all-trans Retinoic Acid Enhances Cisplatin-induced ... · ranged from 0 to 15 p.M for ATRA in all cell lines, 0 to 12 p.M DDP in 2008, 0 to 60 p.M DDP in 2008/Cl3*5.25, 0 to 15 p.M

Vol. 3, 2033-2038, November 1997 Clinical Cancer Research 2033

all-trans Retinoic Acid Enhances Cisplatin-induced Apoptosis in

Human Ovarian Adenocarcinoma and in Squamous Head and

Neck Cancer Cells1

Stefan Aebi,2’3 Relef Kroning,2 Bruno Cenni,

Ashima Sharma, Daniel Fink, Gerrit Los,

Robert Weisman, Stephen B. Howell, and

Randolph D. ChristenDepartment of Medicine and the Cancer Center, University of

California at San Diego, La Jolla, California 92093-0058 [R. K..

B. C.. A. S.. D. F., G. L.. R. W., S. B. H., R. D. C.] and Institute ofMedical Oncology, University of Bern. Inselspital, CH-3010 Bern,

Switzerland [S. A.]

ABSTRACTCisplatin exerts its cytotoxicity by inducing apoptosis.

Similarly, all-trans retinoic acid (ATRA) causes apoptosis in

certain cells. We studied the interaction of cisplatin and

ATRA in human ovarian adenocarcinoma cells 2008, in

human head and neck squamous carcinoma cells

UMSCC1Ob, and in their respective cisplatin-resistant sub-

lines. ATRA enhanced the cytotoxicity of cisplatin. The

interaction of the drugs was synergistic in combination in-

dex-isobobogram analyses (combination index

2034 Synergy between all-trans Retinoic Acid and Cisplatin

mathematically rigorous technique of CI analysis to examine the

nature of the interaction between ATRA and DDP in human

ovarian carcinoma and squamous head and neck carcinoma cell

lines and investigated potential mechanisms of the observed

synergy.

MATERIALS AND METHODS

Cell Lines. The experiments were performed with the

human ovarian serous adenocarcinoma cell line 2008 (15) and

its DDP-resistant subline 2008/C 13*5.25 ( 16). In addition, the

interaction of ATRA with DDP was studied in the human

squamous head and neck cancer cell line UMSCC 1Ob ( 17) and

its DDP-resistant subline UMSCC1Ob-Pt/l5S (18). The cells

were maintained in a 5% CO2 atmosphere at 37#{176}Cin RPM!

1640 (Irvine Scientific, Irvine, CA) supplemented with 2 mM

glutamine and 10% heat-inactivated FCS without antibiotics.

The plating efficiencies of 2008, 2008/C13*5.25, UMSCC lOb,

and UMSCC1Ob-Pt/1SS were 63, 68, 61, and 54%. All experi-

ments were performed with exponentially growing cells. All cell

lines tested negative for contamination with Mvcop!as�na spp.

Chemicals. DDP was a gift from Bristol-Myers Squibb

(Princeton, NJ). ATRA and all other chemicals used were pur-

chased from Sigma Chemical Co. (St. Louis, MO).

Drug Treatment. DDP was dissolved in 0.9% (w/v) sa-

line. ATRA was dissolved in DMSO: stock.s of ATRA were kept

frozen under nitrogen until use. AThA was added to the cells in

various dilutions such that the concentration of DMSO was con-

stant and did not exceed 0. 1 % (v/v): control cells were treated with

equal concentrations of DMSO. The concentration of 0.1% DMSO

was previously shown not to interfere with the growth of 2008 and

UMSCCIOb cells. The exposure to ATRA was continuous starting

either at the same time as the treatment with DDP or 72 h prior to

DDP. The medium was replaced with new ATRA-containing me-

dium after 3 days. In contrast, the cells were exposed to DDP for

1 h, after which the medium was replaced.

