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Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030-4095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that 4HPR can promote apoptosis in tumor cell lines implies
a possible common cellular event that may be important with respect to
both the chemopreventive and therapeutic effects of this compound
(5, 6, 7, 8)
. The mechanism through which 4HPR induces apoptosis
is not well understood. Retinoids are believed to act via nuclear
receptor-mediated transactivation of target genes by retinoic acid
receptors 
, ß, and 
and retinoid X receptors , ß, and
(9)
. In vitro studies have shown that 4HPR can
induce the transcription of retinoic acid response elements by retinoic
acid receptors, particularly retinoic acid receptor (10
, 11)
. Yet, the ability of 4HPR to induce apoptosis in cell lines
refractory to ATRA (12, 13, 14)
, the natural ligand for
nuclear retinoic acid receptors (9)
, points to the
possibility that 4HPR may trigger apoptosis in a receptor-independent
manner (6)
. This is supported by recent findings that 4HPR
acts as a prooxidant in leukemia cell lines and in carcinoma cell lines
derived from the cervix and prostate (5
, 7
, 8)
. Work with
these cell lines has shown that 4HPR can enhance the production of ROS,
and its ability to induce apoptosis can be inhibited by exogenous
antioxidants like ascorbic acid, pyrrolidine dithiocarbamate, and
butylated hydroxyanisole. The ability of 4HPR to promote ROS does not
appear to be receptor-mediated (5
, 7)
.
This study was conducted to determine whether 4HPR could promote apoptosis in human SCC cells. Furthermore, ways to modulate the effects of 4HPR in SCC cells were investigated to gain a clearer understanding of the way in which 4HPR functions at the cellular level. Data are presented that further substantiate the possibility that 4HPR can operate in a novel pathway with respect to therapeutic effectiveness (15) , supporting its usefulness as a treatment for skin neoplasms and perhaps for other hyperproliferative cutaneous disorders.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retinoids and Reagents.
4HPR was obtained from Dr. Ronald Lubet (Division of Cancer Prevention
and Control, National Cancer Institute, Bethesda, MD). ATRA and
9-cis RA were obtained from Dr. Werner Bollag (F. Hoffman-La
Roche, Basel, Switzerland). The retinoic acid receptor ß,
antagonist CD2665, and retinoic acid receptor agonist CD437
(18
, 19)
were obtained from Dr. Braham Shroot (Galderma
Research and Development, Sophia Antipolis, France).
Vit-C, CsA, DMSO, and hydrogen peroxide 30% solution were purchased
from Sigma Chemical Co. (St. Louis, MO). R123 and
2',7'-dichlorofluorescin diacetate were purchased from Molecular
Probes, Inc. (Eugene, OR). Cyp was purchased from Calbiochem (La Jolla,
CA). Retinoids, CsA, R123, Cyp, and 2',7'-dichlorofluorescin diacetate
were dissolved in DMSO. Vit-C was dissolved in sterile deionized water,
and hydrogen peroxide 30% solution was diluted in sterile deionized
water. All solutions were stored at -20°C before use. BA, 
30
mg/ml in 2 M NH4OH, was purified as
described (20)
and kindly provided by Dr. J. A. Duine
(Delft University of Technology, Delft, The Netherlands).
SCC Cell Culture and Proliferation Inhibition Study.
For experimental manipulation, SCC cells were cultured in
keratinocyte growth medium consisting of keratinocyte basal medium
supplemented with 100 ng/ml human recombinant epidermal growth
factor and 0.4% bovine pituitary extract (BioWhittaker/Clonetics, San
Diego, CA). The calcium concentration in the keratinocyte growth medium
was 0.15 mM. Cell cultures were incubated at 37°C in
humidified air containing 5% CO2.
Proliferation rates for SCC cells were obtained by seeding 400,000
cells in 10-cm diameter dishes. After allowing the cells to attach and
proliferate for 24 h, cells were detached after a brief incubation
with 0.025% trypsin/0.01% EDTA at 6, 12, 24, 36, or 48 h later
and counted with a hemacytometer. The doubling times obtained for the
SCC cell lines were COLO 16, 23.9 h; SCC-13, 24.5 h; SRB-1,
38.6 h; and SRB-12, 26.9 h. Treatment with retinoids and
other agents was conducted on 50% confluent cultures.
