Mol Cancer Ther. 2006;5:2218-2224
© 2006 American Association for Cancer Research
Research Articles: Therapeutics
Antiangiogenic activity of 4'-thio-ß-D-arabinofuranosylcytosine
Anshu M. Roy,
Kamal N. Tiwari,
William B. Parker,
John A. Secrist, III,
Rongbao Li and
Zhican Qu
Drug Discovery Division, Southern Research Institute, Birmingham, Alabama
Requests for reprints: Zhican Qu, Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205. Phone: 205-581-2552; Fax: 205-581-2093. E-mail: qu{at}sri.org
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Abstract
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4'-Thio-ß-D-arabinofuranosylcytosine (T-araC), a new-generation deoxycytidine nucleoside analogue, showed significant efficacy against numerous solid tumors in preclinical studies and entered clinical development for cancer therapy. It is a structural analogue of cytarabine (araC), a clinically used drug in the treatment of acute myelogenous leukemia, which has no or very limited efficacy against solid tumors. In comparison with araC, the excellent in vivo activity of T-araC against solid tumors suggests that, in addition to inhibition of DNA synthesis, T-araC may target cellular signaling pathways, such as angiogenesis, in solid tumors. We studied T-araC and araC for their antiangiogenic activities in vitro and in vivo. Both compounds inhibited human endothelial cell proliferation with similar IC50s. However, only T-araC inhibited endothelial cell migration and differentiation into capillary tubules. T-araC also abrogated endothelial cell extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, a key signaling molecule involved in cellular processes of angiogenesis. Results from chick chorioallantoic membrane angiogenesis assays revealed that T-araC significantly inhibited the development of new blood vessels in vivo, whereas araC showed much less effect. The findings of this study show a role of T-araC in antiangiogenesis and suggest that T-araC combines antiproliferative and antiangiogenic activity in one molecule for a dual mechanism of drug action to achieve the excellent in vivo efficacy against several solid tumors. This study also provides important information for optimizing dosage and sequence of T-araC administration in clinical investigations by considering T-araC as both an antiproliferative and an antiangiogenic agent. [Mol Cancer Ther 2006;5(9):2218–24]
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Introduction
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Nucleoside analogues constitute an important class of antimetabolites used in the treatment of hematologic malignancies and, more recently, in solid tumors (1–3). These therapeutic compounds mimic physiologic nucleosides incorporated into newly synthesized DNA, leading to termination of DNA chain elongation and interference with DNA repair mechanisms, resulting in inhibition of rapidly proliferating cancer cells and induction of cell apoptosis. Key enzymes in nucleoside metabolic pathways are also targets of these nucleoside analogues, such as ribonucleotide reductase, an enzyme involved in the recycling of the cellular nucleotide pool. It has been found recently that nucleoside analogues are also able to modulate functions of important proteins in cellular signaling pathways, such as protein kinase C (4, 5) and mitogen-activated protein kinases (6).
The drug discovery program of Southern Research Institute (Birmingham, AL) has rationally designed and synthesized a new-generation nucleoside analogue, 4'-thio-ß-D-arabinofuranosylcytosine (T-araC; ref. 7), which exhibits improved antitumor activity related to cytarabine (araC), a clinically used chemotherapeutic. T-araC is structurally related to araC, with the replacement of the oxygen atom in the arabinose sugar ring by a sulfur atom. Both agents are analogues of the natural nucleoside deoxycytidine. However, the minor structural difference between the two analogues results in a significant difference in their antitumor activities. T-araC showed remarkable efficacy against numerous human solid tumors in xenograft mice, including renal, non–small cell lung, colon, pancreatic, prostate, and breast cancers (8), whereas araC has no or very limited effect on any of these solid tumors in vivo. T-araC, a promising antitumor compound, has completed two phase I clinical trials (9) and is now being considered for additional clinical trials.
