J Cancer Res Clin Oncol (2005) 131: 205–213 DOI 10.1007/s00432-004-0642-z
O R I GI N A L P A P E R
Hiroshi Kamiyama Æ Shingo Takano Æ Koji Tsuboi Akira Matsumura
Anti-angiogenic effects of SN38 (active metabolite of irinotecan): inhibition of hypoxia-inducible factor 1 alpha (HIF-1a)/vascular endothelial growth factor (VEGF) expression of glioma and growth of endothelial cells
Received: 30 August 2004 / Accepted: 18 October 2004 / Published online: 4 December 2004 Springer-Verlag 2004
Abstract Purpose: Inhibition of angiogenesis is an important new treatment modality for malignancies, including gliomas. Vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1a (HIF-1a) have been investigated as potent mediators of tumor angiogenesis. We investigated whether four major chemotherapeutic agents (ACNU, cisplatin, etoposide, and SN38) showed an angiosuppressive effect in vitro. Method: The effects of ACNU, cisplatin, etoposide, and SN38 for endothelial cells were assessed by cell growth inhibition assay (WST-8 assay) and vessel formation assay (angiogenesis kit). The inhibitory effects of the HIF-1a and VEGF expression of glioma cells after SN38 treatment were assessed by real-time RT-PCR, Western blot, and ELISA. Results: SN38, but not other chemotherapeutic agents, selectively inhibited endothelial cell proliferation and three-dimensional tube formations at the 0.01 lM. Furthermore, SN38 significantly decreased the HIF-1a and VEGF expression of glioma cells in a dose- and time-dependent manner under normoxic and hypoxic conditions. SN38 has dual angiosuppressive actions, including both the inhibition of endothelial proliferation and tube formation, and the inhibition of the angiogenic cascade in glioma cells. Conclusion: SN38 is an attractive agent as both a direct and indirect angiogenesis inhibitor and provides the anti-glioma agents with an angiosuppressive function. This study was supported by a Grant-in-Aid for Scientific Research from Japanese Ministry of Education, Science, and Culture and by a grant provided by the Tsukuba Advanced Research Alliance H. Kamiyama Æ S. Takano (&) Æ K. Tsuboi Æ A. Matsumura Department of Neurosurgery, Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba City, Ibaraki 305-8575, Japan E-mail:
[email protected] Tel.: +81-298-533220 Fax: +81-298-533214
Keywords SN38 Æ Irinotecan Æ HIF-1a Æ VEGF Æ Angiogenesis inhibitor
Introduction Angiogenesis, the growth of new blood vessels from those pre-existing in tissue, is crucial to glioma growth (Zagzag 1995). Glioblastoma is the most common and most malignant brain tumor in humans, and the feature that distinguishes glioblastoma from low-grade astrocytoma and normal brain is the presence of vascular endothelial proliferation and necrosis caused by tissue hypoxia (Bacher et al. 2003). Among many angiogenic factors, vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis in gliomas (Takano et al. 1996). VEGF expression is induced under hypoxic conditions, and the induction of VEGF is a multistage process in which the a subunit of hypoxia inducible factor-1 (HIF-1a) plays an important role (Christopher and Peter 2003; Safran and Kaelin 2003). HIF-1a rapidly decreases under normoxia because HIF-1a is bound to the tumor suppressor Von Hippel-Lindau (VHL) protein, and its interaction causes HIF-1a to become ubiquitylated and targeted to proteosome. However, because tumors generally become hypoxic, HIF-1a is stabilized and induces transcriptional activation of VEGF expression (Harris 2002; Carmeliet and Rakesh 2000). Therefore, the inhibition of HIF-1a becomes a valid therapeutic marker of tumor angiogenesis (Pili and Donehower 2003). Common anti-neoplastic agents, which are usually used for neoplasms with standard protocols, are one of the attractive angiosuppressive agents. Scheduled and long-acting alternative usage of these agents has been effective for some neoplasms (Gasparini 1999). The inhibitory mechanisms involved with these usages have
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been considered to be angiosuppression (Hayot et al. 2002; Bisacchi et al. 2003). In the present study, we investigated the anti-angiogenic effects of four antineoplastic agents (ACNU, cisplatin, etoposide, and SN38) that are commonly used for glioma patients on a scheduled basis (Yoshida et al. 1994; Ashby and Shapiro 2001; Parney and Chang 2003). Among them, CPT11 {7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin}, in which the active metabolite is SN38, has been recently intensively used for malignant glioma patients (O’Leary and Muggia 1998; Buckner et al. 2003; Reardon et al. 2003), but its inhibitory effects are limited (Parker et al. 2004). In addition, the investigation of its anti-proliferative mechanisms is limited to neoplastic cells. In this study, we demonstrated the anti-angiogenic effects of CPT11 and proposed a schedule for its use as an angiosuppressive agent for malignant glioma patients.
