Advertisement
Cancer Therapy: Preclinical

Targeting Tumor Angiogenesis with Histone Deacetylase Inhibitors: the Hydroxamic Acid Derivative LBH589

David Z. Qian, Yukihiko Kato, Shabana Shabbeer, Yongfeng Wei, Hendrik M.W. Verheul, Brenda Salumbides, Tolib Sanni, Peter Atadja and Roberto Pili
DOI: 10.1158/1078-0432.CCR-05-1132 Published January 2006

Abstract

Purpose: Angiogenesis is required for tumor progression and represents a rational target for therapeutic intervention. Histone deacetylase (HDAC) inhibitors have been shown to have activity against various tumor cell types by inhibiting proliferation and inducing apoptosis both in vitro and in vivo. HDAC inhibitors have also been reported to inhibit angiogenesis. The goal of this study was to characterize the antiangiogenic and antitumor activity of a recently developed HDAC inhibitor, the hydroxamic derivative LBH589.

Materials and Methods: To evaluate the antiangiogenesis activity of LBH589, we did cell cycle analysis, cell proliferation, tube formation, invasion assays in vitro, and Matrigel plug assay in vivo. To determine the antitumor activity of LBH589, we established human prostate carcinoma cell PC-3 xenografts in vivo. To evaluate the effect of LBH589 on endothelial signaling pathways, gene expression, and protein acetylation, we did Western blots and reverse transcription-PCR in human umbilical vein endothelial cells (HUVEC). Immunohistochemical analysis was done to evaluate new blood vessel formation in vivo.

Results: LBH589 induced acetylation of histone H3 and α-tubulin protein in HUVECs. Histone and nonhistone protein acetylation correlated with induction of G2-M cell cycle arrest, inhibition of HUVEC proliferation, and viability. Noncytotoxic concentrations of LBH589 inhibited endothelial tube formation, Matrigel invasion, AKT, extracellular signal-regulated kinase 1/2 phosphorylation, and chemokine receptor CXCR4 expression. In vivo dosing of mice with LBH589 (10 mg/kg/d) reduced angiogenesis and PC-3 tumor growth.

Conclusion: This study provides evidence that LBH589 induces a wide range of effects on endothelial cells that lead to inhibition of tumor angiogenesis. These results support the role of HDAC inhibitors as a therapeutic strategy to target both the tumor and endothelial compartment and warrant the clinical development of these agents in combination with angiogenesis inhibitors.

  • Histone deacetylase inhibitor
  • tumor angiogenesis
  • gene regulation
  • prostate cancer

Histone deacetylase (HDAC) inhibitors represent an emergent class of anticancer drugs. The targets of these agents are enzymes (HDACs), which induce histone and nonhistone protein deacetylation at the lysine residues, chromatin condensation, and modulation of gene expression. Altered HDAC activity is associated with cancer, and inhibitors targeting HDACs have been shown to induce tumor cell cytostasis, differentiation, and apoptosis in various hematologic and epithelial tumors (1, 2). Five classes of HDAC inhibitors have been characterized, including short-chain fatty acids (i.e., sodium butyrate), hydroxamic acids (i.e., thrichostatin A, SAHA, and LAQ824), cyclic peptides containing a 2-amino-8-oxo-9,10-epoxy-decanoyl moiety (i.e., trapoxin A), cyclic peptides without the 2-amino-8-oxo-9,10-epoxy-decanoyl moiety (i.e., FK228), and benzamides (i.e., MS-275). These agents induce a dose-dependent inhibition of either class I (1, 2, 3, and 8) or class II (4-10) HDACs, or both. Depending on the specific drug, the cancer cell line tested, the dose, and time of exposure, HDAC inhibitors can induce G1 or G2 cell cycle arrest and/or cell death (3, 4).

The reported p53-independent induction of the p21 gene and protein expression is probably due to the nature of epigenetic regulation of the p21 promoter and, along with histone lysine acetylation, represents a common molecular observation associated with drug exposure (5, 6). There have also been reports of up-regulation of proapoptotic pathways and down-regulation of prosurvival pathways associated with the HDAC inhibitors, which depend on the agent and the tumor cell line used (7, 8).

We have previously shown that the HDAC inhibitors phenylbutyrate and MS-275 induce the expression of the putative tumor suppressor gene RARβ2 and increase the retinoid sensitivity in retinoid-resistant prostate and renal cell carcinoma cell lines (9, 10).

In addition to the inhibitory effect on cancer cell proliferation, HDAC inhibitors have been reported to inhibit the process of new capillary blood vessel formation or angiogenesis (6, 11–13). Tumor-initiated angiogenesis takes place following an “angiogenic switch.” During the initial avascular tumor growth, there is an accumulation of hypoxia-inducible factor-1α (HIF-1α) protein, which activates an array of gene transcriptions, including vascular endothelial growth factor (VEGF), one of the most proangiogenic factors (14). VEGF-A (the principal isoform of VEGF) binds to VEGF receptor 2 (VEGFR2) on the surface of quiescent endothelial cells in the surrounding vessel walls and triggers the signaling pathways for endothelial activation and downstream angiogenesis (15).

During the multistep angiogenesis process, several ligand/receptor interactions are required. The interaction of angiopoietins and endothelial receptor Tie-2 affects endothelial cell survival and vessel stability (16). Stromal-derived factor-1 (SDF-1) and endothelial chemokine receptor CXCR4 influence the homing of mobilized/activated endothelial cells or endothelial progenitor cells to active sites of neovascularization (17, 18). Overall, the combined net result of ligand/receptor interaction activates intracellular pathways, leading to endothelial cell survival, proliferation, mobilization, and invasion of the extracellular matrix. This culminates in recognition and homing to the sites of new blood vessel formation.

In our previous work, we identified two different HDAC inhibitors, phenyl butyrate (short-chain fatty acid) and LAQ824 (hydroxamic acid), which have antiangiogenesis activity both in vitro and in vivo (3, 6). Angiogenesis inhibition induced by LAQ824 was associated with modulation of angiogenesis-related genes both in cancer cells (inhibition of HIF-1α and VEGF) and in endothelial cells (inhibition of Tie-2 and survivin, an inhibitor of apoptosis). Other groups have also reported that other HDAC inhibitors, including SAHA, TSA, FK228, valproic acid, and apicidin, have antiangiogenic activity (11–13).

In the current study, we investigated the antiangiogenic effect of a recently developed HDAC inhibitor, LBH589 that is currently in phase I clinical trial (19, 20). We identified altered gene expression and protein phosphorylation induced by LBH589 in endothelial cells. This data lead us to explore the potential of LBH589 as therapeutic agent with dual activity against both tumor proliferation and angiogenesis.

Materials and Methods

Cell lines, reagents, and animals. Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Walkersville, MD) and cultured in complete EGM-2 medium containing EGM-2 bullet kit (Cambrex) with growth factors and 2.5% fetal bovine serum supplied by the vendor. All experiments were done using endothelial cells between passages 3 and 8. A human prostate (PC-3) carcinoma cell line was originally obtained from the American Type Culture Collection (Manassas, VA), maintained in RPMI 1640 with l-glutamine supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). LBH589 was kindly provided by Novartis Pharma AG (Cambridge, MA). Stock solutions for in vitro assays were prepared in DMSO with final concentrations of <0.01%. For the in vivo animal experiments, LBH589 was dissolved in saline solution. Six-week-old male athymic nude mice and C57/BL6J mice (National Cancer Institute, Bethesda, MD) were housed under pathogen-free conditions. All animal studies were done according to the protocol approved by the Animal Care and Use Committee at the Johns Hopkins University.

Cell viability assay. The effect of LBH589 on HUVEC growth and survival was assessed by a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay (Roche, Indianapolis, IN) as previously described (6). In brief, 1 × 104 cells were seeded into 96-well plates. After an overnight incubation, the medium was replaced by fresh EGM-2 with either complete growth factors (EGM-2 bullet kit), 50 ng/mL VEGF-A (R&D Systems, Minneapolis, MN), or 50 ng/mL basic fibroblast growth factor (bFGF; R&D Systems). LBH589 was added at 0 to 800 nmol/L final concentrations. After 48 hours of incubation, the viable cells (a net result of both cell growth and cell death) were measured by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt reagent at UV 490 nm as described by the manufacturer's protocol. The UV readings of solvent-treated controls were normalized to 100%, and the readings from LBH589-treated cells were expressed as % controls.

Cell proliferation assay. The proliferation assay was run as previously described (3, 6). Briefly, cancer and endothelial cells were seeded (1 × 104 per well) in six-well plates and incubated at 37°C, 5% CO2 for 24 to 48 hours. The medium was then replaced with appropriate basal medium without growth factors. After overnight starvation, cells were counted in triplicate with a Coulter counter and designated as day 0 values. Then, cells were treated with different concentrations of LBH589 (triplicate) in complete media with growth factors and serum. Forty-eight hours later, the viable cells from each condition were harvested and counted by Coulter counter. Cell number at day 0 was normalized to 100%, and cell proliferation following 48 hours of treatment was adjusted to the % day 0 controls. The experiments were repeated thrice with similar results.

Cell cycle and cell death analysis. Proliferating HUVECs were starved overnight and then treated with increasing concentrations of LBH589 with complete media for 24 hours. After drug treatment, both starved and treated cells were harvested, fixed, and stored in 70% ice-cold ethanol. The percentage of the cell cycle that was in G0-G1, S, and G2-M phase was quantitated by using a propidium iodide–based cellular DNA flow cytometry analysis kit (Roche) following manufacturer's instructions. For cell death analysis, Annexin V and propidium iodide costaining followed by flow cytometry was done according to manufacturer's instructions (BD Biosciences, San Jose, CA).

Matrigel angiogenesis assay in vitro (tube formation). HUVECs were cultured in complete EGM-2 media before being plated in 24-well plates (5 × 104 per well) previously coated with 300 μL of growth factor–reduced Matrigel (BD Biosciences), in the presence of LBH589 or solvent control. The morphology of capillary-like structures formed by HUVECs 15 hours after culturing was visualized using an inverted microscope (Zeiss Axioskop) and photographed with a digital camera. Tube formation was analyzed with an imaging system (Image-Pro) as previously described (6).

Matrigel invasion assay. The Matrigel invasion assay was done with precoated Matrigel inserts following manufacturer's instructions (BD Biosciences). Proliferating HUVECs were pretreated with indicated doses of LBH589 overnight, and then the cells were harvested and resuspended in basal EGM-2 media with 0.5% fetal bovine serum and the indicated dose of LBH589. An equal number of cells (4 × 105) were added to the invasion chambers in triplicate. The chemoattractants were either VEGF-A or SDF-1a (50 ng/mL, R&D Systems). Twenty-four hours later, the membranes containing migrated cells were fixed and stained, and cell numbers were counted under a microscope. Five random fields were chosen for each membrane, and the results were expressed as migrated cells per field for each condition.

Reverse transcription-PCR and Western blot analysis. The details of reverse transcription-PCR and Western blots have been described previously (6). The antibodies for AKT and extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphorylation were purchased from Cell Signaling (Beverly, MA); antibodies for HIF-1α and CXCR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies for histone H3 and acetylated H3 were purchased from Upstate (Lake Placid, NY); and antibodies for tubulin and acetylated tubulin were purchased from Sigma (St. Louis, MO).

Matrigel angiogenesis assay in vivo. Four to 6-week-old C57/BL6J mice were pretreated with LBH589 (10 mg/kg/d, i.p.) for 3 days and injected s.c. in the abdomen with 500 μL of Matrigel (BD Biosciences, San Jose, CA) supplemented with VEGF-A (150 ng/mL) and bFGF (50 ng/mL; R&D Systems). Treatment was continued for 10 days after the Matrigel injection. Then, the mice were sacrificed, and the plugs were retrieved for immunohistochemical analysis. The plugs were fixed in PBS-buffered 10% formalin containing 0.25% glutaraldehyde and were processed for Masson's Trichrome staining.