Clonogenic Assays and Drug Interactions. For clono-

genic assays. the cells were trypsinized, cell clusters were dis-

rupted by repetitive pipetting, and the absence of cell clusters

was confirmed by microscopy. Three hundred cells were seeded

on 60-mm plastic dishes in 5 ml of media. Clones of more than

50 cells were scored visually after 9-10 days. Drug interactions

were analyzed by the CI method (19). The drug concentrations

ranged from 0 to 15 p.M for ATRA in all cell lines, 0 to 12 p.M

DDP in 2008, 0 to 60 p.M DDP in 2008/Cl3*5.25, 0 to 15 p.M

in UMSCCIOb, and 0 to 40 p.M DDP in UMSCCIOb-Pt/l5S

cells. The median-effect principle was applied to determine the

dose-effect parameters for DDP, ATRA, and a mixture of both

drugs corresponding to the ratio of the ICS() for each individual

drug in each cell line. The median effect equation is expressed

as f,/fu(D/Dm)’#{176}(Eq. A) and can be linearized as bog(f,,/f�)inlog(D) - �rrlog(D�,) (Eq. B), where D is the concentra-tion of the drug or the sum of the concentration of both drugs,

respectively, Dm is the median effective dose,f., is the fractional

inhibition of colony formation,f� is the fraction unaffected ( I -

fa)’ and ,n is the exponent defining the sigmoidal shape of thedose-effect curve. The parameters of the median effect equation

(Dm, � for both drugs alone and in combination were deter-

mined from Eq. B by linear regression analysis. The isoeffective

2008

� 10 � � 2008/C13*5.25:� -#{149}#{149}-2008 (RA-3d).� I

�.� 0.1

(5 �0.01

0.2 0.4 0.6 0.8

UMSCCIOb

� 10 - - UMSCCIOb/15S:� -#{149}#{149}-UMSCCIOb

0.01

0.2 0.4 0.6 0.8

Fractional inhibition

of colony formation

Fig. I CI analysis of the interaction of DDP ( 1 -h exposure) and ATRA[continuous exposure starting simultaneously with the DDP treatment or

72 h prior to DDP (PA-3d)]. A CI < I indicates synergy. whereas >1denotes antagonism and = 1 denotes additivity. Upper panel, ovarian

adenocarcinoma cell line 2008 and the DDP-resistant subline 2008/C13*5.25. Lower panel, squamous head and neck carcinoma

UMSCCIOb and the DDP-resistant subline UMSCCIOb-PtJl5S. Thecurves represent the means of at least two independent experiments.

each conducted with triplicate cultures for each data point.

dose D� for any effect bevel for each drug and their combination

were calculated by rearranging the median effect Eq. A as

follows: D� = D�[fj(l f�,)]�#{176}’ (Eq. C). Synergism or antag-

onism for ATRA plus DDP was determined on the basis of the

multiple drug equation of Chou and Talalay (19) and was

quantitated by the CI. The CI for x% inhibition of colony

formation was calculated for mutually exclusive effects as CI =

(D)ATRAI(DX)ATRA+(D)DD�/(DX)DDp (Eq. D), where (DX)ATRA

and (DX)I)[)p are the doses of single drug ATRA or DDP

required to produce x% inhibition; DATRA and DDDP are the

doses in combination that can also inhibit colony formation by

.v%. If CI = 1 , Eq. D describes a classical isobole. A C! of < 1,

= 1 , or > I signifies synergism, additivity, or antagonism, re-

spectively. The CI formula (Eq. D) allows the construction of

fa’�� plots such as shown in Fig. 1 . Each experiment wasperformed with triplicate cultures for each data point and was

repeated independently at least twice.

Cellular Pharmacokinetics. The cellular pharmacoki-

netic studies were performed with [3HIDEP, a radiolabeled

analogue of DDP. [3H]DEP produces the same type of DNA

adducts as DDP (20). Its cellular pharmacokinetics have been

well characterized and shown to parallel those of DDP (21).

