Enhancing the anti-angiogenic action of histone deacetylase inhibitors
© Kuljaca et al; licensee BioMed Central Ltd. 2007
Received: 25 July 2007
Accepted: 25 October 2007
Published: 25 October 2007
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.
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 p21WAF1 expression and response to TSA and IFNα 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 IFNα. In vitro 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.
Our results indicate that combination TSA and IFNα 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.
Acetylation and deacetylation of histones by histone acetyltransferases and histone deacetylases (HDACs) alter chromatin structure and modulate transcriptional regulation (reviewed in [1–3]. Inhibitors of HDACs (HDACIs) are emerging as a new class of anticancer agents. HDACIs induce cancer cell differentiation, growth arrest, programmed cell death, and inhibit tumour-driven angiogenesis [1, 3]. Clinical trials with HDACIs in cancer patients demonstrate that HDACI treatment leads to tumour regression and symptomatic improvement in some heavily pre-treated and multiply relapsed patients, with a surprisingly low side-effect profile [1, 4]. However, a large proportion of the patients are not sensitive to the treatment, demonstrating the need to examine the effectiveness of HDACIs in combination with other anti-cancer agents.
Angiogenesis is vital for tumor progression and metastasis [5, 6]. As anti-angiogenic therapy is generally less toxic and better tolerated than conventional cytotoxic chemotherapy, strategies which combine anti-angiogenic agents with other anti-cancer drugs have been the focus of current clinical trials to widen the therapeutic index. The interferons (IFNs) are a family of naturally occurring cytokines with anti-proliferative and anti-angiogenic effects [7, 8]. Through inhibiting pro-angiogenic gene expression and acting directly on endothelial cells, α-interferon (IFNα) suppresses angiogenesis and tumour growth in vitro and in vivo [7, 9]. Rapamycin and its derivatives also inhibit tumour cell proliferation and angiogenesis by acting on the mammalian target of rapamycin and suppressing the transcriptional activity of pro-angiogenic hypoxia-inducible factor 1α (HIF1α), (reviewed in ). While clinical trials with IFNα, rapamycin and its derivatives used as single agents have shown some effects, none of the drugs are effective alone in the majority of patients.
It has been reported that a combination therapy with the HDACI, valproate (VPA), and IFNα exerts synergistic anti-cancer effects in neuroblastoma BE(2)-C cells both in vitro and in vivo [11, 12]. Here we evaluated the anticancer actions of combination therapy with HDACIs (Trichostatin A [TSA] or VPA) and anti-cancer agents with anti-angiogenic function (IFNα, rapamycin), and, sought to determine their mechanism of action.
TSA and IFNα exerted co-operative cytotoxic effects in cancer cell lines from a range of different tissue origins
SAHA and IFNα exerted co-operative cytotoxic effects in cancer cell lines, but not in normal cells
The effects of other HDACIs and anti-cancer agents used in combination
Absence of p21WAF1 expression correlated with sensitivity to TSA and IFNα combination therapy
To determine the role of p21WAF1 expression in cancer cell sensitivity to TSA and IFNα combination therapy, MCF-7 cells were transfected with control, scrambled siRNA or siRNA specifically targeting p21WAF1, and, then treated with control, TSA and/or IFNα. RT-PCR and immunoblot analysis revealed that p21WAF1 mRNA and protein were knocked down by approximately 75% by the p21WAF1 siRNA, compared with scrambled control (Figure 4B). The p21WAF1 siRNA significantly increased the sensitivity of MCF-7 cells to TSA and IFNα alone, and, in combination, as measured by cell viability assays (p < 0.01) (Figure 4C).
HDACI and IFNα co-operatively inhibit endothelial cell functions and pro-angiogenic gene expression in cancer cells in vitro
We next evaluated whether the combination of TSA [15, 16] and IFNα [7, 17] represses pro-angiogenic gene expression, as measured by RT-PCR, in neuroblastoma BE(2)-C cells. Compared with treatment with TSA or IFNα alone, the combination therapy significantly down-regulated gene expression of HIF1α, VEGF and MMP-9 under normoxic conditions at 72 hours after treatment, while no co-operative effects were observed on the expression of MMP-2, activin A, thrombospondin-1, von Hippel-Lindau protein and bFGF (data not shown). Suppression of HIF1α, VEGF and MMP-9 gene expression by TSA and IFNα was more significant, when compared with TSA or IFNα alone, under hypoxic conditions (Figure 5E). In the case of HIF1α and VEGF, IFNα alone repressed gene expression, however, the combination still had a more significant repressive effect, compared with IFNα alone (p < 0.05). Although MMP-9 gene expression was stimulated by IFNα and TSA alone, the combination suppressed its expression, when compared with control-treated samples (p < 0.05).