Tumor growth in vivo. PC-3 tumor cells were resuspended in HANKS solution and mixed with Matrigel (1:1) in a final volume of 0.1 mL. Approximately two million cells were injected bilaterally and s.c. into male athymic mice. Once the tumors became palpable (50-100 mm3), mice were randomly assigned to control group (n = 10) and experimental group (n = 10). The experimental group was treated with LBH589 (10 mg/kg/d, i.p.). Control animals were given vehicle consisting of saline solution with 5% DMSO and 1% Tween 80. The mice were treated 7 days/wk. Tumor volume was measured with a caliper and calculated according to the formula:MathThe efficacy of treatment was evaluated by the change in tumor volume during treatment period. Following 3 to 4 weeks of treatment, the mice were sacrificed, and the tumors were harvested for histologic studies and Western blots.

In vivo cell death analysis. DeadEnd Fluorometric Terminal Deoxynucleotidyl Transferase–Mediated Nick-End Labeling System (Promega, Madison, WI) was used to evaluate the cell death in sectioned tumor xenografts from control and LBH589-treated animals according to the manufacturer's instructions. The green signal was considered being positive for cell death. The positive controls were obtained by treating cells with DNase, which causes DNA fragmentation.

Immunohistochemistry study. Frozen tissue was generated from each tumor to quantify differences in microvessel density between control and experimental groups. Sections were incubated (18 hours at 4°C) with anti-CD31 antibody (PharMingen, San Diego, CA), a specific marker for endothelial cells. Sections were incubated with a secondary biotin-conjugated rabbit anti-goat IgG antibody (1:100) for 30 minutes at room temperature. Sections were incubated with avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA) as per manufacturer's instructions. Sections were then incubated with 3,3′-diaminobenzidine solution, washed, and counterstained with methyl green.

Imaging analysis for angiogenesis. Measurement of the level of angiogenesis in the in vivo Matrigel plug experiment was based on the Masson's Trichrome staining, and CD31 staining was used in the PC-3 tumor experiment. The regions containing the most intense area of neovascularization (“hotspots”) were chosen for analysis. Eight hotspots were identified for each Matrigel or tumor section. The ImagePro Plus analysis system was used to quantify the percentage of area occupied by the vessel-like structures in each field. The mean and the SE of mean from each group were compared. The negative control was obtained by tissue staining with secondary antibody only.

Statistical analysis. Differences between the means of unpaired samples were evaluated by the Student's t test using the SigmaPlot and SigmaStats program. Ps < 0.05 were considered statistically significant. All statistical tests were two sided.

Results

LBH589 increased histone and nonhistone protein acetylation and inhibited HUVEC proliferation in vitro. The hydroxamic acid HDAC inhibitor LBH589 was very effective in increasing the acetylation level of histone H3 protein in endothelial cells (Fig. 1A). LBH589 treatment of HUVECs also increased the acetylation level of nonhistone proteins. α-Tubulin is deacetylated by cytoplasmic HDAC6 in several cell types of mouse and human origin (21, 22). In the current study, the acetylation level of α-tubulin was increased after LBH589 treatment (Fig. 1A), and this is an indication of HDAC6 inhibition. The increase in protein acetylation was also associated with perturbation of the endothelial cell cycle. Using our experimental tissue culture conditions, growth factor starvation induced HUVECs cell cycle to arrest in the G0-G1 phase. Twenty-four hours following treatment with complete EGM-2 growth media, the previously starved HUVECs showed a decrease in the G0-G1 phase and an increase in the S-phase population (Fig. 1B). However, simultaneous incubation with increasing doses of LBH589 significantly induced G2-M arrest and decreased S-phase cells (Fig. 1B).

Fig. 1.
Fig. 1.

Effect of LBH589 on HUVECs protein acetylation and cell cycle. A, proliferating HUVECs were treated for 24 hours with increasing doses of LBH589 (i.e., 0, 50, 100, 200 nmol/L). Total proteins were isolated, and the acetylations of histone H3 and α-tubulin were evaluated by Western blot analysis. B, proliferating HUVECs were first starved overnight and then restimulated with complete media (EGM-2 bullet kit) and increasing doses of LBH589 for 24 hours. Then, cell cycle analysis was done by propidium iodide staining and flow cytometry. % Cells in G0-G1, S, and G2-M. Points, mean (n = 3) from triplicate experiments; bars, SE. *, P < 0.05 versus solvent-treated controls.

To investigate the effects of different growth factors on LBH589 inhibition of HUVECs, proliferating cells were maintained in either complete EGM-2 growth media, or EGM-2 basal media with 50 ng/mL bFGF/0.5% fetal bovine serum, or 50 ng/mL VEGF-A/0.5% fetal bovine serum. Under these culture conditions, cells were treated with increasing doses of LBH589 for 48 hours, and inhibition of cell viability and growth was evaluated by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay (Fig. 2A). These results showed that LBH589 had similar dose-dependent inhibitory effects on HUVECs regardless of the growth factor used to induce cell proliferation. Using the complete EGM-2 bullet kit growth media, the inhibition of endothelial cell proliferation was also determined by counting viable cell numbers as previously described (6). Following 48 hours of treatment, the solvent-treated control HUVECs had a 1.5-fold increase in cell number, whereas ≤200 nmol/L LBH589 inhibited cell growth. Cytotoxicity was observed with ≥400 nmol/L LBH589 (Fig. 2B) as evidenced by a sharp decrease in cell number. This dose-dependent response was rather unique for endothelial cells. LBH589 inhibited cell proliferation in LNCaP and PC-3 human prostate carcinoma cells at concentrations up to 1 μmol/L (Fig. 2C) without significant cell death and induced primarily G2-M cell cycle arrest (data not shown). This difference between HUVECs and PC-3 was confirmed by caspase assay. A significant induction of caspases was observed only in HUVECs with concentration of >400 nmol/L (Fig. 2D). Costaining of cells with Annexin V and propidium iodine followed by flow cytometry analysis confirmed a significant cell death in HUVECs but not in PC-3 following treatment with LBH589 (Fig. 2E).

Fig. 2.
Fig. 2.

Effect of LBH589 on HUVEC proliferation and viability. A, proliferating HUVECs, cultured in complete media (EGM-2 bullet kit), or 50 ng/mL VEGF-A, or 50 ng/mL of bFGF, were treated with increasing concentrations of LBH589 for 48 hours. Cell growth and viability were measured by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay. Points, mean % solvent-treated control (n = 6); bars, SD. B-C, proliferating HUVECs and prostate carcinoma cells PC-3 were counted by Coulter counter and treated with the indicated doses of LBH589 for 48 hours. The viable cells were counted and expressed as % day 0 control. The cell numbers at the beginning of incubation (day 0) were normalized to 100%. For treated and untreated cells after 48 hours, cell number >100% meant net cell gain, <100% meant net cell loss. Columns, mean from three separate experiments; bars, SE. D, HUVECs were treated as in (B) and (C), and the total homogenous caspases activity from each condition was measured with a commercial kit based on the manufacturer's protocol. Normalized to 1 with solvent-treated controls. Columns, mean of triplicate experiments; bars, SE. *, P < 0.001 versus solvent-treated controls. E, under similar conditions, cells were stained with Annexin V and propidium iodide and analyzed by flow cytometry. Dead cell population from each condition was plotted. Columns, mean of three separate experiments; bars, SD. *, P < 0.05 versus solvent-treated controls.

LBH589 inhibited in vitro angiogenesis. In the presence of high concentrations of VEGF-A, endothelial cells form tube and capillary-like structures on the surface of basement membrane matrices (Matrigel), through a process involving attachment, alignment, and migration. Treatment with a noncytotoxic dose of LBH589 (50-200 nmol/L) significantly reduced this process (Fig. 3A). We also tested the effect of LBH589 on endothelial chemotaxis. Proliferating HUVECs were starved for 24 hours and pretreated with different concentrations of LBH589 for the last 12 hours of starvation. The cells were then tested in the Matrigel invasion assay in the presence of LBH589. The chemokines used in this assay were VEGF-A and SDF-1α, a growth factor that attracts CXCR4 receptor–positive endothelial cells and endothelial precursor cells. Both VEGF-A and SDF-1α induced solvent-treated control cells to invade and migrate through Matrigel (Fig. 3B). Noncytotoxic doses of LBH589 (50-200 nmol/L) induced a dose-dependent inhibition of HUVECs migration and invasion.

Fig. 3.
Fig. 3.

Effect of LBH589 on HUVEC tube formation and Matrigel invasion. A, proliferating HUVECs were plated on the surface of Matrigel in complete media with indicated doses of LBH589 or solvent controls. The tube formation was evaluated 15 hours later. The solvent-treated controls (n = 4) were normalized to 100%. % Controls (n = 4) from drug-treated groups. *, P < 0.05; **, P < 0.01 versus controls. B, HUVECs were pretreated with the indicated dose of LBH589 overnight, and then equal numbers of cells were plated into Matrigel invasion chambers in triplicate with the indicated dose of LBH589 and 0.5% serum. Chemotaxis was achieved with 50 ng/mL VEGF-A or SDF-1α. Twenty-four hours later, the membrane containing cells migrated through the Matrigel was stained, and six random fields from each experiment were counted under the microscope. Columns, mean (n = 4); bars, SD. * and **, P < 0.05 versus solvent-treated control.

LBH589 modulated VEGF-A signaling. VEGF-A has been shown to be an important stimulator for endothelial cell functions, such as survival, proliferation, migration, and differentiation. Following VEGF/VEGFR binding, endothelial cell activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase/ATK signaling pathways mediates the downstream effect of VEGF (23–26). To study the effect of LBH589 on this signaling pathway, proliferating HUVECs were growth factor starved and treated with the indicated doses of LBH589 for 24 hours. A high concentration of VEGF-A (100 ng/mL) was added to restimulate the cells for 10 minutes and 24 hours. The phosphorylation of AKT and ERK1/2 and the transcription of three VEGF-responsive genes were evaluated (Fig. 4A). VEGF rapidly induced the phosphorylation of AKT and ERK1/2 (indicative of AKT and mitogen-activated protein kinase activation) in control cells. LBH589 treatment prevented protein phosphorylation in a dose-dependent fashion but had no effect on total AKT and ERK1/2 protein stability.

Fig. 4.
Fig. 4.

Effect of LBH589 on HUVEC protein phosphorylation, HIF-1α stability, and CXCR4 expression. A, HUVECs were starved and treated with the indicated dose of LBH589 for 24 hours. Then, VEGF-A (100 ng/mL) was added to restimulate HUVECs for 10 minutes. Phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204) were assessed using total cellular protein lysates. AKT and ERK1/2 phosphorylation was determined in growth factor–depleted HUVECs, and following the addition of VEGF-A in untreated and 24-hour LBH589-treated cells. B, HUVECs were treated under the same conditions as in (A). VEGF restimulation was conducted for 24 hours. Total mRNA from cells under different conditions (starvation, VEGF restimulation, and VEGF + 150 nmol/L LBH589) were isolated, and gene expression for Ang-2, survivin, and CXCR4 was evaluated using reverse transcription-PCR. C, HUVECs were treated with 150 nmol/L LBH589 for 24 hours. The hypoxia mimetic CoCl2 was added to induce HIF-1α stabilization for the last 6 hours of treatment. Total cellular protein extracts were used to determine HIF-1α levels. D, proliferating HUVECs were treated with LBH589 and CoCl2 for 24 hours, and CXCR4 expression was evaluated by reverse transcription-PCR and Western blot.