Previous studies have shown that T-araC and araC exerted cytotoxic activity with similar IC50s in vitro against the same panel of human solid tumor cell lines that were used for in vivo mouse studies (7, 8). The basic mechanism of the antiproliferative action of these two agents is similar (10). araC is phosphorylated to its triphosphate, araCTP, which competes with dCTP as a substrate for incorporation into DNA (11, 12). Once it is incorporated, araCTP either stalls the replication fork or causes chain termination. T-araC, the 4'-thionucleoside analogue of araC, has been shown to have a similar mechanism of action in inhibiting DNA replication after the phosphorylation to its triphosphate form. Some important differences between these two agents have been recognized. T-araC is phosphorylated to active metabolites at 1% the rate of araC; however, the phosphorylated form of T-araC, T-araCTP, is a 10- to 20-fold more potent inhibitor of DNA synthesis than araCTP. In addition, T-araCTP has 10-fold the retention time as araCTP in solid tumor cells (10, 13, 14). The longer half-life and more potent inhibitory activity of T-araCTP may contribute to the differences in the antitumor efficacy of the two compounds in vivo. However, there may still be other molecular mechanisms involved, which contribute to the profound differences in antitumor activity of the two analogues against solid tumors.
It has not been determined if T-araC is active against tumor angiogenesis, which is required for a solid tumor to grow beyond 2 mm in diameter. The progression of solid tumors requires formation of new blood vasculature, and therefore, tumors gain access to the host vascular system for an adequate supply of oxygen and nutrients as well as for elimination of toxic waste products (15). Experimental evidence suggests that, in the absence of vascular support, tumors can remain dormant or eventually become necrotic or apoptotic (16). Several studies have shown that nucleobase and nucleoside analogues, such as 6-thioguanine (17) and a series of 5-and 6-substituted uracil derivatives (18), can be potent antiangiogenic agents. Many conventional chemotherapy agents, such as 5-fluorouracil, paclitaxel, and cyclophosphamide, used at low doses for prolonged periods (metronomic chemotherapy) display clinical activities presumably mediated via the inhibition of angiogenesis through their cytotoxic effects on proliferating endothelial cells (19). To address the possibility of angiosuppression, we examined the antiangiogenic activities of the nucleoside analogues T-araC and araC to further understand possible mechanisms of T-araC activity in solid tumors. The results showed that T-araC inhibited angiogenesis in different in vitro assays that represent different steps of the angiogenic cellular process and exerted a potent in vivo antiangiogenic activity in the chick chorioallantoic membrane assay. Based on these results, it is suggested that T-araC targets two important pathways of solid tumor progression at the same time, tumor cell proliferation and neoangiogenesis. A dual mechanism of drug action that combines antiproliferative and antiangiogenic activity in one molecule may contribute to excellent in vivo efficacy of T-araC against several solid tumors.
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Materials and Methods
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Cell Culture
Human umbilical vascular endothelial cell (HUVEC) cultures were obtained from Clonetics (San Diego, CA) and cultured in HUVEC growth medium, which is the endothelial cell basal medium, supplemented with 2% fetal bovine serum, 12 µg/mL bovine brain extract, 1 µg/mL hydrocortisone, and 1 µg/mL GA-1000 (gentamicin-amphotericin B). HUVEC cultures, between passages 4 and 8, 
70% confluent, were used for most of the experiments. A human lung fibroblast cell line (LL47) and a human prostate cancer cell line (DU145) were obtained from the American Type Culture Collection (Manassas, VA) and cultured in improved modified Eagle's medium (Invitrogen Corp., Carlsbad, CA) supplemented with 50 µg/mL gentamicin (CellGro, Herndon, VA) and fetal bovine serum in the recommended percentage. ML20 is an MCF-7-derived breast cancer cell line that contains a stably transfected LacZ gene (20). ML20 cells were maintained in improved modified Eagle's medium supplemented with 10% fetal bovine serum. All cells were cultured at 37°C with 5% CO2.
