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Endocrinology, doi:10.1210/en.2002-0180
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Endocrinology Vol. 144, No. 7 2822-2828
Copyright © 2003 by The Endocrine Society

Valproic Acid Increases the Stimulatory Effect of Estrogens on Proliferation of Human Endometrial Adenocarcinoma Cells

Grazia Graziani, Lucio Tentori, Ilaria Portarena, Matteo Vergati and Pierluigi Navarra

Department of Neuroscience (G.G., L.T., I.P., M.V.), University of Rome "Tor Vergata," 00133 Rome, Italy; Institute of Pharmacology (P.N.), Catholic University Medical School, 00168 Rome, Italy

Address all correspondence and requests for reprints to: Grazia Graziani, M.D., Department of Neuroscience, University of Rome "Tor Vergata," Via Montpellier 1, 00133 Rome, Italy. E-mail: graziani{at}uniroma2.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Long-term use of valproic acid (VA), a well-tolerated anticonvulsant agent widely used for treating epilepsia, has been recently shown to inhibit histone deacetylases, which in turn are involved in the regulation of the expression of estrogen receptor {alpha} (ER{alpha}) by suppressing gene transcription. Because estrogens are known to increase cell proliferation of human endometrial tumors, in this study we investigated whether treatment with VA may increase the proliferative response of human endometrial adenocarcinoma cells to 17-ß-estradiol through induction of ER{alpha}. The results clearly show that VA, at concentrations of clinical interest, significantly enhanced the proliferative activity exerted by 17-ß-estradiol in the endometrial adenocarcinoma Ishikawa cell line. Moreover, in these cells treatment with VA resulted in increased ER{alpha} gene expression. Similar effects of VA on cell proliferation were also observed in an ER{alpha}-positive breast cancer cell line (MCF-7). These findings indicate that VA might favor proliferation of estrogen-dependent human tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS HAVE BEEN shown to increase cell proliferation of human endometrial tumors. Conditions characterized by increased exposure to endogenous or exogenous estrogens not opposed by progesterone or synthetic progestins are considered risk factors for endometrial adenocarcinoma (1, 2). Moreover, prolonged activation of the estrogen receptor (ER) {alpha} by the synthetic compound tamoxifen has been associated with the development of endometrial malignancies in patients with breast cancer treated with this agent (1, 3). In fact, although in breast tissue the competitive binding of tamoxifen to ER{alpha} prevents the estrogen/ER{alpha}-mediated breast cancer cell growth, tamoxifen functions as an estrogen agonist on the human endometrial tissue.

In tumor cells ER{alpha} expression is regulated by the level of DNA methylation of the corresponding gene and the level of acetylation of nucleosomal histones (4, 5, 6). Acetylation of nuclear histone proteins is an important regulatory mechanism of gene expression. Histone deacetylases (HDACs) cleave acetyl groups from their binding to histones, thereby allowing interaction of the latter to DNA, and the inhibition of gene transcription (7). Noteworthy, it has been recently demonstrated that the antifungal agent trichostatin A (TSA) acts as HDAC inhibitor and is capable of reactivating ER{alpha} gene expression in ER-negative breast cancer cells (5, 8). On the contrary, the HDAC inhibitor sodium butyrate has been shown to cause a decrease in ER{alpha} gene transcription (9).

Valproic acid (VA), a drug largely used as an anticonvulsant and mood stabilizer, has been recently shown to inhibit HDAC at concentrations of clinical interest (10, 11). Although treatment with VA is well tolerated by patients, it should be noted that long-term use of this agent has been associated with reproductive endocrine disorders such as menstrual irregularity, polycystic ovary, and hyperandrogenism (12, 13, 14, 15, 16). However, the pathogenic mechanism responsible of the endocrine side effects of this agent still needs to be clarified.