Materials and methods Cells and reagents Human glioma cell lines U87-MG and U251-MG, and calf pulmonary artery endothelial cell line (CPAE) were obtained from the American Type Culture Collection (Rockville, Md., USA). Mouse glioma cell line, GL261, was donated by Dr. Toda (Keio University). Immortalized human umbilical vein endothelial cell line, TE-1, was donated by Dr. Mitsui (Tokushima Bunri University) (Kobayashi et al. 1991; Tanaka et al. 1998). Cells were maintained in Eagle’s minimum essential medium supplemented with 10% fetal calf serum and 5% penicillin-streptomycin solution in a humidified atmosphere containing 5% CO2 at 37 C. At each passage, cells were harvested as single cell suspensions using trypsin/EDTA. U87-MG, U251-MG, and GL261 cells were cultured in falcon flask (Becton Dickinson, Franklin, N.J., USA). TE-1 and CPAE were cultured in Collagen type I coated flask (IWAKI, Tokyo, Japan). ACNU (1-(4-amino-2methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea, nimstine) was donated by Sankyo. Cisplatin (cis-diamminedichloroplatinum) and etoposide (4’demethlepipodophyllotoxin-9-(4,6-O-ethylidene-b-D-glucopyranoside)) were donated by Nippon Kayaku. SN38 was donated by Daiichi Pharmaceutical. Cell growth inhibition assay Cell survival was determined by a WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) assay kit (Kishida Kagaku, Osaka, Japan). This is based on the conversion of the tetrazolium salt WST-8 to highly water-soluble formazan by viable cells. Briefly, cells (5·103 cells/well) in 96-well plates were incubated overnight. Then, the medium was changed to the new medium with
various concentrations (0 lM, 0.01 lM, 0.1 lM, 1 lM, 5 lM, 10 lM, 50 lM, and 100 lM) of ACNU, etoposide, cisplatin and SN38. After 48 h incubation, WST-8 reagents were added to the culture. After 2 h incubation, the absorbance at 450 nm was measured with a Model 550 microplate reader (BioRad, Hercules, Calif., USA). Absorbance correlates with the number of living cells. The number of living cells (% Control) was calculated with the following formula: % Control = (Each absorbance Absorbance of blank well)/Absorbance of 0 lM well·100. In vitro angiogenesis assay Neovascularization was assessed in vitro using an angiogenesis kit (Kurabo, Osaka, Japan). Evaluation of angiogenesis, using angiogenesis kit, in vitro was performed by assessing the occurrence of microvessel structures from human umbilical vein endothelial cells (HUVEC) pre-seeded in a 24-well plate and co-cultured with fibroblasts cells. When HUVEC reached the early stage of neovascular formation, the medium was changed to an angiogenesis medium containing 10 ng/ml VEGF-A for control well. At the same time, the medium with various concentrations of ACNU, etoposide, cisplatin, and SN38 (0 lM, 0.001 lM, 0.01 lM, 0.1 lM, 1 lM, 5 lM, 10 lM, 50 lM, and 100 lM) were made with the angiogenesis medium and replaced. Furthermore, suramin at 50 lM, which is known to be an angiosuppressive drug, was assayed as a positive control. The medium was changed at days 2 and 5 of culture. On day 7 of incubation, cells were fixed 70% ethanol, and washed with PBS containing with 1% BSA. Cells were incubated for 1 h with anti-human CD31 antibody at the dilution of 1:4,000 and goat anti-mouse IgG at the dilution of 1:500. Cells were washed with distilled water, and added BCIP/NBT solution to stain vascular wall. Neovascularization was assessed under the microscope with ·200 magnification. Five fields (3.303 mm2) per well were photographed, and vessel lengths and branch-points were assessed using Win ROOF analyzing software (Mitani, Fukui, Japan) (Arthur et al. 1998; Yabushita et al. 2003). SN38 treatment of cells U87-MG and U251-MG cells were seeded at a concentration of 2·105 cells in 6-well plates at the day before treatment. Then, the medium was replaced with a new medium, containing SN38 at concentrations of 0 lM, 0.001 lM, 0.01 lM, 0.1 lM, 1 lM, and 10 lM. The cells were incubated with 5% CO2 at 37 C for 8 h, 16 h, and 24 h under hypoxic (0.1% O2) or normoxic conditions (20% O2). Hypoxic conditions were performed by placing cells in GasPak Pouch (Becton Dickinson, Sparks, Md., USA). Cells were harvested for RNA and protein expression and conditioned medium was collected for ELISA at each point.