Five hundred thousand cells were seeded into a 60-mm dish and

exposed to S p.M ATRA or an equal volume of DMSO (controls)

for 72 h, at which point they were treated with S p.M [3H]DEP

with a specific activity of 500 p.Ci/p.mol for 60 mm. Thereafter,

the cells were washed with ice-cold PBS and lysed overnight in

2 ml of NaOH (1 M). [3H]DEP was measured by liquid scintil-

lation counting, and cellular protein was assayed by the Brad-

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Table I Cytotoxicity of DDP and ATRA in cbonogenic assays inhuman ovarian adenocarcinoma (2008) and squamous head and neck

carcinoma (UMSCC1Ob) cells and in their respective DDP-resistant

C

w0)C

0.

a)

I’ 1 -h exposure.b Continuous exposure.

Ca)‘O

w0)C

0.

Caa)a: 0.1

0.0 0.5 3.0 4.5 6.0 7.5

DDP (pM)

Clinical Cancer Research 2035

sublines

1C5() DDP (jiM)” ATRA (jiM)”

2008 7.3 ± 2.9 3.8 ± 1.02008/C13*5.25 50 ± 5.9 1.3 ± 0.3

UMSCCIOb 7.7 ± 1.5 4.9 ± 1.1UMSCCIOb-Pt/15S 15.5 ± 1.2 1.4 ± 1.0

ford Coomassie Blue method. Each experiment was performed

with triplicate cultures.

Intracellular Glutathione. Cytosolic GSH was assayed

as described previously (22). Briefly, after labeling with mono-

bromobimane, the cellular thiols were separated by reversed

phase HPLC and were detected and quantitated with on-line

fluorometry. Calibration curves with known amounts of GSH

were constructed daily.

Immunoblots. Cells were lysed on ice in 150 mrsi NaC1

containing S mM EDTA, 1% Triton X-lOO, 10 msi TrisfHCl (pH

7.4), 5 flhlsl DTT, 0. 1 mM phenylmethylsulfonylfluoride, and S msi

#{128}-aminocaproic acid. After centrifugation, 50 jig of protein were

denatured by boiling in an equal volume of 130 mi�i TrisfI-ICI (pH

6.8) containing 20% glycerol, 4.6% SDS, and 0.02% bromphenol

blue. For the assay of PARP, the cells were lysed in a buffer

containing 63.5 msi Tris (pH 6.8), 6 M urea, 10% glycerol, 2%

SDS, 0.003% bromphenol blue, and 5% �3-mercaptoethanol. Nu-

clear PARP was released by somcation for 20 s, and the proteins of

100,000 cells were denatured by heating to 65#{176}Cfor 15 mm. Theproteins were separated by SDS-PAGE on an 8% gel for PARP or

a 12% gel for Bcl-2 and Bax, respectively. They were electrotrans-

ferred onto a polyvinylidene fluoride membrane (Immobibon P;

Millipore, Bedford, MA). Bcl-2 and Bax were detected using the

monoclonal antibodies Bcl-2(l0O) and Bax(Pl9), respectively

(Santa Cruz Biotechnology, Santa Cruz, CA). PARP was detected

with the monocbonal antibody C-2-l0 (23) purchased from Dr. Guy

G. Poiner (Universitd Laval, Quebec, Quebec, Canada). The bind-

ing of antibody was detected by peroxidase-conjugated sheep anti-

mouse antibodies (Amersham Corp., Arlington Heights, IL) and

generation of chemoluminescence by ECL (Amersham). X-ray

films were used to create a permanent record, and the bands of

interest were quantitated with a LKB Ultroscan XL densitometer.

Morphological Assay for Apoptosis. Cells (5 X l0�)

were seeded into 10-cm dishes and were treated with drugs as

described above. The medium containing floating cells and the

adherent cells after trypsin treatment were centrifuged and re-

suspended in 100 p.1 of PBS. The cells were immediately stained

with acridine orange and ethidium bromide (X25 solution:

0.01% w/v of both stains in PBS) and examined independently

by two investigators (B. C. and S. A.) using fluorescent micros-

copy (24). Apoptotic cells showed condensed and often frag-

mented nuclei and could be easily distinguished from normal

and from necrotic cells. In addition, this method permits the

distinction between early apoptotic cells with intact cytoplasmic

membranes (green fluorescent condensed nuclei) and late apop-

totic cells (red fluorescent condensed nuclei).

