4-HPR modulates gene expression in ovarian cells

Authors


Abstract

Ovarian cancer has a high rate of recurrence and subsequent mortality following chemotherapy despite intense efforts to improve treatment outcomes. Recent trials have suggested that retinoids, especially 4-(N-hydroxyphenyl) retinamide (4-HPR), play an important role as a chemopreventive agent and are currently being used in clinical trials for ovarian cancer chemoprevention as well as treatment. This study examines the mechanism of its activity in premalignant and cancer cells. We investigated the modulation of gene expression by 4-HPR in immortalized ovarian surface epithelial (IOSE) cells and ovarian cancer (OVCA433) cells with DNA microarray. Real time RT-PCR and western blotting were used to confirm the microarray results and metabolic changes were examined with optical fluorescence spectroscopy. 4-HPR resulted in an up-regulation of expression of proapoptotic genes and mitochondrial uncoupling protein in OVCA433 cells and modulation of the RXR receptors in IOSE cells, and down-regulation of mutant BRCA genes in both IOSE and OVCA433 cells. 4-HPR had a larger effect on the redox in the 433 cells compared to IOSE. These findings suggest that 4-HPR acts through different mechanisms in premalignant ovarian surface cells and cancer cells, with a preventive effect in premalignant cells and a treatment effect in cancer cells. © 2006 Wiley-Liss, Inc.

Epithelial ovarian cancer is the leading cause of death from the gynecologic cancers.1 In 2003, an estimated 25,400 women were diagnosed with ovarian cancer, and 14,300 women died from the disease.1 It is most commonly diagnosed in Stage III or IV where the mortality rate is 70% or greater.2 Currently, there is no generally accepted screening test in which sensitive and reliable biomarkers can be used to identify women destined to develop ovarian cancer. Although initial treatment for ovarian cancer has an excellent response rate, the recurrence rate is high following chemotherapy, and drug resistance is a common problem. Better strategies for prevention and treatment of ovarian cancer are thus strongly warranted.

Retinoids have been intensively investigated as chemopreventive agents and have been shown to inhibit ovarian carcinogenesis based on both laboratory data and clinical trials.3, 4, 5, 6, 7 The potential of the synthetic retinoid 4-(N-hydroxyphenyl) retinamide (4-HPR) to prevent ovarian cancer was recognized in a large Italian breast cancer chemoprevention clinical trial.5, 6, 7 Patients on the 4-HPR arm demonstrated a decreased incidence of ovarian cancer,7 with 6 patients developing ovarian cancer in the control group for the duration of drug ingestion but none in the 4-HPR group (p = 0.0327).6 After cessation of the clinical trial, 6 patients in the 4-HPR group developed ovarian cancer compared to 4 in the control arm (p = 0.7563).6 This difference was not statistically significant suggesting that the effect of the retinoid was not durable.6 The mechanism of action of 4-HPR's cancer chemoprevention is unclear. It may act partly through modulation of gene expression via retinoid receptors although modulation of retinoid receptors is still controversial.8, 9, 10, 11, 12, 13, 14 Retinoid receptors are members of the steroid hormone receptor superfamily. Two types of receptors have been identified: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Each type includes 3 subtypes with distinct amino- and carboxyl-terminal domains. The RARs bind to all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9cRA), a natural retinoic acid isomer, whereas the RXRs bind only to 9cRA.15, 16, 17, 18 RARs can form heterodimers with RXRs and bind to retinoic acid response elements (RAREs), specific DNA sequences that are characterized by direct repeats of (A/G)GGTCA separated by 2 or 5 nucleotides that act as ligand-dependent transcriptional regulators for retinoic acid-responsive genes. Some investigators hypothesize that both ATRA and 4-HPR bind to RARE and regulate gene expression (Zou and Lotan, unpublished data).19, 20

DNA microarray is widely used in identifying gene expression in normal and cancer cells, and in evaluating molecular changes before and after treatment with drugs.21, 22, 23, 24 Use of array technology allows simultaneous evaluation of expression of many (up to thousands) genes. The challenge of such a powerful technique is to develop rigorous, quantitative methods for interpretation of such a wealth of data to identify the expression profile providing maximal biologic information.