For the proliferation inhibition study with 4HPR, SCC cells were seeded in 10-cm dishes (750,000 cells/dish). After seeding, the cells were allowed to attach and proliferate for 24 h (COLO 16, SCC-13, and SRB-12) or 36 h (SRB-1) before treatment with 10 µM 4HPR. In addition, SCC-13 cells also received 1 and 5 µM 4HPR for characterization of doseresponse relationships. All control dishes received the same amount of the vehicle, DMSO, as the retinoid-treated cultures. After 6, 12, 24, or 48 h, both the floating and the attached cells were harvested and counted with a hemacytometer. The trypan blue exclusion assay indicated that viability exceeded 90% in both control and treatment populations at 6 and 12 h.
Detection of Apoptotic Cells and Cell Cycle Evaluation.
Detection of intranucleosomal DNA fragmentation was performed using a
flow cytometry apoptosis detection kit (Phoenix Flow Systems, Inc., San
Diego, CA), which is based on the TUNEL technique and labels the
3'-hydroxyl termini of DNA fragmented during apoptosis
(21)
. Cell samples were also stained with propidium iodide
to indicate DNA content as a relative indicator of cell cycle
progression. SCC cells were treated with 4HPR and harvested at the
respective time points after the procedure described above for the
proliferation inhibition study.
Cells were fixed and stained using the protocol provided in the apoptosis detection kit, with limited modifications. The first modification involved additional permeabilization of fixed cells in 1 ml of 1% Triton X-100 (Sigma Chemical Co.) in deionized water for 10 min on ice before rinsing and suspension in 70% (v/v) ethanol/deionized water. The second modification involved passing the cells slowly three times through a 1-ml syringe fitted with a 25-gauge needle to disperse cell clumps before incubation in the TUNEL reaction solution. The third modification required an overnight incubation at room temperature in the TUNEL reaction solution.
Flow cytometric analysis was conducted using a Coulter EPICS Profile II flow cytometer (Coulter Corp., Miami, FL). Approximately 10,000 events (cells) were evaluated for each sample. Gating of control nonapoptotic populations (cells treated with DMSO) was used as a reference to compare with treatments with 4HPR. An internal control (HL-60 cells treated with camptothecin to induce apoptosis) provided in the apoptosis detection kit was also used to insure the TUNEL reaction was occurring during the staining procedure.
The flow cytometer detected fluorescence at 623 nm for propidium iodide and at 520 nm for fluorescein using an excitation wavelength of 488 nm provided by an argon laser. These were recorded as single-parameter histograms along the X-axis as an indication of DNA content (propidium iodide) and DNA strand breaks (fluorescein-labeled dUTP incorporation), relative to population frequency. The appearance of a sub-G1 population in the propidium iodide histogram was used to provide a second indicator of DNA degradation (22) . The fluorescence values for each probe were also recorded as a dual-parameter scatter diagram with DNA content (linear propidium iodide fluorescence) along the X axis and fluorescein-labeled dUTP incorporation (log fluorescein fluorescence) on the Y axis. This diagram provided the option of determining the phase of the cell cycle, if any, during which the cells were most sensitive to apoptosis induction after treatment (23) .
Quantitative Determination of ROS Generation.
Generation of intracellular ROS was measured using
2',7'-dichlorofluorescin diacetate. This nonpolar compound is taken up
by cells and converted to the nonfluorescent derivative DCF by cellular
esterases. DCF is membrane impermeable, localizing in the cytosol where
it can be oxidized to the fluorescent compound
2',7'-dichlorofluorescein by reactions with ROS, primarily
hydroperoxides, via cellular peroxidases (24)
. SCC cells
were seeded in six-well tissue culture plates (90,000 cells/2 ml/well)
and allowed to reach 50% confluence. The wells were washed twice with
2 ml of buffer A [Krebs-Ringer buffer containing 10 mM
D-glucose, 120 mM NaCl, 4.5 mM KCl,
0.15 mM CaCl2, 0.7 mM
Na2HPO4, 1.5 mM
NaH2PO4, and 0.5
mM MgCl2 (pH 7.4) at 37°C]. The
wells were then covered with 2 ml of buffer A containing 10 µg/ml
2',7'-dichlorofluorescin diacetate and the appropriate concentration of
the agent of choice. The plates were rocked for 2 min to insure
adequate mixing. Fluorescence emission at 530 nm (representing
oxidation of DCF) after excitation at 485 nm was measured at time 0
(immediately after mixing) and subsequently at 30-min intervals over a
150-min period using a CytoFluor 4000 spectrofluorimeter (Perseptive
Biosystems, Inc. Framingham, MA). Cultures were incubated at 37°C
between the 30-min intervals for fluorescence determination.
