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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Histone deacetylase (HDAC) is an attractive target for cancer therapy because it plays a key role in gene expression and carcinogenesis. N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) is a novel synthetic HDAC inhibitor (HDACI) that shows better pharmacological properties than a known HDACI present in the human fibrosarcoma cell: suberoylanilide hydroxamic acid (SAHA). Here, we investigate the anti-cancer activity of HNHA against breast cancer both in vitro and in vivo. HNHA arrested the cell cycle at the G1/S phase via p21 induction, which led to profound inhibition of cancer cell growth in vitro. In addition, HNHA-treated cells showed markedly decreased levels of VEGF and HIF-1α than SAHA and fumagillin (FUMA) when accompanied by increased histone acetylation. HNHA significantly inhibited tumor growth in an in vivo mouse xenograft model. HNHA-treated mice survived significantly longer than SAHA- and FUMA-treated mice. Dynamic MRI showed significantly decreased blood flow in the HNHA-treated mice, implying that HNHA inhibits tumor neovascularization. This finding was accompanied by marked reductions of proangiogenic factors and significant induction of angiogenesis inhibitors in tumor tissues. We have shown that HNHA is an effective anti-tumor agent in breast cancer cells in vitro and in breast cancer xenografts in vivo. Collectively, these findings indicate that HNHA may be a potent anti-cancer agent against breast cancer due to its multi-faceted inhibition of HDAC activity, as well as anti-angiogenesis activity. (Cancer Sci 2011; 102: 343–350)

Cancer cells contain multiple defects in genes that regulate activities such as tumor suppression, cell differentiation, DNA repair and cell cycle progression. These genes can be silenced by aberrant epigenetic transcriptional repression.(1–3) Recent studies have shown that inappropriate histone acetylation alters chromatin structure and results in the dysregulation of cell growth and death, leading to neoplastic transformation.(2–4) Histone deacetylase (HDAC) plays a key role in gene expression and carcinogenesis, and is overexpressed in several types of tumor cells.(5,6) Numerous studies have shown that the expression of tumor suppressors, such as p53 and p21, is downregulated in cells that overexpress HDAC.(7–9) Furthermore, tumor activators such as hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) are upregulated in such cells.(10) Accordingly, epigenetics has been recognized as a new target for treating cancer. In fact, HDAC inhibitors (HDACI) can induce G1 or G2 cell cycle arrest or apoptosis in addition to directly modulating gene expression in cancer cells.(11–16) It has also been demonstrated that HDACI inhibit cancer cell invasion(17,18) and angiogenesis.(19,20) Moreover, they may have potent antitumor activity in vivo,(21–23) as HDACI treatments lead to tumor regression and symptom improvement in some heavily pre-treated and multiple-relapse patients, with surprisingly few side-effects.(3,24)

Angiogenesis is the multistep process of forming new blood vessels and a crucial factor in tumor progression and metastasis.(25,26) When tumors adapt to hypoxia, HIF-1α protein activates a number of angiogenesis-responsive genes such as VEGF, one of the most pro-angiogenic factors.(27) Additionally, degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMP) such as MMP-2 and MMP-9 releases basement membrane-sequestered angiogenic factors.(28) Strategies that combine anti-angiogenesis therapy with chemotherapy are the focus of current clinical trials, as anti-angiogenic drugs are relatively less toxic and more effective than chemotherapy drugs. It has been discovered recently that HDAC are overexpressed under hypoxia, hypoglycemia and serum deprivation, and that HDACI have been shown to inhibit angiogenesis.(10,29)

A number of HDACI from natural sources and chemical libraries have been used to study post-translational modification with regards to acetylation. Among these compounds, suberoylanilide hydroxamic acid has recently been launched as the first clinical anti-tumor drug of this class of inhibitors; others such as FK228 and MS-27-275 are being investigated in clinical trials.(30) However, there is a continuous need to develop new HDACI with better pharmacological efficacy and novel modes of action.