2008

.\

0.11- , , , , I

0.0 0.5 3.0 4.5 6.0 7.5

DDP (pM)

Fig. 2 Modification of the dose-response curve of DDP ( I -h exposure,open symbols) by simultaneous treatment with ATRA (5 p.M. continuous

exposure, closed symbols) in 2008 ovarian adenocarcinoma cells ( I I .4jiM for DDP alone versus 5.6 jiM for combined ATRA and DDP) and inUMSCC1Ob (15.2 jiM versus 10.0 jiM) squamous head and neck cancercell lines. Means of three independent experiments are shown: bars, SD.

Statistical Analysis. One-way factorial ANOVA and re-

peated measures ANOVA were calculated with Statview 4.5 for

Windows (Abacus Concepts, Berkeley, CA). Post-hoc compar-

isons were performed using the Scheff#{233} procedure.

RESULTS

Interaction of DDP with ATRA. DDP and ATRA were

cytotoxic as single agents and inhibited colony formation in

human ovarian adenocarcinoma (2008) and head and neck squa-

mous carcinoma (UMSCC1Ob) cells and in their respective

DDP-selected sublines 2008/C 13*5.25 and UMSCC lob-Pt/l 5S

(Table 1). The nature of the interaction of DDP and ATRA was

analyzed by the CI method (19). This procedure is based on the

median effect principle and on isobobogram analysis and allows

the characterization of drug interactions with a single number,

the CI. A CI < 1, = 1, and > 1 implies synergy, additivity, and

antagonism, respectively. Fig. 1 shows that a significant syner-

gistic interaction was consistently observed in the ovarian and

the head and neck carcinoma cell lines at most levels of inhi-

bition of colony formation as demonstrated by a CI < 1 . Synergy

was present when the exposure to DDP (1 h) and to ATRA

(continuous) was started simultaneously and also when the cells

were preincubated with ATRA for 72 h prior to the exposure to

DDP and continuously thereafter (CIs for the simultaneous start

of treatment at a fractional inhibition of 0.5: 2008, 0.39; 2008/

C13*5.25, 0. 17; UMSCC1Ob, 0.41 ; UMSCC1Ob-Pt/15S, 0.16).

However, in all cell lines tested, the interaction of the drugs was

not synergistic when ATRA was present only prior to but not

after DDP (data not shown). It is evident from Fig. 1 that a

higher degree of synergism resulted from pretreating the cells

with ATRA for 72 h prior to the exposure to DDP (plots marked

“ATRA-3d”; CIs at a fractional inhibition of 0.5: 2008, 0.08;

UMSCC1Ob, 0.31). This schedule, consisting of a continuous

exposure to ATRA starting 72 h prior to a 1-h exposure to DDP

and continuing thereafter, was used for the subsequent studies.

As an example, Fig. 2 illustrates that continuous treatment with

5 p.M ATRA starting 72 h before DDP increased the slope of the

dose-response curve of DDP in both ovarian (2008) and head

and neck (UMSCC1Ob) carcinoma cells.

Cellular Pharmacokinetics. The accumulation of DDP is

a major determinant of its cytotoxicity (4). [3H]DEP is an analogue



2008 UMSCCIOb

40 -

30 -

C,)

a)

0 20 -

10�


0��0.�0.��

0 � 0

I- I-<

L��J

I � � I

Fig. 3 Fraction of apoptotic cells after treatment with DDP (20()8. 5

jiM: UMSCC1Ob. 10 psi: I h exposure. ATRA (2008. 5 jiM:UMSCCIOb. I jisi: continuous exposure). or the combination of ATRAand DDP: DDP was added after a 72-h preincubation with ATRA.