Examination of the fluorescence characteristics of cells treated in
buffer A containing 4HPR without 2',7'-dichlorofluorescin diacetate
present, and buffer A containing 4HPR and 2',7'-dichlorofluorescin
diacetate without cells present in a six-well tissue culture plate
revealed a signal of 2 fluorescence units at time 0, which remained
unchanged during the exposure period described above (not shown).
Treatment Protocol.
To determine whether the effects of 4HPR could be modulated in SCC
cells, the treatment effects of other agents were examined. As an
example of the treatment procedure used for the TUNEL analysis, COLO 16
cells were seeded in 10-cm tissue culture plates and cultured 24 h. Two h before treatment with 4HPR, CD2665 was added directly to the
culture medium to give final concentrations of 10 µM.
Vit-C was diluted to 500 µM in keratinocyte growth medium
at the initial seeding for a 24-h exposure. 4HPR was added directly to
the medium at a final concentration of 10 µM for a 12-h
exposure. CsA and Cyp were added to the culture medium of SCC-13 cells
at the same time as 4HPR. BA was added to the culture medium of SCC-13
cells 2 h before 4HPR. After the appropriate exposure period, the
cells were harvested for TUNEL staining as described previously. The
same treatment procedures were used for ROS and
m determinations.
Measurement of m.
Quantitation of m was determined by R123
retention. R123 is a cationic fluorescent dye, which localizes in the
mitochondria of viable cells because of the relatively high negative
electric potential across the mitochondrial inner membrane
(25)
. SCC cells were seeded in six-well tissue culture
plates and cultured as described for the ROS determination. Treatment
with 4HPR and other agents followed the protocol describe above. The
cultures were incubated at 37°C for the appropriate time, after which
R123 was added to each well to a final concentration of 1 µg/ml. The
plates were incubated an additional 15 min at 37°C. The media was
removed from each well, which was then washed once with 2 ml of buffer
A, and replaced with 2 ml of buffer A. Fluorescence emission at 530 nm
(representing R123 retention as a function of
m) after excitation at 508 nm was measured
with a CytoFluor 4000 spectrofluorimeter. As an internal control, the
mitochondrial uncoupling agent 2,4-dinitrophenol (26)
at a
concentration of 100 µM was used to diminish
m. In COLO 16 cells, this treatment
resulted in 60% reduction of R123 fluorescence relative to control
after a 2-h exposure (not shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The treatment
concentration of 4HPR was determined empirically based on achieving
maximum reduction of cell number relative to controls over 48 h
among all of the cell lines examined without causing necrosis,
determined by trypan blue exclusion, at the 6- or 12-h time points.
This concentration has been used by other investigators for in
vitro treatment of various cell types. Decreased cell survival
became apparent between 12 and 24 h, with the exception of SRB-1
cells. SCC-13 cells were the most sensitive to 4HPR treatment, with
50% cell survival observed at 12 h. SRB-1 cells did not show a
similar effect until 36 h later. This differential response to 4HPR
is likely attributable to the extended doubling time observed for SRB-1
cells with respect to other SCC cell lines (see "Materials and
Methods").
