Research Article

Thrombospondin-1 Peptide ABT-510 Combined with Valproic Acid
Is an Effective Antiangiogenesis Strategy i...
Valproic Acid and TSP-1 Inhibit Neuroblastoma Growth

has also been seen when VPA is combined with all-trans retinoic    ...
Cancer Research

similar. Each terminal deoxynucleotidyl transferase–mediated dUTP nick       endothelial cells. Primary ...
Valproic Acid and TSP-1 Inhibit Neuroblastoma Growth

   Evan’s blue dye assay. The Evan’s blue dye assay was used to ass...
Cancer Research

shown in Fig. 2C, control tumor volumes increased 3.5-fold over        the highest number of apoptotic c...
Valproic Acid and TSP-1 Inhibit Neuroblastoma Growth

Cancer Research

                                                                                     VPA-treated tumors,...
Valproic Acid and TSP-1 Inhibit Neuroblastoma Growth

was recently granted Food and Drug Administration approval for     ...
Cancer Research

References                                                    the thrombospondin-1-mimetic angiogenesis ...
Upcoming SlideShare
Loading in …5

Yang et al. Cancer Research 2007


Published on

Antiangiogenic treatment effects on neuroblastoma xenografts

Published in: Health & Medicine
  • Be the first to comment

  • Be the first to like this

No Downloads
Total views
On SlideShare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide

Yang et al. Cancer Research 2007

  1. 1. Research Article Thrombospondin-1 Peptide ABT-510 Combined with Valproic Acid Is an Effective Antiangiogenesis Strategy in Neuroblastoma 1 1 1 1 1 Qiwei Yang, Yufeng Tian, Shuqing Liu, Rana Zeine, Alexandre Chlenski, 1 3 2 Helen R. Salwen, Jack Henkin, and Susan L. Cohn 1 The Robert H. Lurie Comprehensive Cancer Center and 2Department of Pediatrics, Northwestern University, Feinberg School of Medicine, Chicago, Illinois and 3Abbott Laboratories, Abbott Park, Illinois Abstract substantial progress has been made in the treatment of children with low- and intermediate-risk neuroblastoma with reduced In the pediatric cancer neuroblastoma, clinically aggressive therapy approaches. However, more effective therapy is still needed disease is associated with increased levels of angiogenesis for children with high-risk disease. Despite intensive multimodality stimulators and high vascular index. We and others have treatment, <30% of high-risk patients are cured (1). Similar to other hypothesized that blocking angiogenesis may be effective solid tumors, angiogenesis is required for neuroblastoma tumor treatment for this pediatric malignancy. However, little is growth and metastasis. The majority of published studies indicate known about the efficacy of antiangiogenic agents in pediatric that angiogenesis contributes to the aggressive behavior of malignancies. Recently, promising results have been reported neuroblastoma tumors, and a positive correlation between poor in an adult phase I study of ABT-510, a peptide derivative of outcome and high levels of factors that stimulate blood vessel the natural angiogenic inhibitor thrombospondin-1. Histone growth and vascular index has been reported (3, 4). In addition, deacetylase inhibitors, such as valproic acid (VPA), have also preclinical experiments have shown that angiogenesis inhibitors been shown to have antiangiogenic activity in several cancer are capable of suppressing neuroblastoma growth, further support- models. In this study, we evaluated the effects of ABT-510 ing the concept that the blood vessels in neuroblastoma tumors are and VPA on neuroblastoma tumor growth and angiogenesis. likely to be clinically relevant therapeutic targets (5, 6). Although only VPA was capable of blocking the proliferation Thrombospondin-1 (TSP-1) is a well-known natural inhibitor of of neuroblastoma cells and inducing neuroblastoma cell angiogenesis, and down-regulation of TSP-1 plays a critical role in apoptosis in vitro, treatment with VPA or ABT-510 alone the angiogenic switch in several tumor types (7–9). In neuroblas- significantly suppressed the growth of neuroblastoma xeno- toma, we have previously shown that TSP-1 is epigenetically grafts established from two different MYCN-amplified cell silenced in a subset of undifferentiated, advanced-stage tumors and lines. Combination therapy more effectively inhibited the cell lines, whereas it is expressed in tumors with morphologic growth of small neuroblastoma xenografts than single-agent evidence of neuroblast differentiation (10). The antiangiogenic treatment, and in animals with large xenografts, total effect of TSP-1 is mediated in part by the CD36 receptor, which cessation of tumor growth was achieved with this treatment triggers a signaling cascade that leads to apoptosis in activated approach. The microvascular density was significantly re- endothelial cells via the CD95 death receptor. The inhibitory duced in the xenografts treated with combination therapy activity of TSP-1 has been mapped to the properdin type 1 repeats compared with controls or tumors treated with single agents. in the NH2-terminal third of the molecule, and a single substitution In addition, the number of structurally abnormal vessels was of D-isoleucine for L-isoleucine in one properdin-region heptapep- reduced, suggesting that these agents may ‘‘normalize’’ the tide leads to a 1,000-fold increase in the antiangiogenic activity tumor vasculature. Our results indicate that ABT-510 com- (11, 12). ABT-510 is a modified analogue of the active TSP-1 bined with VPA may be an effective antiangiogenic treatment heptapeptide, which has slowed tumor growth in both xenograft strategy for children with high-risk neuroblastoma. [Cancer and syngeneic models (13). Administration of ABT-510 to TSP-1 Res 2007;67(4):1716–24] knockout mice for 5 consecutive days has been shown to reduce by f5-fold both elevated circulating endothelial cells and endothelial Introduction precursor cells to near wild-type levels (14). Although no pediatric The clinical hallmark of neuroblastoma is heterogeneity, with the cancer studies have been done to date, a recently completed phase likelihood of tumor progression varying widely according to age at I trial of ABT-510 in adults with advanced cancer has shown a diagnosis and extent of disease (1). In addition to these clinical favorable toxicity profile and a decrease in the median serum basic factors, biological markers, such as MYCN gene status, tumor cell fibroblast growth factor (bFGF) levels. Stable disease lasting for six ploidy, and tumor histology, have also been shown to be strongly cycles or more was seen in 6 of the 39 patients enrolled on the predictive of risk of relapse (2). Modern treatment strategies are phase I study, and additional response data are being collected on stratified according to these clinical and biological classifiers, and ongoing phase II studies (15). Valproic acid (VPA), a branched chain fatty acid initially used for the treatment of epilepsy, has also been shown to have anticancer Note: Supplementary data for this article are available at Cancer Research Online activity and is currently being tested in clinical trials. VPA inhibits ( Current address for S.L. Cohn: University of Chicago, Chicago, Illinois. the growth of many types of cancer, including glioma, neuroblas- Requests for reprints: Susan L. Cohn, Institute for Molecular Pediatric Sciences, toma (16, 17), breast cancer (18), acute myelogenous leukemia University of Chicago, 5841 Maryland Avenue, MC 4060, Room N114, Chicago, IL (19, 20), thyroid cancer (21), and endometrial carcinoma (22). 60637. Phone: 773-702-2571; Fax: 773-834-1329; E-mail: I2007 American Association for Cancer Research. Recently, VPA and IFN-a have been reported to synergistically doi:10.1158/0008-5472.CAN-06-2595 inhibit neuroblastoma growth, and increased anticancer activity Cancer Res 2007; 67: (4). February 15, 2007 1716
  2. 2. Valproic Acid and TSP-1 Inhibit Neuroblastoma Growth has also been seen when VPA is combined with all-trans retinoic The experiment was done in duplicate. The cell cycle data were analyzed acid (16, 17). Although the mechanisms responsible for the anti- with an Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL), with cancer activity of VPA are not completely understood, like other System II (version 3.0) software (Beckman Coulter). Further analysis of cell short chain fatty acids, it inhibits histone deacetylase (HDAC) cycle distribution was determined by using ModFit LT (Verity Software House, Topsham, ME). activity and plays a role in the regulation of gene expression. Annexin V/propidium iodide staining. Apoptosis and/or necrosis were Recent studies have indicated that HDAC inhibitors are also evaluated using two-color flow cytometric analysis with fluorescein-labeled capable of blocking hypoxia-induced and vascular endothelial Annexin V and propidium iodide (10). Cells were fluorescein labeled using growth factor–induced angiogenesis (23). methods provided by the manufacturer of the Annexin V-FITC Apoptosis In this study, we investigated the effects of the