We synthesized a novel HDAC inhibitor, N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA). As we previously reported, HNHA is a new synthetic HDACI with better pharmacological properties than a known HDACI found in human fibrosarcoma cells, suberoylanilide hydroxamic acid (SAHA).(31) Here, we have evaluated two HDACI: SAHA and HNHA and an antiangiogenic agent: fumagillin (FUMA), all of which inhibit tumor growth and angiogenesis in cancer cells. We conducted dynamic MRI of a mouse xenograft model to accurately determine their effects. We found that, in particular, HNHA dramatically inhibited histone hypoacetylation, tumor growth and downregulation of HDAC target genes related to proliferation in breast cancer cells. In addition, HNHA induced gene expression of anti-angiogenic factors while suppressing pro-angiogenic factors both in vitro and in vivo. These promising results encourage exploration of HNHA’s potential as a therapeutic agent with dual activity against tumor proliferation and angiogenesis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cell culture.  Cancer cell lines FM3A, C1300, LA-N-1, LA-N-2, LA-N-5, NB16, NB19, NB69, SK-N-SH and MCF-7 were grown in RPMI 1640 medium and HT-29 in DMEM medium containing 10% FBS (Gibco-BRL, NY, USA) at 37°C in a 5% CO2 humidified atmosphere. Hypoxia incubations were carried out at a controlled oxygen tension (1%) using a Pro-ox 110 oxygen controller and Pro-ox in vitro chamber (BioSpherix, Redfield, NY, USA).

Cell proliferation assay.  Cells viability was determined by trypan blue exclusion. After a 24-h pre-incubation, the growth medium was replaced with an experimental medium containing FUMA, HNHA or SAHA at final concentrations ranging from 10−12 M to 10−5 M in log dilutions or in a growth medium containing 0.1% (v/v) saline as a vehicle control. After a 96-h incubation, cell proliferation was estimated using the sulforhodamine B colorimetric assay.

IC50 estimation.  A logistic model, modified from Van Ewijk and Hoekstra,(32) was adjusted for our data.

Cell viability assay.  The effect of HNHA on breast cancer cell growth and survival was assessed by a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT)(32) assay (Roche, Indianapolis, IN, USA), as previously described. HNHA was added at 0–30 μM final concentrations. After 48 h of incubation, viable cells were measured by XTT reagent at UV 490 nm, as described by the manufacturer’s protocol.

Cell cycle phase distribution by flow cytometric DNA analysis.  FM3A and MCF-7 cells were cultured in each medium. The DNA content of the cells (10 000 cells/experimental group) was analyzed using a Beckton–Dickenson FACS Vantage flow cytometer system (Beckton–Dickenson, San Jose, CA, USA), and the cell cycle distribution was analyzed using Cell Quest software version 3.2 (Beckton–Dickenson).

Mouse breast cancer cell xenograft.  FM3A breast cancer cells (1.0 × 107/mouse) were cultured in vitro and subcutaneously injected into the upper left flank region of the mice. After 10 days, tumor-bearing mice were randomly grouped (= 25/group) and an intraperitoneal injection of each of the three drugs (FUMA, HNHA or SAHA) was given once every 2 days for a total of six injections (20 μM/mouse) when the complete tumor size reached (4/3 × π × [0.7 cm × 0.4 cm]3 × ½). Tumor volume was estimated using the following formula: 4/3 × π × (a cm × b cm)3 × ½, where a and b are the two perpendicular diameters. Animals were maintained under specific pathogen-free (SPF) conditions. All experiments were approved by the Animal Experiment Committee of Yonsei University.

In vivo toxicity study. In vivo toxicity was performed on C3H/HeJ-FasL mice. Six-week-old mice were caged for 1 week before the experiment to acclimatize them to the environment. For the experiment, each group, consisting of 25 mice, was injected intraperitoneally with one of the three different drugs (FUMA, HNHA or SAHA) at a dose of 20 μM per mouse. The animals were monitored regularly for external signs of toxicity or lethality.

Dynamic MRI.  Magnetic resonance images were acquired using a 1.5T scanner (Gyroscan Intera; Philips Medical Systems, Best, the Netherlands). For each tumor, perfusion and vascular permeability were measured using a bolus intravenous injection of 0.2 mM/kg Gd-DTPA (Magnevist; Schering, Berlin, Germany) mixed with 0.3 mL saline. The MRI speed was measured for both the control and experimental groups. The gradient represents expression of blood vessel growth.

Western blot analysis.  Total proteins were electrophoresed under denaturing conditions in SDS-PAGE gels using a discontinuous procedure. Primary antibody bindings for TIMP-1 (Abcam, Cambridge, UK), TIMP-2 (Biomeda, Foster City, CA, USA), MMP-2 (Abcam), MMP-9 (Abcam), HIF-1α (Novus, Littleton, CO, USA), VEGF (R&D Systems, MN, USA), p21 (Abcam) and β-actin (Biomeda) were performed by incubating the membrane in a moist chamber at 4°C overnight.