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. This is especially evident
at the 24-h time point, where 10 µM 4HPR caused
90% reduction in cell number relative to control, whereas 5 and 1
µM 4HPR promoted 50% and 20% reduction,
respectively. COLO 16 cells treated with 1 and 5
µM 4HPR required 48 h to experience similar
effects in proliferation inhibition to those observed in SCC-13
cells at 24 h (not shown). SRB-12 and SRB-1 cells were not
examined using lower concentrations of 4HPR for this reason. Within 6–12 h after exposure to 10 µM 4HPR, SCC cells exhibited early morphological changes (specifically, chromatin condensation and cell shrinkage) typical of apoptotic cells (27) . Over time, SCC cells exposed to 4HPR would shrink to approximately one-half of their normal size and detach from the tissue culture dish. To determine whether this was actually apoptosis and to possibly quantify the event, the TUNEL assay was conducted to measure DNA fragmentation associated with apoptosis induction (21) .
Fig. 2
depicts typical flow cytometry
scatter diagrams obtained for SCC-13 cells. Control populations were
gated, and apoptosis induction was assessed as 4HPR-treated cells
migrated above the gate, indicating increasing degrees TUNEL staining
[presented in the upper right corner of each panel as a
percentage of the total population (SD)]. SCC-13 cells treated with
increasing concentrations of 4HPR exhibited apoptosis induction in both
a concentration- and time-dependent fashion. Secondary necrosis
(27)
was evident in SCC-13 cells after a 48-h exposure to
5 µM 4HPR and after a 24-h exposure to 10
µM 4HPR. This advanced degradation prevented
additional TUNEL evaluation because the remaining intact cells were not
sufficient in quantity for the labeling procedure (1–1.5 x
106 cells/sample were required for labeling).
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. All of the SCC cell
lines exhibited apoptosis induction in 80% of the cell population
between 12 and 24 h after exposure to 10 µM 4HPR
(Fig. 3
A–D). Interestingly, COLO 16 cells (Fig. 3A)
exhibited latency from the initial time of exposure to
10 µM 4HPR until the 12-h time point before
TUNEL staining was markedly different from control cells. In the other
SCC cell lines exposed to 10 µM 4HPR (Fig. 3
B, C, and D), apoptosis induction was
between 30 and 60% at the 6-h time point. Secondary necrosis was
evident in SRB-12 cells after a 48-h exposure to 10
µM 4HPR, which prevented additional TUNEL
evaluation. Although initially SCC-13 cells did not respond to 4HPR,
eventually apoptosis induction was evident at the 24-h time point for
exposure to 5 µM 4HPR (Fig. 3
E) or
at the 48-h time point for exposure to 1 µM
4HPR (Fig. 3
F).
|
. The
appearance of a sub-G1 population, indicating
cells or apoptotic bodies with less than G1 DNA
content via incorporation of propidium iodide, can be used as an
indicator of apoptosis (22)
. The maximal
sub-G1 values for 4HPR-treated cells did not
exceed 36% of the total sample population at any of the times for the
concentrations of 4HPR examined. As a secondary indicator of apoptosis,
this was typically <50% of the values obtained from the TUNEL
procedure. It should be stressed that the apoptosis detection method
(using propidium iodide as a counterstain) used in this work is
optimized for TUNEL-mediated fluorescein-labeled dUTP incorporation.
The cells were fixed with paraformaldehyde before the permeabilization
procedures with Triton X-100 and 70% ethanol. Thus, any soluble
intracellular histone-associated DNA fragments would be covalently
cross-linked to intracellular matrix and unable to escape the cell
after permeabilization. As such, a substantial
sub-G1 population would not be expected until
perhaps the formation of apoptotic bodies that would ultimately shift
into the sub-G1 population as a result of their
reduced DNA content. This shift may require more time to develop, or
may occur to variable degrees in keratinocytes (28)
.
SCC-13 cells exposed to 1 µM 4HPR have less cells in
G1 and more cells in G2-M
than do controls at the 12- and 24-h time points; and cells exposed to
5 µM 4HPR have less cells in G1,
more cells in S phase, and less cells in G2-M
than do controls. This shows that 4HPR is altering progression
through the cell cycle before apoptosis induction.
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demonstrates the ability of
various concentrations of 4HPR to promote the oxidation of DCF in
SCC-13 cells. After 60 min of exposure to 4HPR, a linear increase in
fluorescence, as a measure of ROS production, was observed.