antiangiogenic Detection kit (Immunotech, Marseille, France). The experiment was done agent ABT-510 on neuroblastoma growth and angiogenesis. in duplicate. In brief, cultured neuroblastoma cell lines were treated with Because the efficacy of angiogenesis inhibitors is greatly improved 10, 100, and 1,000 nmol/L of ABT-510 for 24 h. Cells were washed twice when they are combined with other anticancer drugs (24, 25), we with PBS and resuspended at 1  106/mL in binding buffer with FITC- conducted additional experiments with ABT-510 combined with conjugated Annexin V and propidium iodide. Samples were analyzed using VPA. VPA was selected for the combination studies because it is the Epics XL-MCL flow cytometer. Endothelial cell migration assay. Endothelial cell migration assays capable of inhibiting neuroblastoma cell proliferation and tumor were done in a modified Boyden chamber (NeuroProbe, Gaithersburg, MD) growth and can block HDAC activity and angiogenesis. In addition, with human microvascular endothelial cells (Cambrex Corp., East VPA is known to have a low toxicity profile. We found that VPA, Rutherford, NJ) in EBM medium (Cambrex) containing 0.01% bovine serum but not ABT-510, was capable of blocking the proliferation of albumin as described previously (26, 27). Test substances were assayed with neuroblastoma cells and inducing neuroblastoma cell apoptosis or without 5 ng/mL bFGF (National Cancer Institute Preclinical Repository, in vitro. However, treatment with either ABT-510 or VPA alone Bethesda, MD). To generate dose-response curves, the data were normalized significantly suppressed the growth of neuroblastoma xenografts as percentage of maximum migration using the difference between bFGF/ established from two different MYCN-amplified cell lines. ABT-510 EBM-induced migration and background migration in EBM alone as 100% combined with VPA more effectively inhibited the growth of small control. The experiment was done in triplicate. tumors than single-agent therapy, and in animals with large cDNA synthesis and Taqman assay. Total RNA (2 Ag) was used for reverse transcription using SuperScript III (Invitrogen) and 50 Amol/L tumors, combination therapy stabilized tumor growth. In addition oligo(dT)20 in a 20 AL reaction volume according to standard protocols to blocking angiogenesis, tumor cell apoptosis and inhibited tumor supplied by the manufacturer. cDNA synthesis was done at 50jC for cell proliferation were seen in the tumors treated with combina- 50 min. Following heat inactivation at 85jC for 5 min and RNase H tion therapy. Our results, together with the known low toxicity treatment at 37jC for 20 min, 480 AL distilled water was added and 3 AL of profile of both agents, suggest that VPA combined with ABT-510 diluted cDNA were used for Taqman analysis. Taqman reactions were set may be an effective, well-tolerated antiangiogenic treatment up containing 1 Taqman Fast Universal PCR Master Mix (Applied strategy for children with high-risk neuroblastoma. Biosystems, Foster City, CA), 250 nmol/L FAM-labeled specific probe, and 900 nmol/L forward and reverse primers in a 20 AL reaction. All assays were carried out in a 96-well format. Real-time fluorescent detection of PCR Materials and Methods products was done with an Applied Biosystems 7500 Fast Real-Time PCR Cell lines. The biological and genetic characteristics of the SMS-KCNR System using the following thermocycling conditions: 1 cycle of 50jC for and NMB MYCN-amplified neuroblastoma cell lines used in this study have 2 min and 95jC for 20 s; 40 cycles of 95jC for 3 s and 60jC for 30 s. The been previously described (10). Cells were grown at 5% CO2 in RPMI 1640 sequences for the primers and probes for MYCN, TSP-1, SPARC, and (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal b2-microglobulin were described previously (10, 27, 28). Data were analyzed bovine serum (FBS), L-glutamine, and antibiotics. using the comparative method (DDC t) to calculate relative quantities of Peptide. ABT-510 (Abbott Laboratories, Abbott Park, IL) is a capped a nucleic acid sequence. Nontreated SMS-KCNR and NMB were used as the nonapeptide based on the linear TSP-1 heptapeptide sequence containing a calibrator sample, and h2-microglobulin was used as an endogenous fragment of the second type I TSP-1 repeat. The sequence of ABT-510 is control detector. acetyl-sarcosine-glycine-valine-D-alloisoleucine-threonine-norvaline-isoleu- Neuroblastoma xenograft studies. Four- to 6-week-old female cine-arginine-proline-ethylamide. Lyophilized peptides were dissolved in homozygous athymic nude mice (Harlan, Madison, WI) were inoculated sterile 5% dextrose and stored at 4jC. s.c. into the right flank with 1  107 SMS-KCNR or 1.25  107 NMB cells. Proliferation assay. The in vitro proliferation of SMS-KCNR cells was Once tumors were palpable (70 mm3), mice were treated once daily for determined by using the CellTiter 96 AQ nonradioactive cell proliferation 20 days with either ABT-510 (i.p., 40 mg/kg), VPA (i.p., 400 mg/kg), or ABT- assay (Promega Corp., Madison, WI). SMS-KCNR cells were seeded into 510 and VPA in combination. In separate experiments, treatment was 96-well plates at a density of 7.5  103 per well. After 24 h, ABT-510 or VPA started after the animals developed larger tumors z380 mm3 in size and was added to quadruplicate wells at various concentrations in medium continued for 10 days. Tumor volumes were measured twice weekly and containing either 10% or 2% FBS. The lower concentration of serum was calculated using the following formula: tumor volume = (length  width2) / used to ensure that effects of the drugs were not counteracted by excessive 2 (29). The Student’s t test was used to compare tumor size in the control growth factors present in the serum. Following treatment for 24 to 72 h, and treatment groups. Mice were sacrificed after 20 or 10 days of treatment, MTS reagent was added and cells were further incubated for 3 h. The respectively. Animals were treated according to NIH guidelines for animal absorbance of the samples was measured using a Synergy HT Microplate care and use, and protocols were approved by the Animal Care and Use Reader (Bio-Tek Instruments, Winooski, VT). Committee at Northwestern University. Measurement of cell cycle phase distribution. Cell cycle distribution Quantification of apoptosis. In situ detection of cleaved, apoptotic was determined by flow cytometric analysis. Briefly, SMS-KCNR cells were DNA fragments was done using the In Situ Cell Death Detection kit (Roche cultured in RPMI 1640 containing various concentrations of ABT-510 or Diagnostics Corp., Indianapolis, IN) as described previously (30). Sections of VPA for 24 h. Control cells were cultured in medium lacking ABT-510 or human colon mucosa processed similarly to the xenografts were included in VPA. Cells were then washed with PBS, fixed in 70% ethanol, and each assay. To check the intra-assay and interassay consistency, the hypotonically lysed in 1 mL of DNA staining solution [0.05 mg/mL apoptotic bodies in colonic mucosa, stained as the first and last slide of propidium iodide (Sigma, St. Louis, MO) and 0.1% Triton X-100]. The cells each assay, were assessed. The assays were considered adequate only when were incubated, while protected from light, at 4jC overnight before analysis. the frequency of apoptosis on the two sections of colonic mucosa was 1717 Cancer Res 2007; 67: (4). February 15, 2007
