Valproic acid inhibits tumor angiogenesis in mice transplanted with Kasumi-1 leukemia cells

Histone deacetylase (HDAC) inhibitors have been reported to inhibit tumor angiogenesis via the downregulation of angiogenic factors. Our previous in vitro studies demonstrated that valproic acid (VPA) exerted antitumor effects on Kasumi-1 cells, which are human acute myeloid leukemia cells with an 8;21 chromosome translocation. In the present study, the effects of VPA on tumor angiogenesis were investigated in mice transplanted with Kasumi-1 cells. Semi-quantitative reverse transcription-polymerase chain reaction, western blotting and immunohistochemistry were used to detect the expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR2) and basic fibroblast growth factor (bFGF). The tumor microvessel density was measured following staining with an anti-CD34 antibody. Chromatin immunoprecipitation was used to study the effect of VPA-induced histone hyperacetylation on VEGF transcription. An intraperitoneal injection of VPA inhibited tumor growth and angiogenesis in mice transplanted with Kasumi-1 cells. The mRNA and protein expression of VEGF, VEGFR2 and bFGF were inhibited by VPA treatment. In addition, VPA downregulated HDAC, increased histone H3 acetylation and enhanced the accumulation of hyperacetylated histone H3 on the VEGF promoters. The findings of the present study indicate that VPA, an HDAC inhibitor, exerts an antileukemic effect through an anti-angiogenesis mechanism. In conclusion, the mechanism underlying VPA-induced anti-angiogenesis is associated with the suppression of angiogenic factors and their receptors. VPA may increase the accumulation of acetylated histones on the VEGF promoters, which possibly contributes to the regulation of angiogenic factors.


Introduction
Chromosomal translocations are frequent events that occur in leukemia. The translocation t(8;21)(q22;q22) is one of the most frequent chromosomal translocations in leukemia and accounts for 12-15% of acute myeloid leukemia (AML) and ~40-50% of M2 AML (French-American-British classification) (1). This translocation involves the AML1 gene at q22 on chromosome 21 and the ETO gene at q22 on chromosome 8, resulting in an AML1/ETO fusion gene. This fusion gene encodes a chimeric protein, AML1/ETO. The chimeric protein silences target gene transcription by recruiting histone deacetylases (HDACs), which remove acetyl groups from histone lysine residues. The abnormal recruitment of HDAC due to chromosomal rearrangements often occurs in the development of malignant tumors and contributes to their pathogenesis (2). Several studies have demonstrated that the abnormal AML1/ETO protein and the silencing of hematopoietic genes contribute to the hematopoietic developmental abnormalities of AML with t(8;21) (3)(4)(5)(6). Inhibition of HDAC activity has been reported to restore the abnormal histone acetylation in tumors, thus resulting in the growth arrest, differentiation and/or apoptotic cell death of tumor cells (7). Therefore, HDAC inhibitors represent a promising treatment for patients with AML with t(8;21), as they may enhance histone acetylation via inhibition of HDAC activities, thus restoring the disrupted gene transcripts in AML (8).
Angiogenesis is critical for tumor growth and metastasis. Vascular endothelial growth factor (VEGF), VEGF receptors (VEGFRs) and basic fibroblast growth factors (bFGFs) are the most potent pro-angiogenic factors and are critical in tumor angiogenesis (9,10). Anti-angiogenic approaches are a novel strategy to treat AML. It has been reported that the HDAC inhibitor, FK228, inhibits the expression of angiogenic factors, including VEGF and bFGF, in PC-3 xenografts implanted in nude mice, indicating that the antitumor effects of FK228 are mediated through the inhibition of angiogenesis (11). Valproic acid (VPA), which is widely used clinically for the treatment of epilepsy, has been demonstrated to be a strong HDAC

Valproic acid inhibits tumor angiogenesis in mice
transplanted with Kasumi-1 leukemia cells inhibitor (12). Our previous in vitro studies revealed that VPA exerted antitumor effects on Kasumi-1 cells, human acute myeloid leukemia cells with an 8;21 chromosome translocation, via downregulation of VEGF and VEGFR (13,14). The purpose of the present study was to investigate the effect of VPA on tumor growth and the expression of angiogenic factors in mice transplanted with Kasumi-1 cells, and also to analyze the histone acetylation on VEGF promoters in these cells.