Techniques based on quantitative optical fluorescence spectroscopy have shown promise to improve detection of epithelial lesions in the colon, cervix, bladder, head and neck, esophagus and other epithelial surfaces.25, 26, 27, 28 Certain molecules within a cell can be excited using light in the visible and UV range. This principle can be used to optically interrogate endogenous fluorophores with quasi monochromatic excitation light. Natural intracellular fluorophores include electron carriers nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) and the aromatic amino acids tryptophan, tyrosine and phenylalanine, as well as structural proteins, each of which have a characteristic wavelength for excitation with an associated characteristic emission.29 In particular, FAD and NADH can provide an estimate of mitochondrial metabolic activity through an estimate of cellular redox.30 Fluorescence spectroscopy of endogenous fluorophores has been used as a marker for both early detection and chemoprevention.31, 32, 33

Identification of biomarkers is important to detect abnormal cells so that invasive cancer can be prevented through chemoprevention. Furthermore, intermediate end point biomarkers are valuable in timely evaluation of the drug efficacy during chemoprevention trials. However, potentially useful biomarkers for evaluating ovarian cancer and 4-HPR's effect on the ovary are currently limited. To identify biomarkers in response to 4-HPR treatment and to investigate the mechanism of its action in ovarian cancer treatment and/or prevention, we used the in vitro model of normal cells [immortalized ovarian surface epithelial (IOSE) cells] and ovarian cancer OVCA433 cells. Ovarian surface epithelial (OSE) cells originated from ovarian epithelial carcinomas in which women with a strong family history of ovarian carcinomas or with a mutation in one of the two known cancer suppressor genes—BRCA1 and BRCA2.34, 35 Since OSE cells are thought to be the site of origin of epithelial ovarian cancer, these cells are important to study their molecular and cellular properties compared to ovarian cancer cells to enhance our understanding of malignancy in ovarian cancer.

We compared changes in gene expression from treatment with 4-HPR in normal and malignant ovarian cells by microarray and examined 4-HPR action in the context of growth inhibition, apoptosis induction, and mitochondrial permeability transition (MPT) changes. The gene expression changes were verified further by real-time RT-PCR and Western blot, while the metabolic status was evaluated with fluorescence spectroscopy.

Material and methods

Cell lines and retinoids

IOSE and ovarian cancer OVCA433 cells were grown as previously described.8 The cells were incubated for 3 days with 4-HPR with different concentration of 4-HPR (1, 5 and 10 μM). Control cultures contained dimethylsulfoxide (DMSO). N-(4-hydroxyphenyl) retinamide (4-HPR) was purchased from Sigma Chemical (St. Louis, MO), dissolved in DMSO at stock solutions of 10−2 M, and stored in an atmosphere of N2 at −80°C.

RNA preparation and microarray

Total RNA was extracted as previously described.8 We used DNA chip technology to identify gene expression in the Genomics Core Laboratory at the University of Texas Health Sciences Center. The cDNA chips (Agilent Technologies, Palo Alto, CA) with 8,000 human cDNA's, which had dual-labeled cDNA hybridization for use in high density cDNA microarrays. IOSE and OVCA433 cells were treated with 1 μM 4-HPR for 3 days. The RNA samples were evaluated for degradation and DNA contamination on an Agilent 2100 Bioanalyser (Agilent) using an RNA 6000 NanoKit. RNA from the control and the treated cells were submitted for comparison to each microarray chip. These RNA's were used with a Micromax TSA Labeling Kit (Perkin Elmer Life Sciences, Boston, MA) and Cyanine-3 or Cyanine-5 dyes. The recommended wash and labeling procedures for the Perkin Elmer TSA kit were followed except for increasing the post Cyanine 5 Tyramide labeling washes to 10 min each. The washed chips were read on a ScanArray Lite (Perkin Elmer) and the digital image output analyzed using their software (QuantArray). The resulting values were then compiled using either Excel or File Maker Pro macros written for the Microarray Core Laboratory.

Effect of 4-HPR on cell proliferation in monolayer cultures

IOSE and OVCA433 cells were placed in 96-well plates at 105 cells per well and grown for 24 hr. The cells were incubated for 5 days with 4-HPR in 1, 5 and 10 μM concentrations. Growth inhibition was determined using the crystal violet method as previously described.8 All experiments were performed in triplicate and the mean ± standard deviations calculated.

Analysis of apoptosis induced by 4-HPR

Terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein–deoxyuridine–triphosphate nick-end labeling assay was used.8 Flow cytometry used a FACScan flow cytometer (Epics Profile, Coulter, Hialeah, FL) with a 15 mW Argon laser used for excitation at 488 nm. Fluorescence was measured at 570 nm. Computer analysis of the data provided information on the percentage of apoptotic cells. All experiments were performed in triplicate and the mean ± standard deviations calculated.