Materials
T-araC was chemically synthesized at Southern Research Institute following our published procedure (7, 21), and araC was purchased from Sigma Chemical Co. (St. Louis, MO). Biocoat angiogenesis endothelial cell migration inserts (BD Biosciences Discovery Labware, Bedford, MA) and Hoechst 33342 (Molecular Probes, Inc., Eugene, OR) were used for the migration assay. Extracellular matrix from Chemicon International, Inc. (Temecula, CA) was used for the endothelial cell tube formation assay. Antibodies against p44/42 mitogen-activated protein kinase [extracellular signal-regulated kinase (ERK) 1/2] and phosphorylated p44/42 mitogen-activated protein kinase (Thr202/Tyr204) were obtained from Cell Signaling Technology, Inc. (Beverley, MA). All other chemicals used were of pure analytic grade and procured from Sigma Chemical.
Cell Viability Assay
Sensitivity of HUVEC culture to T-araC and araC was determined by CellTiter-Glo Luminescent Cell Viability Assay (Promega Corp., Madison, WI). As described in detail (22), this assay, generating luminescent signals, is based on quantification of ATP levels in cell cultures. Amount of ATP produced in cell culture reflects the number of viable cells. Hence, this assay is often used to estimate cell proliferation and cytotoxic effects of test compounds. Endothelial cells were seeded in 96-well plates in the growth medium, 5 x 103 per well. After 24 hours, various doses of araC and T-araC were added to the endothelial cultures, having six replicates for each dose. After 72 hours of treatment, CellTiter-Glo reagent was added to the cultures following the manufacturer's instruction and luminescence was measured with EnVision Multilabel Reader (Perkin-Elmer, Wellesley, MA). The control groups were given DMSO vehicle only. IC50s of T-araC and araC for endothelial cell proliferation in the growth medium were determined based on the dose response curve and plotted against a range of 1 nmol/L to 20 µmol/L concentrations.
Migration Assay
The BD Biocoat Angiogenesis System was used for the endothelial cell migration assay, which was a 24-Transwell chamber plate containing 3-µm pore size inserts coated with human fibronectin. The inserts were incubated at 37°C with 0.1% bovine serum albumin containing endothelial cell basal medium for 1 hour. Endothelial cells were starved with 0.1% bovine serum albumin in endothelial cell basal medium for 4 to 5 hours before the cell harvest and then seeded (1 x 105 per well) in upper chambers of the Transwell plate with various treatments in 100 µL of 0.1% bovine serum albumin in endothelial cell basal medium. The growth medium that contained various chemoattractants was added in the lower chambers. The cells were allowed to migrate for 22 ± 1 hours at 37°C. Unmigrated cells at the inside of the inserts were carefully removed with a Q-tip. Migrated cells at the lower part of the Transwell inserts were then fixed with 4% paraformaldehyde, stained with Hoechst 33342, and photographed under a fluorescent microscope (Zeiss Axiovert 200 M, Carl Zeiss MicroImaging, Inc., Thornwood, NY). Data were expressed as average number of migrated cells per microscopic view field of x10 objective magnification. Three view fields per insert were analyzed for each triplicate treatment condition. IC50s of T-araC and araC for endothelial cell migration were determined based on the dose response curve of the plotted data.
Endothelial Cell Tube Formation Assay
Endothelial cells were seeded (1.5 x 104 per well) in a 96-well plate coated with extracellular matrix with different conditions in the growth medium in triplicate. Cells were allowed to form endothelial tubes at 37°C for 12 to 18 hours and photographed under an inverted light microscope. The tubule lengths were quantified using image analysis software, Image-Pro Plus (Media Cybernetics, Inc., Silver Spring, MD). Data were expressed as average tube length of three view fields for each well and triplicate wells for each treatment condition.
Chick Chorioallantoic Membrane (CAM) Assay
Fertilized chicken eggs (S&G Poultry Co., LLC, Clanton, AL) were incubated at 37.5°C in a humidified egg incubator with forced air circulation. On embryonic day 4, eggs were cracked open and embryos were transferred into 100-mm3 Petri plates to continue their development in a cell culture incubator at 37.5°C with 1.5% CO2. Test compound in stock solution was mixed with 0.5% methylcellulose in water. Drops of this solution (10 µL per drop) were allowed to dry on a Teflon-coated surface forming methylcellulose discs of 2 mm in diameter. The methylcellulose discs were gently implanted on top of chicken chorioallantoic membrane on embryonic day 6, and the embryos were incubated for 2 more days. The chorioallantoic membranes were then examined and photographed under a stereomicroscope. An average of 10 embryos was used to assay each compound. Drug effect on angiogenesis was analyzed by using Image-Pro Plus software to calculate the vascular density index, which represents the number of intersections made by blood vessels with three equidistant concentric circles on the area covered by methylcellulose discs. Vascular density index under different treatments was calculated and plotted as a percentage of the control.
