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[Cancer Research 66, 5394-5402, May 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Potent In vivo Anti–Breast Cancer Activity of IN-2001, a Novel Inhibitor of Histone Deacetylase, in MMTV/c-Neu Mice

Ki E. Joung1, Kyung N. Min1, Jin Y. An1, Dae-Kee Kim1, Gu Kong2 and Y. Yhong Sheen1

1 College of Pharmacy, Ewha Womans University; 2 Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea

Requests for reprints: Yhun Yhong Sheen, College of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-ku, Seoul 120-750, Korea. Phone: 82-2-3277-3028/3025; Fax: 82-2-3277-3028; E-mail: yysheen{at}ewha.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A novel synthetic inhibitor of histone deacetylase (HDAC), 3-(4-dimethylaminophenyl)-N-hydroxy-2-propenamide (IN-2001), was examined for its antitumor activity and for the underlying molecular mechanisms of any such activity. IN-2001 effectively inhibited cellular HDAC activity (IC50, 5.42 nmol/L) in MCF-7 human breast cancer cells. Based on the Western blot analysis, this HDAC inhibitory effect of IN-2001 was confirmed by an increase in histone H4 acetylation from the IN-2001-treated breast cancer cells. IN-2001 suppressed mammary tumor growth in MMTV/c-Neu transgenic mice and also showed higher apoptotic index and lower lymphatic invasion compared with controls. In human breast cancer cells (MCF-7, T47D, MDA-MB-231, and MDA-MB-468), IN-2001 induced cell cycle arrest at G2-M phase through up-regulation of p21WAF1 and p27KIP1 and eventually caused apoptosis. IN-2001-induced apoptosis was caspase dependent and seems mediated through an increase in Bax/Bcl-2 ratio. Taken together, our data indicate that this novel HDAC inhibitor is a promising therapeutic agent against human breast cancer. (Cancer es 2006; 66(10): 5394-402)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer is the most common form of cancers among Korean women (1). Animal models have been useful for studying the biology of breast cancer and for the evaluation of new approaches for treatment and prevention of breast cancer (2). Female rats with mammary tumors induced by carcinogens, such as 12-dimethylbenz(a)anthracene or N-methylnitrosourea (NMU), have been the most commonly used animal models for the study of mammary carcinogenesis and evaluation of therapies (3–5). However, most human breast cancers seem to occur from genetic and environmental factors rather than carcinogens. Recently, transgenic mouse models featuring the v-Ha-ras, c-myc, and activated c-Neu (ErbB2) oncogenes under the control of mouse mammary tumor virus regulatory signals have become available for the study of breast cancer (6–10). In this study, we used the MMTV/c-Neu transgenic mice model. This model has a high incidence of mammary tumor development, which parallels that of human breast cancer, metastasizes by 7 months (6, 11), and does not develop other histologic types of tumors in other tissues, making it highly suitable for the evaluation of intervention strategies to delay/prevent human breast cancer.

Although the precise mechanisms of action have not been elucidated, histone deacetylase (HDAC) inhibitors show various antitumor effects, such as growth inhibition, induction of apoptosis and/or differentiation, and blocking of angiogenesis in vivo (12, 13). They induce histone hyperacetylation associated with transcriptional modulation of a set of genes, which control numerous cellular events. HDAC inhibitors have been reported to elicit up-regulation of the cell cycle inhibitor p21WAF1 in malignant cells, and apoptosis induced by HDAC inhibitor treatment most likely is consistent with the cleavage and activation of the proapoptotic Bcl-2 family member Bid (14, 15). Thus, in recent years, an increasing number of structurally diverse HDAC inhibitors have been identified as an exciting new class of potential anticancer agents.

Preclinical experiments using small-molecule inhibitors of HDACs, such as MS-27-275, suberoylanilide hydroxamic acid (SAHA), and NVP-LAQ824, exhibited efficacy against several human tumor xenografts in athymic mice (16–18). In addition, the natural product HDAC inhibitor trichostatin A was also effective in the NMU-induced rat mammary carcinoma model (19). These inhibitors of HDACs seem to be less toxic among agents used in the treatment of human cancers.