Caspase 3 activity assay and protein analysis

The cells were plated in 96-well tissue culture plates at densities ranging from 0.5 to 1 × 105 cells per well and treated with 4-HPR in 1, 5 and 10 μM concentrations for 12, 24, 48 and 72 hr. Control cultures and treated cultures contained the same amount of DMSO. The method for analysis of Caspase 3 activity as previously described.8

Mitochondrial permeability transition assay

IOSE and OVCA433 cells were treated with 4-HPR in 1, 5 and 10 μM concentrations for 3 days to determine the time of maximal MPT. Cells were washed and resuspended in 40 nM MitoFluor medium, then incubated at 37°C for 30–45 min. Cells were visualized under the fluorescence microscope at 490 nm excitation, 576 nm emission. A field of 20–30 cells was chosen using a photo amplifier to measure light intensity. Once the time of maximal mitochondrial permeability was determined, the remainder of the experiments were carried out using the predetermined times of incubation.

Western analysis of p53, p21 and p16 gene expression modulated by 4-HPR

Genes showing alterations in expression by 4-HPR (>2-fold increase or >2-fold decrease) were validated by real-time Q-RT-PCR or Western blotting. Nuclear and cytoplasmic protein extracts were prepared as previously described.8

Real time Q-RT-PCR analysis for mRNA expression of RARs and BRCA genes

Real time Q-RT-PCR performed in the University of Arizona Core facility by utilizing the 7700 sequence detector (Applied Biosystems, Foster City, CA) with a similar protocol as previously described.8

Optical spectroscopic analysis of redox ratio FAD/(FAD + NADH)

Fluorescence emission was measured on IOSE cells and OVCA433 cells. Cells were treated with 1, 5 or 10 μM 4-HPR 24 hr before fluorescence measurements, described previously.36

Results

Expression of genes altered by 4-HPR in IOSE and OVCA433 cells detected by microarray

Microarray analysis was performed using total RNA purified from treated and untreated cells. The expression of genes modulated by 4-HPR was evaluated. Genes with a change of expression >2-fold were recorded in Table I. In IOSE cells, there was up-regulation of apoptotic related genes and differentiation genes, as well as genes on chromosome 3 and 9. Cancer cells showed up-regulation of fewer genes associated with apoptosis and showed similar effects on up-regulation of the antioncogene segment on chromosome 9. Mitochondrial, NAD, NADH and NADPH genes were modulated by 4-HPR in both IOSE and OVCA433 cells (Table II).

Table I. Gene Response to 4-HPR (>2-Fold Changes) Detected by DNA Array1
Gene responseIOSEOVCA433
  •  H, Human; P, protein; R, receptor.

  • 1

    Ovarian cells were grown in 10% FBS and DMEM/F 12 medium treated with 1 μM of 4-HPR. Total RNA was purified and microarray was analyzed.

Twofold up
 Hormone-related  or retinoid relatedH β-carotene mRNATR4 orphan receptor mRNA H corticotropin-release factor 1 R
 Apoptosis-repair relatedH p53 tumor suppressor binding P mRNAH p53 tumor suppressor binding P mRNA
H RbAp48mRNA encoding Rb binding PRbAp48 mRNA encoding Rb binding P
H RB-associated KRAB mRNA
H cyclin-selective ubiquitin carrier P mRNAUbiquitin-P ligase E3-a
H apoptosis regulater BCL-G long form mRNA 
H mRNA for BCL7AP 
H TNF-a & TNF-like P (ZTNF4) 
H mRNA for TRAF & TNFr associate P 
H jun dimerization P gene, cfos gene 
H GADD 45 mRNAH GADD45 mRNA
H PDGFr b-like tumor suppressorDNA repair system, specific for alkylated DNA
H mRNA for protein kinase PAK5
 Differentiation-relatedH 13Kd diff mRNA associated P 
H microtubule-associate P tau gene
 Growth factor and homeoboxH EGF-r geneH mRNA for serum response factor-related P (RSRFC2)
H homeobox P (PHOX1)
 Mitochondrial-related (table