To further assess the effect of LBH589 on VEGF signaling, we measured the gene expression level of angiopoietin-2 (Ang-2), survivin, and CXCR4, which have been reported to be up-regulated by VEGF stimulation in HUVECs (27–29). Following 24 hours of restimulation with VEGF-A, the mRNA level of all three genes was increased by reverse transcription-PCR. LBH589 treatment (150 nmol/L) inhibited Ang-2, survivin, and CXCR4 gene expression (Fig. 4B).

We have previously reported that LAQ824, another hydroxamic HDAC inhibitor, down-regulates Ang-2, survivin, HIF-1α, and VEGF (6). Besides VEGF, another factor capable of up-regulating the expression of CXCR4 is hypoxia, which results in HIF-1α–initiated transcription (30). CXCR4 promoter has been reported to contain hypoxia response elements. Using the hypoxia mimetic cobalt chloride (CoCl2), we first tested whether LBH589 was able to inhibit HIF-1α protein accumulation in HUVECs, similar to LAQ824 in tumor cells (Fig. 4C). Then, following the treatment of HUVECs with LBH589 (150 μmol/L) in the presence of CoCl2, we observed the inhibition of CXCR4 at both mRNA and protein levels (Fig. 4D).

LBH589 inhibited in vivo angiogenesis and prostate tumor growth. The in vitro antiangiogenic property of LBH589 was tested in vivo. First, the effect on neovascularization in responses to VEGF-A and bFGF was evaluated in the Matrigel plug assay. Matrigel supplemented by VEGF-A and bFGF was injected s.c. into the abdomen of C57/BL6 mice. Ten days after the injection, the neovascularization within the Matrigel plug was evaluated by histology. The Matrigel plugs in the control groups were visually bloodier than the LBH589 (10 mg/kg/d)–treated ones, suggesting a higher level of angiogenesis (Fig. 5A). Masson's Trichrome staining of the vasculature within the plugs identified more vessel-like structures within the control than the LBH589 treated (Fig. 5B). By imaging analysis, there was a ∼50% reduction in the number of blood vessels in mice treated with LBH589 compared with controls (Fig. 5C). These data suggested that LBH589 attenuated the angiogenic response initiated by both VEGF and bFGF.

Fig. 5.
Fig. 5.

Effect of LBH589 on Matrigel plug in vivo angiogenesis. In vivo angiogenesis was measured in mice. Matrigel mixed with 150 ng/mL VEGF-A and 50 ng/mL bFGF was injected into the abdomen of C57/BL6J mice. Ten days later, the Matrigel plugs were harvested and compared between solvent control and daily LBH589 (10 mg/kg/d, i.p.)–treated groups. A, the macroscopic appearance of Matrigel plugs from both groups before harvest. B, examples of Masson's Trichrome staining of Matrigel plug cross-sections from control and LBH589-treated mice. C, imaging and statistical analysis of neovascularization. Eight hotspot fields (×200) were taken from each plug. Columns, mean % microvessel area (n = 10); bars, SE. *, P < 0.05 versus control.

In a prostate tumor (PC-3) mouse xenograft model, daily treatment with LBH589 (10 mg/kg/d) induced a statistically significant inhibition of the tumor growth (Fig. 6A), without overt toxicity (no statistically significant weight loss). Western blot analysis of histone protein extract recovered from the tumors revealed induction of histone acetylation in vivo following LBH589 treatment. (Fig. 6B). In vivo tumor angiogenesis was evaluated by immunohistochemical staining of the tumor blood vessel. Using a rat anti-mouse CD31/platelet/endothelial cell adhesion molecule 1 antibody, we observed that control tumors had more CD31+ vessel structures in terms of size and number per field than the LBH589-treated group (Fig. 6C). Quantitative analysis of the percentage of the CD31-positive vessel area in each high power field (×200) confirmed reduction of tumor angiogenesis in the LBH589-treated group (Fig. 6C). Terminal deoxynucleotidyl transferase–mediated nick-end labeling staining of tumor sections for detection of in vivo apoptosis did not reveal significant difference between the control and LBH589-treated animals (Fig. 6D). The apoptotic index calculated based on the ratio of terminal deoxynucleotidyl transferase–mediated nick-end labeling–positive cells per 100 4′,6-diamidino-2-phenylindole–stained cells under high power (×200) fluorescence microscope were 11.5 ± 3.5% SD and 7.5 ± 5.7% SD for control and LBH589-treated group, respectively.

Fig. 6.
Fig. 6.

Effect of LBH589 on prostate tumor growth and angiogenesis in vivo. Prostate carcinoma cells (PC-3) were injected s.c. into male nude mice. As the tumors became palpable, the mice were randomly divided into solvent control and daily LBH589-treated (10 mg/kg/d, i.p.) groups. A, the tumor volumes of two groups during the treatment. Points, mean (n = 10); bars, SE. *, P < 0.05 versus control. B, tumor samples from both control and LBH589-treated group were homogenized, and whole-cell protein lysates were used for Western blot analysis of histone H3 acetylation. C, representative pictures of CD31-positive immunohistochemical staining of tumor cross-sections in the control and LBH589-treated mice and quantitative analysis. Eight hotspot fields (×200) were taken from each tumor section. Columns, mean % microvessel area; bars, SE. *, P < 0.05. D, representative pictures of terminal deoxynucleotidyl transferase–mediated nick-end labeling assay for tumor samples from control and LBH589-treated groups. The green signal was considered being positive for cell death.

