EFFECT OF RETINOIC ACID AND INTERFERON �-2a ONTRANSITIONAL CELL CARCINOMA OF BLADDER

CHANGPING ZOU,* SANJAY RAMAKUMAR, LIXIN QIAN, CHANGCHUN ZOU, RONGYU ZANG,JIAN WANG, H. BARTON GROSSMAN,† REUBEN LOTAN AND MONICA LIEBERT‡

From the Department of Obstetrics/Gynecology and Urology, University of Arizona (CZ, SR, LQ, CZ, RZ, JW), Tucson, Arizona, M. D.Anderson Cancer Center (HBG, RL), Houston, Texas, and American Urological Association (ML), Baltimore, Maryland

ABSTRACT

Purpose: Retinoids modulate the growth and differentiation of normal and malignant epithelialcells in vitro and in vivo. Retinoids and their analogues have been used in animal models andclinical trials of chemoprevention and superficial bladder cancer treatment. Interferons arecytokines that have antiviral, antiproliferative and immunomodulatory function. They are usedin many clinical trials for the treatment of different cancers. To identify new effective agents anddevelop novel approaches for the chemoprevention and treatment of superficial bladder cancerwe investigated the effects of a combination of retinoids and interferon �-2a (IFN) on growth andapoptosis in bladder cancer cell lines.Materials and Methods: The 4 bladder cancer cell lines UM-UC-6, UM-UC-9, UM-UC-10 and

UM-UC-13 were treated with 2 retinoids, namely all-trans-retinoic acid (ATRA) and 9-cis retinoicacid (9cRA), as well as with IFN or with combinations of retinoids and IFN. The ability of theseagents used alone and in combination to inhibit growth, induce apoptosis and modulate geneexpression was investigated. The effects of retinoids on an INF related gene were also examined.Results: Most bladder cancer cell lines were resistant to growth inhibition and apoptosis

induction by ATRA and 9cRA, even at a high concentration. The effects of these retinoids on cellgrowth and apoptosis were enhanced by IFN. The combination of ATRA and IFN induced retinoicacid receptor �, and signal transducer and activator of transcription 1 expression in 3 bladdercancer cell lines, as detected by reverse transcriptase-polymerase chain reaction and Westernblot analysis. Retinoids increased IFN-related gene expression detected by microarray analysisand real-time reverse transcriptase-polymerase chain reaction.Conclusions: The results demonstrate that IFN acts synergistically with ATRA and 9cRA in

the growth and apoptosis of bladder cancer cells in vitro and suggest that this combination hasa potential for the treatment of transitional cell carcinoma of the bladder.

KEY WORDS: bladder; carcinoma, transitional cell; retinoids; interferons; apoptosis

More than 80% of patients with a first bladder cancer havetumors that are superficial. These types of tumors have lowinvasive potential but 50% to 80% of these patients experi-ence recurrence within 12 months after initial resection.1,2

Primary chemoprevention of bladder cancer and secondarychemoprevention of recurrent bladder cancer are goals forphysicians and researchers that have not yet been achieved.Retinoids regulate the growth and differentiation of nor-

mal and malignant epithelial cells in vitro and in vivo, andthey have been intensively investigated as chemopreventiveagents for the treatment and prevention of various can-cers.3,4 Retinoids have been shown to inhibit bladder carci-nogenesis in animal models, although data in man have beenless convincing.5,6 Retinoid receptors were thought to havean important function in mediating the effects of retinoids.7,8

Two types of receptors have been identified, namely retinoicacid receptors (RARs) and retinoid X receptors (RXRs). RARsbind to all-trans-retinoic acid (ATRA), while 9-cis-retinoicacid (9cRA) (Sigma Chemical Co., St. Louis, Missouri), a

natural retinoic acid isomer, binds to RARs and RXRs.7,8

Because each subtype of RAR or RXR shows distinct patternsof expression and different distributions, each one is thoughtto regulate the expression of distinct genes.7,8