II

)
Mitochondrial uncoupling P 5 long formH inner mitochondrial membrane peptidas 2 mRNA
 Omithine decarboxylase  antizymeORF1 and ORF2ORF2
 Chromosome 21H genomic DNA Chr21q, sct 57/105H genomic DNA Chr21q, sct 15/105
Chromosome 21 segmentHS21C100Chromosome 21 segmentHS21C100
Chromosome 21 segmentHS21C047Chromosome 21 segmentHS21C101
 Chromosome 22Chromosome 22q11.2 Cat Eye Syn region 22q11.2, clone KB1269O1Chromosome 22q11.2 Cat Eye Syn region 22q11.2, clone KB1125A3
Novel H gene mapping to chromosome2222q11.2, clone KB1896H10
 Chromosome 3Chromosome 3p21.3 anti-oncogene seg2/5 
 Chromosome 9Chromosome9 DNA.321bpChromosome9q32 anti-oncogenceflat epithelium cancer seg6/10
> Twofold down
 Hormone-related; or retinoid-related H thyroid hormone R-associated P TRAP95 mRNA
 H lactate dehydrogenase-A gene
 Apoptosis-relatedH MDM2 gene 
H mRNA for Fas-asso factor (FAF1) 
H cyclin-dependent kinase 5 activator 
 BRCA geneBRCA1 associated RING domain P geneBRCA2 mRNA sequence CG016
 Chemokins cytokineH IL4R mRNA for IL4 receptorGermline T-cell receptor bH monocyte/macrophage Ig-related R MIR-10 mRNA
Table II. Microarray Analysis of all Genes Relaped to NADH and NAD Modulated by 4-HPR
Y00764Human mRNA for mitochondrial hinge protein
AF030162Human inner mitochondrial membrane translocase Tim23 (TIM23) mRNA, nuclear gene encoding mitochondrial protein
AA524277Mitochondrial intermediate peptidase
AF043253Human mitochondrial outer membrane protein (Tom40) gene, nuclear gene encoding mitochondrial protein, exon 10
BG527393Minichromosome maintenance deficient (S. cerevisiae) 3-associated protein
AF192559Human mitochondrial carrier homolog 1 isoform b (MTCH1) mRNA, partial cds; nuclear gene for mitochondrial product
D28500Human mRNA for mitochondrial isoleucine tRNA synthetase, partial cds
M62810Human mitochondrial transcription factor 1 mRNA, complete cds
AF050639Human NADH-ubiquinone oxidoreductase AGGG subunit mRNA, nuclear gene encoding mitochondrial protein, complete cds
AF087660Human NADH:ubiquinone oxidoreductase SDAP subunit mRNA, complete cds, nuclear gene encoding mitochondrial protein
AW006760NADH dehydrogenase (ubiquinone) 1 α subcomplex, 1 (7.5 kDa, MWFE)
BG236381NADH dehydrogenase (ubiquinone) 1 α subcomplex, 9 (39 kDa)
BG700635NADH dehydrogenase (ubiquinone) 1 β subcomplex, 6 (17 kDa, B17)
BC003417Human, NADH dehydrogenase (ubiquinone) 1 α subcomplex, 10 (42 kDa), clone MGC:5103 IMAGE:3451514, mRNA, complete cds
BC001016Human, NADH dehydrogenase (ubiquinone) 1 α subcomplex, 8 (19 kDa, PGIV), clone MGC:793 IMAGE:3345138, mRNA, complete
AU133256NADH dehydrogenase (ubiquinone) 1 α subcomplex, 5 (13 kDa, B13)
BC000266Human, NADH dehydrogenase (ubiquinone) 1 α subcomplex, 1 (7.5 kDa, MWFE), clone MGC:2066 IMAGE:3352028, mRNA
BC002595Human, NADH dehydrogenase (ubiquinone) 1 β subcomplex, 7 (18 kDa, B18), clone MGC:2480 IMAGE:3140806, mRNA, complete
AI056375NADH dehydrogenase (ubiquinone) 1 β subcomplex, 3 (12 kDa, B12)
J03934Diaphorase (NADH/NADPH) (cytochrome b-5 reductase)
J03934Diaphorase (NADH/NADPH) (cytochrome b-5 reductase)
AF020038Human NADP-dependent isocitrate dehydrogenase (IDH) mRNA, complete cds
X16396Human mRNA for NAD-dependent methylene tetrahydrofolate dehydrogenase cyclohydrolase (EC 1.5.1.15)
AU138067ADP-ribosyltransferase (NAD+; poly (ADP-ribose) polymerase)
AF020038Human NADP-dependent isocitrate dehydrogenase (IDH) mRNA, complete cds

Growth inhibition and apoptosis induction by 4-HPR in ovarian cell lines

IOSE and OVCA433 cells treated with different concentrations of 4-HPR, the growth inhibitory effect were compared in monolayer culture. Increasing the concentration of 4-HPR resulted in dose-dependent growth inhibition (Fig. 1).

Figure 1.

Effect of 4-HPR on growth and apoptosis in OVCA433 and IOSE cells. Cells were grown in the absence (control) or presence of 4-HPR in concentrations of 1, 5 and 10 μM. Growth inhibition assay were performed with crystal violet on day 5. The percentage of growth inhibition was calculated, as described in Materials and Methods. The data was presented as the mean ± SE of triplicate determinations.

Apoptosis induction by 4-HPR in ovarian cells

Apoptosis induction in IOSE and OVCA433 cells were analyzed by TdT-labeling and flow cytometry after 3 days of treatment. Results showed 4-HPR apoptosis induction was dose-dependent (Fig. 2). Cell-cycle analysis demonstrated that 4-HPR increased the percentage of cells in the G1 phase in OVCA433 cells, also in a dose-dependent manner (Fig. 2).