In this framework, the present study was set to investigate whether treatments with VA may increase the proliferation of estrogen-dependent malignancies. We used a well-characterized in vitro model, i.e. human endometrial adenocarcinoma cells, Ishikawa cells (IK) (17, 18, 19), and carried out treatments with VA alone or in combination with 17ß-estradiol (E2) and estrogen receptor antagonists. The expression of ER{alpha} was also assessed. Further experiments were conducted in the human breast cancer cell line MCF-7. Our findings clearly show that VA is devoid of intrinsic stimulatory effect on cell growth but strongly potentiates the proliferative activity exerted by E2, most likely via a mechanism involving increased ER{alpha} gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell line and culture conditions
The human endometrial adenocarcinoma IK cell line (17) was kindly provided by Prof. Gigliola Sica (Institute of Histology, Catholic University Medical School, Rome). The human breast cancer MCF-7 cell line was purchased from the American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM (Life Technologies, Inc., Paisley, Scotland) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Inc.), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Flow Laboratories, McLean, VA) at 37 C in a 5% CO2 humidified atmosphere. To remove estrogenic components present in the serum and estrogenic contaminants in phenol red indicator, all experiments were performed in phenol red-free DMEM (Life Technologies, Inc.) containing 1% or 10% charcoal/dextran-stripped FBS (Hyclone Laboratories, Inc., Logan, UT), as specified, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Drug treatment and cell growth evaluation
VA, TSA, sodium butyrate, and E2 were purchased from Sigma (St. Louis, MO). Raloxifene was kindly provided by Dr. Maurizio Civelli (Chiesi Farmaceutici, Parma, Italy). Sodium butyrate and VA were dissolved in sterile water at stock concentrations of 5 M and 500 mM, respectively, whereas TSA, E2, and raloxifene were dissolved in 95% ethanol at the concentration of 3 mM, 1 mM, and 100 mM, respectively. Five days before starting an experiment, cells were cultured in phenol red-free DMEM supplemented with 10% heat-inactivated charcoal/ dextran-stripped FBS. Cells were then harvested with 0.05% EDTA, counted, plated (5 x 106) in T175 flasks and incubated with 1% estrogen-deprived FBS. After 24 h, cells were treated with VA (0.1–2 mM), sodium butyrate (5 mM), or TSA (165 nM) for an additional 48 h. At the end of the incubation period, cells were harvested, counted, plated in T25 flasks (3 x 105), and treated with E2 (0.1–10 nM). In selected experiments, the estrogen antagonist, raloxifene (0.01–1 µM), was added to cell cultures 20 min before E2 treatment.

Cells were then incubated at 37 C for 3 d and growth was evaluated, every 24 h, by counting cells in quadruplicate. Cell viability was determined by trypan blue dye exclusion. Long-term cell survival was assessed by means of colony-forming ability (CFA) assay. Cells (5 x 102) were seeded into 10-cm plastic Petri dishes to allow colony formation. After 15 d, untreated and drug-treated colonies were fixed and stained with rhodamine B basic violet 10 (ICN Biomedicals, Inc., Aurora, OH).

RNA isolation and RT-PCR of ER{alpha}
Total cellular RNA was extracted using the TriPure isolation reagent (Roche, Milan, Italy). RNA integrity was confirmed by electrophoresis on a formaldehyde-containing 1.2% agarose gel after addition of ethidium bromide to the RNA gel-loading buffer. RNA (3 µg) was subjected to reverse transcription (RT) using sensiscript RT kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. PCR was performed by adding cDNA samples (one fourth of the RT reaction volume) to a solution (total volume 50 µl) containing 1x PCR buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin) and 200 µM (each) deoxy (d)CTP, dATP, dGTP, and dTTP. Ten picomoles each of two synthetic ER{alpha}-specific oligonucleotide primers [5'-GCACCCTGAAGTCTCTGGAA-3' (position 1751–1770) and 5'-TGGCTAAAGTGGTGCATGAT-3' (position 2201–2220)] (accession no. NM_000125) were added to the mixture. These primers map in exons 7 and 8, respectively, of the ER{alpha} gene and yielded an amplified product of 470 bp (20). For ERß analysis the primers used were: 5'-CTGTTACTGGTCCAGGTTCA-3' (position 825–844) and 5'-CCAGCTGATCATGTGTACCA-3' (position 1335–1354). These primers are located in exons 2 and 4 of ERß gene (MN_001437). The primers used for amplification of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5'-TGGTATCGTGGAAGGACTCATGAC-3' and 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3') amplified a 190-bp product. Amplification was performed using Taq DNA polymerase (Roche Diagnostics Corp., Indianapolis, IN) for 30 cycles in a DNA thermal cycler (Perkin Elmer Cetus, Norwalk, CT). Each cycle consisted of denaturation at 94 C for 45 sec, annealing at 55 C (ER{alpha} and GAPDH) or 56 C (ERß) for 90 sec and extension at 72 C for 120 sec. Twenty microliters of PCR were electrophoresed through a 2% agarose gel containing ethidium bromide. The signal was quantified by bidimensional densitometry using a scanning (Bio-Rad Laboratories, Inc., Richmond, CA) apparatus (imaging densitometer, GS-670, Molecular Analyst software).

Western blot analysis
Cell lysates were prepared as previously described (21). Eighty micrograms protein per sample were electrophoresed in 8% sodium dodecyl sulfate-polyacrylamide minigels. Then proteins were transferred to nitrocellulose membranes (Schleicher \|[amp ]\| Schuell, Keene, NH). Equal protein loading was visualized by Ponceau S staining. Filters were blocked with blocking buffer (Roche) and incubated overnight with monoclonal antibody directed against human ER{alpha} (F-10) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or actin (Sigma). Histone hyperacetylation was determined using a rabbit polyclonal antibody against acetylated histone H3 (Upstate Biotechnology, Inc., Lake Placid, NY). Immune complexes were visualized using a chemiluminescence kit (Amersham Pharmacia Biotech, Amersham, UK), according to the manufacturer’s instructions. Filters were exposed to X-OMAT AR autoradiographic film (Kodak, Rochester, NY) for 10–45 sec, depending on the intensity of the signal. Bidimensional densitometry of the blots was performed using an imaging densitometer (GS-670, Bio-Rad Laboratories, Inc.).

