Enhancing the anti-angiogenic action of histone deacetylase inhibitors

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Trichostatin A (TSA) and α-interferon (IFNα) 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 p21^WAF1expression and response to TSA and IFNα treatment. Moreover, inhibition of p21^WAF1expression in a p21^WAF1-expressing breast cancer cell line by a specific siRNA increased the cytotoxic effects of TSA and IFNα. <it>In vitro </it>assays of endothelial cell function showed that TSA and IFNα decreased endothelial cell migration, invasion, and capillary tubule formation, without affecting endothelial cell viability. TSA and IFNα co-operatively inhibited gene expression of some pro-angiogenic factors: vascular endothelial growth factor, hypoxia-inducible factor 1α and matrix metalloproteinase 9, in neuroblastoma cells under hypoxic conditions. Combination TSA and IFNα therapy markedly reduced tumour angiogenesis in neuroblastoma-bearing transgenic mice.</p> <p>Conclusion</p> <p>Our results indicate that combination TSA and IFNα therapy has potent co-operative cytotoxic and anti-angiogenic activity. High basal p21^WAF1expression appears to be acting as a resistance factor to the combination therapy.</p

Mechanisms and treatment strategies to overcome resistance to non-cytotoxic therapy in cancer

As anti-cancer agents, retinoid induce cell growth arrest and differentiation, while HDACIs cause cell differentiation, growth inhibition, death and inhibit angiogenesis in many cancer types. However, a proportion of patients respond poorly to these therapies.My studies, presented here, aimed to improve the anti-cancer effects of these agents by identifying key factors which mediate cancer cell sensitivity or resistance to their action. In this study I have found that the clinically used retinoid, 13-cis RA, exerts its anti-cancer signal in a manner similar to atRA, by modulating the transcriptional response of retinoid-regulated genes.HDACI-induced cytotoxicity is significantly enhanced when combined with IFN&#945; in 8 out of 9 cancer cell lines from various organ origins. Sensitivity to the combination treatment correlated with an absence of basal p21 protein expression, and cell cycle arrest. Knocking-down p21 gene expression further sensitized cancer cells to the combination therapy. Moreover, IFN&#945; and HDACI co-operatively inhibited pro-angiogenic gene expression in cancer cells, and the combination therapy decreased endothelial cell migration, invasion, and capillary tubule formation.Further experiments on p21 as a resistance factor to anti-cancer treatment demonstrated that conditioned media from breast cancer MCF-7 cells transfected with p21 siRNA, induced significantly less endothelial cell migration, invasion and vascular sprouting, compared with media from cells transfected with scrambled siRNA. LC/MS analysis of the conditioned media revealed that Trx secretion was significantly reduced after p21 knockdown. The reduction in Trx secretion following p21 knockdown was due to a direct effect of p21 siRNA on the expression of intracellular TBP2 in neuroblastoma, prostate and lung cancer cells. Consistent with this result, media from MCF7 cells transfected with TBP2-specific siRNA alone, promoted endothelial cell invasion and vascular sprouting, Trx knockdown resulted in opposite effects, and the anti-angiogenic effect of p21 siRNA was offset by simultaneous TBP2 siRNA transfection. ChIP assay revealed that p21 directly bound to an E2F1-bindng site in the TBP2 gene promoter. These data indicate that p21 promoted tumour-driven angiogenesis through transcriptional repression of TBP2. Collectively, my experiments indicate several potential treatment targets directed toward enhancing the effectiveness of HDACIs and retinoids

SAHA and IFNα exerted co-operative cytotoxic effects in cancer cell lines, but not in normal non-malignant cells

<p><b>Copyright information:</b></p><p>Taken from "Enhancing the anti-angiogenic action of histone deacetylase inhibitors"</p><p>http://www.molecular-cancer.com/content/6/1/68</p><p>Molecular Cancer 2007;6():68-68.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2173905.</p><p></p> 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

TSA and IFNα co-operatively suppress tumour-driven angiogenesis in neuroblastoma bearing transgenic MYCN mice

<p><b>Copyright information:</b></p><p>Taken from "Enhancing the anti-angiogenic action of histone deacetylase inhibitors"</p><p>http://www.molecular-cancer.com/content/6/1/68</p><p>Molecular Cancer 2007;6():68-68.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2173905.</p><p></p> 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

HDACI and IFNα co-operatively inhibit endothelial cell functions, and pro-angiogenic gene expression in cancer cells under hypoxic conditions

<p><b>Copyright information:</b></p><p>Taken from "Enhancing the anti-angiogenic action of histone deacetylase inhibitors"</p><p>http://www.molecular-cancer.com/content/6/1/68</p><p>Molecular Cancer 2007;6():68-68.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2173905.</p><p></p> 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% O) 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

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

<p><b>Copyright information:</b></p><p>Taken from "Enhancing the anti-angiogenic action of histone deacetylase inhibitors"</p><p>http://www.molecular-cancer.com/content/6/1/68</p><p>Molecular Cancer 2007;6():68-68.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2173905.</p><p></p> 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

Absence of p21expression correlated with sensitivity to TSA and IFNα combination therapy

<p><b>Copyright information:</b></p><p>Taken from "Enhancing the anti-angiogenic action of histone deacetylase inhibitors"</p><p>http://www.molecular-cancer.com/content/6/1/68</p><p>Molecular Cancer 2007;6():68-68.</p><p>Published online 25 Oct 2007</p><p>PMCID:PMC2173905.</p><p></p> 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- p21antibody, and, an anti-actin antibody as a loading control. B. MCF-7 cells were transfected with control scrambled or p21siRNA 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 p21gene 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