Enzyme-linked immuno-sorbent assays (ELISA).  Blood was centrifuged for 5 min at full speed in a microcentrifuge; the supernatant was removed and placed in a new tube, which was centrifuged again for 5 min at full speed. The supernatant was removed and tested for VEGF by mouse VEGF Quantakine ELISA according to the manufacturer’s instructions (R&D Systems). The results are expressed as μg/mL of total extracted protein.

Immunohistochemistry.  Following a washing step with PBS, the avidin–biotin complex (Strept ABComplex; DAKO, CA, USA) was applied and the sections were rinsed in PBS, developed with diaminobenzidine tetrahydrochloride substrate for 3 min and counterstained with hematoxylin. The primary antibodies used were monoclonal mouse anti-CD34 (Biomeda), monoclonal mouse anti-HIF-1α (Novus) and monoclonal mouse anti-VEGF (R&D Systems).

Statistical analysis.  Statistical analysis was performed with SPSS software version 11.5 (SPSS, Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

HNHA inhibits cancer cell proliferation.  HNHA showed stronger inhibition at lower concentrations on all cancer cell lines when compared with SAHA, especially on breast cancer cells, mouse FM3A (suspension cell) and human MCF-7 (adherent cell; Table 1). It is known that the effects of HDACI on solid tumors are low; thus, we selected the more sensitive FM3A and MCF-7 from among the 11 cell lines as target cell lines to test HNHA and evaluate the newly developed HDACI in solid tumor.

Table 1.   Effects of HNHA and SAHA on proliferation of cancer cell lines
Cell lineTissue derivedAnimalCell proliferation IC50† (μM)
HNHA (±SD)SAHA (±SD)
  1. Each data point represents the mean of three experiments performed in triplicate using sulforhodamine B, as described in the Materials and Methods. †IC50 (half maximal inhibitory concentration) estimation described in the Materials and Methods. ±SD, standard deviation.

FM3ABreastMouse15.70 (±0.9)24.49 (±0.4)
C1300NeuroblastomaMouse55.63 (±1.6)78.22 (±2.1)
LA-N-1Bone marrowHuman22.78 (±1.1)47.53 (±1.3)
LA-N-2Nervous systemHuman23.18 (±1.2)32.56 (±2.3)
LA-N-5NeuroblastomaHuman26.70 (±1.1)34.58 (±0.9)
NB16Bone marrowHuman19.64 (±0.8)30.95 (±1.7)
NB19Bone marrowHuman21.26 (±1.7)32.12 (±1.9)
NB69Nervous systemHuman22.31 (±2.1)30.12 (±0.7)
SK-N-SHNeuroblastomaHuman65.09 (±0.7)67.32 (±1.6)
MCF-7BreastHuman14.33 (±0.9)19.95 (±1.1)
HT-29ColorectalHuman16.98 (±1.4)24.91 (±1.5)

HNHA suppresses breast cancer cell growth.  Treatment of breast cancer cells with FUMA, HNHA and SAHA results in lower viability than in control cells. In addition, HNHA showed the highest suppression in terms of cell population and viability in mouse breast cancer cells (Fig. 1a) and dose-dependent inhibition of viability in mouse and human breast cancer cells (Fig. 1b). Cell population and adhesion ability were lower in the HNHA-treated group than in the control group, and exhibited a more elongated shape. This is similar to the morphological changes seen in the SAHA or FUMA treatment (Fig. 1c).

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Figure 1.  Effect of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) on proliferation, cell viability and cell adhesion of breast cancer cells. (a) Cell growth assay. Saline was added every 12 h to the media of FM3A culture that included one of three drugs (fumagillin [FUMA], HNHA and suberoylanilide hydroxamic acid [SAHA]), each at a concentration of 10 μM. The positive control consisted of saline. (b) Cell viability assay. FM3A and MCF-7 were cultured in complete media with increasing concentrations of HNHA for 48 h. Points, mean % solvent-treated control. (a) and (b) experiments were repeated more than three times with similar results. (c) Cell adhesion assay. MCF-7 cell adhesion was measured. The yellow circles indicate observed viable activity, while the red circles indicate observed non-viable activity.