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Real-time reverse-transcriptase polymerase chain reaction Total RNA from U87-MG and U251-MG cells were obtained using Rneasy Mini Kit (Qiagen, Maryland, Md., USA). RNA of 1 lg/ll or less was used to perform RTPCR using GeneAmp RNA PCR kit (Applied Biosystems, Foster, Calif., USA). The RT-PCR conditions were following: 15 min at 42 C, 5 min at 99 C, and 5 min at 5 C. To measure mRNA of human HIF-1a, VEGF, and 18S rRNA, real-time PCR was performed using an ABIPrism 7700 Sequence Detector (Applied Biosystems). Primers and specific probes of HIF-1a and VEGF were used Assays-on-Demand Gene Expression products (Applied Biosystems). 18S rRNA, a commonly used human housekeeping gene, was amplified using ribosomal RNA internal control reagents (Applied Biosystems). A master mix was made for each well: 10.3 ll DEPC-distilled water, 12.5 ll qPCRTM Mastermix (Eurogentec, Belgium), 1.2 ll primers and specific probes of HIF-1a, VEGF or 18S rRNA. A total of 24 ll of master mix and 1 ll cDNA of each sample were added to 96-well reaction plate. To generate a standard curve, template cDNA from U251-MG cells under hypoxic conditions was used. Realtime PCR cycles started with 2 min at 50 C, 10 min at 95 C, and then 40 cycles of 15 s at 95 C and 1 min at 60 C. Quantification of gene expression relative to HIF1a and VEGF was calculated by the standard curve and the cycle threshold of each sample.
used were monoclonal anti-HIF-1a clone H1a67 (Novus Biologicals, Littleton, Colo., USA) at the dilution of 1:500, polyclonal anti-VEGF clone A-20 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) at 1:200, and monoclonal anti-b-actin clone AC-74 (Sigma, Saint Louis, Mich., USA) at 1:5,000. After washing three times in TTBS buffer, membranes were incubated for 1 h at room temperature with a secondary antibody (diluted 1:2,000 in TTBS buffer), which was goat anti-mouse HRP conjugate antibody for HIF-1a and b-actin and goat anti-rabbit HRP conjugate antibody for VEGF. Membranes were washed three times in TTBS buffer, and chemiluminescence detection was performed using ECL reagents (Amersham Biosciences, Piscataway, N.J., USA). The protein expression was quantified with densitometric data of the each band using NIH image. Determination of VEGF levels in culture medium U87-MG cells were seeded in a 24-well plate at a density of 5·104 cell/well and incubated overnight. Cells were treated with SN38 for 24 h and 48 h under normoxic and hypoxic conditions. VEGF concentrations in the conditioned media were quantified by using the Quantikine human VEGF Immunoassay kit (R&D Systems, Minneapolis, Minn., USA).
Western blot analysis Cells were washed with PBS and added a nuclear extract (NE) buffer consisted of 20 mM dithiothreitol, 6% SDS, 0.25 M Tris[hydroxymethyl]aminomethane pH 6.8, 10% glycerol and bromophenol blue or a whole cell extract (WCE) buffer consisted of 25 mM Tris[hydroxymethyl]aminomethane pH 7.4, 100 mM NaCl, 20 mM NH4HCO3. Then 20% complete mini (Roche Diagnostics, Mannheim, Germany), which was dissolved with distilled water, was added to the NE buffer as a proteinase inhibitor. Cells were immediately collected from dishes. NE and WCE were prepared by sonicating of 5–10 s each on ice. The pellet was obtained after centrifugation at 11,000 rpm at 4 C for 30 min and the supernatants were collected. After the total protein quantities in the extracts were measured with DC Protein Assay Kit (BIO-RAD, Richmond, Calif., USA), the supernatants were stored at 20 C. Samples (30–60 lg) from the NE or WCE were subjected to protein electrophoresis with 7.5% SDS-PAGE and transferred to nitrocellulose membranes (BIO-RAD) by electrotransfer for 1 h. Membranes were washed with TTBS buffer (10 mM Tris[hydroxymethyl]aminomethane, 150 mM NaCl, 0.1% Tween 20) and blocked for 1 h at room temperature with TTBS buffer in 5% nonfat milk. Then, the membranes were developed with primary antibodies and incubated for overnight at 4 C. Primary antibodies
Fig. 1 Effect of increasing concentrations of ACNU, cisplatin, etoposide, and SN38 on cell proliferation. (a) Effect of four different chemotherapeutic agents against TE-1 cell line. SN38 selectively inhibited the proliferation of TE-1. (b) Effect of four different chemotherapeutic agents against GL261
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Statistical analyses The number of living cells (% of Control), vascular areas, numbers, branch-points, real-time PCR mRNA value of (HIF-1a/18S rRNA) and (VEGF/18S rRNA), and the densitometric value of HIF-1a and VEGF were expressed as mean±SD. Statistically significant differences between the groups were determined using a oneway analysis of variance and the Tukey-test. All P-values were 2-sided; values were considered statistically significant for P