Figure 2.

Effect of 4-HPR on apoptosis induction in OVCA433 and IOSE cells. Cells were treated with the indicated 4-HPR concentrations for 3 days. The cells were then stained with fluorescein-labeled dUTP to label DNA fragments by the TUNEL method, as described in Materials and Methods. The percentage of apoptosis cell population and DNA contents, including cell cycle, were calculated.

Effect of 4-HPR on Caspase 3 activity and Caspase 3 and 9 protein expression

Caspase 3 activity is a central mediator of apoptosis. Caspase 3 activity was measured in IOSE and OVCA433 cells at different time points with different concentration of 4-HPR. Caspase 3 enzyme activity was slightly increased at day 3 in the different concentration groups in OVCA433 cells (Fig. 3a), which correlated with maximal apoptosis and growth inhibition in these cells. However, Caspase 3 and Caspase 9 protein were not changed by4-HPR in either IOSE or OVCA433 cells (Fig. 3b).

Figure 3.

(a) Effect of 4-HPR on Caspase 3 Activity in OVCA433 and IOSE cells. Cells were grown in 96-well plate with absence (control) or presence of 4-HPR in concentrations of 1, 5 and 10 μM for 12, 24, 48 and 72 hr and incubated in Caspase 3 buffer, as described previously in Materials and Methods. The plates were read at 400 nm excitation, 505 nm emission using a fluorescence plate reader immediately after adding Caspase 3 fluorescent substrate conjugate. (b) The Western blot on Caspase 3 and 9 expression shown in the upper right corner were treated with same concentration of 4-HPR for 3 days.

Effect of 4-HPR on mitochondrial permeability transition

MPT changes are associated with mitochondrial mediated apoptosis. To investigate the mechanism of 4-HPR induced apoptosis in ovarian cancer cells, experiments were carried out to investigate the effect of 4-HPR in mitochondrial potential in IOSE and OVCA433 cells. 4-HPR decreased mitochondrial inner-membrane potential, which increased MPT in IOSE and OVCA433 cells (Fig. 4). An inverse relationship in mitochondrial potential correlated in a dose-dependent manner with the increase in apoptosis and growth inhibition by 4-HPR in IOSE and OVCA433 cells (Figs. 1 and 4), suggesting that these activities were mediated by changes in the mitochondrial membrane.

Figure 4.

Effect of 4-HPR on Mitochondrial Permeability Transition in IOSE and OVCA433 cells. Cells were treated with 4-HPR in concentrations of 1, 5 and 10 μM for 3 days and resuspended in 40 nM MitoFluor™ medium. Cells were visualized under the fluorescence microscope at 490 nm excitation, 516 nm emission. A field of 20–30 cells was chosen using a spectrophotometer to measure light intensity.

Expression of apoptosis-associated genes modulated by 4-HPR

The effect of 4-HPR on the expression of the apoptosis-associated genes p53, p21, p16 and Rb were examined along with BRCA genes with Western blot and real-time PCR method. The expression of these genes was detected in both IOSE and OVCA433 cells (Fig. 5). 4-HPR increased expression after 3 days of treatment in a dose-dependent manner in OVCA433 cells (Fig. 5), which correlated with the microarray results, showing an increase in human p53 binding protein mRNA in these cells (Table I).

Figure 5.

Effect of 4-HPR on p53, p21 and other protein levels in IOSE and OVCA433 cells. Nuclear proteins were extracted from cells treated with 1 μM 4-HPR for 3 days. Thirty microgram per lane of nuclear proteins were subjected to SDS-PAGE. The p53, p21, p16 and Rb proteins were identified by blotting with monoclonal antibodies. Immunoreactive bands were visualized using the enhanced chemiluminescence method described in Materials and Methods. The blots were stripped and reblotted to mouse anti-β-actin antibody for assessment of loading in each lane.

4-HPR modulating retinoid receptors and BRCA genes detected by Q-RT-PCR

Microarray data showed that retinoid receptors were modulated by 4-HPR. Some receptors were induced by 4-HPR and others were suppressed by 4-HPR. We verified the effect of 4-HPR on receptor expression and induction by Q-RT-PCR. RARs were not significantly changed by 4-HPR in either cell line (data not shown); however, RXRs were modulated by 4-HPR in IOSE cells. 4-HPR increased RXRα and RXRβ expression and decreased RXRγ expression in IOSE cells (Fig. 6a). The expression of RARs and RXRs were not altered by 4-HPR in cancer cells (Fig. 6a).

Figure 6.