Interferons (IFNs) are cytokines that have antiviral, anti-proliferative and immunomodulatory functions.9 There are 2types of IFNs, namely types I (IFN-� and IFN-�) and II(IFN-�). IFN-� and IFN-� use different cell surface receptors,and partially different tyrosine kinase, and signal transducerand activator of transcription (STAT) in their signal trans-duction. The anticarcinogenic effects of IFN are the result ofinduced changes in cell growth and differentiation caused bychanges in the expression of specific genes, such as onco-genes, growth factors and growth factor receptors.9,10 IFNsexert their effect on gene expression by activating a signaltransduction pathway in which STAT protein has an impor-tant role. IFN-� and IFN-� can activate STAT1 protein. IFNregulate factor regulates the expression of IFN and IFN induc-ible genes that can be activated by IFNs and retinoids.9,10

We have previously reported that abnormalities in recep-tor expression were found in human bladder cancer celllines.11 RAR� expression was suppressed in most bladdercancer cell lines.11 Abnormalities in RAR� expression sug-gest that this receptor may be involved in bladder cancer. Acombination of retinoids and IFN has been used for phase IItreatment of invasive cervical cancer and renal cancer.12,13

Our group as well as others has reported that a combination

Submitted for publication April 21, 2004.Supported by National Institutes of Health/National Cancer Insti-

tute CA75966.* Correspondence: Department of Obstetrics/Gynecology, College

of Medicine, University of Arizona Cancer Center, Tucson, Arizona85724 (telephone: 520-626-8883; FAX: 520-626-9287).† Financial interest and/or other relationship with Fujirebio Diagnos-

tics, PhotoCure, Abbott/Vysis, AstraZeneca, Pharmacia and UroCor.‡ Financial interest and/or other relationship with UroCor and

Department of Defense.

0022-5347/05/1731-0247/0 Vol. 173, 247–251, January 2005THE JOURNAL OF UROLOGY® Printed in U.S.A.Copyright © 2005 by AMERICAN UROLOGICAL ASSOCIATION DOI: 10.1097/01.ju.0000141584.50650.26

247

of retinoids and IFN had a synergistic effect on growth inhi-bition of cervical cancer cells.14 A combination of these 2agents also had signaling cross-talk in normal and retinoidresistant acute promyelocytic leukemia.15 We now describethe effects of ATRA (RAR pathway) and 9cRA (RXR pathway)used alone and in combination with IFN�-2a on growth in-hibition, apoptosis, and RAR� and STAT1 expression in blad-der cancer cell lines.

MATERIALS AND METHODS

Cell lines and reagent. The bladder cancer cell lines UM-UC-6, UM-UC-9, UM-UC-10 and UM-UC-13 were derivedfrom transitional cell carcinomas.16 Cells were grown in a 1:1(volume per volume) mixture of Dulbecco’s modified Eagle’smedium (DMEM) with 10% fetal bovine serum (FBS) at 37Cin a humidified atmosphere of 95% air and 5% CO2.Retinoids used in the study were ATRA and 9cRA. Retin-

oids were dissolved in dimethyl sulfoxide as stock solutions of0.1 mM and stored in an atmosphere of N2 at �80C.Effects of IFN and ATRA on cell proliferation in mono-

layer cultures. Cells were plated in 96-well plates at aconcentration of 104 or 2 � 104 cells per well and grown for24 hours. Cells were then incubated with 100, 500 and1,000 U IFN�-2a, and 1 �M concentrations of ATRA and9cRA. Control cultures contained dimethyl sulfoxide.Growth inhibition was determined using the crystal violetmethod, as described previously.11 Fixed and stained cellswere read by using a plate reader (BioRad Laboratories,Hercules, California). Growth inhibition was calculated accord-ing to the equation, inhibition % � (1 � Nt/Nc) � 100, where Ntrepresents the number of cells in treated cultures and Nc rep-resents the number of cells in control cultures. All experimentswere performed in triplicate and the mean � SD was calcu-lated.Analysis of apoptosis induced by IFN and retinoids. The