Selected significance levels are indicated below the bars: �. P � 0.001:* � < 0.03 (ANOVA. Scheff#{233}test). The number of apoptotic cells was

not significantly different between controls and cells treated with ATRA

alone. Means of three independent experiments are shown: bars, SD. �.early apoptosis: D. late apoptosis.

of DDP that forms the same DNA adducts as DDP and has very

similar cellular pharmacokinetic properties (2 1 ). Preincubation

with 5 p.M ATRA for 72 h reduced the accumulation of [3H]DEP

by 22-33% in three of four cell lines tested (2008: 59.6 ± 5.2 in

controls versus 46.6 ± 2.2 pmol/mg protein in cells treated with

AThA, P < 0.01; 2008/Cl3*5.25: 38.4 ± 8.6 versus 42.8 ± 2.1

pmoL/mg protein. P = 0.37; UMSCC1Ob: 74.0 ± 6.5 versus

49.4 ± 3.1 pmol/mg protein, P < 0.01: UMSCC1Ob-PtJ1SS:

50.5 ± I .6 versus 41.3 ± 3.8 pmollmg protein, P = 0.02; a = 3,

mean ± SD, two-sided unpaired t test). These results indicate that

the synergy between ATRA and DDP is not mediated by increased

cellular accumulation of DDP.

Intracellular GSH. Treatment of 2008 cells with ATRA

(5 jiM, 72 h) had no significant effect on the intracellular content

of GSH (20 ± 2.3 p.mol/mg in control cells versus 22 ± 9.3

p.mol/mg protein in ATRA-treated cells; mean ± SD, ii = 4,

P = 0.75).

Mechanism of Cell Death. ATRA is known to induce

apoptosis in certain ovarian carcinoma cells ( I 1 ). Similarly, DDP

exerts its cytotoxic action by activating the apoptotic pathway of

cell death ( 1 ). Therefore. we studied the induction of apoptosis in

the ovarian (2008) and in head and neck carcinoma (UMSCC1Ob)

cells. Both cell lines were treated with DDP (5 p.M in 2008, 10 p.M

in UMSCC1Ob), ATRA (5 p.M in 2008, 1 p.M in UMSCC1Ob) and

the combination of both drugs; the typical morphology of apoptosis

was evident 48 h after treatment with DDP when stained with

acridine orange and ethidium bromide and was examined by fiuo-

rescent microscopy (24). Fig. 3 shows that in both cell lines, the

- - � - PARP- -85kD

Control DDP ATRA ATRA+DDP

Fig. 4 Immunoblot of PARP in 2008 human ovarian adenocarcinomacells. The cells were treated as follows: DMSO + saline (Control):

DMSO + DDP 5 jiM for I h (DDP): ATRA 5 jiM continuous exposurefor 72 h + saline (ATRA): combined treatment with ATRA and DDP(ATR4+DDP). The cells were lysed 48 h after the treatment with DDPor saline. respectively. in the control and ATRA-treated cells. The

appearance of the Mr 85,000 fragment indicates cleavage of PARP by

apopain/CPP32.

combined treatment with ATRA and DDP resulted in about 1.5

(UMSCC1Ob) to 2 (2008) times the number ofapoptotic cells when

compared to either drug alone. In addition, the fraction of cells in

late apoptosis was significantly higher after combined treatment,

suggesting an earlier onset or a more rapid completion of apoptosis

after pretreatment with ATRA. Necrotic cells constituted less than

1% of the cells following any treatment and in controls.

To further confirm that apoptosis was the mechanism of

cell death induced by DDP and ATRA in 2008 human ovarian

adenocarcinoma cells, we studied the cleavage of PARP. PARP

is cleaved specifically by apopain/CPP32. a protease that is

necessary to initiate apoptosis (25). Fig. 4 demonstrates that

cleavage of PARP was not detected in untreated cells. However,

the typical Mr 85,000 cleavage product (23) was present in the

nuclear extracts 48 h after treatment with DDP and with the

combination of ATRA and DDP. In agreement with the mor-

phological finding of only modest induction of apoptosis, it was

almost undetectable after treatment with ATRA alone.