(a) Estimated fluorescence redox ratios are presented from the OVCA433 and IOSE cells with increasing concentrations of 4-HPR. Standard error bars are shown and the value above each bar represents the number of times the treatment group was measured. (b) Comparisons relative redox fluorescence ratio between the control and treatment measurements for both cell lines. The mean ratios for the treatment groups are normalized to the control group's mean ratio.

BRCA1 and BRCA2 gene expressions were decreased by 4-HPR in both IOSE and OVCA433 cells (Fig. 6b). Real time RT-PCR result was consistent with microarray analysis (Table I and Fig. 6b).

Redox ratio changed by 4-HPR detected by optical spectroscopic analysis

As shown in Figure 7a, the IOSE cell line exhibited a highly variable redox related fluorescence ratio compared to the OVCA433 cell line in which the estimated redox increased in a linear fashion. The OVCA433 cells demonstrated a strong sensitivity to 4-HPR treatment and Figure 7a illustrates that dose dependence as a linear increase with a slope of 0.0059/μM 4-HPR (p < 0.001). An increased redox ratio suggests less oxidative metabolism indicating that the cells may be entering quiescence. When considering the relative ratios to untreated cells, as shown in the Figure 7b, the OVCA433 cells had a higher value at each drug dosage. At higher concentrations of 4-HPR the redox related fluorescence ratio increased for the IOSE cells but never reached the level of the OVCA433 cells. This is consistent with the result that the IOSE cell line was variable in response to 4-HPR treatment.

Figure 7.

(a) Estimated fluorescence redox ratios are presented from the OVCA433 and IOSE cells with increasing concentrations of 4-HPR. Standard error bars are shown and the value above each bar represents the number of times the treatment group was measured. (b) Comparisons relative redox fluorescence ratio between the control and treatment measurements for both cell lines. The mean ratios for the treatment groups are normalized to the control group's mean ratio.

Discussion

The goal of chemoprevention is to prevent the progression of precancerous cells to cancer. In practice, to achieve this goal, surrogate endpoint biomarkers are needed, because the biologic endpoint (cancer development) may take many years and may be difficult to detect precisely. Large numbers of patients would have to be entered into such a trial to reach statistically significant conclusions. Using biomarkers that reliably predict progression and differentiation, a study can be completed with fewer patients in a reasonable length of time.37, 38 Unfortunately, only a limited number of potentially useful biomarkers for chemoprevention studies in ovarian cancer have been described.39, 40, 41, 42, 43, 44, 45, 46, 47, 48

An Italian trial that evaluated 4-HPR for prevention of secondary breast cancers demonstrated a decreased incidence of ovarian cancer in women receiving 4-HPR, suggesting that retinoids prevented the development of ovarian cancer.5, 6, 7 After cessation of 4-HPR treatment, new ovarian cancers occurred in the treatment group, suggesting that this prevention was not durable.7 Experimental studies have demonstrated that retinoids can affect human ovarian cancer cell growth by inhibiting proliferation and inducing apoptosis,4, 11, 12, 13 which are thought to be important mechanisms in cancer prevention, as well as in cancer treatment. However, the detailed mechanism of retinoid activity, including 4-HPR, in cancer chemoprevention has remained unclear. We have used DNA microarray to examine genes whose expression is modulated by 4-HPR in immortalized normal ovarian epithelial and ovarian cancer cells. The results are verified by real-time RT-PCR and western blot to further strengthen our conclusions.

p53, A tumor suppressor protein and transcription factor, is deleted or altered in many human cancers. p53 Binds to DNA of cell cycle related genes to induce G1 arrest and allows cells to repair DNA damage or undergo apoptosis if DNA damage is too large for repair. p53 binds to DNA in response to DNA damage, a process that is redox sensitive and is inhibited by oxidizing conditions. Mutation of the p53 protein decreases DNA binding and inhibits activity in induction of apoptosis of genetically altered cells.49 In our study, microarray data demonstrated that 4-HPR increases human p53 binding protein mRNA expression and Rb binding protein in both normal and cancer cells (Table I), and p53/Rb expression were found in both IOSE and ovarian cancer cells. However, Western blot analysis showed p53 expression increased in OVCA433 cells only. In OVCA433 cells, p53 as well as downstream p21 and p16 proteins increase in a dose-dependent manner, suggesting that the cells carry wild-type p53 (Fig. 5). The concentration of 1 μM 4-HPR was chosen in microarray study and western analysis because most of clinical trials used this concentration.5, 6, 7 A concentration of 1 μM is approximately equivalent to the plasma concentration when a dose of 200 mg/day is administered. However, our results suggested that this dose may not be effective for ovarian cancer prevention. Although microarray results showed increasing gene expression, there were few changes at the protein level (Table I and Fig. 5).