Interestingly, SCC-13 cells responded similarly in ROS production to
both the 5- and 10-µM concentrations of 4HPR,
indicating possible saturation of the source of ROS generation. The
other SCC cell lines were equally or more effective producers of ROS
after a 10-µM 4HPR treatment, with COLO 16
cells exhibiting the greatest degree of ROS generation. ATRA,
9-cis RA, and the retinoic acid receptor -specific ligand
CD437 (all at 10 µM) were unable to enhance ROS
production after short-term treatment of SRB 12 cells (Fig. 5B)
. Furthermore, as observed in SCC-13 cells, there was a
similar response to both the 5- and 10-µM 4HPR
treatments with respect to ROS production in SRB-12 cells.
|
. This
antioxidant has the ability to scavenge free radicals and hydrogen
peroxide (30)
. Because Vit-C was unable to completely
suppress 4HPR-induced apoptosis, we cannot exclude the possibility that
some other mechanism, not associated with ROS production, could
contribute to the overall effects of 4HPR in COLO 16 cells.
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(31)
, and this receptor is expressed to
varying degrees in the SCC cell lines examined in this study (not
shown). Treatment with the retinoic acid receptor ß- and -specific
antagonist CD2665 (32)
was unable to influence
4HPR-mediated apoptosis at the 12-h time point (Fig. 6A)
.
Treatment with CD2665 at concentrations higher than 10
µM caused necrosis when combined with 10
µM 4HPR. Trypan blue exclusion was restricted
to 60% of the total cell population after treatment with 20
µM CD2665 and treatment with 10
µM 4HPR for 12 h (not shown). This limited
the use of this antagonist at higher concentrations with respect to
possibly blocking apoptosis induction in COLO 16 cells. CD2665 at 10
µM and Vit-C at 500 µM
were unable to promote appreciable degrees of apoptosis as single
agents in COLO 16 cells after 24 h of exposure (not shown).
Treatment with Vit-C could inhibit apoptosis in COLO 16 cells exposed
to 4HPR. This prompted reexamination of ROS production to determine
whether Vit-C was influencing the prooxidant activity of 4HPR as a
means of modulating apoptosis induction. Fig. 6B
illustrates that treatment with Vit-C could diminish 4HPR-mediated ROS
production in COLO 16 cells by 50%. ROS can affect various
mitochondrial components, ultimately resulting in the loss of
m and apoptosis induction
(33)
. To investigate if 4HPR alone or in combination with
Vit-C could modulate m in SCC cells,
retention of the cationic mitochondrial dye R123 (25)
was
examined. Fig. 6C
illustrates that COLO 16 cells treated
with 10 µM 4HPR showed a gradual sustained
decrease in R123 fluorescence (as a measure of
m dissipation) over time. When 4HPR was
combined with Vit-C, dissipation of m was
inhibited relative to the degree exhibited by 4HPR alone. Vit-C
treatment promoted a slight reduction in m
relative to DMSO-treated cells.
MPT Antagonists Rescue SCC Cells from the Proapoptotic Effects of
4HPR.
Having observed that R123 retention and the oxidation of DCF were
directly associated with apoptosis or cell survival in SCC cells
treated with 4HPR alone or in combination with Vit-C, the role of MPT
in 4HPR-induced apoptosis was investigated. MPT is characterized as the
opening of mitochondrial megachannels that can be triggered by ROS or
other agents, resulting in the dissipation of
m, ATP depletion, caspase/endonuclease
activation, and ROS production (reviewed in Refs. 33
and
34
). CsA has been used to inhibit MPT triggered by various
agents, including those capable of promoting oxidative stress (35
, 36)
. Therefore, the effects of CsA on SCC-13, the cell line most
sensitive to the apoptotic effects of 4HPR, were examined to determine
whether MPT was an essential characteristic of 4HPR-induced apoptosis.
