki M, Kato M, Wagai N. Sequential operation of ceramide synthesis and ICE cascade in CPT-11-initiated apoptotic death signaling. Exp Cell Res 1997;233: 41-7.[Medline]
76 Kalemkerian GP, Slusher R, Ramalingam S, Gadgeel S, Mabry M. Growth inhibition and induction of apoptosis by fenretinide in small-cell lung cancer cell lines. J Natl Cancer Inst 1995;87:1674-80.[Abstract]
77 Zou CP, Kurie JM, Lotan D, Zou CC, Hong WK, Lotan R. Higher potency of N-(4-hydroxyphenyl)retinamide than all-trans-retinoic acid in induction of apoptosis in non-small cell lung cancer cell lines. Clin Cancer Res 1998;4:1345-55.[Abstract]
78 Springer LN, Stewart BW. N-(4-Hydroxyphenyl)retinamide-induced death in human lymphoblastoid cells: 50 kb DNA breakage as a means of distinguishing apoptosis from necrosis. Cancer Lett 1998;128:189-96.[Medline]
79 Keshelava N, Yang YJ, Tsai JM, Seeger RC, Reynolds CP. Drug resistance patterns in human neuroblastoma cell lines correlates with clinical therapy. Eur J Oncol 1997;33:2002-6.
80 Merrill AH Jr, Wang E, Vales TR, Smith ER, Schroeder JJ, Menaldino DS, et al. Fumonisin toxicity and sphingolipid biosynthesis. Adv Exp Med Biol 1996;392:297-306.[Medline]
Manuscript received October 27, 1998; revised May 6, 1999; accepted May 10, 1999.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |