Antiangiogenic Therapy with Mammalian Target of Rapamycin ...

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Feb 1, 2008 - cer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radio- therapy (5 x 2 or 5 ... Development of a tumor microvascular network by angio- ...... amplify the direct anti-endothelial action of radiation.

CancerTherapy: Preclinical

Antiangiogenic Therapy with Mammalian Target of Rapamycin Inhibitor RAD001 (Everolimus) Increases Radiosensitivity in Solid Cancer Philipp C. Manegold,1Carmen Paringer,1Ulrike Kulka,2 Klaus Krimmel,2 Martin E. Eichhorn,1Ralf Wilkowski,2 Karl-Walter Jauch,1 Markus Guba,1 and Christiane J. Bruns1


Purpose: Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth. Experimental Design: Effects of radiation combined with mTOR inhibitor RAD001were studied on proliferation of murine colon cancer CT-26, human pancreatic cancer L3.6pl, and human umbilical vascular endothelial cells in vitro. In vivo tumor growth of subcutaneous colon cancer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radiotherapy (5  2 or 5  4 Gy) with or without the addition of the mTOR inhibitor RAD001. RAD001 (1.5 mg/kg/d) was administered until the end of experiments beginning before or after radiotherapy. Results: A single dose of 2 Gy reduced in vitro proliferation of L3.6pl (-16%), CT-26 (-70%), and human umbilical vascular endothelial cells (HUVEC; -72%). The mTOR inhibitor RAD001 (10 ng/mL) suppressed proliferation of HUVEC (-83 %), L3.6pl (-8%), and CT-26 (-82 %). Combination of even low concentrations of 0.01 ng/mL RAD001 and 0.25 Gy radiation significantly reduced proliferation of HUVECs (-57%), whereas additive effects of RAD001 and radiation on tumor cells were seen only at the highest concentrations tested. In vivo, RAD001 introduced before radiotherapy (5  2 Gy) improved tumor growth control in mice (L3.6pl: 326 mm3 versus 1144 mm3; CT-26: 210 mm3 versus 636 mm3; P < 0.05 versus control). RAD001turned out to possess a dose-modifying effect on radiotherapy. Conclusion: Endothelial cells seem to be most sensitive to combination of mTOR inhibition and radiotherapy. Additive tumor growth delay using the mTOR inhibitor RAD001 and radiotherapy in vivo therefore might rely on combined antiangiogenic and antivascular effects.

Radiotherapy is one of the most widely used therapeutic modalities in treatment of cancer. Local control of tumor growth is achieved by radiation-induced cell death as a result of damage to cell membranes and DNA. DNA damage can be a consequence of direct radiation effects or indirectly induced through reactive oxygen species (1, 2). These effects are not

limited to tumor cells but also affect microvascular endothelial cells within the tumor stroma (3, 4). Therefore, radiosensitivity of solid tumors is not only defined by intrinsic factors such as metabolic activity and cell cycle, but also by the tumor microvascular network providing oxygen supply. Development of a tumor microvascular network by angiogenic processes is inevitable for tumor growth and metastasis. Tumor cells produce growth factors that stimulate proliferation and migration of endothelial cells, and finally the formation of new blood vessels within the tumor tissue (5). The irregular architecture and the high permeability of tumor microvessels cause heterogeneous blood flow, high interstitial fluid pressure within the tumor, and hypoxic tumor areas. These hypoxic tumor areas are more resistant to radiotherapy despite their high vascular density (6). Radiotherapy damages existing tumor microvessels by induction of endothelial cell apoptosis (3). These antivascular effects significantly contribute to tumor growth control by radiotherapy. In response to the endothelial damage and subsequent hypoxia, however, tumor cells increase their expression of proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (7, 8). This, in

Authors’Affiliations: Departments of 1Surgery and 2Radiation Oncology, Klinikum Grosshadern, University of Munich, Munich, Germany Received 4/21/07; revised 10/2/07; accepted 11/8/07. Grant support: Wilhelm Sander Stiftung (no. 2003.133.1), the Deutsche Forschungsgemeinschaft (SPP1190:64 489/3-1), and the Fo«FoLe Research Program (no. 436) of the University of Munich, Munich, Germany. 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. Note: P.C. Manegold and C. Paringer contributed equally to this work. Requests for reprints: Philipp Manegold, Department of Surgery, Klinikum Grosshadern, University of Munich, Marchioninistrasse 15, 81377 Munich, Germany. Phone: 49-89-7095-3430; Fax: 49-89-7095-6433; E-mail: Philipp. [email protected] F 2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-0955