Fig. 7A
shows that the
addition of CsA with 4HPR could preserve R123 fluorescence in SCC-13
cells as compared with exposure to 4HPR alone. In addition, CsA
promoted a discernable increase in R123 retention in SCC-13 cells,
indicating a possible hyperpolarization of
m. CsA was also able to decrease the
oxidation of DCF when combined with 4HPR (Fig. 7B)
,
indicating a reduction in ROS production. To determine whether this
reduction could be directly attributed to possible reactions between
ROS and CsA, hydrogen peroxide was combined with CsA. This resulted in
no decrease in the oxidation of DCF. This would indicate that CsA was
not directly competing with DCF for oxidation by ROS. Fig. 7C
demonstrates that the CsA/4HPR treatment combination
could completely block the DNA fragmentation promoted by 4HPR alone
after 6 or 12 h as measured by the TUNEL assay. This combination
promoted even less apoptosis than the CsA treatment. CsA is also an
inhibitor of the Ca2+-regulated protein
phosphatase calcineurin (37
, 38) . However, a
10-µM concentration of the calcineurin
inhibitor Cyp (39)
was unable to protect SCC-13 cells from
4HPR-induced apoptosis after a 6-h exposure (Fig. 7D)
. A
specific inhibitor of MPT, BA (40)
, at a concentration of
50 µM, was also able to block apoptosis in
SCC-13 cells exposed to 4HPR (Fig. 7D)
. BA also inhibited
dissipation of m, and was slightly more
effective in reducing 4HPR-induced ROS production compared with
CsA in SCC-13 cells (not shown). The ability of CsA and BA to inhibit
dissipation of m, ROS production, and
apoptosis points strongly to the mitochondria and MPT as the primary
regulators of 4HPR-induced cell death in SCC cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of 4HPR to increase oxidation of DCF in all of the SCC cell lines implies that oxidative stress is associated with apoptosis induction. This is consistent with the observed inhibitory effects of Vit-C on 4HPR-induced ROS production and apoptosis in COLO 16 cells. Still, the ability of COLO 16 cells to generate more ROS than the other SCC cell lines after exposure to 10 µM 4HPR, and to display a latency to apoptosis induction, points to the possible involvement of intracellular defenses against ROS, possibly enzymes responsible for hydrogen peroxide production from superoxide anions, specifically superoxide dismutases (29) . This tenet is reflected by the similar responses in DCF oxidation obtained from both the 5- and 10-µM concentrations of 4HPR in SCC-13 and SRB-12 cells, as well as by the ability of SCC-13 cells to tolerate 5 µM 4HPR for at least 12 h before the majority of the cell population became apoptotic.
We speculate that 4HPR promotes two distinct types of cell death depending on the duration of exposure and the treatment concentration. This is especially evident in SCC-13 cells exposed to 1, 5, or 10 µM 4HPR. The 10 µM exposure completely inhibited proliferation by promoting interphase cell death (44) , where apoptosis occurred in the entire cell population without cell cycle progression or mitosis. Lower concentrations of 4HPR partially inhibited proliferation but ultimately resulted in mitotic or delayed reproductive cell death. This type of cell death is believed to be attributable to secondary changes in cellular metabolism or to genetic damage causing unbalanced cell cycle progression and proliferation (44) . SCC-13 cells treated with 1 or 5 µM 4HPR exhibited decreased proliferation and alterations in cell cycle progression relative to controls before apoptosis induction. The 5-µM 4HPR treatment promoted more ROS production than the 1-µM 4HPR treatment. Thus, it would appear that the basis of the delayed apoptosis after exposure to decreasing concentrations of 4HPR was associated in some respect with varying degrees of oxidative stress.
Observations in cervical carcinoma cells suggest that 4HPR-induced ROS
production is linked to components of the mitochondrial respiratory
chain (45)
. In this study, COLO 16 cells treated with 4HPR
showed the greatest increase in ROS production along with a gradual
dissipation of m. Interestingly, the
uncoupling agent 2,4-dinitrophenol was able to reduce mitochondrial
R123 retention in COLO 16 cells by 60% after a 2-h exposure.
However, this exposure was unable to promote ROS generation in the same
cell line (not shown). This would indicate that 4HPR-induced ROS
production was associated with the loss of
m. 4HPR-induced dissipation of
m in COLO 16 cells could be prevented with
Vit-C, and this was accompanied by a decrease in ROS production. In the
case of the Vit-C/4HPR treatment combination, the maintenance of
m can potentially be linked to the
inhibition MPT via reduction of ROS, assuming MPT can be triggered by
ROS (33
, 46)
.