Flow cytometry analysis of ER{alpha}
Untreated or drug-treated cells were washed with PBS, fixed in ethanol (70%) for 10 min, rehydrated with PBS, and then incubated with the anti-ER{alpha} monoclonal antibody (F-10, 2 µg) at 4 C for 30 min. After addition of a secondary antimouse Ig-fluorescein isothiocyanate (FITC) antibody (DakoCytomation, Milan, Italy), cells were washed twice in PBS containing 0.05% sodium azide and analyzed immediately using a FACSscan flow cytometer (Becton Dickinson and Co., San Jose, CA). Data were collected on 1 x 104 viable cells as determined by forward and side-angle light scatter. For each sample, control staining of cells with secondary antibody only was used to obtain background fluorescence values. The reported percentage values indicate positive fluorescence over background. All data were recorded and analyzed using Cell Quest software (Becton and Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of VA on proliferation of endometrial adenocarcinoma cells induced by estrogen
IK cells were treated with concentrations of VA ranging between 0.12 mM and 2 mM for 48 h and then exposed to 10 nM E2. The results of cell count indicate that VA was devoid of intrinsic stimulatory effect on cell growth and at the concentrations of 1–2 mM induced weak, not significant, antiproliferative effects. Treatment with VA significantly increased the proliferative action of E2 at concentrations of 0.25 mM or more (Fig. 1AGo). In IK cells VA was capable of increasing the level of histone H3 acetylation in a dose-dependent manner (Fig. 1BGo).



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  		Figure 1. Effect of VA on histone acetylation and the growth of endometrial adenocarcinoma cells. A, IK cells were exposed to the indicated concentrations of VA for 48 h and then treated with 10 nM E2, and cell growth was analyzed by cell count. The results are expressed as percentage of cell number increase in tumor cells treated with VA or E2 only or VA+E2 with respect to untreated control 24 h after E2 addition. Values represent the mean (±SE) of the percentages calculated following angular transformation of the percentage increase values evaluated from quadruplicate counts. The results are representative of one of three repeated experiments with comparable results. Statistical analysis was performed according to t test. Differences between groups treated with VA+E2 and the sample treated with E2 only were statistically significant in the case of VA concentrations of 0.25 mM or more (P < 0.05 in the case of 0.25 mM VA and P < 0.01 from 0.5 mM VA onward). B, Histone acetylation was detected by Western blot analysis of whole-cell extracts prepared from IK cells treated with the indicated concentrations of VA, 165 nM TSA, or 5 mM sodium butyrate (NaBu), using a polyclonal antibody against acetylated H3. Equal loading was confirmed by Coomassie blue staining of polyacrylamide gel (not shown).

 
The effect of VA on IK responsiveness to E2 reached the plateau at the concentration of 0.5 mM (Fig. 1AGo); this value is within the therapeutic range (0.35–0.75 mM) and close to the IC50 of HDAC inhibition (0.4 mM) (10). Thus, subsequent experiments were conducted with a fixed concentration of VA (0.5 mM).

In further experiments, IK cells, after pretreatment with VA, were challenged with graded concentrations of E2 (0.1–10 nM), and the proliferative response was monitored in the time frame of 24–72 h. The dose-dependent proliferative effect of E2 was significantly potentiated by pretreatment with VA (Fig. 2AGo). The influence of VA on long-term survival of IK cells exposed to E2 was assessed by CFA assay. The results, illustrated in Fig. 2BGo, showed a significant increase in the number of colonies in the groups receiving E2 after pretreatment with VA with respect to groups treated with E2 only.



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  		Figure 2. VA potentiates the concentration-dependent increase of IK cell proliferation induced by E2. IK cells were exposed to 0.5 mM VA for 48 h and then treated with graded concentrations of E2. Cell growth was analyzed by cell count (A) or CFA assay (B). Statistically significant differences between groups treated with VA+E2 and those treated with E2 only are indicated by asterisks (* and **, P < 0.05 and P < 0.01, respectively). Statistical analysis was performed according to t test. A, The results are expressed as percentage of cell number increase in tumor cells treated with VA or E2 only or with VA+E2 with respect to untreated control 24 h after E2 addition. Histograms represent the mean of the percentages calculated following angular transformation of the percentage increase values evaluated from quadruplicate counts. The results are representative of one of three repeated experiments with comparable results. B, For the CFA assay, untreated or drug-treated cells were seeded in Petri dishes. After 20 d, colonies were stained and counted. Results are expressed as percent increase in the number of colonies formed by treated cells with respect to untreated controls. Histograms represent the mean of two independent experiments (three plates per group of treatment in each experiment).