Discussion

Preclinical tumor models have shown the potential therapeutic role of HDAC inhibitors. This approach has been targeting tumor cell proliferation and viability by inducing cell cycle arrest and/or cell death. In previous studies, we reported that the HDAC inhibitors phenylbutyrate and LAQ824 have inhibitory activity on endothelial cell proliferation in vitro and inhibited angiogenesis in vivo (3, 6). These initial observations and similar results from other groups suggested that targeting endothelial cells with HDAC inhibitors may directly contribute to inhibition of tumor angiogenesis and eventually cancer progression. In the current study, our results suggest that LBH589, a recently developed hydroxamic HDAC inhibitor, is also an effective inhibitor of angiogenesis. LBH589 induced G2-M cell cycle arrest in both cancer (PC-3) and endothelial cells (HUVEC) at nanomolar concentrations. The IC50 for LBH589 in HUVEC was 1-fold lower than the IC50 for the HDAC inhibitor LAQ24 (6). Under these conditions, LBH589 was also very effective in inhibiting differentiation processes of endothelial cells, such as tube formation, migration, and invasion. The LBH589 concentrations used in our in vitro studies are achievable in mice. Pharmacokinetic data show that LBH589 dose at 25 mg/kg in mice bearing PC-3 tumors resulted in an AUC of 463 ng h/mL (0.96 μmol/L). In mice bearing HCT116 tumors, the AUC was 533 ng h/mL (1.11 μmol/L).3

In our study, we report for the first time that the HDAC inhibitor LBH589 attenuates VEGF-induced signaling in endothelial cells and reduces endothelial cell chemotaxis and invasion. A recent report has shown that class I HDACs regulate the expression of extracellular matrix proteins, and treatment of cancer cells with HDAC inhibitor impaired cancer cell invasion (31). Our results also show for the first time that an HDAC inhibitor inhibits HIF-1α and CXCR4 expression in human endothelial cells. HDAC inhibitor induced repression of HIF-1α not only in tumor cells but also in endothelial cells, and consequent reduction in CXCR4 expression may contribute to the antiangiogenesis properties of these agents.

The down-regulation of CXCR4 by LBH589 is of interest because of the role of this chemokine receptor in the homing of bone marrow progenitor and circulating endothelial cells to active sites of angiogenesis (32). Interestingly, HDAC activity has been reported to be involved in endothelial progenitor cell biology, and HDAC inhibition resulted in impaired endothelial progenitor cell differentiation (33). Taken together, our results provide both potential mechanisms for antiangiogenic activities of LBH589 and the rationale for clinical testing of HDAC inhibitors as angiogenesis inhibitors.

Although the angiogenesis-related gene modulation, such as HIF-1α, eNOS, VEGF, VEGFRs, and Tie-2 by HDAC inhibitors both in vitro and in vivo, is consistent, the underlying molecular mechanisms have yet to be elucidated (6, 34). One possibility is that through chromatin histone acetylation and gene transcription, HDAC inhibitors up-regulate tumor suppressor genes, such as p53 and VHL, which negatively regulate HIF-1α and VEGF (11).

HDAC inhibitors may also up-regulate some yet to be identified transcriptional repressors, which subsequently inhibit specific gene expression. Another possibility is that HDAC inhibitors induce acetylation of nonhistone proteins and subsequently affect the gene expression (both up-regulation and down-regulation) and protein stability. For example, the acetylation of transcription factor YY1 modifies its transcriptional activity (35), and the acetylation of androgen receptor has been associated with its increased nuclear localization and transcriptional activity (36). Therefore, it is conceivable that unidentified transcription repressors are activated upon acetylation and may contributed to angiogenesis-related gene regulation.

Protein acetylation may have a direct effect on endothelial cell biology. The acetylation of α-tubulin through HDAC6 inhibition has been associated with the change of tubulin dynamics and cell motility (37, 38). This observation may provide insights for the LBH589-induced inhibition of endothelial cell tube formation and Matrigel invasion, because both processes require cell motility.

HIF-1α acetylation by acetyl transferase ARD1 promotes HIF-1α degradation (39) and HSP90 acetylation compromises its chaperone function (40). Class I and II HDACs (HDAC1, HDAC6, and HDAC10) have been shown to physically associate with protein phosphatase 1, and the inhibition of HDAC by TSA disrupts their disassociation (41). All these events may be potentially associated with HDAC inhibitors and responsible for their biological activity.

The future direction of pharmacogenetic research on HDAC inhibitors will need to expand from chromatin acetylation and epigenetic regulation to nonhistone protein acetylation and its functional consequences. It is very likely that changes in protein acetylation and function will provide molecular links for inhibition of gene expression and attenuation of signaling pathways.

Major cellular signaling pathways, such as the phosphatidylinositol 3-kinase/AKT and ERK1/2 pathways, receive input from growth factors, including bFGF and VEGF, and integrins in endothelial cells (42). LBH589-induced inhibition of AKT and ERK 1/2 phosphorylation may be responsible for the antiproliferative and proapoptotic effect observed in HUVEC. The mechanisms underlying this inhibition remain to be elucidated and may involve either upstream regulation or direct kinase modification.

Both VEGF and bFGF significantly reduce the proapoptotic potency of chemotherapy on endothelial cells by a phosphatidylinositol 3-kinase–dependent mechanism (43). Downstream gene activation by phosphatidylinositol 3-kinase and survivin has been shown to play a pivotal role in VEGF-mediated endothelial cell protection by preserving the microtubule network (44). Survivin up-regulation may represent a novel mechanism of endothelial cell “resistance” (43). Thus, exploiting HDAC inhibitors as antiangiogenesis agents by impairing endothelial cell survival should enhance the antiangiogenesis activity of chemotherapy agents, such as microtubule inhibitors.

Several HDAC inhibitors are currently in clinical trials both in solid and hematologic malignancies (45). These clinical studies will provide the opportunity to test the dual function (both antitumor and antiangiogenesis) of some HDAC inhibitors, such as LBH589, SAHA, depsipeptide, MS-275, and valproic acid. A therapeutic approach where a drug simultaneously targets both the tumor cell and the endothelial compartment seems to be a rational strategy.

The HDAC inhibitors currently undergoing clinical trials have a safer toxicity profile than traditional chemotherapeutic agents. These characteristics raise the possibility of combining the HDAC inhibitors with other anticancer agents for targeted therapy. For example, we recently reported that the combination of LAQ824 and the VEGFR tyrosine kinase inhibitor PTK787 has an additive inhibitory activity on VEGF-induced angiogenesis in vitro and is more effective than single agents in controlling the progression of both prostate and breast cancer without overt toxicity. The mechanisms underlying the observed additive and/or synergistic effect may due to the inhibition of multiple independent and/or converging signaling pathways (6).