CsA, as an inhibitor of MPT, is believed to bind cyclophilin associated
with the mitochondrial adenine nucleotide translocator, thereby
inhibiting its function by promoting a closed-matrix conformation
(46
, 47)
. BA is believed to bind directly to the
mitochondrial adenine nucleotide translocator, also inhibiting its
function by promoting a closed-matrix conformation (47)
.
CsA had the ability to maintain m when
combined with 4HPR in SCC-13 cells. This observation would suggest that
4HPR was not functioning simply as a protonophore, because CsA combined
with aristolochic acid was unable to preserve R123 retention in
fibroblasts treated with carbonyl cyanide
m-chlorophenylhydrazone (38)
.
The ability of CsA to enhance R123 retention in SCC-13 cells
demonstrates that CsA was functioning at the mitochondrial level. CsA
and BA also had the ability to reduce ROS generation when combined with
4HPR in SCC-13 cells. As such, a substantial amount of the ROS
production after exposure to 4HPR alone could be attributed to
hyperproduction of ROS resulting from MPT (33)
,
represented by the rapid dissipation of m.
The ROS production detected in the CsA/4HPR treatment combination was
apparently independent of MPT, indicating ROS was responsible for MPT
as observed in COLO 16 cells. Most notably, CsA and BA could inhibit
4HPR-induced DNA fragmentation, illustrating that MPT was required for
apoptosis.
This study has highlighted the effects of 4HPR on ROS production and promotion of MPT, which appear to be the major mechanisms associated with apoptotic cell death in the SCC cell lines examined. It is noteworthy that in other cell types (e.g., neuroblastoma) other mechanisms may be important, such as increased production of ceramide (48) . In neuroblastoma cells, 4HPR promoted necrosis in addition to apoptosis. A shift from predominately apoptotic to predominately necrotic cell death has been observed in SCC cells exposed to 20 µM 4HPR for 12 h (not shown). However, other potential treatment effects of this concentration were not examined because these conditions precluded accurate mechanistic evaluation of apoptotic cell death, which was the focus of this study.
The mechanism of action associated with the antitumor effects of 4HPR in vivo remains to be elucidated. This retinoid is appealing for chemopreventive and therapeutic application because it is effective without many of the undesirable side effects associated with the prolonged use of other synthetic and natural retinoids. In addition, it is conceivable that 4HPR could be developed for topical delivery in a cosmetic application. Such an application would be ideally suited as a prophylactic measure to prevent skin cancer development or to resolve premalignant actinic damage. Together, results obtained in clinical trials combined with in vitro findings lend support for the continued examination of 4HPR as a chemopreventive and therapeutic agent in skin.
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Acknowledgments |
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Footnotes |
|---|
1 Supported in part by the USPHS Program Project
Grant PO1 CA68233 from the NCI, and by a cancer prevention fellowship
by the NCI Grant R25 CA57780, Robert M. Chamberlain, Ph.D., Principal
Investigator. 
2 To whom requests for reprints should be
addressed, at the Department of Thoracic/Head and Neck Medical
Oncology, Box 80, University of Texas M. D. Anderson Cancer Center,
1515 Holcombe Boulevard, Houston, TX 77030-4095, Telephone:
(713) 792-7480, Fax: (713) 794-0209,
E-mail:rlotan{at}mdanderson.org
3 The abbreviations used are: 4HPR,
N-(4-hydroxyphenyl)retinamide; ATRA,
all-trans retinoic acid; Vit-C, L-ascorbic
acid; BA, bongkrekic acid; 9-cis RA,
9-cis retinoic acid; CsA, cyclosporin A; Cyp,
cypermethrin; DCF, 2',7'-dichlorofluorescin; m,
mitochondrial inner transmembrane potential; MPT, mitochondrial
permeability transition; R123, rhodamine 123; ROS, reactive oxygen
species; SCC, squamous cell carcinoma; TUNEL, terminal deoxynucleotidyl
transferase dUTP nick end labeling.