TSA and IFNα co-operatively suppress tumour-driven angiogenesis in neuroblastoma-bearingN-Myc transgenic mice
HDACIs have shown great promise in clinical trials in cancer patients. However, a majority of patients have been insensitive to the treatment. In this study, we found that the combination of IFNα with the HDACI TSA induced co-operative cytotoxic effects in almost all cancer cell lines of diverse tissue types, and demonstrated little cytoxicity in normal non-malignant cells. The combination of IFNα with the HDACI SAHA, already in clinical use, also exerted co-operative anti-cancer effects, with little effect on normal cells. The combination of IFNα with another HDACI, VPA, was less effective than IFNα and TSA, but more effective than VPA and rapamycin. These results suggest that HDACI and IFNα combination therapy may be an effective anti-cancer strategy for future clinical trials.
Our data identified p21WAF1 expression as a key factor responsible for cancer cell resistance to the cytotoxic effects of combination HDACI and IFNα therapy. While IFNα can both induce or suppress p21WAF1 gene transcription in different cells , it is the most common transcriptional target of HDACIs (reviewed in ). Previous literature suggested that up-regulation of p21WAF1 by HDACIs may mediate HDACI-induced cell cycle arrest and growth inhibition . However, recent publications have cast doubt on the role of p21WAF1 in the action of HDACIs, and, conversely demonstrated that inducible p21WAF1 reduced HDACI-induced cell death [20–24]. Our data suggests p21WAF1 expression in some cancer cells acts as a resistance factor for the cytotoxic effects of TSA and IFNα combination therapy.
The individual effects of HDACIs and IFNα on angiogenesis predict a co-operative therapeutic role in blocking tumour angiogenesis. Expression of HDACs is often up-regulated under angiogenic stimuli such as hypoxia in cancer cells, and HDACIs can suppress HIF1α expression and its down-stream targets, including VEGF. HDACIs have been recently demonstrated to inhibit endothelial cell migration, invasion, vascular sprouting in vitro, and vasculature formation in animal models of cancer [14, 16, 26]. IFNα can repress VEGF and MMP-9 gene expression, endothelial cell functions, and, inhibit tumour-driven angiogenesis in vivo [9, 27]. In our endothelial cell migration experiments, we found in contrast, that either TSA or IFNα alone stimulated migration. We cannot fully explain the discrepancy between our data and previously published migration assays , however, this may be due to different characteristics of the migration chamber used. Importantly, the combination of HDACI and IFNα suppressed all endothelial cell functions, indicating a possible role for this drug combination as a therapy for cancer patients at the point of minimal residual disease.
In summary, we have found that the combination of HDACIs, TSA, SAHA and VPA, with IFNα have significant cytotoxic effects on a wide variety of cancer cells, with little toxicity to normal non-malignant cells. Inhibition of p21WAF1 expression sensitizes p21WAF1-expressing cancer cells to the combination therapy. Furthermore, HDACI and IFNα co-operatively suppress pro-angiogenic gene expression in cancer cells, multiple endothelial cell functions in vitro, and tumour-driven vasculature formation in vivo. Our results provide a basis for further in vivo studies and eventual clinical trials using the combination of HDACIs and IFNα.
Cell culture and reagentsThe neuroblastoma cell line, BE(2)-C, was generously supplied by Dr J Biedler (Memorial Sloan-Kettering Cancer Center, NY, USA). Breast (MCF-7 and MDA-MB-468), lung (Calu-6 and H460), prostate (DU-145 and LNCaP), and, colon (HT-29 and Caco-2) cancer cells were purchased from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, with the exception of H460 and LNCaP, which were cultured in Roswell Park Memorial Institute Medium, supplemented with 10% fetal calf serum. All cell lines were maintained in a humidified incubator at 37°C and 5% CO2 in air.
TSA (Sigma, St. Luis, MO, USA) was dissolved in ethanol, and SAHA (BioVision, Mountain View, CA) in dimethylsulfoxide (Sigma). IFNα (Sigma) was diluted in serum free cell culture medium and aliquoted as a stock solution of 100 000 units/ml. For studies in animals, TSA was dissolved in dimethyl sulfoxide (Sigma) and further diluted with saline solution to give the final concentration of 30% dimethyl sulfoxide and 1 mg/ml TSA.
Endothelial cell culture
Human umbilical vein endothelial cells (HUVECs) were a gift from Dr K MacKenzie (Children's Cancer Institute Australia, Sydney, Australia). HUVECs were maintained in 0.1% gelatin coated tissue culture flasks or wells with medium 199 (Invitrogen, Carlsbad, CA, USA) supplemented with 20% fetal bovine serum, 5% human serum (Sigma), 10 U/ml heparin (Pharmacia & Upjohn, Peapack, NJ, USA), 5 ng/ml basic fibroblast growth factor (bFGF) (Sigma) and 20 ug/ml endothelial growth factor (Roche, Mannheim, Germany). Only passages 5 and 6 were used in the experiments. Hypoxic conditions were maintained in a chamber filled with 1% oxygen.
Alamar blue cell viability assay
After plating in 96 well plates, cells were allowed to attach for 24 hours, followed by treatment with various drugs for 72 hours. Before the end of treatment, cells were incubated with Alamar blue (Invitrogen) for 5 hours, and plates were then read on a micro-plate reader at 570/595 nm. Relative cell viability was calculated according to the readings and expressed as optical density (OD) absorbance units.