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HNHA arrests cancer cells at the G1/S phase of the cell cycle and rescues strongly protein acetylation.  Increasing the dose of HNHA arrested FM3A and MCF-7 cells in the G1/S phase more than SAHA did (Fig. 2a). This means that increased HNHA caused a greater reduction of cells in the G1/S phase, confirming our previous finding that HNHA inhibits cancer cell proliferation through loss of cell adhesion. Interestingly, HNHA decreased the G2–M and S phase populations as well. Moreover, p21, a cell proliferation arrestor, was activated in the HNHA-treated group (Fig. 2b). The activation of p21 by HNHA was comparable to that observed with SAHA and is consistent with the observed effects of SAHA on cell viability and apoptosis. These observations suggest that HNHA arrested the cell cycle at the G1/S phase via the induction of p21, which led to profound inhibition of cancer cell growth. p21 was increased by the HDAC inhibitor. Effects of HNHA on histone acetylation were assessed in FM3A and MCF-7 cells after exposure to increasing doses of SAHA and HNHA under hypoxia (Fig. 3a). The cells were treated with SAHA and HNHA for 1, 6, 24, 48 or 72 h. The acetylation of histone H3 was much stronger in HNHA treated cells than in SAHA treated cells. The most effective dose point for acetylation of histone H3 was 10–20 μM. Histone H3 acetylation peaked after 1 h of exposure to the drugs and remained stable for 1–6 h (Fig. 3b), after which point the effect decreased. HNHA increased histone and non-histone protein acetylation and inhibited FM3A and MCF-7 proliferation in vitro, and was very effective in increasing the acetylation level of histone H3 protein in FM3A and MCF-7.

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Figure 2.  Effects of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) on the cell cycle progression of breast cancer cell lines. (a) Cell cycle phase distribution by flow cytometric DNA analysis. FM3A and MCF-7 cells were treated at IC50 with HNHA and suberoylanilide hydroxamic acid (SAHA) for 24 h. Cells were stained with propidium iodide to measure the DNA content by FACS analysis. (b) p21 protein levels. FM3A and MCF-7 cells were treated with FUMA, HNHA and SAHA at IC50. Total cell lysates from four groups were subjected to western blot analysis and probed with antibodies against p21.

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image

Figure 3.  Effects of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) on protein acetylation of breast cancer cell lines. (a) FM3A and MCF-7 were treated for 24 h with increasing doses of HNHA (0.1, 1, 10 and 20 μM) under hypoxia conditions. (b) FM3A and MCF-7 were treated with suberoylanilide hydroxamic acid (SAHA) and HNHA (15 μM) for 1, 6, 24, 48 and 72 h under hypoxia conditions, respectively. The total proteins were isolated, and the acetylation of histone H3 and α-tubulin were evaluated by western blot analysis.

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HNHA inhibits angiogenic proteins in breast cancer cells.  The HNHA-treated group showed the strongest induction of TIMP-1 and TIMP-2 and the weakest expression of MMP-2, MMP-9, HIF-1α and VEGF (Fig. 4). When compared with the other agents, HNHA most effectively inactivated MMP-2, MMP-9, VEGF and HIF-1α. This suggests that HNHA might be the most effective in reducing angiogenesis in the analyzed cell lines among the three HDACI.

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Figure 4.  Downregulation of vascular endothelial growth factor (VEGF) and HIF-1α by N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA). Fumagillin (FUMA), HNHA and suberoylanilide hydroxamic acid (SAHA) (15 μM) was added every 12 h to the media of FM3A and MCF-7 cultures under hypoxic conditions. The positive control consisted of saline.

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HNHA reduced tumor burden and extended the survival rate in a mouse tumor xenograft model.  HNHA, FUMA and SAHA were injected intraperitoneally to evaluate their effect in vivo. The HNHA-treated group had smaller tumor volumes and survived longer than the other groups (Fig. 5a). In addition, the survival rate of the HNHA-treated group was higher than the other groups (Fig. 5b). There was no evidence of systemic toxicity attributable to HNHA, SAHA or FUMA (Fig. 5c).