In OSE cells, however, immortalized IOSE cells with catalytic subunit of telomerase (hTERT) and a SV40 Large T antigen inactivity of the p53/Rb pathway, the expression of p53 were detected but diminished when the temperature increased to 39°C for 5 and 7 days,50 supporting our findings. In downstream genes, p21 expression showed increased expression with the higher temperature but p16 expression was not changed by temperature.50 The hTERT and a SV40 Large T antigen affected not only expression of p53, but also p16 in OSE cells.50 4-HPR treatment increased p21 expression in IOSE cells but the expression of p53 and p16 were not changed by 4-HPR, further confirming that the large T antigen affects p53 expression and thus is not modulated by4-HPR (Fig. 5). This is a limitation of this cell line. However, primary cell cultures give highly variable results, limiting their usefulness.

p53/Rb mediates the action of 4-HPR on ovarian cancer cells which carry a functional or wild type p53. Up-regulation of p53, p21 and other downstream genes may be one of the mechanisms of retinoid-response in ovarian cancer cells, as is seen in other cell lines.51, 52 A previous study has reported52 that ovarian cancer cell lines that are sensitive to retinoic acid have a higher expression of p53, p27, p21 and p16 compared to the retinoid resistant lines,52 which are concordant with our data. In OVCA433, which is sensitive to 4-HPR, there is an increase in p53, p21 and p16 expression in a dose-dependent manner, which correlates with growth inhibition and apoptosis. In early cancers, wild type p53 may still be present because p53 mutations may be a late event in carcinogenesis, specifically in ovarian cancer, suggesting that one of the major effects of 4-HPR may require an intact p53 gene. This would also suggest that 4-HPR may be more active as a preventive agent rather than a treatment agent if a p53 mutation has occurred.

There is thought to be a role for nuclear retinoid receptors in mediating the retinoid regulation of growth, apoptosis and gene expression.15, 16, 17, 18 RAR and RXR expression have different patterns in different tissues and organs. Retinoid treatment increased certain RAR mRNA levels in several normal and cancer cell lines9, 10, 11, 12, 13, 14 including several ovarian cancer cell lines.4, 11, 12, 19, 20, 53 However, 4-HPR modulation of expression of retinoid receptors in ovarian cancer is still controversial. In this study, 4-HPR increased RXRα and RXRβ expression and decreased RXRγ expression in IOSE cells. Formelli's group reported that RARβ basal level expression and induction by 4-HPR play an important role in mediating4-HPR response in ovarian cancer cells.53 The over expressing RARα clone and RARβ clone increased the tumor-suppressive effect in ovarian tumors.53 They also found that the most sensitive cell lines had RARβ expression and the highest levels of RARα and RARγ expression.4 Moreover, ATRA inhibited ovarian cancer cell growth through RARα and RXRα,54, 55 with RXRα playing a critical role in mediating the growth inhibition in ovarian cancer cells.54 In this study, the RXRs were regulated by 4-HPR in IOSE, but not in ovarian cancer cells, suggesting ovarian carcinogenesis may block some of the receptor expression and induction, and further study on blocking these receptors to evaluate whether the effect of 4-HPR is altered will be forthcoming.

Retinoids, particularly 4-HPR, have been shown to increase aerobic glycolysis by increasing mitochondrial permeability to the coenzymes NADH and FAD, as well as activity of the electron transport chain characterized by an increase in reactive oxygen species and cytochrome oxidase.56, 57 There has been an increased interest in mitochondrial function in both normal and cancer cells; in particular, the mitochondria may be the site of induction of apoptosis by many preventive agents. 4-HPR induces a change in the mitochondrial permeability of the membrane permeability transition,57, 58, 59 which we hypothesize is one of the mechanisms of its suppressive activity in growth inhibition. Permeability of the mitochondrial inner membrane is increased by thiol agents and oxidative stress-inducing agents and is thought to be dependent on the opening of a nonselective pore58; intracellular redox potential increased along with increases in 4-HPR induced growth inhibition and apoptosis.59 A shift towards a more oxidized condition increases membrane permeability, while the opposite occurs with reducing agents. Change in mitochondrial permeability allows cytochrome c to be released into the cytosol and is thought to initiate the Caspase system, ultimately with activation of Caspase 3 activity.58, 60 Caspase 3 activity was investigated in this study because it is a pivotal step in both Caspase 9 and mitochondrial-induced apoptosis. We could not detect any significant change in Caspase genes after treatment with 4-HPR in our microarray and western analysis. Other Caspase genes besides Caspase 3 and 9 may be involved in 4-HPR induced apoptosis in ovarian cells and this requires further investigation.