Immunoblotting Analysis
HUVECs and other cell lines were harvested and lysed with ice-cold cell lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerophosphate, 1 mmol/L sodium orthovanadate, 1 µg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride]. The cell lysates were then cleared by centrifugation at 14,000 x g for 10 minutes at 4°C. Total proteins (50–75 µg per sample) of each lysate sample were resolved on a 4% to 12% SDS-PAGE and transferred onto nitrocellulose membrane. Appropriate concentrations of primary antibody in blocking buffer (5% nonfat dry milk in TBS with 0.1% Tween 20) were used to probe the membrane overnight at 4°C after blocking in blocking buffer at room temperature. The detected protein bands were visualized using the enhanced chemiluminescence detection system (Amersham Life Science, Inc., Piscataway, NJ) after probing with secondary antibodies. After probing with phosphorylated ERK1/2 antibodies, the blots were stripped with Restore stripping solution (Pierce, Rockford, IL) and reprobed with antibodies against total ERK1/2.
Statistical Analysis
Statistical analysis was done by using Student's t test method in SigmaPlot software to determine differences between various treatment conditions in angiogenesis assays. The differences were considered to be statistically different when P < 0.05 as calculated by the software.
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Results
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T-araC Inhibits Angiogenesis In vitro
In solid tumors, adjacent endothelial cells are activated by angiogenic growth factors released from tumor cells to initiate a complex neoangiogenic process, including endothelial cell proliferation, migration, and differentiation into capillary vessels. Inhibition of tumor angiogenesis has been proven to be an effective treatment for human solid tumors. T-araC and araC are closely related nucleoside analogues with different antisolid tumor activities. We examined and compared their effects on angiogenesis, including endothelial cell proliferation, migration, and tube formation, in vitro. The growth medium for endothelial cell culture is supplemented with fetal bovine serum and bovine brain extract, which contain several angiogenic growth factors that stimulate endothelial cell proliferation in vitro. To investigate possible angiosuppressive activity of T-araC and araC, both compounds were tested for their capacity to inhibit endothelial cell growth using an ATP-based cell viability assay with concentrations from 1 nmol/L to 20 µmol/L. T-araC and araC showed similar inhibitory activity on the endothelial cell proliferation (Fig. 1
). The IC50 for both compounds was 2 µmol/L, which is in the same range as that observed previously with several human solid tumor cell lines (7). For these solid tumor cell lines, the cell viability was measured using the neutral red assay or the sulforhodamine B assay, and the IC50 for T-araC was in a range of 2 to 40 µmol/L, whereas that for araC was in a range of 0.6 to 10 µmol/L. Therefore, the observed inhibitory activity of T-araC and araC in growth-stimulated endothelial cells seems to be similar to that in the solid tumor cell lines. This antiproliferative activity of T-araC and araC for endothelial cells is likely a result of their inhibitory effect on DNA synthesis, a mechanism of drug action previously recognized for these two agents in tumor cells.
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Figure 1. araC and T-araC inhibit endothelial cell viability. HUVEC cultures were treated with araC or T-araC at varying concentrations (1 nmol/L to 20 µmol/L), and the cell viability, in terms of ATP-based luminescence intensity, was examined 3 d after the treatments. The data were plotted as percentage inhibition of endothelial cell viability compared with the control. The IC50s were determined based on the dose response curve.