TUNEL assay was performed, as previously described.11 Fol-lowing incubation with 1 �M ATRA or 9cRA, or 1,000 UIFN�-2a alone and in combination for 3 days cells were fixedin 1% formaldehyde. Cells were first suspended in 1 ml washbuffer from a flow cytometry kit (Phoenix Flow Systems, SanDiego, California). Approximately 106 cells were resuspendedin 50 �l staining buffer. Cells were stained with 500 �lpropidium iodide/ribonuclease A solution and then analyzedby flow cytometry using a FACScan flow cytometer (EpicsProfile, Coulter Corp., Hialeah, Florida) with a 15 mW argonlaser used for excitation at 488 nm. Fluorescence was meas-ured at 570 nm. Computer analysis of the data providedinformation on the percent of apoptotic cells.Analysis of nuclear retinoid receptor � and IFN� 1 mRNA

expression by quantitative reverse transcriptase-polymerasechain reaction (RT-PCR). Real-time quantitative RT-PCRwas performed using a 7700 Sequence Detector (AppliedBiosystems, Foster City, California). Specific quantitativeassays for RAR� and IFN� were developed using PrimerExpress software (Applied Biosystems). Total RNA was ex-tracted from bladder cells by Tri-reagent. Resulting RT-PCRdata were analyzed using SDS software (Applied Biosys-tems). Final data were normalized to the housekeeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH) andthey are presented as molecules of transcript/molecules ofGADPH � 100 (percent GAPDH). For this type of analysis itis critical that the housekeeping gene does not change acrossexperimental conditions. �Ct was calculated for each gene byExcel (Microsoft, Redmond, Washington) according to theequation, �Ct � Cttreatment condition � Ctcontrol condition2ˆ(- ��Ct) � fold change, where Ct represents the cycle atwhich the reaction crossed the threshold. If the fold changewas greater than 1.0, the gene was up-regulated relative tothe control (equal to 1). If the fold change was less than 1.0,the gene was down-regulated relative to the control.

Microarray analysis. UM-UC-6 cells were treated with 1�M ATRA and synthetic retinoic acid (4HPR) for 3 days.RNA was purified as previous described.11 RNA sampleswere assessed for degradation and DNA contamination onan Agilent 2100 Bioanalyzer (Agilent Technologies, PaloAlto, California). Two separate RNAs (control and treatedgroup) were submitted for each microarray chip. Dual labelcDNA hybridization chips (10,000 cDNAs per chip) for highdensity cDNA microarrays were used in the assay to iden-tify transcripts subject to differential expression. An ErieScientific Lifterslip (Erie Scientific, Portsmouth, NewHampshire) was used for hybridization. Hybridization wasperformed in a regular hybridization chamber, as recom-mended by the manufacturer. The recommended washeswere followed. Washed chips were read on a Scan ArrayLite and the digital image output analyzed using Quan-tArray software (Perkin-Elmer, Foster City, California).All experiments were performed in triplicate and themean � SD was calculated.Western blot analysis of STAT gene expression. Nuclear

and cytoplasmic extracts were prepared from control, andATRA and IFN treated bladder cancer cells, as previouslydescribed.11 Nuclear or cytoplasmic protein (30 �g per lane)was electrophoresed on 8% polyacrylamide gels and transferredto a nitrocellulose membrane. Membranes were incubated withmouse IgG monoclonal antibodies against STAT1 to 3 (SantaCruz Biotechnology, Santa Cruz, California), washed and incu-bated with peroxidase conjugated antimouse antibody as a sec-ond antibody (Amersham International, Arlington Heights, Il-linois). Immunoreactive bands were developed using anenhanced chemiluminescence reagent (Amersham Interna-tional). The blots were stripped and then re-incubated withmouse anti-�-actin antibody (Sigma Chemical Co., St. Louis,Missouri) to normalize for loading differences.

RESULTS

Growth inhibitory effect of retinoids on human bladdercancer cell lines. The combination of ATRA, 9cRA and IFN aswell as each drug used alone was compared for the effect onthe growth of 4 human bladder cancer cell lines grown inmonolayer cultures. Cells were treated for 5 days at differentconcentrations of IFN alone and IFN combined with retin-oids. Bladder cancer cells were resistant to most retinoids(ATRA and 9cRA), as we have previously found.11 Dose de-pendent responses to IFN were observed in UM-UC-6, UM-UC-9 and UM-UC-13 cells (fig. 1). UM-UC-10 cells wereresistant to ATRA and IFN (fig. 1). The combination of IFNand ATRA had a greater growth inhibitory effect than anydrug used alone (fig. 1). There were no significant changes inmorphology when cells were treated with ATRA and IFNalone. Cell number decreased significantly after combinationdrug treatment (fig. 2).IFN and retinoid induced apoptosis.We tested the effect of