  3. 3. Cancer Research similar. Each terminal deoxynucleotidyl transferase–mediated dUTP nick endothelial cells. Primary antibodies were incubated in a humidity end labeling (TUNEL) in situ–labeled section was quantified using a chamber overnight at 4jC. The staining for Ki-67 was developed using Leica (Heidelberg, Germany) DM IRB inverted fluorescence microscope the DAKO Envision+ System-HRP (DakoCytomation) according to the equipped with Image-Pro Plus version 4.5 software (Media Cybernetics, manufacturer’s instructions. The CD31 goat anti-mouse primary antibody Silver Spring, MD) at a magnification of Â400. The frequency of labeled was bridged by biotinylated anti-goat IgG (1:200) and detected with apoptotic cells was obtained by quantifying 10 consecutive fields starting streptavidin (1:400; Vector Laboratories, Burlingame, CA). Sections were with areas with the highest number of TUNEL-labeled nuclei, avoiding areas counterstained with Gill’s hematoxylin. In addition, the primary antibodies of necrosis, and expressed as apoptosis per 10 high-power fields (HPF). were omitted in each assay to ensure the specificity. Immunohistochem- Immunohistochemical studies. Xenograft tissue was fixed in 10% istry for TSP-1 expression was done as described previously (10). Mean formaldehyde/zinc fixative (Electron Microscopy Sciences, Hatfield, PA) vascular density (MVD) was quantified by measuring pixels in 10 and embedded in paraffin. Serial sections (4 Am thick) were deparaffinized consecutive fields at Â200 magnification. and rehydrated as described previously (31). Sections of each tumor were Morphologic evaluation of blood vessels. Blood vessel morphology and stained with H&E, and selected sections were stained with Masson’s tumor histology were evaluated by a pathologist (R.Z.) on H&E-stained trichrome special staining. Additional sections were used for immunohis- sections. The presence or absence of hemorrhage was evaluated, and tumor tochemistry. Antigen retrieval was done with 0.01 mol/L citrate buffer blood vessels were classified morphologically into four types. Vessels were (pH 6.0) for Ki-67 or 1 mmol/L EDTA (pH 8.0) for CD31 antibody, heated counted, and the percentage of each vessel type in the tissue section was in a boiling steamer for 20 min, and then cooled down to room calculated. Type 1 vessels had disorganized cellular proliferation with poorly temperature for 20 min. For analysis of proliferation rate, slides were formed lumina and vascular endothelial proliferation (VEP). Type 2 vessels incubated with Ki-67 (clone MIB-1, 1:200; DakoCytomation, Carpinteria, were structurally more normal, thin walled, and capillary like. Type 3 CA) mouse anti-human monoclonal antibody. CD31 (platelet/endothelial vessels were structurally similar to type 2 vessels but were embedded in cell adhesion molecule 1, M-20, 1:100; Santa Cruz Biotechnology, Santa fibrocollagenous stroma. Type 4 vessels had concentric layers of cells within Cruz, CA) polyclonal goat anti-mouse antibody was used to highlight a thickened wall often accompanied by fibrinoid necrosis and vasculitis. Figure 1. Analysis of SMS-KCNR cell apoptosis, necrosis, cell cycle, and endothelial cell migration after treatment with ABT-510 or VPA. A, flow cytometric analysis of SMS-KCNR cell apoptosis and necrosis after treatment with ABT-510 for 1 d. Top left quadrant, necrotic cells; top right quadrant, late apoptotic cells; bottom right quadrant, early apoptotic cells; bottom left quadrant, live cells. B, sub-G1 apoptotic analysis of SMS-KCNR cells treated with VPA. C, cell cycle analysis of control, ABT-510–treated, and VPA-treated neuroblastoma cells. SMS-KCNR was treated with either vehicle or various concentrations of ABT-510 and VPA for 24 h and analyzed by flow cytometry. D, ABT-510 and VPA inhibited angiogenesis in vitro . Cancer Res 2007; 67: (4). February 15, 2007 1718
  4. 4. Valproic Acid and TSP-1 Inhibit Neuroblastoma Growth Evan’s blue dye assay. The Evan’s blue dye assay was used to assess tumor microvascular permeability as described previously (32). Evan’s blue dye (2%, 100 AL; MP Biomedicals, Aurora, OH) was given via tail vein injection of tumor-bearing mice and allowed to circulate for 20 min. To remove remaining intravascular dye, mice were perfused with 25 mL saline through the left ventricle with a right atrial vent. Tumors were excised, cut into pieces, and weighed. Formamide was added at a ratio of 1 mL/100 mg of tissue and let stand for 72 h at room temperature to facilitate the extraction of Evan’s blue dye. Tissues were then removed and resultant extract was centrifuged. The levels of Evan’s blue were quantified using a spectrophotometer at a wavelength of 620 nm. The amount of Evan’s blue dye remaining in the tumor reflects the degree of intratumoral vessel permeability. All samples were run in duplicate and compared with those of standards. Statistical analysis. The two-tailed Student’s t test was used to compare the statistical significance of differences between the mean values of quantification of apoptosis, proliferation, CD31-positive vessels, and permeability in the neuroblastoma xenografts comparing vehicle-treated with ABT-510, VPA, and combination-treated groups. ANOVA followed by Dunnett’s multiple comparison procedure was used to compare the statistical significance of differences between control and treatment groups for sub-G1 and cell cycle analysis. Results VPA, but not ABT-510, inhibits neuroblastoma cell prolifer- ation, blocks cell cycle progression, and induces apoptosis. To investigate the effects of VPA and ABT-510 as single agents on neuroblastoma cell growth in vitro, SMS-KCNR neuroblastoma cells were cultured in a range of drug concentrations. As shown in Supplementary Fig. S1, treatment with ABT-510 did not inhibit the proliferation of SMS-KCNR neuroblastoma cells even at concen- trations as high as 1,000 nmol/L (Supplementary Fig. S1A and C). In contrast, VPA significantly inhibited neuroblastoma cell proli- feration at concentrations of 5 and 10 mmol/L of VPA when the cells were cultured in medium containing either 10% or 2% serum (P < 0.01, respectively; Supplementary Fig. S1B and D). Further- more, 2.5, 5.0, and 10 mmol/L of VPA induced neuroblastoma cell Figure 2. Growth of neuroblastoma xenografts. SMS-KCNR (A ) or NMB (B ) apoptosis as shown by the increased population of sub-G1 cells mice were treated once the tumors were palpable with vehicle control, VPA alone, ABT-510 peptide alone, or ABT-510 and VPA in combination. (P = 0.0014, P = 0.0008, and P < 0.0001, respectively; Fig. 1B), and C, SMS-KCNR xenografts were treated once they grew to z380 mm3 in size with cell cycle analysis revealed that treatment with VPA at concen- ABT-510, VPA, or combination therapy. trations of 2.5, 5.0, and 10 mmol/L induced G2 growth arrest (P = 0.0003, 0.0002, and 0.0004, respectively; Fig. 1C). Neither apoptosis (Fig. 1A) nor changes in cell cycle (Fig. 1C) were seen antiangiogenic activity of VPA was due, at least in part, to up- with ABT-510 (P > 0.01). regulation of endogenous inhibitors of angiogenesis in neuroblas- VPA and ABT-510 inhibit bFGF-induced endothelial cell toma cells, we also evaluated the level of expression of TSP-1 and migration. To test the antiangiogenic activity of VPA and ABT-510, SPARC by RT-PCR in the SMS-KCNR neuroblastoma cells following endothelial cell migration assays were done using a range of drug treatment with VPA. We found that treatment with 1 mmol/L VPA concentrations in the presence or absence of bFGF. bFGF-induced for 24 h resulted in up-regulation of the level of TSP-1 and SPARC endothelial cell migration was inhibited by ABT-510 and VPA in a expression (Supplementary Fig. S2C and D). dose-dependent manner at concentrations ranging from 0.1 to 100 VPA and ABT-510 inhibit neuroblastoma xenograft growth. nmol/L and 10 nmol/L to 1 mmol/L, respectively (Fig. 