The combination of HDAC inhibitors with anti-VEGF therapies (i.e., VEGF blocking agents or VEGFR tyrosine kinase inhibitors) is of particular interest. Treatment with the anti-VEGF monoclonal antibody bevacizumab in combination with chemotherapy has been shown to increase overall survival in patients with metastatic colon carcinoma (46). Moreover, molecular targeted therapies with VEGFR tyrosine kinase inhibitors have been reported to have clinical activity in metastatic renal cell carcinoma patients (47). However, there are preclinical and clinical evidences that tumor “escape” to anti-VEGF therapy occurs (48). It is conceivable that angiogenesis-related gene modulation by HDAC inhibitors may affect tumor cell and endothelial cell adaptation to anti-VEGF therapies and prevent or delay the escape.

In conclusion, the HDAC inhibitor LBH589 has shown the dual function of targeting both tumor cells and proliferating endothelial cells and to inhibit tumor angiogenesis by gene modulation. Rational clinical testing of these anticancer agents as single agents or in combination with angiogenesis inhibitors and other biological therapies is warranted and should include angiogenesis-related correlative studies as potential markers of drug efficacy.

Acknowledgments

We thank Dr. John Isaacs and Michael Carducci for helpful discussions and Bonnie Gambichler for technical assistance with histology.

Footnotes

  • ↵3 Unpublished data.

  • Grant support: Prostate Cancer Foundation Award, Sidney Kimmel Research Award (R. Pili), and Aegon fellowship (D. Qian).

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Accepted October 28, 2005.
    • Received May 23, 2005.
    • Revision received October 14, 2005.