 
The results of time-course experiments indicate that the enhancing effect of VA was maintained throughout 72 h of culture (Fig. 3AGo). The effect of TSA, another HDAC inhibitor, was also tested in this experimental system at a concentration that has been shown to up-regulate ER{alpha} expression in vitro (8). Similarly to VA, the drug significantly enhanced IK proliferation in response to E2 (Fig. 3BGo). Sodium butyrate at the concentration described to inhibit HDAC (5 mM) (22) profoundly impaired IK cell proliferation (data not shown).



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  		Figure 3. Time course analysis of cell growth in IK tumor cells treated with VA or TSA and then exposed to E2. Cells were treated with 0.5 mM VA (A) or 165 nM TSA (B) for 48 h and then exposed to 10 nM E2. Cell growth was evaluated in terms of number of viable cells at daily intervals during 3 d of culture. Each symbol value represents the mean of cell counts performed in quadruplicate. Bars, SE. The results are representative of one of three repeated experiments with comparable results. Regression line analysis applied to growth curve of cells exposed to VA+ E2 or VA+TSA showed statistically significant differences (P < 0.01) with respect to untreated control or groups treated with E2 at all time points. The enhancing effect of VA was maintained throughout 72 h of culture also when cells were exposed to 0.1 and 1 mM E2 (data not shown).

 
Addition of raloxifene, a well-known ER{alpha} receptor antagonist (3), to cell culture counteracted the increase of cell proliferation induced by VA+E2 in a dose-dependent fashion (Fig. 4Go).



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  		Figure 4. Influence of the ER{alpha} antagonist raloxifene on VA-induced increase of the proliferative response of IK cells to E2. Cells were treated with 0.5 mM VA for 48 h and then to 10 mM E2 or the indicated concentrations of raloxifene (Ralox) ± E2. The results are expressed as percentage of increase in the number of tumor cells with respect to untreated control. Histograms represent the mean of four replicates per group. Statistical analysis was performed according to t test (* and **, P < 0.05 and P < 0.01, respectively). Raloxifene used as single agent induced effects similar to those obtained with raloxifene + VA (data not shown).

 
Influence of VA on ER{alpha} and -ß gene expression in IK cells
Expression of ER{alpha} and -ß genes was assessed by RT-PCR and Western blot analysis in untreated cells or in cells treated with VA. The results of RT-PCR analysis indicate that IK cells express the ER{alpha} transcript under basal conditions, even though at a lesser extent than the breast cancer cell line MCF-7. Treatment with 0.5 mM VA induced a 3-fold increase of ER{alpha} gene expression with respect to untreated control, as assessed by densitometric analysis (Fig. 5AGo). Moreover, exposure of IK cells to VA (0.5 and 1 mM) resulted in an 8- and 14-fold increase of ER{alpha} protein with respect to untreated cells, which expressed barely detectable levels of this receptor (Fig. 5BGo). On the contrary, ERß was not detected in IK cells, in accordance with a previous study (23). Moreover, treatment with VA did not result in ERß induction (data not shown).



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  		Figure 5. Effect of VA treatment on ER{alpha} expression in IK cells. Untreated cells or cells exposed to 0.5 or 1 mM VA for 48 h were analyzed for ER{alpha} expression. The data are representative of one of two independent experiments with similar results. Total RNA and lysate from MCF-7 cells were used as ER{alpha}-positive control. A, RT-PCR analysis of ER{alpha} transcript. The expression of ER{alpha} and GAPDH mRNA were assessed by RT-PCR, as described in Materials and Methods. RNA integrity was confirmed by electrophoresis on a formaldehyde-containing 1.2% agarose gel in the presence of ethidium bromide. B, Western blot analysis of ER{alpha} protein. Eighty micrograms of proteins were loaded in each lane. Actin was probed as protein loading control.

 
Flow cytometry analysis was also carried out to confirm up-regulation of ER{alpha} expression and assess whether VA might increase the percentage of ER{alpha}-positive cells. Untreated cells or cells exposed to 0.5 or 1 mM VA and 165 nM TSA were analyzed for ER{alpha} expression 48 h after treatment. Cells were incubated with an anti-ER{alpha} monoclonal antibody and then with a secondary antimouse Ig-FITC antibody. Cells incubated with antimouse Ig-FITC antibody only were considered as negative control. The data are representative of one of two independent experiments with similar results. The percentages of ER{alpha}-positive cells were as follows: untreated cells, 9%; 0.5 mM VA, 15%; 1 mM VA, 27%; TSA, 18%.

Influence of VA on estrogen responsiveness of breast cancer cells
Breast cancer MCF-7 cells, which express high levels of ER{alpha} (Fig. 5Go), were treated with 0.5 mM VA and then exposed to graded concentrations of E2 (0.1–10 nM). The results indicated that VA was able to enhance the proliferative response of MCF-7 cells to E2 (Fig. 6Go).