1D). To examine the antitumor effects of VPA and ABT-510, nude VPA down-regulates MYCN expression and induces TSP-1 mice with xenografts (f70 mm3 in size) established from two and SPARC expression in neuroblastoma cells. Because MYCN different MYCN-amplified neuroblastoma cell lines were treated plays a critical role in the regulation of neuroblastoma cell with each agent alone and in combination. Tumor growth was proliferation and apoptosis, we examined the levels of MYCN inhibited with single-agent therapy, and enhanced effects were mRNA expression in neuroblastoma cells following treatment with seen with combination therapy. After 20 days of treatment with 1 mmol/L VPA by quantitative real-time reverse transcription-PCR VPA combined with ABT-510, tumor volume was reduced by (RT-PCR). As shown in Supplementary Fig. S2A and B, MYCN 91% (P < 0.001; Fig. 2A) in the SMS-KCNR model and by 87% in the expression was significantly down-regulated in both SMS-KCNR NMB xenografts (P = 0.029; Fig. 2B) compared with controls. and NMB cell lines within 2 h of treatment. In contrast, MYCN Because the effectiveness of some antiangiogenic strategies is expression did not significantly change following treatment with inversely related to tumor burden, we repeated our studies in a set 100 nmol/L ABT-510 (data not shown). To determine if the of animals with large xenografts that were z380 mm3 in size. As 1719 Cancer Res 2007; 67: (4). February 15, 2007
  5. 5. Cancer Research shown in Fig. 2C, control tumor volumes increased 3.5-fold over the highest number of apoptotic cells was seen in the xenografts the subsequent 10 days, and tumors in animals treated with ABT- treated with VPA alone or VPA combined with ABT-510 (93 F 13 510 or VPA, respectively, showed 33% and 44% reduced growth and 141 F 39, respectively, P < 0.001). increases (P < 0.01 and 0.001). However, during this interval, VPA and ABT-510 inhibit neuroblastoma tumor cell prolif- animals receiving combined treatment had total cessation of tumor eration in vivo. Cell proliferation in the xenografts was evaluated by volume increase from the start of treatment through the entire Ki-67 expression, a nuclear protein that is preferentially expressed 10-day period. Thus, the combination of ABT-510 and VPA achieved during active phases of the cell cycle. Ki-67–negative cells represent immediate tumor growth stasis (P < 0.001), whereas the individual quiescent G0 phase cells. A significantly higher percentage of treatments gave only modest inhibition. neuroblasts in G0 were detected in tumors treated with VPA alone Cell apoptosis is increased when VPA is combined with ABT- compared with control tumors (50.6 F 4.7 versus 34.2 F 9, 510. To evaluate whether treatment induced apoptosis, TUNEL respectively, P < 0.01; Fig. 3B). The percentage of neuroblasts in assays were done on the neuroblastoma xenografts treated with G0 in the control tumors and ABT-510–treated tumors was not ABT-510, VPA, or combination therapy. The tumors treated with significantly different (34.2 F 9 versus 42.4 F 5.3, P = 0.076; ABT-510 contained significantly more apoptotic cells than the Fig. 3B). However, the percentage of G0 cells was significantly control tumors (53 F 16 versus 34 F 9, P = 0.015; Fig. 3A). However, increased in the tumors treated with combination therapy compared Figure 3. A, representative photographs of TUNEL fluorescent apoptosis in situ detection assay from tumors treated with VPA alone, ABT-510 alone, or ABT-510 and VPA in combination. Magnification, Â400. Mean apoptosis counts per 10 HPFs in treated tumors versus controls. B, MIB-1– immunostained (Ki-67) sections. Proliferating cells were identified by MIB-1 antibody in control versus ABT-510, VPA, and combination-treated groups. Percentage of G0 (Ki-67–negative cells) per 10 HPFs of the control and treated tumors. C, MVD in SMS-KCNR xenografts after treatment with ABT-510, VPA, or ABT-510 and VPA in combination. Columns, mean MVD measured in SMS-KCNR xenografts following treatment with vehicle, ABT-510, VPA, or combination therapy; bars, SD. P < 0.001, vehicle versus VPA; P < 0.001, vehicle versus ABT-510; P < 0.001, vehicle versus combination. Cancer Res 2007; 67: (4). February 15, 2007 1720
  6. 6. Valproic Acid and TSP-1 Inhibit Neuroblastoma Growth P = 0.00024; Fig. 3C). The MVD was significantly lower in the tumors treated with combination therapy (247,805 F 73,062 versus 31,406 F 18,401; Fig. 3C) than in either single-agent group (P < 0.001). We also evaluated TSP-1 expression in the control and treated SMS-KCNR tumors using immunohistochemistry to deter- mine if the treatment with either agent alone or in combination was capable of inducing expression of this angiogenic inhibitor. No significant up-regulation of TSP-1 was observed in treated tumors. VPA and ABT-510 treatment decreases hemorrhage and modifies tumor blood vessel structure. To investigate the effects of VPA and ABT-510 on tumor growth, treatment was initiated in animals with tumors measuring f70 mm3. Mice were sacrificed after 20 days of treatment, and the tumors were histologically examined. The control NMB neuroblastoma xenografts were grossly hemorrhagic, and extravasated RBCs were seen microscopically. The tumor blood vessels were morphologically classified into four types as described in Materials and Methods. More than half of the vessels in the control neuroblastoma xenografts were structurally abnormal with VEP (type 1 vessels; 58 F 9.04%; Fig. 4, Control, left and right; Table 1A). The remaining vessels were thin walled and capillary like Figure 4. Representative sections from H&E-stained and Masson’s (type 2; Fig. 4, Control, middle; Table 1A). To investigate if the trichrome–stained control and treated NMB xenografts. Hemorrhage is moderate structurally abnormal vessels were related to the large size of the in control tumors, mild in ABT-510–treated group, minimal in VPA-treated group, and absent following combination treatment. Control tumors show predominantly control tumors, we repeated these experiments and sacrificed mice VEP (type 1 vessels; left and right ) and a minority of thin-walled vessels when the tumors were similar in size to the treated tumors (ranging (type 2; middle ). In contrast, tumors from treatment groups show predominantly from 220 to 726 mm3). Similar to the large tumors, significant thin-walled vessels that are either capillary-like or arteriolar with hyaline arteriolosclerosis (middle ) or embedded in fibrocollagenous stroma (type 3 hemorrhage was present in the smaller control tumors, and the vessels; left and right ). Magnification, Â600. majority of vessels were structurally abnormal and with VEP. Tumor hemorrhage was decreased in experiments in which treatment with ABT-510 was started when tumors were f70 mm3 with control tumors or tumors treated with either single agent in size. Significantly fewer type 1 blood vessels were observed (86.4 F 9.1 versus 34.2 F 9, P < 0.01; 86.4 F 9.1 versus 42.4 F 5.3, (7.85 F 5.35%; P = 0.0028) in these tumors, and the majority of the P < 0.01; 86.4 F 9.1 versus 50.6 F 4.7, P < 0.01; Fig. 3B). vessels were thin walled (type 2 and 3 vessels; Fig. 4, ABT-510, right, Treatment with ABT-510 and VPA significantly inhibits middle, and left; Table 1A). Virtually no hemorrhage was seen in neuroblastoma tumor angiogenesis. To investigate the anti- tumors treated with VPA, and the percentage of blood vessels with angiogenic effects of ABT-510 and VPA, the vascularity of tumors VEP (type 1 vessels) decreased to 12.69 F 3.5% (P = 0.0039). The treated early with ABT-510, VPA, or ABT-510 and VPA in majority of vessels in these tumors were thin walled and