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Endothelial cell migration is a key step of the angiogenesis process, which is crucial for on-site recruitment of blood vessel formation. The effect of T-araC and araC on endothelial cell migration toward angiogenic growth factors was investigated by using a Transwell system to examine the chemotactic mobility of the endothelial cells. As shown in Fig. 2A
, T-araC at 1 µmol/L concentration reduced the endothelial cell migration to 42% compared with the control (P < 0.001) under the chemotactic influence of the full growth medium containing numerous angiogenic factors. In contrast, araC showed no significant effect on the endothelial cell migration at the same drug concentrations. The IC50 for T-araC in endothelial cell migration, as computed, was 1.3 µmol/L and that for araC was >10 µmol/L (Fig. 2B). Thereby, T-araC is much more potent than araC in inhibiting endothelial cell migration.
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Figure 2. A, T-araC inhibits human endothelial cell migration. HUVEC culture migration was assayed using a Transwell culture system under treatment with araC or T-araC (0.01–10 µmol/L) for 22 h. The migrated cells were counted in three microscopic fields (x10 objective magnification) for each triplicate condition. Columns, average of migrated cells per field of each treatment condition; bars, SD. B, dose response curve of T-araC and araC. The migration data were plotted as percentage inhibition of endothelial cell migration compared with the control, and the IC50s were computed.
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Following cell migration toward angiogenic factors, an endothelial cell differentiation process is initiated during angiogenesis. The migrated cells start to change shape, adhere to each other, and form a lumen structure of new blood vessels (15). Human endothelial cells can be cultured in vitro on extracellular matrix, which stimulates the attachment, migration, and differentiation of endothelial cells into tubules in a manner that mimics the in vivo situation (23, 24). The endothelial tube formation assay is based on this phenomenon and has been widely used to evaluate antiangiogenic activity of compounds. T-araC and araC were further compared and tested in the tube formation assay with 2 µmol/L concentration, by which both T-araC and araC showed 50% inhibition for the endothelial cell growth (Fig. 1). The experimental results showed that the T-araC treatment reduced average endothelial tubule length >50%, from 98 to 46 µm (P < 0.001); araC did not affect the average tubular length (Fig. 3
). Thus, T-araC effectively interrupted the formation of endothelial tubes in vitro, whereas araC had no inhibitory effect at the same concentration. Therefore, these in vitro studies showed that T-araC inhibited endothelial cell proliferation, migration, as well as differentiation to form capillary tubules, whereas araC showed only inhibitory activity on cell proliferation. These results suggest that, in addition to its inhibitory effect on DNA synthesis in rapidly proliferating cells, T-araC also targets other cellular activities during the angiogenesis process, possibly the signal transduction pathway responsible for endothelial cell migration and differentiation.
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Figure 3. T-araC inhibits human endothelial cell tube formation. Top, representative images of endothelial tube formation under treatment with araC or T-araC (2 µmol/L) for 15 h; bottom, graphic representation of a quantitative analysis of average tubular lengths by using Image-Pro Plus analysis software.
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T-araC Inhibits Angiogenesis In vivo
Studies have shown that compounds that affect endothelial cells in vitro may not necessarily regulate angiogenesis in vivo (25). The in vivo assessment is a vital step in evaluation of angiogenic modulators. Chicken embryo chorioallantoic membrane provides an ideal in vivo model for the physiologic process of angiogenesis. Angiogenic modulators released by methylcellulose discs placed on top of chicken embryo chorioallantoic membrane are able to alter the development of new blood vasculature (26). The angiogenesis chorioallantoic membrane assay is used for in vivo evaluation of the antiangiogenic potential of synthetic compounds and recombinant proteins. Based on our in vitro observations, we further assessed the in vivo antiangiogenic activity of T-araC and araC using the chorioallantoic membrane assay. We have developed a method of quantifying the chorioallantoic membrane blood vessel network by using a vascular density index, which represents the number of intersections made by blood vessels with three concentric circles within the chorioallantoic membrane area covered by a methylcellulose disc. In the control methylcellulose disc, which had only DMSO vehicle, the blood vessels formed a dense and spatially oriented leaf-like branching network as shown in Fig. 4
. Treatment with T-araC (7.5 µg, 28.5 nmol) limited the development of blood vascular network on chicken embryo chorioallantoic membrane. The blood vessels were markedly diminished not only in the chorioallantoic membrane area under the methylcellulose disc but also in a larger area around the disc containing T-araC. Quantitative analysis showed that the chorioallantoic membrane vascular density index was reduced by 70% on the T-araC treatment compared with the control (P < 0.001), whereas araC treatment resulted in a 30% reduction (P < 0.01) of the vascular density index. Therefore, T-araC showed superior in vivo antiangiogenesis activity over its structurally related analogue, araC.