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  		Figure 6. Influence of VA on the proliferative response of breast cancer cells to E2. MCF-7 cells were exposed to 0.5 mM VA for 48 h and then treated with graded concentrations of E2. Cell growth was analyzed by cell count. The results are expressed as percentage of cell number increase in tumor cells treated with VA or E2 only or with VA+E2 with respect to untreated control 48 h after E2 addition. Values represent the mean (±SE) of the percentages calculated following angular transformation of the percentage increase values evaluated from quadruplicate counts. Data are representative of one of two repeated experiments with comparable results. Statistical analysis was performed according to t test. Differences between groups treated with VA+E2 and the sample treated with E2 only were statistically significant (P < 0.01) at all doses tested.

 
Also in this cell type, flow cytometry analysis was used to assess whether VA could increase the percentage of ER{alpha}-positive cells. The data are representative of one of two independent experiments with similar results. The percentages of ER{alpha}-positive cells were as follows: untreated cells, 44%; 0.5 mM VA, 53%; 1 mM VA, 66%.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work we showed that treatments with VA potentiate the proliferative activity of E2 on human endometrial adenocarcinoma cells via a mechanism involving increased expression of ER{alpha} receptor. The increased proliferative response of endometrial adenocarcinoma cells to E2 was also observed at concentrations of VA capable of increasing acetylation of histones and corresponding to the therapeutic levels reached in the serum of patients receiving this drug for the treatment of seizures. The effect was antagonized, in a dose-dependent fashion, by the ER{alpha} antagonist raloxifene, suggesting the involvement of this receptor in the VA-mediated enhancement of proliferation induced by E2 in IK cells. Noteworthy, VA was capable of increasing the expression of ER{alpha} both at the RNA and protein level, whereas it did not affect the ERß isoform. Increased responsiveness to E2 after VA treatment was also observed in an ER{alpha} positive breast cancer cell line (MCF-7).

VA has been widely used for the treatment of various epileptic disorders, including absence, simple, and generalized seizures, and it has also been approved for use in depressive bipolar syndromes. The actions of VA at the central nervous system level have been classically associated with its capability to prolong the recovery of voltage-activated Na+ channels from inactivation as well as to increase {gamma}-aminobutyric acid levels in the brain (24). VA also depletes intracellular inositol trisphosphate (25) and modulates prolyl oligopeptidase activity in neurons (26). It is unlikely, however, that the above mechanisms are related to the effects of VA observed in the present study. Instead, the latter effects are strongly reminiscent of the modulation of ER{alpha} gene expression induced by one other HDAC inhibitor, TSA (5, 8), thereby suggesting that HDAC inhibition is involved. Indeed, VA increases ER{alpha} expression and affects IK proliferative response to E2 at concentrations that increase histone acetylation (Fig. 1BGo). Moreover, it is worth to note that the influence of VA on IK response to E2 was comparable to that induced by nontoxic concentrations of the HDAC inhibitor TSA (Fig. 3Go). Actually, TSA was previously reported to potentiate the estrogen responsiveness of MCF-7 cells and increase the level of histone acetylation, suggesting the involvement of HDAC inhibition (27). Another putative HDAC inhibitor, sodium butyrate, was used in the present study at a concentration (5 mM) known to inhibit HDAC activity; however, at this concentration butyrate proved to be highly toxic in our model.

Human HDACs are a fast-growing family of regulatory enzymes, currently approaching the number of 20 isoforms, and each member of the HDAC family plays a distinct role in regulating gene expression (28). Because of such a large variety of isoforms, a few generalizations are possible. These enzymes are widely expressed in adult human tissues as well as normal and tumor mammalian cells in vitro, but others, HDAC 9 and 11 in particular, are expressed in a restricted number of tissues (kidney, heart, brain, skeletal muscle, and testis) (29, 30). Most HDACs have been shown to be part of transcriptional corepressor complexes; interestingly, the regulation of transcription might not be dependent on HDAC enzymatic activity because the sites of HDAC molecules bridging transcription factor complexes are not related to catalytic domains (31, 32, 33, 34).

HDAC inhibitors have been shown to prevent proliferation and induce differentiation of a number of tumor types, such as neuroblastoma, leukemias, and a variety of carcinomas of different tissue origin (35, 36, 37, 38, 39, 40). For this reason, HDAC inhibitors have been recently considered a novel class of therapeutic agents for cancer treatment, and several of these compounds are currently evaluated in phase I/II clinical studies (41, 42, 43, 44, 45, 46, 47). In regard to VA, it has been demonstrated that this agent is capable of inhibiting cell growth in human leukemia cell lines (11, 48, 49). This effect has been observed at concentrations capable of relieving HDAC-dependent transcriptional repression and causing hyperacetylation of histones (10, 11). Furthermore, it has been reported that VA induces differentiation and suppression of malignant phenotype in neuroblastoma or glioma cell lines (50, 51, 52, 53). However, the antiproliferative or differentiating effects of VA appear to be dependent on the tumor model investigated (54). In our experimental model, represented by endometrial adenocarcinoma IK cells, treatment with concentrations of VA up to 0.5 mM did not induce antiproliferative effects, but concentrations of 1–2 mM induced a modest, not significant, growth inhibition.