many were combination was evaluated. Histologic sections were stained with capillary like (Fig. 4, VPA, right, middle, and left; Table 1A). Some of CD31 to highlight endothelial cells, and a MVD was quantified. the vessels were embedded in fibrocollagenous stroma, highlighted Compared with control tumors, a significantly lower MVD was seen by Masson’s trichrome special stain (type 3 vessels), and others in xenografts following treatment with VPA or ABT-510 (247,805 F were arteriolar with mild hyaline sclerosis (Table 1A). In tumors 73,062 versus 114,597 F 47,568 versus 75,880 F 31,638, respectively, treated with VPA combined with ABT-510, hemorrhage was not Table 1. Effects of ABT-510 and VPA treatment on neuroblastoma tumor blood vessel morphology Vehicle (% F SE) ABT-510 (% F SE) VPA (% F SE) ABT-510 + VPA (% F SE) A. NMB xenografts Treatment started when tumors were <70 mm3 VEP (type 1) 58.00 F 9.04 7.85 F 5.35 12.69 F 3.53 3.55 F 1.12 Thin-walled vessels 42.00 F 9.04 92.15 F 5.35 87.31 F 5.53 96.45 F 1.12 Thin or capillary like (type 2) 69.05 F 9.91 62.04 F 7.11 68.55 F 12.12 59.95 F 9.83 Embedded in fibrocollagenous stroma (type 3) 30.95 F 9.91 37.96 F 7.11 31.45 F 12.12 40.05 F 9.83 Hyperplastic arteriolosclerosis (type 4) 0 0 0 0 B. SMS-KCNR xenografts Treatment started when tumors were z380 mm3 VEP (type 1) 100 (network) 14.68 F 3.83 2.09 F 0.72 0 Thin-walled vessels 0 85.32 F 3.85 97.91 F 0.72 88.14 F 10.58 Thin or capillary like (type 2) 0 19.95 F 4.50 21.33 F 4.96 32.23 F 9.89 Embedded in fibrocollagenous stroma (type 3) 0 80.05 F 4.50 78.67 F 4.96 67.77 F 9.89 Hyperplastic arteriolosclerosis (type 4) 0 0 0 11.86 F 10.58 1721 Cancer Res 2007; 67: (4). February 15, 2007
  7. 7. Cancer Research VPA-treated tumors, hemorrhage was minimal (Fig. 5, VPA, left and middle) and the number of the type 1 vessels was only 2.09 F 0.72% (P < 0.0001; Table 1B). Almost all of the vessels in the VPA-treated tumors were thin walled (Fig. 5, VPA, middle and right; Table 1B). Some vessels were capillary like (Fig. 5, VPA, middle), some had mild hyaline sclerosis (Fig. 5, VPA, middle), and others were embedded in fibrocollagenous stroma (Fig. 5, VPA, right; Table 1B). Minimal hemorrhage was seen in the tumors treated with both VPA and ABT-510 (Fig. 5, ABT-510 + VPA, left and middle), and VEP was absent. The majority of vessels in these tumors were thin walled and classified as type 2 or 3 vessels (Fig. 5, ABT-510 + VPA, middle; Table 1B). In three of the seven tumors treated with combination therapy, there were small numbers of vessels with thickened walls exhibiting concentric lamellar cellular arrangement (‘onionskin’ pattern) with marked luminal narrowing, focal fibrosis, fibrinoid necrosis, and vasculitis (type 4 vessels, hyperplastic arteriolosclerosis; Fig. 5, ABT-510 + VPA, right; Table 1B). VPA and ABT-510 treatment decreases vascular permeability. Tumor blood vessels are functionally abnormal and leaky. To determine if treatment with VPA and ABT-510 modified vessel function, we assessed intratumoral vessel permeability using the Evan’s blue dye assay. In this assay, Evan’s blue dye is given systemically, mice are then perfused with saline to wash out the intravascular dye, and the amount of Evan’s blue dye remaining in the tumor correlates to permeability of the microvasculature. Our results indicate a decrease in intratumoral vessel permeability in all treatment groups (Fig. 6). The decrease was modest in the tumors treated with ABT-510 alone compared with controls and did not reach statistical significance (P = 0.124; Fig. 6). However, the reduction in vascular permeability was statistically significant in tumors treated with either VPA alone or in combination with ABT- 510 compared with controls (P = 0.044 and 0.007, respectively; Fig. 6). Figure 5. Representative H&E-stained sections of SMS-KCNR xenografts treated after reaching z380 mm3 in size. Severe hemorrhage is seen in the control tumors (left and middle ). Hemorrhage is moderate in the tumors treated Discussion with ABT-510 (left and middle ), minimal in VPA-treated tumors (left and middle ), and absent following combination treatment (left and middle ). Control tumors More than 30 years ago, Folkman (33) proposed the concept show extensive VEP (type 1 vessels; middle and right ). Tumors treated with that blocking angiogenesis would result in tumor dormancy. ABT-510 show a few type 1 vessels (right ) and a majority of thin-walled type 2 and 3 vessels (middle ). In the VPA-treated tumors, most vessels are thin walled, Clinical trials in adults with cancer have supported this including capillary like and arteriolar (type 2 vessels; middle ) as well as those hypothesis, and the antiangiogenic agent bevacizumab (Avastin) embedded in fibrocollagenous stroma (type 3 vessels; right ). Tumors from the ABT-510 + VPA combination-treated group show thin-walled vessels surrounded by fibrosis (middle). A subset of these tumors showed small vessel changes with features of hyperplastic arteriolosclerosis (type 4 vessels; right ). Arrows, vessels. seen and the proportion of vessels with VEP was further reduced to 3.55 F 1.12% (P = 0.0007; Table 1A). The vessels in the tumors treated with combination therapy were thin walled and embedded in fibrocollagenous stroma (type 3; Fig. 4, ABT-510 + VPA, left and right) or capillary like (type 2; Fig. 4, ABT-510 + VPA, middle). We also evaluated the effects of ABT-510 and VPA on the growth of large established neuroblastoma tumors. Similar to the previous experiment, significant hemorrhage was seen in the control SMS- KCNR tumors that were z380 mm3 in size when treatment was started (Fig. 5, Control, left and middle). These tumors had a continuous network of structurally abnormal type 1 vessels (Fig. 5, Control, right, middle, and left; Table 1B). Following treatment with ABT-510, the tumors were less hemorrhagic than the controls (Fig. 5, ABT-510, left and middle; Table 1B) and type 1 vessels Figure 6. Evan’s blue dye assay for intratumoral vessel permeability of the constituted only 14.68 F 3.83% (P < 0.0001) of the total (Fig. 5, ABT- SMS-KCNR xenografts. Quantitative analysis shows reduction of Evan’s blue extravasation in mice treated with ABT-510, VPA, and ABT-510 and VPA 510, right). The majority of vessels were thin walled, and some were in combination compared with controls. RQ, relative quantification by embedded in fibrocollagenous stroma (Fig. 5, ABT-510, middle). In spectrophotometry. Cancer Res 2007; 67: (4). February 15, 2007 1722