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Figure 5.  Effect of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) on mouse breast cancer cell tumor xenografts. The graphs show results for (a) changes in cancer volume (black arrows indicate death and red arrows indicate survival) and (b) survival rate between the control and experiment groups. No tumor indicates HNHA-, fumagillin (FUMA)- and suberoylanilide hydroxamic acid (SAHA)-treated mice without tumor in panel (c). (d) Dynamic MRI scans for each group. The image is a cross-section of the mice. The area circled in yellow is a tumor. The measured MRI speed is shown in the yellow circle (e). Magnetic resonance migration speed around the injection site (Y-axis, blood vessel intensity; X-axis, time). (□) The graph has an upward tendency, indicating that blood vessel proliferation is flourishing. However the (•, bsl00066) graph has a shallow slope, indicating that blood vessel proliferation is inhibited by SAHA and HNHA. The experiments were repeated more than three times with similar results; the results for triplicate experiments are presented only as mean ± SD in (a).

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For more accurate tumor tissue analysis in the control and experimental groups, we used dynamic MRI to measure the blood vessel intensity and to avoid killing the animal. The measured MRI speed is shown in the yellow-circled area of Figure 5d. The graph of the saline-treated group has an upward tendency, indicating that tumor angiogenesis is flourishing (Fig. 5e). However, the graph of HNHA- and SAHA-treated groups has a shallow slope, indicating inhibition of blood vessel proliferation.

HNHA administration increased TIMP-1, TIMP-2 and p21 and decreased VEGF, MMP-2, MMP-9, HIF-1α and VEGF protein expression in a mouse model.  The HNHA-treated group showed the strongest expression of TIMP-1 and TIMP-2 and the weakest expression of MMP-2, MMP-9, HIF-1α and VEGF (Fig. 6a). We suggest that the activation of TIMP-1 and TIMP-2 induced inhibition of MMP-2 and MMP-9 in the HNHA group, thereby causing suppression of tumor growth and angiogenesis. HNHA treatment inhibited HIF-1α and VEGF and activated p21, a cell proliferation arrestor. We also investigated the VEGF level in mouse serum by ELISA (Fig. 6b). The HNHA group’s VEGF concentration was the lowest among the four experimental groups. Immunohistochemistry for CD34 (progenitor marker), HIF-1α and VEGF was performed using tumor tissues (Fig. 7a). The distribution of CD34, HIF-1α and VEGF was reduced in the HNHA-treated group compared with the control group (Fig. 7b). These results indicate that HNHA effectively inhibits cancer development and angiogenesis in vivo.

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Figure 6.  Effects of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) on TIMP-1, TIMP-2, MMP-2, MMP-9, HIF-1α, vascular endothelial growth factor (VEGF) and p21 expression in mouse tumor tissue. (a) Western blot analysis on TIMP-1, TIMP-2, MMP-2, MMP-9, HIF-1α, VEGF, p21 and β-actin protein expression. (b) Effect of HNHA on VEGF protein expression analyzed by ELISA. Total mouse protein was extracted from serum and tested for VEGF by ELISA using a total blood vessel density of 9.1 μg/mL. *The HNHA group’s VEGF concentration was lower than the saline group (*P < 0.001).

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image

Figure 7.  Histopathology in the mouse xenograft model. (a) Representative immunohistochemistry for vascular endothelial growth factor (VEGF), CD34 and HIF-1α. Original magnification is ×200. Control slides were treated with isotype-matched IgG serum or antibody. (b) Quantification of VEGF, CD34 and HIF-1α expression. Expression levels of CD34, HIF-1α and VEGF were determined using densitometry. Data are expressed as mean ± SD (n ≥ 3). *P < 0.001. HNHA, N-hydroxy-7-(2-naphthylthio) heptanomide.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

HDACI have recently emerged as promising anti-cancer agents, because they arrest the cell cycle and induce cell differentiation and cell death in a number of cancer cell types, and preclinical tumor models have shown their potential as therapeutic agents.(3–6,33) Moreover, the HDACI currently undergoing clinical trials have better toxicity profiles than traditional chemotherapeutic agents.(4,9,18,22) However, a recent report has shown that a specific type of solid tumor was not sensitive to HDACI.(34) These discoveries demand the development of more potent, less toxic and more selective HDACI. We therefore developed our compound based on its chemistry as a known hydroxamic HDACI, HNHA, which is similar to SAHA. In previous studies, we reported that HNHA and SAHA, two different HDACI, inhibit fibrosarcoma cell proliferation in vitro and angiogenesis in vivo.(31) In this study, we focused on identifying the better pharmacological potential of HNHA as compared with other clinically validated HDACI and its sensitive cancer types.