ATRA, 9cis RA or IFN on apoptosis induction. Based on thegrowth inhibition study 1 �M ATRA and 9cRA, and 1,000 UIFN were used in the experiment. Cells were treated for 3days and apoptosis was analyzed by the method of terminaldeoxynucleotidyl transferase labeling and flow cytometry.Figure 3 shows DNA content and apoptosis induction in theUM-UC-9 bladder cancer cell line. Results demonstrated thatATRA and 9c RA as well as INF alone had little effect oninducing apoptosis (fig. 3). The combination of IFN and reti-noids was more potent in inducing apoptosis than either drugused alone (fig. 3). The combination of IFN and 9cRA (48.9%)was a stronger inducer of apoptosis than the combination ofIFN and ATRA (22.3%). Cell cycle analysis showed thattreatment with IFN and ATRA or 9cRA increased the percentof cells in the G1 phase only slightly compared with controlcells, whereas it was significantly increased by the combina-tion of IFN and 9cRA (fig. 3, right).

RETINOIDS AND INTERFERON IN BLADDER CANCER CELLS248

Expression and induction of nuclear retinoid receptor andIFN� receptor detected by real-time PCR. RAR� may have animportant role in bladder cancer.11 Expression of the nuclearretinoid receptor RAR� was analyzed in these 4 bladder cancercell lines. RAR� was detected in 3 cell lines, namely UM-UC-6,UM-UC-9 and UM-UC-13, by the RT-PCR method (data notshown). RAR� induction by natural and synthetic retinoids,and IFN was detected in UM-UC-6 and UM-UC-9 cells (seetable). Retinoids and IFNwere able to induce RAR� expression.IFN�1 receptor expression was induced by ATRA and the syn-thetic retinoid 4-HPR in UM-UC-6 cells (see table).Retinoids modulated IFN gene expression in bladder cancer

cells. Microarray analysis of natural and synthetic (4HPR)retinoic acid on IFN gene expression was performed in the

bladder cancer cell line UM-UC-6 (see Appendix). Retinoidsincreased the expression of several IFN related genes inbladder cancer cells (see Appendix).Modulation of STAT gene expression by ATRA and IFN.

The effects of retinoids and IFN on the expression of STAT1 to3 gene expression were examined. STAT1 expression was de-tected in UM-UC-6, UM-UC-9 and UM-UC-13 cells. IFN treat-ment increased its expression (fig. 4, a). STAT1 expression wasincreased by the combination of IFN and ATRA or 9cRA inbladder cancer cells (fig. 4). STAT 2 and 3 expression was notsignificantly changed by retinoid or IFN (data not shown).

FIG. 1. Effect of retinoids ATAR (RA) and 9cRA (9c), and IFN�-2a on human bladder cancer cell growth in monolayer cultures. Cells weregrown for 5 days in absence (control) or presence of 1 �M ATRA and 9cRA, different concentrations of IFN�-2a and combination of differentconcentrations of INF with 1 �M ATRA or 9cRA. Culture medium was changed on day 3. Values are shown as means � SD of triplicatecultures. Percent of growth inhibition (GI) was calculated using equation, percent GI � (1 � Nt/Nc) �100, where Nt and Nc represent numberof cells in treated and control cultures, respectively. UC6, UM-UC-6. UC10, UM-UC-10. UC9, UM-UC-9. UC13, UM-UC-13.

FIG. 2. Effect of retinoids and IFN�-2a on human bladder cancercells grown for 3 days in absence (control) or presence of retinoidsand IFN�-2a. Photographs were obtained on day 3 after removal ofmedium containing floating cells. All 4 bladder cancer cell lines weretransitional cell carcinoma of different grades, that is UM-UC-6 wasG2, UM-UC-9 and UM-UC-10 were G3, and UM-UC-13 was G4.