Using the MMTV/c-Neu transgenic mouse model, we investigated the anticancer activity of a novel inhibitor of HDAC, 3-(4-dimethylaminophenyl)-N-hydroxy-2-propenamide (IN-2001; ref. 20). We found that IN-2001 significantly decreased tumor growth; histologically, the MMTV/c-Neu transgenic mice showed higher apoptotic index compared with untreated controls. In addition, p21WAF1 levels in mammary tumor and uterine tissues significantly increased after IN-2001 treatment. In human breast cancer cell lines, IN-2001 showed potent antiproliferative effect, which was manifested by cell cycle arrest at G2-M phase through up-regulation of p21WAF1 and p27KIP1. IN-2001-induced apoptosis seems to be through caspase activation and increase in Bax/Bcl-2 ratio. Our studies indicate that this novel HDAC inhibitor is a promising therapeutic agent against human breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. HDAC inhibitors, such as trichostatin A, IN-2001, SAHA, and MVP-LAQ, were kindly provided by D-K.K. HC toxin was obtained from Sigma Chemical Co. (St. Louis, MO). Apo-One homogenous caspase-3/7 assay kit and HDAC fluorescent activity assay kit were from Promega Corp. (Madison, WI) and Biomol (Plymouth Meeting, PA), respectively.

Cell culture. MCF-7, T47D, and MDA-MB-231 cells were obtained from the Korean Cell Line Bank. MDA-MB-468 cells were obtained from the American Type Culture Collection (Rockville, MD). MCF-7 cells were maintained in MEM supplemented with fetal bovine serum, insulin, and penicillin-streptomycin. T47D, MDA-MB-231, and MDA-MB-468 cells were maintained in RPMI 1640 supplemented with fetal bovine serum and penicillin-streptomycin. Cells were routinely maintained at 37°C in 5% CO2.

Cell proliferation assay. Cells were plated in 96-well plates at a density of 104 per well. After 1 day in culture, the cells were treated with HDAC inhibitors for 72 hours. The number of cells was measured based on the modified sulforhodamine B (SRB) assay of Soto et al. (21). Cells were fixed by treating with cold 10% TCA and incubating at 4°C for 30 minutes and then were washed five times with tap water and let dry. TCA-fixed cells were stained for 30 minutes with 0.4% (w/v) SRB dissolved in 1% acetic acid, after which wells were washed with tap water and air dried. Bound dye was solubilized with 10 mmol/L Tris (pH 10.5) in a shaker for 30 minutes. Finally, absorbances were measured at 570 nm using an ELISA reader (Bio-Rad, Hercules, CA).

Cell cycle analysis. Cells were plated in 60-mm2 dishes and exposed to HDAC inhibitors for 24 or 48 hours. Treated cells were detached from plates using trypsin-EDTA and fixed with 70% ethanol. After centrifugation, cells were treated with RNase A (10 µg/mL) for 20 minutes at 37°C and stained with propidium iodide (2 µg/mL) for 30 minutes at 37°C in the dark. Flow cytometric analysis was done on a FACScalibur instrument (Becton Dickinson, San Diego, CA) and the data were analyzed using the ModFit software program. Percentages of cell populations distributed in the various phases of the cell cycle (G1, S, or G2-M) were calculated and represented using DNA content histograms.

HDAC inhibition assay. HDAC inhibition activity of HDAC inhibitors was assayed by fluorometric method using a fluorescent substrate of {epsilon}-acetyl lysine (HDAC fluorescent activity assay kit). The nuclear extracts of MCF-7 and MDA-MB-231 cells were prepared in buffer [20 mmol/L Tris (pH 7.6), 10 mmol/L KCl, 0.2 mmol/L EDTA, 20% glycerol, 1.5 mmol/L MgCl2, 2 mmol/L DTT, 0.4 mol/L KCl, 0.4 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L NaVO4, and 2 µg/mL each of leupeptin, pepstatin, and aprotinin]. Nuclear extract was incubated with HDAC inhibitors and {epsilon}-acetyl lysine for 10 minutes at 25°C. The fluorescence was monitored at an excitation wavelength of 360 nm and an emission wavelength of 530 nm using fluorescence reader (FL600, Bio-Tek, Winooski, VT).

Caspase-3/7 activity. Caspase-3/7 activity was assayed by fluorometric methods. Cells were plated in 96-well plates and then treated with HDAC inhibitors. After 24 hours, cells were mixed with Apo-One homogenous cell lysis/activity buffer containing the profluorescent substrate Z-DEVD-rhodamine 110 and incubated for 30 minutes at room temperature. On sequential cleavage and removal of the DEVD peptide by caspase-3/7 activity and excitation, the rhodamine 110–leaving group becomes intensively fluorescent. The release of light from rhodamine 110 was monitored at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescence microplate reader (FL600). In the same cells, protein concentrations were determined using fluorescamine. Results were expressed as arbitrary absorbance units per milligram protein.

Western blot analysis. HDAC inhibitor–treated cells or tissues were homogenized in lysis buffer (Pro-prep protein extraction solution, iNtRON, Seoul, Korea) on ice for 10 to 20 minutes. Lysates were centrifuged at 14,000 x g for 5 minutes at 4°C, divided into aliquots, and stored at –80°C. Protein concentration was determined with the Micro-BCA protein assay kit (Pierce, Rockford, IL). For the preparation of cellular histone extract, HDAC inhibitor–treated cells were harvested in ice-cold PBS, centrifuged at 1,000 rpm for 5 minutes, resuspended in 1 mL histone lysis buffer [8.6% sucrose, 1% Triton X-100, 50 mmol/L sodium bisulfite, 10 mmol/L Tris-HCl (pH 6.5), and 10 mmol/L MgCl2], and Dounce homogenized. Cell lysates were centrifuged at 700 rpm for 5 minutes. The nuclear pellet then was washed thrice with lysis buffer and once with 10 mmol/L Tris-HCl (pH 7.4) containing 13 mmol/L EDTA. The acid (H2SO4)–soluble supernatant was precipitated with 10 volumes of cold acetone overnight and histones were collected by centrifugation. The pellet containing histones was dissolved in 50 µL H2O. Lysates containing 30 to 50 µg total protein were separated by electrophoresis on 10% to 15% polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride transfer membranes (Hybond-P, Amersham, Uppsala, Sweden) at 200 mV for 2 to 3 hours. Membranes were blocked with 3% dry milk in PBST (PBS plus 0.1% Tween 20) overnight at 4°C and incubated with specific primary antibodies for 1 to 2 hours at room temperature. Membranes were washed, incubated with secondary antibodies conjugated to horseradish peroxidase for 2 hours at room temperature, washed again, and air dried before enhanced chemiluminescence detection (ECL Plus, Amersham). Membranes were stripped in mild antibody stripping solution (Re-Blot Plus, Chemicon International, Temecula, CA) at room temperature for 30 minutes, washed in PBST, and reprobed.

Tumor growth study in MMTV/c-Neu transgenic mice. ErbB2-overexpressing female FVB/N-TgN (MMTV/c-Neu) mice featuring a FVB background were purchased from The Jackson Laboratory (Bar Harbor, ME) and raised on a fat-controlled diet (5L79; PMI Nutrition International, LLC, St. Louis, MO) in the specific pathogen-free room. Mice were maintained at a room temperature of 21°C with 50% humidity and a 12-hour light/dark cycle. At age 41 weeks, tumor-bearing mice were divided into four separate groups and treated by i.p. injection with either vehicle (saline) or HDAC inhibitors. Following chemical treatment, mice were given a physical examination and their food consumptions and body weight changes were monitored for 4 weeks (observation periods). Afterward, mice were sacrificed and tumors were dissected and measured.

Statistical analysis. All numerical data were expressed as the average of the values obtained ±SD. For all experiments, significance was determined by conducting a paired Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IN-2001 showed potent inhibition of mammary tumor growth in MMTV/c-Neu transgenic mice. IN-2001 (15 mg/kg)–treated mice showed pronounced antitumor activity compared with untreated control mice (Fig. 1A ). The mammary tumor weight decreased by 60% with 15 mg/kg IN-2001 and by 30% with 120 mg/kg SAHA, but 60 mg/kg SAHA did not show any antitumor activity. This indicates that IN-2001 has at least eight times more potent antitumor capacity than SAHA. Because many HDAC inhibitors are known to have toxicity, which limits the dose, we have observed changes in body weight of mice over the experimental period and did necropsy examination. As shown in Fig. 1B, there was no significant difference in body weight changes between control and HDAC inhibitor–treated group and no significant difference in histologic findings of liver, kidney, lung, and uterus from both control and HDAC inhibitor–treated mice (data not shown).


Figure 1
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  		Figure 1. Relative in vivo antitumor activities of IN-2001 and SAHA in MMTV/c-Neu transgenic mice. A, tumor-bearing MMTV/c-Neu transgenic mice were randomly divided into four groups with five mice per group. Indicated concentrations of HDAC inhibitors and vehicle (saline) were given i.p. to mice five times weekly for 1 week. After 4 weeks of observation, mice were sacrificed and the average weight of mammary tumors per mouse was determined. B, animals were treated as in (A) and were monitored for body weight changes during the treatment periods (age 41-42 weeks) and observation periods (age 42-46 weeks). C, tumor-bearing MMTV/c-Neu mice were treated with vehicle (saline) or 30 mg/kg IN-2001 by i.p. injection for the indicated times (0, 24, 48, and 72 hours). Protein extracts were prepared from mammary tumors and uterine tissues and were separated by SDS-PAGE. Blots were probed with p21WAF1 specific antibody. Actin served as a loading control.

 
In addition, mammary tumor tissues from IN-2001-treated mice showed higher apoptotic index and lower lymphatic invasion compared with control mice (Table 1 ). High dose (120 mg/kg) of SAHA showed the same histopathologic findings as IN-2001. To explore the effect of IN-2001 on cell cycle regulation in conjunction with the antitumor effect in vivo, we examined the expression of p21WAF1 in the MMTV/c-Neu transgenic mice. IN-2001 (30 mg/kg) was given to tumor-bearing MMTV/c-Neu transgenic mice by i.p. injection for various times (0-72 hours). After sacrificing, the protein was extracted from both mammary tumors and uterus for Western blot analysis. As shown in Fig. 1C, p21WAF1 expression significantly increased in mammary tumors and uterine tissues of 30 mg/kg IN-2001-treated mice. In mammary tumors, induction of p21WAF1 level started at 24 hours after treatment and peaked at 72 hours after treatment. In the uterus, p21WAF1 began to increase at 24 hours after treatment and reached maximum at 48 hours after treatment. These results suggest that IN-2001 caused a time-dependent increase in p21WAF1 and this may induce cell cycle arrest and its antitumor activity.


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  		Table 1. Histopathology of breast cancer developed in MMTV/c-Neu transgenic mice treated with various HDAC inhibitors

 
IN-2001 induced acetylation of histone. We investigated the effect of IN-2001 and other HDAC inhibitors on histone acetylation in human breast cancer cells. After MCF-7 and MDA-MB-468 cells were treated with 1 µmol/L HDAC inhibitors, such as IN-2001, trichostatin A, SAHA, NVP-LAQ, and HC toxin, cells were lysed. Acetylated histone H4 proteins from these MCF-7 and MDA-MB-468 cells were analyzed by Western blot analysis using antibody against acetylated histone H4. As shown in Fig. 2 , IN-2001 as well as trichostatin A, NVP-LAQ, and HC toxin increased the acetylation of histone H4 in human breast cancer cells. We also examined the ability of IN-2001 to inhibit HDAC activity. As shown in Table 2 , IN-2001 inhibited HDAC activities in the nuclear extracts from MCF-7 cells (IC50, 5.42 nmol/L) and MDA-MB-231 cells (IC50, 227.09 nmol/L). In addition, IN-2001 was as effective as trichostatin A to inhibit HDAC activity in MCF-7 human breast cancer cells. These results confirm that the novel synthetic compound IN-2001 is a HDAC inhibitor.


Figure 2
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  		Figure 2. Accumulation of acetylated histone H4 by IN-2001 and other HDAC inhibitors. MCF-7 (A) and MDA-MB-468 (B) cells were treated with vehicle (0.1% DMSO) or 1 µmol/L HDAC inhibitors for 6 hours. Cells were harvested and histones were prepared as described in Materials and Methods. Histone acetylation was detected by Western blot analysis using antibodies against acetylated H4. Bottom, Coomassie blue–stained gel indicates that the equal amount of histones was loaded. Ac-H4, acetylated histone H4; TSA, trichostatin A.

 

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  		Table 2. In vitro HDAC inhibitory activity of HDAC inhibitors, IN-2001, and trichostatin A

 
IN-2001 inhibited the growth of human breast cancer cells. Human breast cancer cells (MCF-7, T47D, MDA-MB-231, and MDA-MB-468 cells) were treated with IN-2001, other HDAC inhibitors (0.001-10 µmol/L), and their vehicle (0.1% DMSO) for 72 hours and the resulting cell proliferations were measured using the SRB assay. As shown in Fig. 3 , IN-2001 exerted a potent antiproliferative effect in a dose-dependent manner. IN-2001 (IC50, 0.132 µmol/L, T47D) possessed a more potent antiproliferative activity than SAHA (IC50, 1.877 µmol/L, T47D), which is currently undergoing a clinical trial. The other HDAC inhibitors tested, trichostatin A, NVP-LAQ, and HC toxin, are too toxic to be useful in therapy. These data suggest that IN-2001 can serve as a potential potent antitumor drug. Results shown in Fig. 3 indicate that the degree of the antiproliferative effects of different HDAC inhibitors vary depending on the human breast cancer cell lines under study. Our data further show that estrogen receptor (ER)–positive breast cancer cells, such as MCF-7 and T47D cells, are apparently more susceptible to HDAC inhibitors than ER-negative breast cancer cells, such as MDA-MB-231 and MDA-MB-468 cells (Fig. 3).


Figure 3
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  		Figure 3. Inhibition of cell proliferation by IN-2001 and other HDAC inhibitors. MCF-7 (A), T47D (B), MDA-MB-231 (C), and MDA-MB-468 (D) cells were treated with vehicle (0.1% DMSO) or indicated concentrations of HDAC inhibitors for 72 hours. Numbers of cells were determined by SRB assay. All experiments were repeated at least thrice. Columns, mean; bars, SD.

 
IN-2001 induced cell cycle arrest and apoptosis. To elucidate the molecular mechanism of the antitumor activity of IN-2001, we analyzed the cell cycle distribution in several breast cancer cells treated with IN-2001 and other HDAC inhibitors. Cells were treated with 1 µmol/L HDAC inhibitors for 24 or 48 hours and then analyzed for cell cycle distribution by flow cytometric analysis after PI staining. As shown in Fig. 4 , IN-2001 as well as other HDAC inhibitors induced cell cycle arrest at G2-M phase and subsequently increased apoptosis in these cells, such as MCF-7, T47D, MDA-MB-231, and MDA-MB-468 cells. It seems that the growth-inhibitory effect of IN-2001 on human breast cancer cells is associated with the accumulation of cells at G2-M phase and subsequent apoptosis.


Figure 4
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  		Figure 4. Cell cycle changes by IN-2001 and other HDAC inhibitors. MCF-7 (A), T47D (B), MDA-MB-231 (C), and MDA-MB-468 (D) cells were treated with vehicle (0.1% DMSO) or indicated concentrations of HDAC inhibitors for 24 or 48 hours. Cell cycle profiles were analyzed by flow cytometry and the percentages of the cells distributed in the G0-G1, S, and G2-M phases were calculated by the ModFit program.

 
Effect of IN-2001 on cell cycle regulatory proteins. To identify the molecules involved in IN-2001-induced cell cycle arrest, we examined the effects of IN-2001 on p21WAF1 and p27KIP1, which are involved in the control of cell cycle and apoptosis. Whole-cell extracts were prepared after 24-hour treatment with 1 µmol/L IN-2001 or other HDAC inhibitors, and p21WAF1 and p27KIP1 were measured by Western blot analysis. As shown in Fig. 5A , IN-2001 as well as other HDAC inhibitors, such as trichostatin A, SAHA, NVP-LAQ, and HC toxin, increased the expressions of p21WAF1 and p27KIP1 in both ER-positive and ER-negative human breast cancer cells. In ER-positive human breast cancer MCF-7 and T47D cells, the magnitude of increase of p21WAF1 by HDAC inhibitors was much greater than that of p27KIP1, whereas in ER-negative human breast cancer MDA-MB-231 cells, the extent of increase in p21WAF1 was similar to that of p27KIP1 by IN-2001 as well as other HDAC inhibitors. We also investigated the effect of IN-2001 on the expression of cyclin B1 known to be involved in the G2-M checkpoint. As shown in Fig. 5B, cyclin B1 expression showed no remarkable change until 24 hours of IN-2001 treatment. These results suggest that up-regulation of cyclin-dependent kinase (CDK) inhibitors induced by IN-2001 as well as other HDAC inhibitors may lead to cell cycle arrest, ultimately resulting in growth inhibition. With regard to the cell cycle arrest, the main target of IN-2001 as well as other HDAC inhibitors seems to be cell type specific.


Figure 5
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  		Figure 5. Effect of IN-2001 and other HDAC inhibitors on the expression of cell cycle regulatory proteins. Human breast cancer cells were treated with vehicle (0.1% DMSO) or 1 µmol/L HDAC inhibitors for 24 hours. Protein extracts were prepared and 50 µg protein was separated by SDS-PAGE. Blots were probed with the indicated antibodies with actin serving as a loading control.

 
IN-2001 induced caspase-dependent apoptosis. To investigate whether the apoptotic effect of IN-2001 was caspase dependent, we examined the effect of IN-2001 on the activation of the apoptotic process in human breast cancer cells by measuring the activity of caspase-3/7, a main caspase in most mammalian cells. Cells were treated with 1 µmol/L IN-2001 for 24 hours and caspase-3/7 activity was then determined using nonfluorescent Z-DEVD-rhodamine 110 as a substrate. As shown in Fig. 6A , IN-2001 induced an increase in caspase-3/7 activity in human breast cancer cells. This increase in caspase-3/7 activity was more apparent in ER-positive human breast cancer MCF-7 and T47D cells than in ER-negative human breast cancer MDA-MB-231 and MDA-MB-468 cells. Especially, in MDA-MB-468 cells, IN-2001 induced caspase activation was much less potent than in MCF-7, T47D, and MDA-MB-231 cells.


Figure 6
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  		Figure 6. Induction of caspase-mediated apoptosis by IN-2001 and other HDAC inhibitors. Human breast cancer cells were treated with vehicle (0.1% DMSO) or 1 µmol/L HDAC inhibitors for 24 hours. A, induction of caspase-3/7 activity by HDAC inhibitors. Caspase-3/7 activity was determined using the profluorescent substrate Z-DEVD-rhodamine 110. The release of light by fluorescent rhodamine 110 was monitored at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Caspase activity was normalized to protein content and expressed as fold induction compared with the control group. Columns, mean; bars, SD. Western blot analysis of caspase-3 (B) and apoptosis-related proteins (C). Protein extracts were prepared and 50 µg protein was separated by SDS-PAGE. Blots were probed with the indicated antibodies with actin serving as a loading control. For quantification, the band intensity of Bax and Bcl-2 was normalized to that of actin. Data were expressed as percent of control.

 
We also confirmed the effect of IN-2001 treatment on caspase activation by Western blot analysis. As shown in Fig. 6B, IN-2001 treatment resulted in the conversion of the proenzyme form of caspase-3 (p32) to the catalytically active effector proteases p17 and p20, whereas no effector proteases, p17 or p20, was shown in untreated MDA-MB-231 cells.

We further examined cytochrome c release in response to IN-2001 as an upstream signal of caspase-3/7. As shown in Fig. 6C, cytochrome c release varied depending on the cell line and/or the HDAC inhibitors used. IN-2001 increased cytochrome c release in MCF-7 cells but not in T47D or MDA-MB-231 cells. SAHA increased cytochrome c release in all cell lines that we have tested.

Bcl-2 and its dominant inhibitor Bax are key regulators of cell proliferation and apoptosis. Overexpression of Bcl-2 and Bcl-XL enhances cell survival by suppressing apoptosis, but overexpression of Bax accelerates cell death. Therefore, we investigated the expression of Bcl-2 and its dominant inhibitor Bax after treatment with IN-2001 for 24 hours. As shown in Fig. 6C, IN-2001 significantly increased Bax concentrations in T47D and MDA-MB-231 cells. However, IN-2001 slightly decreased the level of Bcl-2 expression in MDA-MB-231 cells and did not change the Bcl-2 expression in T47D cells. When the relative ratio of Bax/Bcl-2 was calculated, IN-2001 and other HDAC inhibitors showed significant induction of the Bax/Bcl-2 ratio in MDA-MB-231 cells as well as in T47D cells, which was less than in MDA-MB-231 cells. However, surprisingly, in MCF-7 cells, IN-2001 and all other HDAC inhibitors tested did not change the Bax/Bcl2 ratio, suggesting that HDAC inhibitor–induced apoptosis in MCF-7 cells is independent of the Bcl-2/Bax pathway.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows the efficacy of a novel inhibitor of HDAC, IN-2001, as a suppressor of breast cancer in MMTV/c-Neu transgenic mice. Recently, the in vivo anticancer activity of hyperacetylating agents has been abundantly documented in animal models (16, 19, 22). However, one has to be cautious in extrapolating the results from studies in animal models to human disease. In this study, we used a combined system of in vivo studies with MMTV/c-Neu transgenic mice and in vitro studies with a variety of human breast cancer cell lines. In our study, IN-2001 seems to be eight times as potent in its antitumor activity as SAHA, which is currently being evaluated in a clinical trial (Fig. 1A). Based on the known role of p21WAF1 as a negative regulator of cell cycle progression, our observation that IN-2001 induced p21WAF1 expression both in vivo and in vitro breast cancer models (Figs. 1C and 5) provides a clue to a possible mechanism for the antitumor activity of this HDAC inhibitor. HDAC inhibitors up-regulate the expression of p21WAF1 and increased p21WAF1 triggers cell cycle arrest, resulting in inhibition of cell proliferation.

In human breast cancer cells, IN-2001 elicited a growth-inhibitory effect via the inhibition of HDAC (Fig. 2; Table 2). cDNA microarray analyses indicate that 4% to 12% of genes are affected by HDAC inhibitors (23–25) and that the altered genes are apparently involved in encoding tumor-associated proteins that mediate cell cycle progression, DNA synthesis, and apoptosis. In agreement with previous data using HDAC inhibitors, such as trichostatin A, SAHA, and NVP-LAQ, in our studies, the antiproliferative effect of IN-2001 was also manifested by cell cycle arrest and apoptosis (Figs. 3 and 4).

Our data show that the induction of p21WAF1 by IN-2001 tends to be greater in cell lines susceptible to the antiproliferative effect of IN-2001. For example, ER-positive breast cancer cell lines, such as MCF-7 and T47D, showed greater induction of p21WAF1 than the ER-negative breast cancer cell line, MDA-MB-231 (Fig. 5A). These results agree with other reports indicating that the accumulation of p21WAF1 was greater and faster in the cell lines sensitive to MS-27-275 (16). This suggests that the induction of CDK inhibitors (p21WAF1 and p27KIP1) through histone acetylation plays a crucial role in the cell cycle arrest at G2-M phase after IN-2001 treatment. However, no change of cyclin B1 expression was observed after treatment of IN-2001 for 24 hours (Fig. 5B). Our findings are consistent with the report by Archer et al. (26) that in human colon adenocarcinoma HT-29 cells NaBu did not change the expression of cyclin B1 after 24-hour treatment. In contrast to early induction of p21WAF1, the decrease in cyclin B1 expression occurred in a delayed fashion (26).

IN-2001 also exhibited potent antiproliferative effects in vitro against human breast cancer cell lines (Fig. 3). Interestingly, ER-positive human breast cancer cell lines (MCF-7 and T47D) were more sensitive to IN-2001 (mean ± SD IC50, 0.210 ± 0.110) than ER-negative cell lines (MDA-MB-231 and MDA-MB-468; mean ± SD IC50, 1.242 ± 0.928); the mechanism underlying this difference is not clear. Previous studies with trichostatin A (27) or sodium butyrate (28), on MCF-7 or MDA-MB-231 cells, also reported a higher sensitivity of ER-positive breast cancer cells than ER-negative breast cancer cells. This differential response was associated neither with a modification of drug efflux via the multidrug resistance system nor with a global modification of histone acetyltransferase/HDAC activities (27, 28). It was suggested that p21WAF1 gene expression, in concert with ER{alpha}, might play a role in this differential response of breast cancer cells to hyperacetylating agents. In ER-positive breast cancer cells, the p21WAF1 gene was more sensitive to trichostatin A regulation and was expressed at higher levels. Moreover, the reexpression of ER{alpha} in ER-negative breast cancer cells increased both growth-inhibitory activity of trichostatin A and p21WAF1 protein accumulation with trichostatin A treatment (27). In the current study, we found that HDAC inhibitors, including IN-2001, increased the expression of CDK inhibitors, such as p21WAF1 and p27KIP1, and that the induction of p21WAF1 was highly correlated with the growth-inhibitory effects of HDAC inhibitors. However, we could not find a strong correlation between ER level and p21WAF1 expression in the HDAC inhibitor–treated cells. Further studies are needed to clarify the implications of ER status in the regulation of p21WAF1 by IN-2001.

Another possible mechanism of growth inhibition could relate to apoptosis. HDAC inhibitors have been reported to induce apoptosis in various human cancer cell lines, such as MCF-7 breast cancer (29), small lung cancer (30), HOS osteosarcoma (31), and HCT116 colon cancer (32). Richon et al. (17) and Rosato et al. (33) have reported that HDAC inhibitor–induced apoptosis involves two different mechanisms, an intrinsic pathway and an extrinsic pathway, and which pathway operates depends on the cell type and class of HDAC inhibitor used. Caspases are key mediators of apoptosis (34–36), and among the 10 distinct caspases, caspase-3 is thought to be the "executioner" farthest downstream in the apoptotic pathway. Caspase-3 is frequently activated by many death signals and cleaves a wide range of cellular proteins with important functions (36, 37). In our study, IN-2001 treatment significantly increased caspase-3/7 activity (Fig. 6A). This was further supported by a Western blot analysis showing the conversion of the proenzyme form caspase-3 (p32) into the catalytically active effector proteins (p17 and p20; Fig. 6B). In addition, in T47D and MDA-MB-231 cells, the expressions of Bax proteins, one of the key components of apoptotic pathways, changed in response to IN-2001. Consistent with other HDAC inhibitors, IN-2001 increased Bax level and eventually increased Bax/Bcl-2 ratio. However, the ratio of these apoptosis regulatory proteins appeared to remain intact in HDAC inhibitor–treated MCF-7 cells (Fig. 6C).

Our data showed the antitumor activity of IN-2001 in both p53 wild-type cells (MCF-7) and p53 mutant cells (T47D, MDA-MB-231, and MDA-MB-468). Thus, IN-2001-induced cell cycle arrest and/or apoptosis seem to proceed through a p53-independent pathway. Such p53-independent cell cycle arrest was also observed with other kinds of HDAC inhibitors, such as trichostatin A, HC toxin, and NVP-LAQ, in agreement with previous reports (18, 38).

In summary, we found that IN-2001 shows potent antitumor activity in vitro as well as in vivo against breast cancer models. Furthermore, IN-2001 induces cell cycle arrest and eventually caspase-dependent apoptosis in human breast cancer cells possibly by modulating p21WAF1, p27KIP1, and Bcl-2/Bax protein. These findings suggest that IN-2001, a novel inhibitor of HDAC, is a promising new therapeutic agent for human breast cancer.


   Acknowledgments
 
Grant support: Korea Food and Drug Administration grant NTP482.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: K.E. Joung and K.N. Min contributed equally to this work.

Received 10/24/05. Revised 2/ 5/06. Accepted 3/22/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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