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. 2007 Oct 25:6:68.
doi: 10.1186/1476-4598-6-68.

Enhancing the anti-angiogenic action of histone deacetylase inhibitors

Selena Kuljaca  1 , Tao Liu, Andrew E L Tee, Michelle Haber, Murray D Norris, Tanya Dwarte, Glenn M Marshall
Affiliations

Enhancing the anti-angiogenic action of histone deacetylase inhibitors

Selena Kuljaca et al. Mol Cancer. .

Abstract

Background: Histone deacetylase inhibitors (HDACIs) have many effects on cancer cells, such as growth inhibition, induction of cell death, differentiation, and anti-angiogenesis, all with a wide therapeutic index. However, clinical trials demonstrate that HDACIs are more likely to be effective when used in combination with other anticancer agents. Moreover, the molecular basis for the anti-cancer action of HDACIs is still unknown. In this study, we compared different combinations of HDACIs and anti-cancer agents with anti-angiogenic effects, and analysed their mechanism of action.

Results: Trichostatin A (TSA) and alpha-interferon (IFNalpha) were the most effective combination across a range of different cancer cell lines, while normal non-malignant cells did not respond in the same manner to the combination therapy. There was a close correlation between absence of basal p21WAF1 expression and response to TSA and IFNalpha treatment. Moreover, inhibition of p21WAF1 expression in a p21WAF1-expressing breast cancer cell line by a specific siRNA increased the cytotoxic effects of TSA and IFNalpha. In vitro assays of endothelial cell function showed that TSA and IFNalpha decreased endothelial cell migration, invasion, and capillary tubule formation, without affecting endothelial cell viability. TSA and IFNalpha co-operatively inhibited gene expression of some pro-angiogenic factors: vascular endothelial growth factor, hypoxia-inducible factor 1alpha and matrix metalloproteinase 9, in neuroblastoma cells under hypoxic conditions. Combination TSA and IFNalpha therapy markedly reduced tumour angiogenesis in neuroblastoma-bearing transgenic mice.

Conclusion: Our results indicate that combination TSA and IFNalpha therapy has potent co-operative cytotoxic and anti-angiogenic activity. High basal p21WAF1 expression appears to be acting as a resistance factor to the combination therapy.