Bcl-2 and Bax are homologous proteins involved in the

regulation of apoptosis with Bax acting as a promoter and Bcl-2

as an inhibitor (26). We measured their expression by immuno-

blot in ovarian cancer cells (2008) after treatment with DDP (5

jiM. I h), ATRA (5 jiM, continuous exposure), and the combi-

nation of ATRA (72 h pretreatment) and DDP. The expression

of Bax remained essentially unchanged after treatment with

either DDP or ATRA alone or in combination. The expression

of Bcb-2 and, therefore, the ratio of Bcb-2:Bax declined rapidly

after treatment with DDP. Fig. SB illustrates that the combined

treatment with ATRA and DDP resulted in further suppression

of Bcb-2 relative to Bax (P = 0.03 for differences between

treatments, repeated measures ANOVA). Fig. 5A shows a rep-

resentative immunoblot demonstrating the decline in Bcl-2 cx-

pression after combined treatment with ATRA and DDP. At the

chosen dose, ATRA by itself produced a decline in the ratio of

Bcl-2:Bax only after prolonged exposure.

DISCUSSION

This study of the interaction of DDP and ATRA produced

two important findings: (a) ATRA synergistically enhanced the

cytotoxicity of DDP in human ovarian adenocarcinoma cells

(2008), in human squamous head and neck carcinoma cells

(UMSCC1Ob), and in their respective DDP-resistant sublines

(2008/C I 3*525 and UMSCC 1Ob-PtJl 55 ): and (h) ATRA

seems to enhance the potency of DDP by increasing the sus-

ceptibibity of the cells to undergo apoptosis.



A- � - � � ,.. _ , BcI-2

� � � . -‘a � � BaxEl E2 E3 El E2 E3 El E2 E3 El E2 E3

B2

x

c� I

(‘sJ

�t5 0.5-�

.2 0.3c�

Clinical Cancer Research 2037

0 24 48 72

Hours after DDP

C) ControlL� DDP

V ATRA

. ATRA+DDPo.i_JI I �

0 24 48 72

Hours after DDPFig. 5 A, immunobbots of Bcl-2 and Bax in 2()08 human ovarian adeno-carcinoma cells after combined treatment with ATRA (5 jiM. continuous

exposure) and DDP(5 jiM, I-h exposure). El. E2. and E3. experiments 1-3.Hours: time after the treatment with DDP. B. ratio of Bcl-2 to Bax afterexposure to ATRA and DDP. The plots represent the normalized geometricmeans of three independent experiments. P = 0.03 for differences betweenthe treatments (repeated measures ANOVA).

Enhancement of the cytotoxicity of DDP by ATRA has been

described previously in human teratocarcinoma cells (27), squa-

mous head and neck carcinoma cells ( 12), and very recently in

ovarian cancer cells ( 13). In the present study, the synergistic nature

of the cytotoxic effects of ATRA and DDP in the ovarian carci-

noma cells (2008 and 2008/Cl 3*5.25) and in the squamous head

and neck cancer cells (UMSCC1Ob and UMSCC1Ob-PtJ15S) was

demonstrated by the mathematically rigorous CI method (19).

Simultaneous treatment with ATRA and DDP followed by contin-

uous exposure to ATRA produced CIs well below I in the wild-

type cells (2008 and UMSCC lOb) and also in the cisplatin-resistant

sublines (2008/Cl3*5.25 and UMSCC1Ob-PtJl5S). The greatest

degree of synergy occurred in the low concentration range for both

drugs. This finding is of interest because the lower drug concen-

trations are readily attainable in the plasma of patients (28, 29).