Received 1/17/00; revised 8/25/00; accepted 9/18/00.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Biochem. Biophys. Res. Commun., 186: 977-983, 1992.[Medline]
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V. K. Puduvalli, J. T. Li, L. Chen, and I. E. McCutcheon Induction of Apoptosis in Primary Meningioma Cultures by Fenretinide Cancer Res., February 15, 2005; 65(4): 1547 - 1553. [Abstract] [Full Text] [PDF]
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 S.-Y. Sun, N. Hail Jr, and R. Lotan Apoptosis as a Novel Target for Cancer Chemoprevention J Natl Cancer Inst, May 5, 2004; 96(9): 662 - 672. [Abstract] [Full Text] [PDF]
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 A. L. Sabichi, M. R. Modiano, J. J. Lee, Y.-M. Peng, M.-J. Xu, H. Villar, W. S. Dalton, and S. M. Lippman Breast Tissue Accumulation of Retinamides in a Randomized Short-term Study of Fenretinide Clin. Cancer Res., July 1, 2003; 9(7): 2400 - 2405. [Abstract] [Full Text] [PDF]
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 I. Korichneva, J. Waka, and U. Hammerling Regulation of the Cardiac Mitochondrial Membrane Potential by Retinoids J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 426 - 433. [Abstract] [Full Text] [PDF]
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 W. F. Holmes, D. R. Soprano, and K. J. Soprano Elucidation of Molecular Events Mediating Induction of Apoptosis by Synthetic Retinoids Using a CD437-resistant Ovarian Carcinoma Cell Line J. Biol. Chem., November 15, 2002; 277(47): 45408 - 45419. [Abstract] [Full Text] [PDF]
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 A.-M. Simeone, S. Ekmekcioglu, L. D. Broemeling, E. A. Grimm, and A. M. Tari A Novel Mechanism by Which N-(4-hydroxyphenyl)retinamide Inhibits Breast Cancer Cell Growth: The Production of Nitric Oxide Mol. Cancer Ther., October 1, 2002; 1(12): 1009 - 1017. [Abstract] [Full Text] [PDF]
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N. Hail Jr. and R. Lotan Examining the Role of Mitochondrial Respiration in Vanilloid-Induced Apoptosis J Natl Cancer Inst, September 4, 2002; 94(17): 1281 - 1292. [Abstract] [Full Text] [PDF]
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S. Bruno, C. Tenca, D. Saverino, E. Ciccone, and C. E. Grossi Apoptosis of squamous cells at different stages of carcinogenesis following 4-HPR treatment Carcinogenesis, March 1, 2002; 23(3): 447 - 456. [Abstract] [Full Text] [PDF]
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N. Hail Jr. and R. Lotan Mitochondrial Respiration Is Uniquely Associated with the Prooxidant and Apoptotic Effects of N-(4-Hydroxyphenyl)retinamide J. Biol. Chem., November 30, 2001; 276(49): 45614 - 45621. [Abstract] [Full Text] [PDF]
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M. A. Smith and B. Anderson Where to Next with Retinoids for Cancer Therapy? Clin. Cancer Res., October 1, 2001; 7(10): 2955 - 2957. [Full Text] [PDF]
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N. Hail Jr., E. M. Youssef, and R. Lotan Evidence Supporting a Role for Mitochondrial Respiration in Apoptosis Induction by the Synthetic Retinoid CD437 Cancer Res., September 1, 2001; 61(18): 6698 - 6702. [Abstract] [Full Text] [PDF]
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, COLO 16; 
, SCC-13; 
, SRB-1; and • SRB-12) were
seeded in 10-cm dishes (750,000 cells/dish) and allowed to achieve 50%
confluence when they were treated with 10 µM 4HPR. Cells
were then harvested at 6, 12, 24, or 48 h and counted with a
hemacytometer. B, SCC-13 cells were seeded, harvested,
and counted as described above after treatment with 1 µM
4HPR (
), SRB-12 (
), and COLO 16
(
) were treated with 10 µM 4HPR. Control cultures from
each cell line responded similarly in ROS production, so only the
SCC-13 control is represented. B, SRB-12 cells were
treated with DMSO (