References

  1. ↵
    Marks PA, Richon VM, Rifkind RA. Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. J Natl Cancer Inst 2000;92:1210–6.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov 2002;1:287–99.
    OpenUrlCrossRefPubMed
  3. ↵
    Pili R, Kruszewski MP, Hager BW, Lantz J, Carducci MA. Combination of phenylbutyrate and 13-cis retinoic acid inhibits prostate tumor growth and angiogenesis. Cancer Res 2001;61:1477–85.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Richon VM, Sandhoff TW, Rifkind RA, Marks PA. Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. Proc Natl Acad Sci U S A 2000;97:10014–9.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Catley L, Weisberg E, Tai YT, et al. NVP-LAQ824 is a potent novel histone deacetylase inhibitor with significant activity against multiple myeloma. Blood 2003;102:2615–22.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Qian DZ, Wang X, Kachhap SK, et al. The histone deacetylase inhibitor NVP-LAQ824 inhibits angiogenesis and has a greater antitumor effect in combination with the vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584. Cancer Res 2004;64:6626–34.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Duan H, Heckman CA, Boxer LM. Histone deacetylase inhibitors down-regulate bcl-2 expression and induce apoptosis in t(14;18) lymphomas. Mol Cell Biol 2005;25:1608–19.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Rosato RR, Almenara JA, Dai Y, Grant S. Simultaneous activation of the intrinsic and extrinsic pathways by histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induces mitochondrial damage and apoptosis in human leukemia cells. Mol Cancer Ther 2003;2:1273–84.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Qian DZ, Ren M, Wei Y, et al. In vivo imaging of retinoic acid receptor β2 transcriptional activation by the histone deacetylase inhibitor MS-275 in retinoid-resistant prostate cancer cells. Prostate 2005;64:20–8.
    OpenUrlCrossRefPubMed
  10. ↵
    Wang X, Qian DZ, Ren M, et al. Epigenetic regulation of retinoic acid receptor β2 by MS-275 in human renal cell carcinoma. Clin Cancer Res 2005;11:3535–42.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Kim MS, Kwon HJ, Lee YM, et al. Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes. Nat Med 2001;7:437–43.
    OpenUrlCrossRefPubMed
  12. Kwon HJ, Kim MS, Kim MJ, Nakajima H, Kim KW. Histone deacetylase inhibitor FK228 inhibits tumor angiogenesis. Int J Cancer 2002;97:290–6.
    OpenUrlCrossRefPubMed
  13. ↵
    Michaelis M, Michaelis UR, Fleming I, et al. Valproic acid inhibits angiogenesis in vitro and in vivo. Mol Pharmacol 2004;65:520–7.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003;3:721–32.
    OpenUrlCrossRefPubMed
  15. ↵
    Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature 2000;407:242–8.
    OpenUrlCrossRefPubMed
  16. ↵
    Holash J, Wiegand SJ, Yancopoulos GD. New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene 1999;18:5356–62.
    OpenUrlCrossRefPubMed
  17. ↵
    Salcedo R, Oppenheim JJ. Role of chemokines in angiogenesis: CXCL12/SDF-1 and CXCR4 interaction, a key regulator of endothelial cell responses. Microcirculation 2003;10:359–70.
    OpenUrlCrossRefPubMed
  18. ↵
    Kucia M, Jankowski K, Reca R, et al. CXCR4-SDF-1 signalling, locomotion, chemotaxis and adhesion. J Mol Histol 2004;35:233–45.
    OpenUrlCrossRefPubMed
  19. ↵
    Lassota PT, Sorensen E, Bao R, et al. NVP-LBH589, a novel histone deacetylase inhibitor, activates caspase-3 and induces apoptosis of human tumor cells in vivo. Proc AACR 2004 # 4013.
  20. ↵
    George P, Bali P, Annavarapu S, et al. Combination of the histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC cells and AML cells with activating mutation of FLT-3. Blood 2005;105:1768–76.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Hubbert C, Guardiola A, Shao R, et al. HDAC6 is a microtubule-associated deacetylase. Nature 2002;417:455–8.
    OpenUrlCrossRefPubMed
  22. ↵
    Zhang Y, Li N, Caron C, et al. HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo. EMBO J 2003;22:1168–79.
    OpenUrlAbstract
  23. ↵
    Yu Y, Sato JD. MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor. J Cell Physiol 1999;178:235–46.
    OpenUrlCrossRefPubMed
  24. Qi JH, Matsumoto T, Huang K, Olausson K, Christofferson R, Claesson-Welsh L. Phosphoinositide 3 kinase is critical for survival, mitogenesis and migration but not for differentiation of endothelial cells. Angiogenesis 1999;3:371–80.
    OpenUrlCrossRefPubMed
  25. Thakker GD, Hajjar DP, Muller WA, Rosengart TK. The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J Biol Chem 1999;274:10002–7.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Yilmaz A, Kliche S, Mayr-Beyrle U, Fellbrich G, Waltenberger J. p38 MAPK inhibition is critically involved in VEGFR-2-mediated endothelial cell survival. Biochem Biophys Res Commun 2003;306:730–6.
    OpenUrlCrossRefPubMed
  27. ↵
    Oh H, Takagi H, Suzuma K, Otani A, Matsumura M, Honda Y. Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells. J Biol Chem 1999;274:15732–9.
    OpenUrlAbstract/FREE Full Text
  28. Salcedo R, Wasserman K, Young HA, et al. Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: In vivo neovascularization induced by stromal-derived factor-1α. Am J Pathol 1999;154:1125–35.
    OpenUrlCrossRefPubMed
  29. ↵
    Tran J, Master Z, Yu JL, Rak J, Dumont DJ, Kerbel RS. A role for survivin in chemoresistance of endothelial cells mediated by VEGF. Proc Natl Acad Sci U S A 2002;99:4349–54.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Schioppa T, Uranchimeg B, Saccani A, et al. Regulation of the chemokine receptor CXCR4 by hypoxia. J Exp Med 2003;198:1391–402.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Whetstine JR, Ceron J, Ladd B, Dufourcq P, Reinke V, Shi Y. Regulation of tissue-specific and extracellular matrix-related genes by a class I histone deacetylase. Mol Cell 2005;18:483–90.
    OpenUrlCrossRefPubMed
  32. ↵
    Avecilla ST, Hattori K, Heissig B, et al. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med 2004;10:64–71.
    OpenUrlCrossRefPubMed
  33. ↵
    Rossig L, Urbich C, Bruhl T, et al. Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells. J Exp Med 2005;201:1825–35.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Rossig L, Li H, Fisslthaler B, et al. Inhibitors of histone deacetylation downregulate the expression of endothelial nitric oxide synthase and compromise endothelial cell function in vasorelaxation and angiogenesis. Circ Res 2002;91:837–44.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Yao YL, Yang WM, Seto E. Regulation of transcription factor YY1 by acetylation and deacetylation. Mol Cell Biol 2001;21:5979–91.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Fu M, Wang C, Zhang X, Pestell RG. Acetylation of nuclear receptors in cellular growth and apoptosis. Biochem Pharmacol 2004;68:1199–208.
    OpenUrlCrossRefPubMed
  37. ↵
    Matsuyama A, Shimazu T, Sumida Y, et al. In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J 2002;21:6820–31.
    OpenUrlAbstract
  38. ↵
    Palazzo A, Ackerman B, Gundersen GG. Cell biology: tubulin acetylation and cell motility. Nature 2003;421:230.
    OpenUrlCrossRefPubMed
  39. ↵
    Jeong JW, Bae MK, Ahn MY, et al. Regulation and destabilization of HIF-1α by ARD1-mediated acetylation. Cell 2002;111:709–20.
    OpenUrlCrossRefPubMed
  40. ↵
    Nimmanapalli R, Fuino L, Bali P, et al. Histone deacetylase inhibitor LAQ824 both lowers expression and promotes proteasomal degradation of Bcr-Abl and induces apoptosis of imatinib mesylate sensitive or -refractory chronic myelogenous leukemia-blast crisis cells. Cancer Res 2003;63:5126–35.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Brush MH, Guardiola A, Connor JH, Yao TP, Shenolikar S. Deacetylase inhibitors disrupt cellular complexes containing protein phosphatases and deacetylases. J Biol Chem 2004;279:7685–91.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Stupack DG, Cheresh DA. Integrins and angiogenesis. Curr Top Dev Biol 2004;64:207–38.
    OpenUrlCrossRefPubMed
  43. ↵
    Tran J, Master Z, Yu JL, Rak J, Dumont DJ, Kerbel RS. A role for survivin in chemoresistance of endothelial cells mediated by VEGF. Proc Natl Acad Sci U S A 2002;99:4349–54.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Tran J, Rak J, Sheehan C, et al. Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells. Biochem Biophys Res Commun 1999;264:781–8.
    OpenUrlCrossRefPubMed
  45. ↵
    Piekarz R, Bates S. A review of depsipeptide and other histone deacetylase inhibitors in clinical trials. Curr Pharm Des 2004;10:2289–98.
    OpenUrlCrossRefPubMed
  46. ↵
    Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;350:2335–42.
    OpenUrlCrossRefPubMed
  47. ↵
    Motzer RJ, Rini BI, Michaelson MD, et al. Phase 2 trials of SU11248 show antitumor activity in second-line therapy for patients with metastatic renal cell carcinoma (RCC). Proc Am Soc Clin Oncol 2005;23:380s.
    OpenUrl
  48. ↵
    Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 2003;111:1287–95.
    OpenUrlCrossRefPubMed
Back to top
Clinical Cancer Research: 12 (2)
January 2006
Volume 12, Issue 2

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Email Article
Citation Tools
Targeting Tumor Angiogenesis with Histone Deacetylase Inhibitors: the Hydroxamic Acid Derivative LBH589
David Z. Qian, Yukihiko Kato, Shabana Shabbeer, Yongfeng Wei, Hendrik M.W. Verheul, Brenda Salumbides, Tolib Sanni, Peter Atadja and Roberto Pili
Clin Cancer Res January 15 2006 (12) (2) 634-642; DOI: 10.1158/1078-0432.CCR-05-1132
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section

Advertisement