Materials and methods
Tumor cells and animals. Tumor generation and VPA treatment. Splenectomies were performed on the BALB/c nude mice. One week after the splenectomies, the mice received whole body irradiation with 137 Cs at a dose of 4 Gy. At 48-72 h post-irradiation, the mice were subcutaneously implanted with Kasumi-1 cells (2x10 7 cells/mouse with 0.15-0.2 ml) in the right axillary region. The mice were randomly assigned to two groups, the VPA (n=6) and control (n=6) groups. When the tumors were ~200 mm 3 in size at ~10 days post-implantation, 0.2 ml VPA (500 mg/kg body weight) or 0.2 ml saline was injected intraperitoneally every day. VPA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in saline at a concentration of 25 mg/ml. The longest diameter (a) and the shortest diameter (b) of the tumor were measured every three days, and the tumor volume (TV) was calculated according to the following formula: TV = 1/2 x a x b 2 . The relative TV (RTV) was calculated as the ratio between the TV on day N and the TV on the day of injection (day 0) according to the following formula: RTV = (TV on day N) / (TV on day 0). The tumor growth inhibition rate (IR) was calculated from the following formula: IR (%) = [1 -RTV (VPA group) / RTV (control) ) x 100, where RTV (VPA group) is the RTV in the VPA-treated group and RTV (control) is the RTV in the control group. Following two weeks of injections, the mice were sacrificed by cervical dislocation and the tumor masses were removed for the following experiments.
Immunohistochemistry. The tumor masses were fixed with 10% formalin and embedded with paraffin. Tissue sections (5-µm thick) were obtained from paraffin-embedded tissue blocks. The tissue sections were immunohistochemically stained for CD34, VEGF, VEGFR2 and bFGF. Briefly, the sections were washed in xylene to remove the paraffin, rehydrated with serial dilutions of alcohol and then washed in phosphate-buffered saline. The samples were then incubated in primary antibodies against CD34, VEGF, VEGFR2 and bFGF overnight at 4˚C. All primary antibodies were rat anti-human antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Subsequent to the primary antibody being washed off, the biotinylated goat anti-rat IgG secondary antibody (Santa Cruz Biotechnology, Inc.) was applied and then reacted with horseradish peroxidase-conjugated streptavidin. The sections were stained with diaminobenzidine solution and counterstained with hematoxylin.
Tumor microvessel density (MVD) analysis performed following staining with the anti-CD34 antibody. Microvessels with brownish staining in the cytoplasm of the endothelium were included. A single endothelial cell was counted as a single vessel. An endothelial cell cluster with a branching structure, which was clearly separated from the adjacent endothelial cells, was also counted as a single vessel. At a low-power field (x40), the tissue sections with the most intense vascular density were selected. At a high-power field (x200), microvessels in five fields were counted in the areas with the most intense vascular density. The mean microvessel count of the five most vascular areas was used as the MVD. Ltd., Ningbo, China) and treated with 1% formalin for 10 min at room temperature. The tissues were then homogenized with lysis buffer and sonicated five times at 80 W for 10 sec/time, at 60 sec intervals. Following centrifugation (5,000 x g, 1 min), the supernatant was removed, and 20 µl supernatant was saved for the controls (DNA input). Rabbit anti-human monoclonal antibodies against Ac-H3 (3 µg) were added into the supernatant and incubated overnight at 4˚C with gentle rotation. Antibodies against RNA polymerase were used as a positive control and mouse IgG was used as a negative control. Protein G agarose beads were added into the solution, and the samples were incubated at 4˚C for 1 h. The beads were washed five times with buffers according to the the ChIP assay kit protocol. The protein-DNA complex was eluted with an elution buffer containing 20% SDS and 1M NaHCO 3 . The immunoprecipitated chromatin was dissolved in 0.9% saline and then incubated at 65˚C for 4 h to reverse the cross-linking and separate the protein-DNA complex. The DNA was extracted, and RT-PCR was performed with primers specific for the VEGF promoter. The primers were as follows: Statistical analysis. Statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). All the data presented are based on an average of at least three independent experiments. The values are presented as the mean ± standard deviation. Student's t-test's were used to compare the differences between groups. P<0.05 was used to indicate a statistically significant difference.