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Figure 4. T-araC inhibits angiogenesis in vivo. Top, representative images of chicken embryo chorioallantoic membrane treated with araC or T-araC (7.5 µg or 28.5 nmol/methylcellulose disc) for 2 d. Dotted circles, area of the chorioallantoic membrane under the methylcellulose disc. Bottom, graphic representation of the quantitative analysis of the vascular density index as described in Materials and Methods. Percentage inhibition by araC and T-araC compared with the control.
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T-araC Inhibits ERK1/2 Phosphorylation in Human Endothelial Cells
As shown in the angiogenesis in vitro study, T-araC inhibited not only the proliferation of endothelial cells but also the cell migration and differentiation. To elucidate the mechanism of T-araC action in antiangiogenesis, T-araC and araC were evaluated for their capacity to affect the activation of the ERK intracellular signaling pathway in endothelial cells. The ERK signaling pathway is involved in many important cellular processes of angiogenesis, including those of endothelial cell survival, migration, differentiation, and morphogenesis (27). To study whether T-araC interfered with ERK signaling, growth factor–stimulated endothelial cells were assessed for the phosphorylation of ERK1/2 by Western blot using an antibody against phosphorylated-specific ERK1/2. T-araC treatment for 2 hours at a concentration (3.8 µmol/L), which is 2-fold of IC50 of T-araC for the endothelial cell migration and tube formation, dramatically reduced the ERK1/2 phosphorylation in the cell culture (Fig. 5
). In contrast, araC treatment at the same concentration did not inhibit ERK phosphorylation; however, there was even a slight increase in the ERK1/2 phosphorylation for 30 minutes of treatment, consistent with the reported study that araC is able to transiently induce ERK1/2 activation in HL-60 cells (28). The observed inhibition of ERK1/2 phosphorylation by T-araC in endothelial cells led us to further compare the effect of T-araC and araC on other cell lines. Experimental results showed that the ERK1/2 phosphorylation remained unchanged on either T-araC or araC treatments in normal human lung fibroblast cells (LL47), human prostate tumor cells (DU145), and MCF-7-derived human breast tumor cells (ML20; Fig. 6
). However, both agents inhibited cell proliferation in these three cell lines (data not shown). These data suggested that T-araC may specifically target the signaling pathway of endothelial cells during the angiogenesis process, although there is no direct evidence yet to prove that the antiangiogenic activity of T-araC is mediated by inhibition of ERK signaling pathway. Further studies on the molecular mechanism of the antiangiogenesis activity of T-araC are warranted.
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Figure 5. T-araC, not araC, inhibits the phosphorylation of ERK1/2 in endothelial cells. Total cell lysates were prepared from cell cultures treated with araC or T-araC (3.8 µmol/L) for various time points. Western blot of the lysates was carried out with antibodies against phosphorylated-specific ERK1/2 (pERK1/2) and protein ERK1/2.
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Figure 6. T-araC does not affect the ERK1/2 phosphorylation in human fibroblast and tumor cells. Western blot of cell lysates prepared from different cell lines treated with araC or T-araC (3.8 µmol/L) for 2 h with antibodies against phosphorylated-specific ERK1/2 and ERK1/2. LL47 is a normal human fibroblast cell line, DU145 is a human prostate cancer cell line, and ML20 is an MCF-7-derived human breast cancer cell line.
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Discussion
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Angiogenesis is a multistep process that is usually initiated by endothelial cell activation. The activated cells proliferate, migrate, and differentiate into capillary tubes with lumens to complete the formation of the new blood vascular network (17). Each step of this process represents a potential target for the inhibitory action of angiosuppressive agents (29). In this article, we describe the antiangiogenic activity of T-araC, a nucleoside analogue in clinical development for cancer therapy, versus its structural analogue, araC. Our results have shown that T-araC inhibits multiple steps in the angiogenic cellular processes and reveal a potential new mechanism of drug action of T-araC against solid tumors, in addition to its potent effect on DNA synthesis of rapidly proliferating cancer cells.