Twenty four hours after treatment with control, TSA and/or IFNα, protein was extracted from whole cells, separated by electrophoresis, and transferred onto nitrocellulose membrane. Membranes were incubated with mouse anti-human p21WAF1 antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) (1:1000), followed by goat anti-mouse antibody (1:2000) conjugated with horseradish peroxidase. Chemiluminescent detection was performed using SuperSignal reagents (Pierce). Membranes were then re-probed with an anti-β-actin antibody (Pierce), as a loading control.
siRNA transfectionMCF7 cells were transfected with a validated scrambled siRNA or siRNA specifically targeting p21WAF1 (SmartPool siRNA CDKN1A, Dharmacon Research, Lafayete, CO) with Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's recommendation. Cells were lysed, and RNA or protein extracted 24 hours later for Reverse Transcription-polymerase Chain Reaction (RT-PCR) or immunoblot analysis of siRNA transfection efficacy.
Semi-quantitative competitive RT-PCR
Semi-quantitative competitive RT-PCR was carried out as described previously  to analyse siRNA transfection efficiency in MCF-7 cells and the effect of TSA and/or IFNα treatment on angiogenic gene expression in BE(2)-C cells. Specific primers used for PCR were as follows: 5'-CAGCAGAGGAAGACCATGTG-3' and 5'-GGCGTTTGGAGTGGTAGAAA-3' for p21WAF1; 5'-TTACAGCAGCCAGACGATCA-3' and 5'-ATTGCCCCAGCAGTCTACAT-3' for HIF1α; 5'-CCTTGCTGCTCTACCTCCAC-3' and 5'-ATGATTCTGCCCTCCTCCTT-3' for vascular endothelial growth factor (VEGF); 5'-TTCCCTGGAGACCTGAGAAC-3' and 5'-AGGGACAGTTGCTTCTGGAG-3' for metalloproteinase-9 (MMP-9); 5'-ACCCCCACTGAAAAAGATGA-3' and 5'-ATCTTCAAACCTCCATGATG-3' for β2-microglobulin (β2M).
Endothelial cell migration assay
HUVEC migration towards the chemo-attractant, VEGF (Sigma), was tested using a BD Biosciences Fluroblok (Becton Dickinson) endothelial cell migration system according to the manufacture's guidelines. Cells were labeled with 1 μM Cell Tracker Green CMFDA fluorescence solution (Invitrogen) for 30 minutes, and migrated through filters into 24 well plates. Thereafter the plate was read with a Fluroscence plate reader at 492/517 nm. The relative cell number was calculated according to the readings and expressed as optical density (OD) absorbance units.
Endothelial cell invasion assay
HUVEC invasion through matrigel towards the chemo-attractant, VEGF, was investigated using BD BioCoat, growth factor-reduced Matrigel, endothelial cell invasion chambers (Becton Dickinson), according to the manufacturer's guidelines. Endothelial cells which invaded through the matrigel to the other side of the inserts, were fixed and stained with Diff Quick staining kit (Baxter) and photographed. The number of cells per 20× objective field was counted under an inverted microscope.
Vascular sprouting (capillary tubule formation) assay
The vascular sprouting assays were performed on 24 well plates coated with 250 μl of polymerized, growth factor-reduced Matrigel matrix (Becton Dickinson) per well. HUVECs were plated on Matrigel and treated with control, TSA and/or IFNα for 18 hours. Quantification of vascular sprouting was determined by counting the number of complete branches per branching point.
Animal model studies
As soon as tumors were confirmed by abdominal palpation, MYCN homozygous transgenic mice , were randomized to four groups (n = 5/group) and injected intraperitoneally daily for 7 days with control, TSA at 20 mg/kg of body weight, mouse IFNα at 1 × 106 IU/kg body weight, or TSA and IFNα. Mice were sacrificed at the end of the week of treatment. Tumors were then removed, formalin-fixed and paraffin-embedded. All studies involving animals were approved by the animal care and ethics committee of the University of New South Wales, Sydney, Australia.
Mouse tissue sections were incubated with goat anti-platelet endothelial cell adhesion molecule 1 (PECAM-1) antibody (1:500) (Santa Cruz Biotechnology), followed by incubation with biotinylated rabbit, anti-goat antibody (1:500) and streptavidin-horseradish peroxidase. Endothelial cells were visualised with 3,3'-diaminobenzidine solution, and micro-vessels were quantified as described previously .
All data for statistical analyses were presented as mean ±standard error. Differences were analyzed for significance using ANOVA among groups. A probability value of 0.05 or less was considered significant.
basic fibroblast growth factor
histone deacetylase inhibitor
hypoxia-inducible factor 1α
human umbilical vein endothelial cells
matrix metalloproteinase 9
platelet endothelial cell adhesion molecule 1
vascular endothelial growth factor
This work was supported by National Health and Medical Research Council, Cancer Institute New South Wales and Cancer Council New South Wales. Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital.
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