When the continuous treatment with AThA was started 72 h prior

to the exposure to DDP, we observed an additional increase in

synergy. This observation is consistent with the multistep mecha-

nism of action of ATRA that involves transport to the nucleus,

activation of the retinoic acid receptor, and binding to retinoic acid

response elements with consecutive transcriptional regulation of

target genes. Furthermore, isomerization of ATRA to 9-cis retinoic

acid with activation of retinoic acid X receptors is possible, but the

extent to which this isomerization occurs in 2008 and UMSCC lOb

cells is presently unknown. The schedule dependence of the inter-

action of ATRA and DDP will have to be considered in future

clinical trials.

Cellular sensitivity to DDP can be modified by a variety of

mechanisms. Diminished drug accumulation is the single most

important determinant of the cytotoxicity of DDP and is present

in about 80% of cisplatin-selected cell lines (4). In this study,

ATRA promoted the toxicity of DDP but did not increase the

accumulation of the DDP analogue [3H]DEP in cells but rather

caused diminished accumulation. Furthermore, depletion of

GSH has been shown to sensitize cells to DDP (16). However,

we did not observe an alteration in the cellular content of GSH

after a 72-h exposure to S p.M ATRA in 2008 cells. Thus,

enhanced accumulation of DDP and changes in the cellular

concentration of GSH did not explain the synergy.

Apoptosis is regulated by numerous proteins (reviewed in

Refs. 5 and 30), among which Bcl-2 inhibits and Bax promotes

apoptosis. DDP induces cell death by apoptosis ( 1-3). In this

study. DDP caused a rapid decline in the ratio of Bcl-2 and Bax

that was further enhanced by concurrent treatment with ATRA.

In 2008 cells, ATRA by itself reduced the expression of Bcl-2

relative to Bax only after prolonged exposure. Whether the

observed decline of the Bcl-2:Bax ratio was the cause or the

effect of apoptotic cell death cannot be decided based on the

present data. In recent reports, ATRA enhanced the toxicity of

cytarabine by inducing apoptosis and down-regulating Bcl-2 in

leukemic stem cells and acute myebogenous leukemia blasts

(3 1 ). This effect may be mediated by retinoic acid X receptors

after isomerization of ATRA to 9-cis retinoic acid (32). DDP-

induced apoptosis as assessed by cellular morphology was en-

hanced by ATRA at a time when ATRA alone induced only a

moderate amount of apoptosis in 2008 cells. These findings are

compatible with the observed temporal changes of the Bcl-2:

Bax ratio, with the PARP cleavage pattern (23), and with a

recent report by Jozan et a!. (33). The temporal and quantitative

relationships between drug exposure, biochemical processes.

and morphological markers of apoptosis will need to be clarified

more in detail to design optimal treatment schedules.

In addition to cisplatin and cytarabine. ATRA also interacts

with IFN-cx by modulating the amounts and state of activation of

some components of the IFN signaling pathways in acute pro-

myebocytic leukemia and breast cancer cells (34-36). To date,

the clinical application of ATRA is limited largely to the treat-

ment of acute promyelocytic leukemia. Similar to 13-cis retinoic

acid (reviewed in Ref. 37), ATRA as a single drug had only

modest activity in phase II studies in solid tumors (38-40).

Taken together. the present data support the conclusion that

ATRA facilitates apoptosis induced by DDP in 2008 ovarian

carcinoma and in UMSCC1Ob head and neck carcinoma cells.

Considering the different clinical toxicities of ATRA and DDP

and the synergistic interaction of the two drugs in vitro, the

combined treatment of malignant tumors with ATRA and DDP

merits further clinical investigation.

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1997;3:2033-2038. Clin Cancer Res S Aebi, R Kröning, B Cenni, et al. neck cancer cells.human ovarian adenocarcinoma and in squamous head and all-trans retinoic acid enhances cisplatin-induced apoptosis in

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all-trans Retinoic Acid Enhances Cisplatin-induced ... · ranged from 0 to 15 p.M for ATRA in all cell lines, 0 to 12 p.M DDP in 2008, 0 to 60 p.M DDP in 2008/Cl3*5.25, 0 to 15 p.M