Nucleoside analogues comprise a major class of chemotherapeutic agents for the treatment of cancer and viral diseases. They are structurally and metabolically related agents that nevertheless have varied biological effects and therapeutic outcomes. Most cytotoxic effects exerted by nucleic acid analogues are the result of affecting DNA synthesis (30). Because endothelial cells are in a mitogenic phase due to growth stimulation by angiogenic growth factors, both araC and T-araC are expected to inhibit endothelial cell proliferation by their known cytotoxic actions. Hence, the observed equivalent inhibition by araC and T-araC on endothelial cell growth most likely results from the capacity of both agents to inhibit DNA synthesis as observed previously in several cancer cell lines.
In addition to being as cytotoxic as araC in proliferating endothelial cells, T-araC significantly inhibited endothelial cell migration and differentiation in vitro, two key steps of the angiogenesis process. In some cases, however, potent in vitro activity of a synthetic compound cannot be realized in vivo. Therefore, an in vivo assessment is important in evaluation of angiogenic modulators. The chicken embryo chorioallantoic membrane provides an in vivo physiologic model for the process of angiogenesis, and the chorioallantoic membrane assay is widely used to evaluate angiosuppressive compounds (26). The in vitro findings of antiangiogenic activities of T-araC were substantiated by the in vivo study with the chorioallantoic membrane assay. The modest inhibitory effect of araC on the new blood vessel formation shown by the chorioallantoic membrane assay is possibly due to its inhibition of endothelial cell proliferation. T-araC was superior in its effect on the inhibition of new blood vessel formation, as it was not only shown to be antiproliferative in endothelial cells but had also inhibited other vital steps of the angiogenic process, such as endothelial cell migration and differentiation.
The results from the in vitro and in vivo angiogenesis experiments suggest that T-araC may target intracellular proteins of endothelial cells that are involved in signal transduction of angiogenic process. The ERK phosphorylation cascade has been characterized as one of the main signaling pathways involved in endothelial cell survival, migration, differentiation, and morphogenesis (31). T-araC is able to inhibit ERK1/2 phosphorylation in activated endothelial cells, but it does not inhibit the same protein phosphorylation in other cell lines, such as human fibroblast and cancer cell lines. These results highlight the specificity of T-araC activity to endothelial cells. Interestingly, araC did not inhibit the ERK1/2 phosphorylation in endothelial cells; instead, it transiently increased the ERK1/2 phosphorylation levels. Currently, we are investigating whether the antiangiogenic activity of T-araC is mediated by its inhibition of ERK activation and other signaling pathways.
T-araC requires conversion to its triphosphate form by intracellular kinases for anti-DNA synthesis activity. It is still unclear which form of T-araC mediated the observed antiangiogenic activities. It is possible that T-araC or one of its metabolite forms, including its monophosphate, diphosphate, and triphosphate forms, interacts with protein(s) in the angiogenic signaling pathway to inhibit the protein phosphorylation cascade. Understanding the molecular mechanism underlying the inhibitory action of T-araC in endothelial cell proliferation, migration, and differentiation is important for designing new generation of nucleoside analogues for effective anticancer therapy. The results of the present study suggest that T-araC exerts its superior antitumor effect in solid tumors by a dual mechanism of drug action. T-araC combines antiproliferative and antiangiogenic activities in one molecule and has shown excellent in vivo efficacy against several solid tumors. More studies have suggested that treatment sequence and dosage are important for the development of an antiangiogenic agent in cancer treatment. The finding of this study provides information for considering T-araC as both an antiproliferative and an antiangiogenic agent in future clinical investigations, especially for optimizing dosage and sequence of administration and combinations with other anticancer drugs for treatment of human solid tumors.
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Footnotes
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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.
Received 1/26/06;
revised 6/16/06;
accepted 6/29/06.
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Abstract
Introduction