FIG. 3. Apoptosis in bladder cancer cells determined by TUNELassay. UM-UC-9 cells were treated with 1 �M ATRA, 1 �M 9cRA,1,000 U IFN�-2a and in combination for 3 days. Cells were har-vested and incubated with terminal deoxyribonucleotidyl trans-ferase in presence of biotin labeled deoxyuridine triphosphate,stained with propidium iodide in ribonuclease A solution for 30minutes in dark and then analyzed by flow cytometry. Left panelsindicate apoptosis induction. Values in left panels indicate per-cent of apoptotic cells. Right panels indicate DNA content. Darkdots represent apoptosis (fluorescence of individual cells) aboveline in upper panels.

RETINOIDS AND INTERFERON IN BLADDER CANCER CELLS 249

DISCUSSION

Superficial bladder cancer has a low probability of metas-tasis but it has a tendency toward multiple recurrences.1,2

Chemoprevention approaches, which may delay or preventrecurrences, are ideal for this type of cancer. Vitamin A andits derivatives, the retinoids, have been studied in the che-moprevention of superficial bladder cancer. However, themechanisms through which they function are still unclear.Knowledge about the molecular mechanisms that underliethe complex biology of retinoid action has increased tremen-dously in since the discovery of the retinoid receptor. Retin-oids can bind and activate heterodimeric receptors and di-rectly affect transcriptional regulation of certain genes.7,8

Retinoid can directly and/or indirectly affect transcriptionalregulation by increasing several transcription factors, suchas RAR� and STAT1, and inhibiting certain transcriptionfactors, such as AP-1 and STAT 3.17,18

Used alone5,6, 19 and in combination12,13 retinoids andIFN-� have been reported to inhibit growth and induce apo-ptosis in several types of cancer in vitro and in vivo. Theseresults are encouraging for potential use in bladder cancer.We have previously reported the effects of ATRA, 9cRA andthe synthetic retinoic acid 4-HPR on growth and apoptosis inbladder cancer cells.11 Most bladder cancer cells were foundto have lost RAR� expression and they showed resistance toATRA and 9cRA at low (1 �M) and high (10 �M) dose treat-ment. However, high concentrations of the synthetic retinoicacid 4-HPR caused growth inhibition and apoptosis.11 Theeffects on growth and apoptosis were enhanced by IFN�-2a inRAR� positive bladder cancer cell lines, accompanied by in-creasing RAR� and STAT1, which are also found in breastcancer cells.17 However, RAR� negative cells (UM-UC-10)showed no synergistic effect on growth inhibition or apoptosisinduction. The effects of combination IFN and retinoids are

regulated by RAR� in breast,17 cervical12 and renal cancer.13

The expression of RAR� and STAT1 is lost in most breastcancer cell lines but it can be induced by retinoids in estrogenreceptor positive cells. The direct involvement of RAR� inATRA induced STAT1 gene activation was demonstrated bytransfection with an antisense RAR� that blocked ATRAinduced STAT1 expression in MCF-7 cells, whereas introduc-tion of a sense RAR� resulted in STAT1 induction by ATRAin MDA-MB 231 cells.17 In addition, it was also reported thatSTAT1 was phosphorylated/activated under ATRA treat-ment of MCF-7 cells, which involved RAR� protein synthesisbut was not due to the activation of JAK1, JAK2 or Tyk 2.17

The clinical trial of renal cancer treatment using a combina-tion of 13cRA and IFN�-2a showed that RAR� expressionwas present in 22 of 23 pretreatment (96%) and 9 of 10on-treatment (90%) specimens, and an increase in RAR�mRNA expression was detected in 4 of 5 patients (80%) whoachieved a major clinical response.13 These data suggest thatRAR� is involved in the response to retinoid based therapy,which it is not only a response to retinoid treatment, but alsomediates the response to the combination of retinoid withIFN. Other reports also support the hypothesis that retinoidreceptor and STAT1 are important for mediating the efficacyof retinoid and IFN treatment in acute promyelocytic leuke-mia.15

Combinations of IFN and ATRA or 9cRA are used in manyclinical trials for cancer treatment and they have shownmorepromise than a single agent, while a dose response effect ofretinoid on STAT1 protein expression has been reported.20

Our results suggest that retinoid induced expression of IFNrelated genes detected by microarray and real-time RT-PCRanalysis support the hypothesis that combining retinoids andIFN� can be effective for bladder cancer treatment, espe-cially in patients with RAR� positive tumors.