The extensive clinical experience indicating that VA has limited side effects and the finding of its ability to inhibit HDAC activity has raised great interest in this compound as a possible alternative to other HDAC inhibitors with less favorable toxicity profile for the treatment of neoplastic patients. However, the results of the present study indicate that an accurate selection of tumor types, which might benefit treatment with VA, must be accomplished. In fact, in the case of ER{alpha}-positive malignancies, such as endometrial or, notably, breast cancers, VA might rather enhance tumor growth favoring the proliferative response of cancer cells to estrogen stimulation.


   Acknowledgments
 
The authors thank M. C. Mastrilli for excellent technical assistance.


   Footnotes
 
This work was supported by a grant from the Italian Association for Cancer Research.

Abbreviations: CFA, Colony-forming ability; E2, 17ß-estradiol; ER{alpha}, estrogen receptor {alpha}; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; IK, Ishikawa; RT, reverse transcription; TSA, trichostatin A; VA, valproic acid.

Received December 23, 2002.

Accepted for publication March 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Akhmedkhanov A, Zeleniuch-Jacquotte A, Toniolo P 2001 Role of exogenous and endogenous hormones in endometrial cancer: review of the evidence and research perspectives. Ann NY Acad Sci 943:296–315[Medline]
  2. Cohen CJ, Thomas GM, Hagopian GS 2000 Endometrial cancer. In: Bast RC, Kufe DW, Pollock RE, Weichselbaum RR, Holland JF, Frei III E, eds. Cancer medicine. Hamilton-London: BC Decker Inc.; 1667–1682
  3. MacGregor JI, Jordan VC 1998 Basic guide to the mechanisms of antiestrogen action. Pharmacol Rev 50:151–196[Abstract/Free Full Text]
  4. Ferguson AT, Lapidus RG, Baylin SB, Davidson NE 1995 Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Res 55:2279–2283[Abstract/Free Full Text]
  5. Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG, Davidson NE 2001 Synergistic activation of functional estrogen receptor (ER)-{alpha} by DNA methyltransferase and histone deacetylase inhibition in human ER-{alpha}-negative breast cancer cells. Cancer Res 61:7025–7029[Abstract/Free Full Text]
  6. Cheung E, Zarifyan AS, Kraus WL 2002 Histone H1 represses estrogen receptor {alpha} transcriptional activity by selectively inhibiting receptor-mediated transcription initiation. Mol Cell Biol 22:2463–2471[Abstract/Free Full Text]
  7. Pazin MJ, Kadonaga JT 1997 What’s up and down with histone deacetylation and transcription? Cell 89:325–328[CrossRef][Medline]
  8. Yang X, Ferguson AT, Nass SJ, Phillips DL, Butash KA, Wang SM, Herman JG, Davidson NE 2000 Transcriptional activation of estrogen receptor {alpha} in human breast cancer cells by histone deacetylase inhibition. Cancer Res 60:6890–6894[Abstract/Free Full Text]
  9. DeFazio A, Chiew YE, Donoghue C, Lee CS, Sutherland RL 1992 Effect of sodium butyrate on estrogen receptor and epidermal growth factor receptor gene expression in human breast cancer cell lines. J Biol Chem 267:18008–18012[Abstract/Free Full Text]
  10. Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS 2001 Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem 276:36734–36741[Abstract/Free Full Text]
  11. Gottlicher M, Minucci S, Zhu P, Kramer OH, Schimpf A, Giavara S, Sleeman JP, Lo Coco F, Nervi C, Pelicci PG, Heinzel T 2001 Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J 20:6969–6978[CrossRef][Medline]
  12. Isojarvi JI, Laatikainen TJ, Pakarinen AJ, Juntunen KT, Myllyla VV 1993 Polycystic ovaries and hyperandrogenism in women taking valproate for epilepsy. N Engl J Med 329:1383–1388[Abstract/Free Full Text]
  13. Isojarvi JI, Laatikainen TJ, Knip M, Pakarinen AJ, Juntunen KT, Myllyla VV 1996 Obesity and endocrine disorders in women taking valproate for epilepsy. Ann Neurol 39:579–584[CrossRef][Medline]
  14. Isojarvi JI, Tauboll E, Pakarinen AJ, van Parys J, Rattya J, Harbo HF, Dale PO, Fauser BC, Gjerstad L, Koivunen R, Knip M, Tapanainen JS 2001 Altered ovarian function and cardiovascular risk factors in valproate-treated women. Am J Med 111:290–296[CrossRef][Medline]