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Figures

Figure 1
Figure 1
TSA and IFNα exerted co-operative cytotoxic effects in cancer cell lines from a range of different tissue origins, but not in normal non-malignant cells. A. Neuroblastoma [BE(2)-C], breast (MCF-7 and MDA-MB-468), lung (H460 and Calu-6), prostate (DU-145 and LNCaP), and colon (HT-29 and Caco-2) cancer cells were treated with control (Cont), 0.02 μM TSA and/or 500 IU/ml IFNα for 72 hours. Cell viability was examined using the Alamar blue assay, measured as optical density (OD) units of absorbance, and expressed as the absorbance of treated over control samples (ie., % viable cells). ** p < 0.01, *** p < 0.001. B. MRC-5 cells were treated with control, 0.02 μM TSA and/or 500 IU/ml IFNα for 72 hours, and cell viability was assessed as above. Moreover, histone protein was extracted and subject to immunoblot analysis with anti-acetylated histone H3 antibody, after 6 hour exposure to control, TSA and/or IFNα.
Figure 2
Figure 2
SAHA and IFNα exerted co-operative cytotoxic effects in cancer cell lines, but not in normal non-malignant cells. Neuroblastoma BE(2)-C, breast cancer MCF-7, and normal non-malignant lung MRC-5 fibroblasts were treated with control, 0.5 μM SAHA and/or 500 IU/ml IFNα for 72 hours. Cell viability was examined using the Alamar blue assay, measured as optical density (OD) units of absorbance, and expressed as the absorbance of treated over control samples (ie., % viable cells). * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
The cytotoxic effects of other HDACI combination therapies. A. Neuroblastoma [BE(2)-C], breast (MCF-7), and lung (Calu-6) cancer cell lines were treated with either control, 500 IU/ml IFNα and/or various dosages of VPA for 72 hours B. In separate experiments, BE(2)-C and MCF-7 cells were treated with control, 1 mM VPA and/or various dosages of rapamycin (RAP) for 72 hours. C. Non-malignant lung fibroblast (MRC-5) cells were treated with a range of VPA doses alone, or in combination with 500 IU/ml IFNα. Cell viability was examined by the Alamar blue assay, measured as optical density (OD) units of absorbance, and expressed as a percentage of absorbance for treated samples, over that for control samples (ie., % viable cells). ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
Absence of p21WAF1 expression correlated with sensitivity to TSA and IFNα combination therapy. A. MCF-7, MDA-MB-468, H460, Calu-6, DU-145, LNCaP, HT-29 and Caco-2 cells were treated with control, 0.02 μM TSA, 500 IU/ml IFNα, or TSA and IFNα for 24 hours. Whole cell protein was extracted and subjected to immunoblot with an anti- p21WAF1 antibody, and, an anti-actin antibody as a loading control. B. MCF-7 cells were transfected with control scrambled or p21WAF1 siRNA for 8 hours, followed by treatment with control, 0.02 μM TSA and/or 500 IU/ml IFNα for 72 hours. The effect of the siRNAs on p21WAF1 gene and protein expression was analysed by semi-quantitative RT-PCR with the house-keeping gene β-2-microglobulin (β2M) as a loading control or by immunoblot, with actin as a loading control. C. Cell viability was examined by the Alamar blue assay, measured as optical density (OD) units of absorbance, and expressed as percentage of absorbance for drug-treated samples over control-treated samples (% viable cells). ** p < 0.01.
Figure 5
Figure 5
HDACI and IFNα co-operatively inhibit endothelial cell functions, and pro-angiogenic gene expression in cancer cells under hypoxic conditions in vitro. A. Human umbilical vein endothelial cells (HUVECs) were treated with control (Cont), 0.1 μM TSA and/or 500 IU/ml IFNα for 18 hours. Cell viability was evaluated with the Alamar blue assay. B. HUVECs were plated in BD Biosciences Fluroblok chambers and treated with control, 0.1 μM TSA and/or 500 IU/ml IFNα for 22 hours. Cells were stained with Cell Tracker Green CMFDA, migrated through chamber filters toward the chemo-attractant VEGF, and then quantified and expressed as optical density (OD) absorbance units. C. HUVECs were plated into BD BioCoat growth factor-reduced matrigel invasion chambers and treated with control, 0.1 μM TSA and/or 500 IU/ml IFNα for 18 hours. Cells which invaded through the Matrigel were fixed, stained with a Diff Quick staining kit, photographed and then quantified. D. HUVECs were plated onto growth factor-reduced Matrigel in 24 well plates and treated with control, 0.1 μM TSA and/or 500 IU/ml IFNα for 18 hours. Vascular sprouting was quantified by counting the numbers of complete branches per branching point. E. Neuroblastoma BE(2)-C cells were treated with control, 0.02 μM TSA and/or 500 IU/ml IFNα for 72 hours under hypoxic (1% O2) conditions. RNA was extracted and subjected to independent semi-competitive RT-PCR analyses using trans-intron PCR primers, together with primers for the house-keeping gene β-2 microglobulin (β2M). Representative gels for each gene at the 72 hour time point were shown, and fold induction of a target gene by treatment was calculated by ascribing the ratio between the level of expression of a target gene and that of β2M as 1.0 for control treated samples. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
TSA and IFNα co-operatively suppress tumour-driven angiogenesis in neuroblastoma bearing transgenic MYCN mice. A. Photomicrographs of neuroblastoma tumour tissue sections from homozygous MYCN transgenic mice treated with either control, TSA, IFNα, or TSA and IFNα, which were subject to immunohistochemical studies using an anti-PECAM-1 antibody. Arrows indicate PECAM-1 positive microvessels (brown colored). B. Quantitation of the number of PECAM positive microvessels per 40× high power field in neuroblastoma tumour cross-sections. *** p < 0.001.
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