Receptor expression changed by retinoids and IFN

Receptor Control ATRA 9cRA 4HPR IFN-�2a

UM-UC-6:RAR� 1.0 (0) 3.12 (0.1) 2.25 (0.5) 2.68 (0.6) 1.51 (0.4)IFN� 1.0 (0) 1.43 (0.1) 0.9 (0.3) 1.19 (0.2) 1.27 (0.2)

UM-UC-9:RAR� 1.0 (0) 4.10 (0.9) 0.9 (0.3) 1.10 (0.1) 2.1 (0.1)IFN� 1.0 (0) 1.00 (0) 1.06 (0.51) 1.96 (0.9) 1.13 (0.1)

To determine RAR� and IFN� receptor expression in UM-UC6 and UM-UC9 bladder cancer cell lines cells were grown in 10% FBS and DMEM/F12 mediumtreated with 1 �M ATRA, 9cRA and synthetic retinoic acid 4HPR for 3 days and harvested, and total RNA was extracted and analyzed by real-time RT-PCR.If fold change was greater than 1.0, gene was up-regulated relative to control (equal to 1) and if fold change was less than 1.0, gene was down-regulated relativeto control.

FIG. 4. STAT1 protein induction by ATRA and IFN�-2a in bladder cancer cell lines determined by Western blot analysis. Bladder cancercell lines UM-UC-6, UM-UC-9 and UM-UC-13 were grown in absence (control) or presence of 1 �M ATRA (ARA), 1 �M 9cRA (9c, RA), 500and 1,000 U IFN�-2a, and combination of 1 �M ATRA and 9cRA with 1.000 U INF�-2a. a, STAT1 protein was identified by blotting withmonoclonal antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence method. Blots were stripped andre-blotted to mouse anti�-actin antibody to assess loading in each lane. b, data quantified for STAT1 protein expression. UC6, UM-UC-6.UC9, UM-UC-9. UC13, UM-UC-13.

RETINOIDS AND INTERFERON IN BLADDER CANCER CELLS250

CONCLUSIONS

In our study the combination of IFN and retinoids inducedgreater growth inhibitory effect and apoptosis than eitherdrug used alone. These effects are related to RAR� expres-sion and induction as well as to STAT1 gene induction. Thesefindings suggest that the effectiveness of combining IFN andretinoids on growth inhibition and apoptosis induction re-quires retinoid receptor mediated regulation and STAT1gene transactivation. Our results demonstrate that the com-bination of IFN and retinoids had a stronger effect on growthinhibition and apoptosis induction than either drug usedalone. A combination of IFN and retinoids might be a prom-ising strategy for the chemoprevention of bladder cancer.

APPENDIX: RETINOIDS MODULATE IFN GENES IN BLADDERCANCER CELLS

Microarray analysis of all IFN related genes induced byretinoid (greater than 2-fold change). Bladder cancer celllines UM-UC6 and UM-UC9 cells were grown in 10% FBSand DMEM/F12 medium treated with 1 �M ATRA. TotalRNA was purified and microarray was analyzed. IFN relatedgenes are reported. Other retinoid modulated gene is notincluded in the Appendix.X57351, Human 1–8D gene from interferon-inducible gene

familyJ03171, Interferon-� receptor (HuIFN-�-Rec) mRNAAF056979, Interferon � receptor 1 mRNAU72882, Interferon-induced protein 35BG535739, Interferon, �-inducible protein 27BG025622, Interferon, �-inducible protein 30J04164, Human interferon-inducible protein 9–27 mRNA,

complete cdsAF024714, Human interferon-inducible protein (AIM2)

mRNA, complete cdsBE886918, Interferon induced transmembrane protein 3

(1–8U)BG506643, Interferon induced transmembrane protein 1

(9–27)U22970, Human interferon-inducible peptide (6–16) gene,

complete cdsAA404652, Interferon-stimulated transcription factor 3, �

(48 kDa)

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