  15. Isojarvi JI, Tauboll E, Tapanainen JS, Pakarinen AJ, Laatikainen TJ, Knip M, Myllyla VV 2001 On the association between valproate and polycystic ovary syndrome: a response and an alternative view. Epilepsia 42:305–310[CrossRef][Medline]
  16. TaubLll E, Isojarvi JI, Harbo HF, Pakarinen AJ, Gjerstad L 1999 Long-term valproate treatment induces changes in ovarian morphology and serum sex steroid hormone levels in female Wistar rats. Seizure 8:490–493[CrossRef][Medline]
  17. Nishida M, Kasahara K, Kaneko M, Iwasaki H, Hayashi K 1985 Establishment of a new human endometrial adenocarcinoma cell line, Ishikawa cells, containing estrogen and progesterone receptors. Acta Obstet Gynecol Jpn 37:1103–1111
  18. Gravanis A, Gurpide E 1986 Effects of estradiol on deoxyribonucleic acid polymerase {alpha} activity in the Ishikawa human endometrial adenocarcinoma cell line. J Clin Endocrinol Metab 63:356–359[Abstract/Free Full Text]
  19. Graziani G, Tentori L, Portarena I, Barbarino M, Tringali G, Pozzoli G, Navarra P 2002 CRH inhibits cell growth of human endometrial adenocarcinoma cells via CRH-receptor 1-mediated activation of cAMP-PKA pathway. Endocrinology 143:807–813[Abstract/Free Full Text]
  20. Ponglikitmongkol M, Green S, Chambon P 1988 Genomic organization of the human oestrogen receptor gene. EMBO J 7:3385–3388[Medline]
  21. Tentori L, Portarena I, Bonmassar E, Graziani G 2001 Combined effects of adenovirus-mediated wild-type p53 transduction, temozolomide and poly (ADP-ribose) polymerase inhibitor in mismatch repair deficient and non-proliferating tumor cells. Cell Death Differ 8:457–469[CrossRef][Medline]
  22. Boffa LC, Vidali G, Mann RS, Allfrey VG 1978 Suppression of histone deacetylation in vivo and in vitro by sodium butyrate. J Biol Chem 253:3364–3366[Abstract/Free Full Text]
  23. Barsalou A, Dayan G, Anghel SI, Alaoui-Jamali M, Van de Velde P, Mader S 2002 Growth-stimulatory and transcriptional activation properties of raloxifene in human endometrial Ishikawa cells. Mol Cell Endocrinol 190:65–73[CrossRef][Medline]
  24. McNamara JO 1996 Drugs effective in the therapy of epilepsies. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Goodman GA, eds. Goodman, Gilman’s the pharmacological basis of therapeutics. 9th ed (international edition). McGraw-Hill International; 476–477
  25. O’Donnell T, Rotzinger S, Nakashima TT, Hanstock CC, Ulrich M, Silverstone PH 2000 Chronic lithium and sodium valproate both decrease the concentration of myo-inositol and increase the concentration of inositol monophosphates in rat brain. Brain Res 880:84–91[CrossRef][Medline]
  26. Williams RS, Cheng L, Mudge AW, Harwood AJ 2002 A common mechanism of action for three mood-stabilizing drugs. Nature 417:292–295[CrossRef][Medline]
  27. Ruh MF, Tian S, Cox LK, Ruh TS 1999 The effects of histone acetylation on estrogen responsiveness in MCF-7 cells. Endocrine 11:157–164[CrossRef][Medline]
  28. Gray SG, Ekstrom TJ 2001 The human histone deacetylase family. Exp Cell Res 262:75–83[CrossRef][Medline]
  29. Zhou X, Marks PA, Rifkind RA, Richon VM 2001 Cloning and characterization of a histone deacetylase, HDAC9. Proc Natl Acad Sci USA 98:10572–10577[Abstract/Free Full Text]
  30. Gao L, Cueto MA, Asselbergs F, Atadja P 2002 Cloning and functional characterization of HDAC11, a novel member of the human histone deacetylase family. J Biol Chem 277:25748–25755[Abstract/Free Full Text]
  31. Zhou X, Richon VM, Rifkind RA, Marks PA 2000 Identification of a transcriptional repressor related to the noncatalytic domain of histone deacetylases 4 and 5. Proc Natl Acad Sci USA 97:1056–1061[Abstract/Free Full Text]
  32. Kao HY, Downes M, Ordentlich P, Evans RM 2000 Isolation of a novel histone deacetylase reveals that class I and class II deacetylases promote SMRT-mediated repression. Genes Dev 14:55–66[Abstract/Free Full Text]
  33. Lemercier C, Verdel A, Galloo B, Curtet S, Brocard MP, Khochbin S 2000 mHDA1/HDAC5 histone deacetylase interacts with and represses MEF2A transcriptional activity. J Biol Chem 275:15594–15599[Abstract/Free Full Text]
  34. Fischle W, Dequiedt F, Hendzel MJ, Guenther MG, Lazar MA, Voelter W, Verdin E 2002 Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR. Mol Cell 9:45–57[CrossRef][Medline]