VPA inhibits tumor growth in mice transplanted with
Kasumi-1 cells. The effect of VPA on tumor growth was investigated in mice transplanted with Kasumi-1 cells. None of the mice died prior to sacrifice at the end of the experiments. The size of the tumor was measured every three days following daily injection of VPA for two weeks. Fig. 1 shows the time-course of tumor growth in the control and VPA-treated groups. The TV in the VPA group increased with time more slowly than that in the control group. The final TV in the VPA group was 699.4±271.01 mm 3 , which was significantly less than the TV of 2235.0±360.21 mm 3 observed in the control group (P<0.05; Table I). Following sacrificing the mice, the size, weight and RVT of the tumors in the VPA group were significantly less than those in the control group (P<0.05; Table I; Figs. 1 and 2), indicating that VPA inhibited the tumor growth in the mice transplanted with Kasumi-1 cells. The IR rate in the VPA group was 57.25% at the end of the experiment.

VPA inhibits tumor angiogenesis in mice transplanted with
Kasumi-1 cells. The effect of VPA on tumor angiogenesis was tested in mice transplanted with Kasumi-1 cells by measuring MVD using CD34 immunostaining, which has been used in previous studies (15,16). Specific staining of capillary-like vessels by anti-CD34 was observed in the control and VPA groups (Fig. 3). The mean MVD in the VPA group (12.23±4.11; number of microvessels) was significantly lower than that in the control group (32.59±5.76) (P<0.05), indicating that VPA inhibited angiogenesis in the mice transplanted with Kasumi-1 cells.
VPA inhibits the mRNA and protein expression of VEGF, VEGFR2 and bFGF. The mechanisms underlying the VPA-induced inhibition of angiogenesis were studied further in the mice transplanted with Kasumi-1 cells. The mRNA and protein expression levels of VEGF, VEGFR2 and bFGF, which have been reported to be involved in tumor angiogenesis, were examined (9,10). RT-PCR demonstrated that the mRNA levels of VEGF, VEFGR2 and bFGF were significantly downregulated in the VPA group compared with those in the control group (Fig. 4). The protein expression of VEGF, VEFGR2 and bFGF was detected using immunohistochemistry (Fig. 5). Consistent with the mRNA levels, the protein expression of VEGF, VEFGR2 and bFGF was suppressed in the VPA group compared with the control group. These results indicated that VPA inhibited tumor angiogenesis most likely through its inhibition of VEGF, VEFGR2 and bFGF. VPA inhibits HDAC activity and increases acetylation of histone H3. As it has already been shown that the histone acetylation of the VEGF promoters regulates VEGF protein expression (11), the present study investigated the effects of VPA on HDAC activity by detecting the nuclear expression of HDAC1 and the acetylation of histone H3 (Fig. 6). Western blotting revealed that the expression of HDAC1 was downregulated, while histone H3 acetylation was increased in the VPA group compared with the control group, indicating that VPA increased the acetylation of histone H3 via the inhibition of HDAC.
VPA enhances the accumulation of hyperacetylated histone H3 on VEGF promoters. To investigate whether VPA induced the hyperacetylation of histone H3 at the VEGF promoter, a ChIP assay was performed using anti-Ac-H3 antibodies to precipitate      (Fig. 7). Anti-Ace-H3 antibodies enriched more VEGF promoter DNA fragments in the VPA group than in the control group. By contrast, non-specific IgG antibodies did not precipitate VEGF promoter DNA in either the VPA group or the control group. Anti-polymerase II antibodies, which served as a positive control, precipitated similar quantities of VEGF promoter DNA in the VPA and control groups.