HNHA, by inducing G1/S cell cycle arrest in breast cancer cells, was found to be most efficacious and showed an IC50 level decreased by half compared with SAHA’s IC50 level. As with the other HDACI, HNHA exposure caused greater accumulation of the cyclin-dependent kinase inhibitor p21, which is essential in cancer cell growth. It is well established that the level of p21 promoter amplified from acetylated histone H3- or H4-associated chromatin is greater in chromatin isolated from HDAC inhibitor-treated groups than that from untreated groups,(35) and that histone hyperacetylation induces the expression of tumor suppressors such as a p21.(15,36,37) HNHA strongly rescued histone acetylation in FM3A and MCF-7 cells, a function possibly linked to its ability to arrest the cell cycle in cancer cells.

We also investigated how HNHA affects the expression of genes involved in angiogenesis, including HIF-1α, VEGF, TIMP-1, TIMP-2, MMP-2 and MMP-9 in breast tumor cells, as compared with the effects of SAHA. VEGF stimulates angiogenesis by promoting endothelial cell proliferation; its expression is known to be regulated by HDAC activity.(25,27) It has also been established that the therapeutic effects of HNHA are associated with its ability to inhibit VEGF secretion; given that HDACI induce repression of tumor HIF-1α, the resultant inhibition of VEGF expression might contribute to their anti-angiogenic properties.(27,31) Our results show for the first time that HNHA inhibits HIF-1a and VEGF expression in a breast cancer model in vitro and in vivo to a higher degree than does SAHA. These results suggest that HNHA is the most effective anti-angiogenesis agent among HNHA, FUMA and SAHA. The combination of HDACI with anti-VEGF therapies (e.g., VEGF-blocking agents or VEGF receptor tyrosine kinase inhibitors) is of particular interest, as there is both preclinical and clinical evidence that tumor cells “escape” from anti-VEGF therapy.(25–27) It is conceivable that HDACI modulation of angiogenesis-related genes affects both tumor cell and endothelial cell adaptation to anti-VEGF therapies, preventing or delaying escape. Another possibility regarding the HDACI antitumor effect is its induction of acetylation of non-histone proteins, which subsequently affects gene expression (both upregulation and downregulation) and protein stability.

It has been suggested that TIMP-1 and TIMP-2 mediate growth-inhibiting signals by inhibiting MMP-2 and MMP-9, which themselves mediate two important effects: ECM degradation and cell proliferation.(38,39) Our study suggests that HNHA activates TIMP-1 and TIMP-2, which induces inhibition of MMP-2 and MMP-9 in the HNHA group, thereby causing the suppression of tumor growth and angiogenesis. Treatment of tumor cell lines with the HDAC inhibitor resulted in the modulation of secreted expression levels of MMP-2, MMP-9 and TIMP, which was accompanied by global histone H3 and H4 hyperacetylation.(40,41) It was also demonstrated that increased histone acetylation by HDAC inhibitor-treated tumor cells decreased mRNA as well as the activity of MMP-2, MMP-9 and TIMP.(41,42) Clinical studies have shown increased TIMP-1 and TIMP-2 expression in many human tumors, a characteristic often associated with a poor prognosis.(39) This raises important questions regarding the roles of TIMP-1 and TIMP-2 in tumor progression. These initial observations and similar results from other groups suggest that targeting endothelial cells with HDACI may directly inhibit tumor angiogenesis and subsequently cancer progression. Here, we used dynamic MRI to determine blood vessel intensity. Specifically, HNHA significantly decreases the cancer population in breast cancer-bearing mice and exhibits a higher potency than SAHA.

In conclusion, our novel HDACI, HNHA, has been shown to have a dual function of suppressing both tumor cell proliferation and tumor angiogenesis by gene modulation. Indeed, HNHA markedly decreased the tumor burden in a mouse xenograft model and exhibited higher potency than SAHA. These findings provide rationale for further testing of HNHA as a single agent or in combination with other angiogenesis inhibitors to treat breast cancer. Our observation that HNHA treatment is associated with relatively low toxicity demonstrates its promise as a new anti-tumor agent in breast cancer.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This work was supported by a faculty research grant from Yonsei University College of Medicine for 6-2007-0104 and the Brain Korea 21 Project for Medical Science, Yonsei University.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References