The redox ratio of a cell defines the level of free radicals divided by the level of antioxidants. It is an indirect measure of the cells metabolic activity and functional ability of the electron transport chain. An increase in the ratio suggests a reduced metabolic activity under normoxic conditions. It is hypothesized that chemopreventive treatments reduce growth rate and induce apoptosis which should result in reduced metabolic activity and an increased redox ratio.32 The availability of free radical versus antioxidants within a cell can be measured by determining the ratio of FAD versus FADH2 or NAD versus NADH. NADH is fluorescent but its oxidized complement NAD is only minimally fluorescent. On the other hand, FADH2 is minimally fluorescent while FAD is fluorescent. Because both fluorophores are oxidized in the electron transport chain, measuring changes in the fluorescence intensity related to FAD serves as an estimate of changes in NAD. Given that the fluorescence from NADH and FAD can be measured noninvasively, a measure approximating the redox ratio can be obtained in vitro on cell cultures without the need for fixation and staining and chemical preparation. This may be an important noninvasive biomarker for the activity of chemopreventive agents in vivo.

Our results suggest the optically approximated redox status of the cells provides evidence of differences between the responses to 4-HPR treatment in the 2 ovarian cell lines. The IOSE cells exhibited a higher redox ratio that did not significantly increase with 4-HPR treatment, while the redox ratio estimated from the OVCA433 cells increased in a dose dependant manner.

The BRCA family of genes regulates apoptosis and often has germ-line mutations in familial breast and ovarian cancer.34, 35 4-HPR down-regulated BRCA1 in the IOSE cells and decreased BRCA2 gene expression in OVCA433 cells detected by both microarray and real-time RT-PCR (Tables I and II). Both BRCA1 and BRCA2 function as tumor suppressor genes in the breast and ovary.34, 35, 61 IOSE cells used in the study are thought to carry a BRCA mutation and originated from a woman with a strong family history of ovarian cancer,34, 61 The ovarian cancer cells OVCA433 had altered expression of BRCA genes, moreover, the expression of BRCA2 transcript in-frame exon 12 deletion (BRCA2Δ12) mRNA was also reported in these cells by Ho and coworkers.61 4-HPR could directly affect mutant BRCA gene to suppress the ovarian carcinogenesis, which is a very important finding in this study and has significant implications for BRCA-related cancer prevention and merits further study.

Our study suggests that 4-HPR may be active in ovarian cancer cells through growth inhibition and apoptosis induction which is mediated by multiple mechanisms. We use both ovarian cancer cells and immortalized normal ovarian epithelium cells in the study. The IOSE cells represent a cell that has been immortalized as a premalignant cell. Although a true premalignant model in the ovary is still unclear, immortalized cells have been used from lung, cervical and other cell lines to mimic premalignant cells.8, 50 In immortalized normal ovarian cells, 4-HPR regulated RXR receptor pathways which increased RXRα and RXRβ expression and decreased RXRγ expression as well as down-regulating the antiapoptotic genes ras and cyclin-dependent kinase and up-regulating the proapoptotic pathways p53 and BCL, although Western blot did not confirm an increase in p53 protein. However, in OVCA433 cells, 4-HPR mediated mitochondrial permeability and induced the proapoptotic p53 and downstream pathway. Early changes that have not undergone p53 mutations, for example, may have a response to 4-HPR with induction of the p53 pathway, while cells that have undergone p53 mutations may not be as responsive to drugs such as 4-HPR. Using biomarkers, such as RXR receptors and p53 up-regulation and downstream protein production as well as fluorescence spectroscopy, to evaluate patient response may be helpful to monitoring 4-HPR or other chemopreventive agents' activity as a preventive agent for ovarian cancer. Hence, this drug merits further study in the ovary, both as a preventive agent and as an agent that might aid in preventing future recurrences.

Natural and synthetic retinoids have been used in many different types of cancers for prevention and treatment. However, the mechanism is still not well studied and the suitable concentration of 4-HPR and/or retinoid has not been determined.62, 63, 64 Negative trials were reported for bladder cancer and cervical cancer.61, 64 A key result from our previous in vitro studies suggested the concentration is important when applying retinoids to different cell types, correlating with different concentrations for various cell populations in vivo, i.e. normal and high risk patients as well as cancer patients need to use specifics concentrations.8, 63, 65, 66, 67 Most of the clinical trials for prevention use the dose of 200 mg/day based on a breast cancer chemoprevention trial.5 Compared with the bladder, cervix and ovary, the breast is fat tissue that stores retinoids; consequently, the local concentration of 4-HPR in the breast is conceivably higher than that in other organs and this local concentration varies from organ to organ. Therefore, it is imperative that the concentration of 4-HPR in different type of cancer needs to be studied carefully before initiating a clinical trial.

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