  35. Inokoshi J, Katagiri M, Arima S, Tanaka H, Hayashi M, Kim YB, Furumai R, Yoshida M, Horinouchi S, Omura S 1999 Neuronal differentiation of neuro 2a cells by inhibitors of cell cycle progression, trichostatin A and butyrolactone I. Biochem Biophys Res Commun 256:372–376[CrossRef][Medline]
  36. Saunders N, Dicker A, Popa C, Jones S, Dahler A 1999 Histone deacetylase inhibitors as potential anti-skin cancer agents. Cancer Res 59:399–404[Abstract/Free Full Text]
  37. Huang H, Reed CP, Zhang JS, Shridhar V, Wang L, Smith DI 1999 Carboxypeptidase A3 (CPA3): a novel gene highly induced by histone deacetylase inhibitors during differentiation of prostate epithelial cancer cells. Cancer Res 59:2981–2988[Abstract/Free Full Text]
  38. Sambucetti LC, Fischer DD, Zabludoff S, Kwon PO, Chamberlin H, Trogani N, Xu H, Cohen D 1999 Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects. J Biol Chem 274:34940–34947[Abstract/Free Full Text]
  39. Minucci S, Nervi C, Lo Coco F, Pelicci PG 2001 Histone deacetylases: a common molecular target for differentiation treatment of acute myeloid leukemias? Oncogene 20:3110–3115[CrossRef][Medline]
  40. He LZ, Tolentino T, Grayson P, Zhong S, Warrell Jr RP, Rifkind RA, Marks PA, Richon VM, Pandolfi PP 2001 Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia. J Clin Invest 108:1321–1330[CrossRef][Medline]
  41. Pandolfi PP 2001 Histone deacetylases and transcriptional therapy with their inhibitors. Cancer Chemother Pharmacol 48(Suppl 1):S17–S19
  42. Kramer OH, Gottlicher M, Heinzel T 2001 Histone deacetylase as a therapeutic target. Trends Endocrinol Metab 12:294–300[CrossRef][Medline]
  43. Richon VM, O’Brien JP 2002 Histone deacetylase inhibitors: a new class of potential therapeutic agents for cancer treatment. Clin Cancer Res 8:662–664[Free Full Text]
  44. Sandor V, Bakke S, Robey RW, Kang MH, Blagosklonny MV, Bender J, Brooks R, Piekarz RL, Tucker E, Figg WD, Chan KK, Goldspiel B, Fojo AT, Balcerzak SP, Bates SE 2002 Phase I trial of the histone deacetylase inhibitor, depsipeptide (FR901228, NSC 630176), in patients with refractory neoplasms. Clin Cancer Res 8:718–728[Abstract/Free Full Text]
  45. Melnick A, Licht JD 2002 Histone deacetylases as therapeutic targets in hematologic malignancies. Curr Opin Hematol 9:322–332[CrossRef][Medline]
  46. Vigushin DM, Coombes RC 2002 Histone deacetylase inhibitors in cancer treatment. Anticancer Drugs 13:1–13[CrossRef][Medline]
  47. Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK 2001 Histone deacetylases and cancer: causes and therapies. Nat Rev Cancer 1: 194–202
  48. Tittle TV, Schaumann BA 1992 Effect of antiepileptic drugs on growth of murine lymphoid tumor cells in single-cell culture. Epilepsia 33:729–735[CrossRef][Medline]
  49. Kawagoe R, Kawagoe H, Sano K 2002 Valproic acid induces apoptosis in human leukemia cells by stimulating both caspase-dependent and -independent apoptotic signaling pathways. Leuk Res 26:495–502[CrossRef][Medline]
  50. Cinatl Jr J, Cinatl J, Scholz M, Driever PH, Henrich D, Kabickova H, Vogel JU, Doerr HW, Kornhuber B 1996 Antitumor activity of sodium valproate in cultures of human neuroblastoma cells. Anticancer Drugs 7:766–773[Medline]
  51. Cinatl Jr J, Cinatl J, Driever PH, Kotchetkov R, Pouckova P, Kornhuber B, Schwabe D 1997 Sodium valproate inhibits in vivo growth of human neuroblastoma cells. Anticancer Drugs 8:958–963[Medline]
  52. Cinatl Jr J, Kotchetkov R, Blaheta R, Driever PH, Vogel JU, Cinatl J 2002 Induction of differentiation and suppression of malignant phenotype of human neuroblastoma BE(2)-C cells by valproic acid: enhancement by combination with interferon-{alpha}. Int J Oncol 20:97–106[Medline]
  53. Driever PH, Knupfer MM, Cinatl J, Wolff JE 1999 Valproic acid for the treatment of pediatric malignant glioma. Klin Paediatr 211:323–328
  54. Li R, El-Mallahk RS 2000 A novel evidence of different mechanisms of lithium and valproate neuroprotective action on human SY5Y neuroblastoma cells: caspase-3 dependency. Neurosci Lett 294:147–150[CrossRef][Medline]



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