The Kasumi-1 cell line was a gift from Dr Jianxiang Wang at the Institute of Hematology, Chinese Academy of Medical Science (Tianjin, China). The cells were maintained in culture with RPMI-1640 medium supplemented with 20% fetal bovine serum in a 37°C incubator with 5% CO₂ and 95% humidity. Female BALB/c nude mice (SPF grade; 10–15 g; 4–6 weeks old) were purchased from Beijing Vital River Lab Animal Technology Co., Ltd. (Beijing, China). The study was approved by the Chengde Medical College Animal Research Ethics Committee.

Splenectomies were performed on the BALB/c nude mice. One week after the splenectomies, the mice received whole body irradiation with ¹³⁷Cs at a dose of 4 Gy. At 48–72 h post-irradiation, the mice were subcutaneously implanted with Kasumi-1 cells (2×10⁷ 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 mm³ 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 × a × b². 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)) × 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.

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.

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.

Total RNA was isolated from the tumor masses using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The final RNA concentration was adjusted to 1 μg/μl. RNA was reverse transcribed into complementary DNA using the reverse transcriptase of Moloney murine leukemia virus (Bio Basic Canada Inc., Markham, ON, Canada). PCR was performed using Taq DNA polymerase [Takara Biotechnology, Co., Ltd., Dalian, China]. The primers were as follows: Sense: 5′-GAAGTGGTGAAGTTCATGGATGTC-3′ and antisense: 5′-CGATCGTTCTGTATCAGTCTTTCC-3′ for VEGF (size, 260 bp); sense: 5′-AGAGCGACCCTCACATCAAG-3′ and antisense: 5′-TCGTTTCAGTGCCACATACC-3′ for bFGF (size, 224 bp); and sense: 5′-GGGGATTGACTTCAACTGG-3′ and antisense: 5′-GACCCTGACAAATGTGCTG-3′ for VEGFR2 (size, 211 bp). β-actin (size, 453 bp) was used as an internal control. The reaction conditions consisted of 38 cycles of 95°C for 45 sec, 61°C for 45 sec and 72°C for 60 sec. The PCR products were analyzed by electrophoresis on a 1.8% agarose gel containing ethidium bromide, and the gel was visualized with a digital imaging system (Fujifilm; Fuji, Tokyo, Japan). The relative mRNA expression of VEGF, bFGF and VEGFR2 was normalized to the β-actin concentration.

For western blotting, the tumor masses were homogenized on ice in RIPA lysis buffer [50 mM Tris-HCl, 150 mM NaCl and 1% NP-40 (pH 7.4)]. Nuclear proteins were extracted using the Nucleoprotein Extraction kit (Sangon Biotech, Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. The protein concentrations were determined with the bicinchoninic acid method. Equal quantities of proteins were loaded and separated by electrophoresis in 120 g/l SDS-PAGE and then transferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies against HDAC1 (rabbit anti-human polyclonal antibodies; Proteintech Group, Chicago, IL, USA) or acetylated histone H3 (Ac-H3; rabbit anti-human monoclonal antibodies; Epitomics, Burlingham, CA, USA) at 4°C overnight. Blots were stained with horseradish peroxidase-linked goat anti-rabbit Ig secondary antibodies and developed with a chemiluminescence detection system (Fujifilm). The nuclear protein, lamin B served as a loading control.

ChIP was performed using the ChIP assay kit (Upstate Biotechnology, Billerica, MA, USA) according to the manufacturer’s instructions. Briefly, tumor masses were cut into small sections (Xinzhi Co. 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 × 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 NaHCO₃. 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: Sense: 5′-CTT CGA GAG TGA GGA CGT GTG T-3′ and antisense: 5′-GGA GCA GGA AAG TGA GGT TAC G-3′ for P1; sense: 5′-CCA GAC TCC ACA GTG CAT ACG T-3′ and antisense: 5′-TGGGAC TGG AGT TGC TTC ATG-3 for P2; sense: 5′-TGC TGC ATT CCC ATT CTC AGT-3′ and antisense: 5′-ATC TTC CCTAAG TGC TCC CAA AG-3′ for P3; sense: 5′-CAG GGA AAG GAT GAT CAC TGT CA-3′ and antisense: 5′-TGC CTT TCA CCA GGA CAA AGT-3′ for I1; and sense: 5′-ATG GAT GTC TAT CAG CGC AGCT-3′ and antisense: 5′-TGG TGA TGT TGG ACT CCTCAG T-3′ for E3. The reaction conditions were 94°C for 3 min, followed by 32 cycles of 94°C for 20 sec, 57°C for 30 sec and 72°C for 30 sec and a final extension of 72°C for 2 min.

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.

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 mm³, which was significantly less than the TV of 2235.0±360.21 mm³ 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.

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.

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.

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.

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 chromatin from Kasumi-1 cell-induced tumors (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.