  8. 8. Valproic Acid and TSP-1 Inhibit Neuroblastoma Growth was recently granted Food and Drug Administration approval for block angiogenesis and that TSA specifically inhibits hypoxia- use in metastatic colon cancer (34). ABT-510, HDAC inhibitors, induced angiogenesis in the Lewis lung carcinoma model. and other promising antiangiogenic agents, either alone or in In the experiments done on both small and large neuroblastoma combination with cytotoxic agents, are currently being tested in xenografts, the combination of ABT-510 and VPA immediately and adult clinical trials. Although we and others have reported that fully stabilized the size of the tumors for 20 and 10 days, the growth of common pediatric cancers, such as neuroblastoma respectively, in the NMB (early treatment) and SMS-KCNR (late and Wilms’ tumor, is also angiogenesis dependent (4, 35), to treatment) models during which time vehicle controls substantially date, the experience with antiangiogenic agents in childhood increased in size. The MVD was significantly lower in xenografts malignancies is limited. In this study, we evaluated the effects of treated with combination therapy compared with tumors treated ABT-510 and VPA on neuroblastoma angiogenesis and tumor with single agents or controls. Furthermore, ABT-510 combined growth. As expected, both agents blocked bFGF-induced with VPA induced significant apoptosis and inhibited neuroblas- endothelial cell migration in vitro. In addition to inhibiting toma cell proliferation. Previous studies done in our laboratory angiogenesis, VPA also inhibited neuroblastoma cell proliferation with TNP-470 showed that this antiangiogenic agent was capable of and induced neuroblastoma apoptosis. Furthermore, MYCN inhibiting the growth of small neuroblastoma xenografts but did expression levels were significantly down-regulated by VPA, not affect the rate of growth of large neuroblastoma tumors (5). In suggesting that this agent is capable of affecting the biology of contrast, we found that the combination of ABT-510 and VPA was neuroblastoma cells. Similar changes in MYCN expression occur also capable of stabilizing the growth of large xenografts. in neuroblastoma following treatment with retinoic acid, a drug Hemorrhage was reduced in the neuroblastoma xenografts that is of clinical benefit in children with high-risk neuroblas- treated with single-agent therapy compared with control tumors, toma (36). We also found that VPA induced the expression of and virtually no hemorrhage was seen in the tumors treated with endogenous inhibitors of angiogenesis, including TSP-1 and combination therapy. In addition, striking changes in blood vessel SPARC. Previous work from our laboratory has shown that morphology were seen following treatment with ABT-510 and SPARC is a potent inhibitor of angiogenesis in neuroblastoma VPA alone or in combination. Control tumors contained large (27). In contrast, ABT-510 had no effect on the neuroblastoma numbers of structurally abnormal blood vessels with VEP. VEP is an cell growth in vitro. As single agents, both ABT-510 and VPA established unfavorable factor in several cancers, including inhibited neuroblastoma tumor growth in vivo, and enhanced melanoma, breast, prostate carcinomas, and glioblastoma (43, 44), antitumor effects were seen with combination therapy. and it is one of the criteria for raising the WHO grade of astrocytic ABT-510 has been extensively studied in cancers that are neoplasms (45). The number of vessels with VEP was significantly common in adults (15), but to our knowledge, this is the first decreased in the treated tumors, whereas the proportion of thin- report showing that this agent has antiangiogenic activity in the walled, capillary-like vessels was increased. Some of the thin-walled pediatric cancer neuroblastoma. TSP-1 and ABT-510 act by inducing vessels in the treated tumors were embedded in fibrocollagenous endothelial cell apoptosis, and recent studies suggest that TSP-1 stroma, whereas others exhibited hyaline sclerosis, a feature that is generates CD95L, a ligand for the CD95 death receptor (37). usually seen in hypertensive disease and attributed to endothelial However, because CD95 expression on the vascular endothelial cell cell injury (46–48). In the large SMS-KCNR tumors treated with is independent of TSP-1, the efficacy of the drug is limited. In an combination therapy, rare vessels with hyperplastic arterioloscle- effort to develop a more effective antiangiogenic treatment strategy, rosis were also seen. Using the Evan’s blue dye assay, we were also ABT-510 has been combined with other agents in preclinical models able to show that treatment with ABT-510 and VPA resulted in of adult cancer (38). We selected VPA for our combination studies modification of vessel function with decreased permeability of the for several reasons. First, VPA is a potent inhibitor of angiogenesis. tumor vasculature. Although its mechanism of action remains unclear, VPA has been Our results indicate that, in addition to reducing the number of shown to relieve HDAC-dependent transcription repression of blood vessels, the combination of ABT-510 and VPA modifies the several antiangiogenic proteins (39). Second, VPA directly affects structure and function of the vasculature in neuroblastoma tumors. the growth of neuroblastoma cells by inhibiting cell cycle Transient ‘‘normalization’’ of tumor vasculature following treatment progression and inducing differentiation (16). Third, VPA has been with other antiangiogenic agents has recently been reported (25). shown to have synergistic effects with other drugs that regulate These changes create a vasculature that is more efficient for oxygen angiogenesis, including IFN-a and retinoic acid (17, 18). Finally, VPA and drug delivery and may thereby lead to chemosensitization (34). has limited toxicities even during long-term treatment. Thus, in addition to inhibiting neuroblastoma tumor growth directly, Other HDAC inhibitors have also been shown to inhibit it is possible that ABT-510 and VPA may also enhance the antineu- neuroblastoma tumor growth. The hybrid polar HDAC inhibitor roblastoma activity of cytotoxic agents. Additional studies evaluat- m-carboxycinnamic acid bis-hydroxamide (CBHA) induces apo- ing the clinical efficacy of this antiangiogenic treatment strategy ptosis in neuroblastoma cells in vitro and suppresses the growth in children with high-risk neuroblastoma are warranted. of neuroblastoma xenografts. The combination of CBHA and all- trans retinoic acid led to an even greater inhibition of tumor Acknowledgments growth (40). Jaboin et al. (41) reported that the synthetic Received 7/14/2006; revised 11/14/2006; accepted 12/14/2006. benzamide derivative MS-27-275 (MS-275) inhibited [3H]thymidine Grant support: NIH/National Institute of Neurological Disorders and Stroke grant uptake in a series of pediatric tumor cell lines and suppressed the NS049814; Neuroblastoma Children’s Cancer Society; Friends for Steven Pediatric Cancer Research Fund; Elise Anderson Neuroblastoma Research Fund; Neuroblastoma growth of undifferentiated sarcoma, Ewing’s sarcoma, and Kids Fund; Abbott Laboratories; and Robert H. Lurie Comprehensive Cancer Center, neuroblastoma in vivo. These investigators reported a marked NIH, National Cancer Institute Core grant 5P30CA60553. decrease in the vascularization of neuroblastoma xenografts 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 following treatment with MS-275. Others (42) have indicated that with 18 U.S.C. Section 1734 solely to indicate this fact. trichostatin A (TSA) and suberoylanilide hydroxamic acid can We thank Dr. Alfred W. Rademaker for the statistical analyses. 1723 Cancer Res 2007; 67: (4). February 15, 2007