Discussion
In the present study, a mouse model of leukemia was established using Kasumi-1 cells, which are human acute myeloid leukemia cells with an 8;21 chromosome translocation. Kasumi-1 cells express the AML-1/ETO fusion protein and are ideal for the study of AML with t(8;21) (17). The in vitro study by Liu et al (18) in Kasumu-1 cells revealed that VPA had pronounced antileukemic effects that were associated with an inhibition of the protein expression of HDAC in the nuclei, a disruption of the physical interaction between AML1/ETO and HDAC1, an increase in their dissociation from the promoters of AML1/ETO target genes and a translocation of these nuclear proteins to the perinuclear region. In agreement with these reported in vitro antileukemic effects of VPA, the present in vivo study demonstrated that intraperitoneal injections of VPA reduced tumor growth in mice implanted with Kasumi-1 cells, and the tumor weights and volumes were significantly smaller in the VPA group compared with those in the control group.
It has been reported that AML with t(8;21) and Kasumi-1 cell lines demonstrate enhanced expression of VEGF and VEGFR2, which is associated with the growth of Kasumi-1 cells and the angiogenesis of AML (19,20). Blockade of the VEGFR2 signaling pathway inhibits the growth of Kasumi-1 cells (20). Furthermore, several in vivo and in vitro studies have revealed that HDAC inhibitors, including NaB, SAHA, TSA and FK228, inhibit tumor angiogenesis via downregulation of the mRNA and protein expression of VEGF (21)(22)(23)(24). In addition, our previous study revealed that VPA exerted antitumor effects on Kasumi-1 cells, most likely through the downregulation of the mRNA and protein expression of VEGF, VEGFR2 and bFGF (14). In the present study, the effects of VPA were directly tested on tumor angiogenesis in mice transplanted with Kasumi-1 cells by measuring the MVD with CD34 immunostaining. VPA was observed to decrease the MVD and downregulate the VEGF, VEGFR2 and bFGF mRNA and protein levels compared with the control. Thus, the present study provided direct evidence that VPA inhibited tumor angiogenesis in mice transplanted with Kasumi-1 cells.
Several mechanisms have been reported to be involved in HDAC inhibitor-induced anti-angiogenesis, including the inhibition of HDAC activity, the overexpression of HIF-1α, the downregulation of tumor suppressor genes, including VHL and p53, and the downregulation of VEGF expression (25)(26)(27). In the present study, VPA, a HDAC inhibitor, was detected to downregulate HDAC protein expression and increase the acetylation of histone H3 in Kasumi-1 cell-induced tumors. In addition, VPA enhanced the accumulation of hyperacetylated histone H3 on the VEGF promoters. However, it remains unclear how histone acetylation inhibits gene transcription.
There are two possible mechanisms underlying the regulation of gene transcription by histone acetylation: i) Histone has several acetylation sites, and the acetylation state of each lysine is different in each site. It has been reported that the patterns of histone acetylation, which are termed the 'histone code' reveal the sign of transcription (28); and ii) histone acetylation by HDAC inhibitors induces alterations in the chromatin structure, which may lead to the disruption of the binding of transcription factors to gene promoters. It has been reported that VPA may alter the chromatin structure by regulating chromatin modulation proteins (29). Although the mechanism underlying the alteration of the chromatin structure by histone acetylation has not been clarified, it may be important to explain how HDAC inhibitors regulate gene expression.
In conclusion, VPA inhibits tumor growth and tumor angiogenesis in mice implanted with Kasumi-1 cells. This antitumor effect of VPA is possibly due to the inhibition of VPA on the expression of angiogenic factors. In addition, VPA may increase the accumulation of acetylated histones on the promoters of the genes, which may contribute to the regulation of the expression of angiogenic factors.