  9. 9. Cancer Research References the thrombospondin-1-mimetic angiogenesis inhibitor 33. Folkman J. Tumor angiogenesis: therapeutic implica- ABT-510 in patients with advanced cancer. J Clin Oncol tions. N Engl J Med 1971;285:1182–6. 1. Cheung NK, Cohn SL, editors. Neuroblastoma. Heidel- 2005;23:5188–97. 34. Kerbel RS. Antiangiogenic therapy: a universal chemo- berg: Springer-Verlag; 2005. 16. Cinatl J, Cinatl J, Driever PH, et al. Sodium valproate sensitization strategy for cancer? Science 2006;312:1171–5. 2. Brodeur GM. Neuroblastoma: biological insights into a inhibits in vivo growth of human neuroblastoma cells. 35. Abramson LP, Grundy PE, Rademaker AW, et al. clinical enigma. Nat Rev Cancer 2003;3:203–16. Anticancer Drugs 1997;8:958–63. Increased microvascular density predicts relapse in 3. Eggert A, Ikegaki N, Kwiatkowski J, Zhao H, Brodeur 17. Cinatl J, Kotchetkov R, Blaheta R, Driever PH, Vogel Wilms’ tumor. J Pediatr Surg 2003;38:325–9. GM, Himelstein BP. High-level expression of angiogenic JU, Cinatl J. Induction of differentiation and suppression 36. Matthay KK, Villablanca JG, Seeger RC, et al. factors is associated with advanced tumor stage in of malignant phenotype of human neuroblastoma Treatment of high-risk neuroblastoma with intensive human neuroblastomas. Clin Cancer Res 2000;6:1900–8. BE(2)-C cells by valproic acid: enhancement by combi- chemotherapy, radiotherapy, autologous bone marrow 4. Meitar D, Crawford SE, Rademaker AW, Cohn SL. nation with interferon-a. Int J Oncol 2002;20:97–106. transplantation, and 13-cis -retinoic acid. N Engl J Med Tumor angiogenesis correlates with metastatic disease, 18. Mongan NP, Gudas LJ. Valproic acid, in combination 1999;341:1165–73. N-myc amplification, and poor outcome in human with all-trans retinoic acid and 5-aza-2¶-deoxycytidine, 37. Quesada AJ, Nelius T, Yap R, et al. In vivo upregulation neuroblastoma. J Clin Oncol 1996;14:405–14. restores expression of silenced RARh2 in breast cancer of CD95 and CD95L causes synergistic inhibition of 5. Katzenstein HM, Rademaker AW, Senger C, Salwen H, cells. Mol Cancer Ther 2005;4:477–86. angiogenesis by TSP1 peptide and metronomic doxoru- Nguyen N, Cohn SL. Effectiveness of the angiogenesis 19. Bug G, Ritter M, Wassmann B, et al. Clinical trial of bicin treatment. Cell Death Differ 2005;12:649–58. inhibitor TNP-470 in reducing the growth of human valproic acid and all-trans retinoic acid in patients with 38. Yap R, Veliceasa D, Emmenegger U, et al. Metronomic neuroblastoma in nude mice inversely correlates with poor-risk acute myeloid leukemia. Cancer 2005;104: low-dose chemotherapy boosts CD95-dependent anti- tumor burden. Clin Cancer Res 1999;5:4273–8. 2717–25. angiogenic effect of the thrombospondin peptide ABT- 6. Shusterman S, Grupp SA, Barr R, Carpentieri D, Zhao 20. Tang R, Faussat AM, Majdak P, et al. Valproic acid 510: a complementation antiangiogenic strategy. Clin H, Maris JM. The angiogenesis inhibitor TNP-470 inhibits proliferation and induces apoptosis in acute Cancer Res 2005;11:6678–85. effectively inhibits human neuroblastoma xenograft myeloid leukemia cells expressing P-gp and MRP1. 39. Blaheta RA, Michaelis M, Driever PH, Cinatl J. growth, especially in the setting of subclinical disease. Leukemia 2004;18:1246–51. Evolving anticancer drug valproic acid: insights into Clin Cancer Res 2001;7:977–84. 21. Catalano MG, Fortunati N, Pugliese M, et al. Valproic the mechanism and clinical studies. Med Res Rev 2005; 7. Bornstein P. Diversity of function is inherent in acid induces apoptosis and cell cycle arrest in poorly 25:383–97. matricellular proteins: an appraisal of thrombospondin differentiated thyroid cancer cells. J Clin Endocrinol 40. Coffey DC, Kutko MC, Glick RD, et al. The histone 1. J Cell Biol 1995;130:503–6. Metab 2005;90:1383–9. deacetylase inhibitor, CBHA, inhibits growth of human 8. Grossfeld GD, Ginsberg DA, Stein JP, et al. Thrombo- 22. Takai N, Desmond JC, Kumagai T, et al. Histone dea- neuroblastoma xenografts in vivo, alone and synergistically spondin-1 expression in bladder cancer: association cetylase inhibitors have a profound antigrowth activity in with all-trans retinoic acid. Cancer Res 2001;61:3591–4. with p53 alterations, tumor angiogenesis, and tumor endometrial cancer cells. Clin Cancer Res 2004;10:1141–9. 41. Jaboin J, Wild J, Hamidi H, et al. MS-27-275, an progression. J Natl Cancer Inst 1997;89:219–27. 23. Michaelis M, Michaelis UR, Fleming I, et al. Valproic inhibitor of histone deacetylase, has marked in vitro and 9. Zabrenetzky V, Harris CC, Steeg PS, Roberts DD. acid inhibits angiogenesis in vitro and in vivo . Mol in vivo antitumor activity against pediatric solid tumors. Expression of the extracellular matrix molecule throm- Pharmacol 2004;65:520–7. Cancer Res 2002;62:6108–15. bospondin inversely correlates with malignant progres- 24. Adams GP, Weiner LM. Monoclonal antibody therapy 42. Deroanne CF, Bonjean K, Servotte S, et al. Histone sion in melanoma, lung, and breast carcinoma cell lines. of cancer. Nat Biotechnol 2005;23:1147–57. deacetylases inhibitors as anti-angiogenic agents alter- Int J Cancer 1994;59:191–5. 25. Jain RK. Normalization of tumor vasculature: an ing vascular endothelial growth factor signaling. Onco- 10. Yang QW, Liu S, Tian Y, et al. Methylation-associated emerging concept in antiangiogenic therapy. Science gene 2002;21:427–36. silencing of the thrombospondin-1 gene in human 2005;307:58–62. 43. Cavenee WK, Furnari FB, Nagane M, et al. Diffusely 26. Huang D, Rutkowski JL, Brodeur GM, et al. Schwann infiltrating astrocytomas. In: Kleihues P, Cavenee WK, neuroblastoma. Cancer Res 2003;63:6299–310. cell-conditioned medium inhibits angiogenesis. Cancer editors. Pathology and genetics of tumors of the nervous 11. Dawson DW, Volpert OV, Pearce SFA, et al. Three Res 2000;60:5966–71. system. WHO classification of tumors. Lyon (France): distinct D-amino acid substitutions confer potent 27. Chlenski A, Liu S, Crawford SE, et al. SPARC is a key IARC Press; 2000. p. 9–54. antiangiogenic activity on an inactive peptide derived Schwannian-derived inhibitor controlling neuroblasto- 44. Birner P, Piribauer M, Fischer I, et al. Vascular from a thrombospondin-1 type 1 repeat. Mol Pharmacol patterns in glioblastoma influence clinical outcome and ma tumor angiogenesis. Cancer Res 2002;62:7357–63. 1999;55:332–8. 28. Manohar CF, Short ML, Nguyen A, et al. HuD, a neuronal- associate with variable expression of angiogenic pro- 12. Huang HH, Campbell SC, Bedford DF, et al. specific RNA-binding protein, increases the in vivo stability teins: evidence for distinct angiogenic subtypes. Brain Peroxisome proliferator-activated receptor g ligands of MYCN RNA. J Biol Chem 2002;277:1967–73. Pathol 2003;13:133–43. improve the antitumor efficacy of thrombospondin 29. Chen SY, Yang AG, Chen JD, et al. Potent antitumour 45. Straume O, Chappuis PO, Salvesen HB, et al. peptide ABT510. Mol Cancer Res 2004;2:541–50. activity of a new class of tumour-specific killer cells. Prognostic importance of glomeruloid microvascular 13. Haviv F, Bradley MF, Kalvin DM, et al. Thrombo- Nature 1997;385:78–80. proliferation indicates an aggressive angiogenic pheno- spondin-1 mimetic peptide inhibitors of angiogenesis 30. Liu SQ, Tian YF, Chlenski A, et al. Cross-talk type in human cancers. Cancer Res 2002;62:6808–11. and tumor growth: design, synthesis, and optimization between Schwann cells and neuroblasts influences the 46. Mandal AK, Frohlich ED, Nordquist J. An ultrastruc- of pharmacokinetics and biological activities. J Med biology of neuroblastoma xenografts. Am J Pathol 2005; tural analysis of renal arterioles in benign and malignant Chem 2005;48:2838–46. 166:891–900. essential hypertensions. Ann Clin Lab Sci 1977;7:158–68. 14. Shaked Y, Bertolini F, Man S, et al. Genetic 31. Yang QW, Liu S, Tian Y, et al. Methylation-associated 47. Kashgarian M. Pathology of small blood vessel disease heterogeneity of the vasculogenic phenotype parallels silencing of the heat shock protein 47 gene in human in hypertension. Am J Kidney Dis 1985;5:A104–10. angiogenesis: implications for cellular surrogate marker neuroblastoma. Cancer Res 2004;64:4531–8. 48. Spector S, Ooshima A, Iwatsuki K, Fuller G, Cardinale analysis of antiangiogenesis. Cancer Cell 2005;7:101–11. 32. Crone C. Permeability of capillaries in various organs G, Udenfriend S. Increased vascular collagen biosynthe- 15. Hoekstra R, De Vos FYFL, Eskens FALM, et al. Phase I as determined by use of indicator diffusion method. sis by hypertension and reversal by antihypertensive safety, pharmacokinetic, and pharmacodynamic study of Acta Physiol Scand 1963;58:292–305. drugs. Blood Vessels 1978;15:176–82. Cancer Res 2007; 67: (4). February 15, 2007 1724