Clin Cancer Res 2008;14(3) February 1, 2008


mTOR Inhibition Increases Radiosensitivity in Solid Tumors culture medium. mTOR inhibitor RAD001 (Everolimus; kindly provided by Novartis Institutes for Biomedical Research, Basel, Switzerland) was added to cultured cells 1 h before radiation or 24 h after radiation in escalating concentrations of 0.01, 0.1, 1, 10, or 20 ng/mL. Medium was changed every 48 h with and without addition of the mTOR inhibitor. Radiation was applied in single doses of 0.25, 0.5, 1, or 2 Gy (Mueller RT250, 225 kV, 15 mA). Cells were also treated with either the mTOR inhibitor RAD001 or radiation alone; untreated cells served as controls. Cell proliferation was assessed 5 to 6 days after radiation using the colorimetric WST-1 10% proliferation assay (Roche Diagnostics GmbH). Absorbance was measured in an ELISA reader at 450 nm 1 h after addition of WST-1. Immunocytochemistry. For analysis of the PI3K/Akt/mTOR pathway activation in HUVECs and tumor cells, phosphorylation of the s6 ribosomal protein (s6rp) as a downstream target of mTOR and p70s6K was assessed by immunocytochemistry of cultured cells. HUVECs and tumor cells were plated on glass coverslips in completely supplemented medium; 24 h later, cells were starved in FBS and supplement-depleted diet medium for 16 h. After this conditioning period, cells were stimulated with 10% FBS; 10 ng/mL insulin-like growth factor (tumor cells); or 10 ng/mL VEGF (HUVECs) or single-dose radiation of 0.5 Gy (HUVEC), 1 Gy (CT-26), or 2 Gy (L3.6pl) with or without pretreatment with 20 ng/mL RAD001 over 1 h. Thirty minutes after stimulation, cells were fixed in methanol acidic acid. Primary rabbit anti – phospho-S6 ribosomal protein (Ser235/236) antibody (Cell Signalling Technology) was added. Detection of the primary antibody was done with a goat anti-rabbit biotinylated antibody and streptavidin (Vector). Vectashield 4¶,6-diamidino-2-phenylindole (Vector) was used for staining of cell nuclei. VEGF ELISA. For in vitro measurement of VEGF production, 4  104 L3.6pl human pancreatic cancer cells or CT-26 colon cancer cells were cultured in six-well culture plates for 24 h. Fresh medium only or medium supplemented with 10 ng/mL RAD001 was added, and cells were incubated for a further 1 h. Tumor cells were treated with single-dose radiation of 2, 4, or 10 Gy (Mueller RT250, 225 kV, 15 mA). Cell supernatants were harvested 72 h after radiation, and quantitative measurements of VEGF were done by ELISA (R&D Systems) according to the instructions of the manufacturer. VEGF concentration was determined spectrophotometrically at 450 nm in combination with a wavelength correction at 570 nm. Animals. Immunodeficient male NMRI nu/nu mice (30-35 g; Harlan Winkelmann) and male BALB/c mice (20-25 g; Charles River) were used for the in vivo experiments. Immunodeficient NMRI nu/nu mice were kept under continuous laminar flow. The animals had free access to water and standard laboratory food throughout the experiments. All experimental procedures done on mice were approved by the local authorities (Regierung von Oberbayern, 55.2-1-54-2531-67/05 and 209.1-211-2531-38/04). Orthotopic pancreatic tumor model. Human pancreatic cancer cells L3.6pl were injected orthotopically in the pancreas of immunodeficient NMRI nu/nu mice according to the method described previously (20). Briefly, after a small left abdominal incision, the spleen was exteriorized and 8  105 tumor cells were injected in the subcapsular region of the pancreas beneath the spleen. Tumor growth was monitored throughout the experiments by abdominal palpation and two-dimensional measurements using a caliper. Tumor volume was calculated according to the formula for ellipsoids: tumor volume = p/6  diametershort2  diameterlong. Subcutaneous tumor model. Subcutaneous tumors were generated by injection of 8  105 tumor cells s.c. in the mid-dorsal region of the animals. CT-26 cancer cells were injected in syngenic BALB/c mice. The tumor volumes were estimated by three-dimensional measurement of subcutaneous tumors using a caliper and calculation of the volumes according to the following equation: tumor volume = a  b  c. If tumor necrosis was present, the volume of necrosis was calculated by measuring the long and short diameter of the necrotic area using the following equation: volume of necrosis = p/6 

turn, leads to a rebound effect in angiongenesis after cessation of radiotherapy. Consequentially, tumors expressing high concentrations of proangiogenic growth factors are more resistant to radiotherapy (9). Therefore, it is reasonable to believe that a combination of antiangiogenic therapy and radiotherapy may improve tumor growth control. Indeed, antiangiogenic therapies such as angiostatin, VEGF/VEGF receptor inhibitors, or epidermal growth factor receptor (EGFR) inhibitors, haven been combined with radiotherapy and have been proven to have at least additive effects on tumor growth control (8, 10 – 12). These additive effects have been explained by three approaches. First, as the so-called ‘‘normalizing effect’’ on tumor microcirculation, antiangiogenic therapy results in improved tissue perfusion and oxygen supply due to reduction of immature tumor blood vessels and reduction of oxygen-consuming endothelial cells and tumor cells (13). The second approach suggests that antiangiogenic therapy can diminish tumor repopulation after chemotherapy and radiotherapy due to interruption of the increased oxygen needs. Third, antiangiogenic therapy might suppress vasculogenesis by preventing recruitment of endothelial precursor cells in response to chemotherapy and radiotherapy (14). Resistance of tumors to radiotherapy has been further associated with activation of distinct intracellular signaling pathways in tumor cells in response to radiation (15). In particular, radiation induces tumor cell proliferation by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway most likely through stimulation of EGFR on tumor cells (16 – 18). A critical downstream effector of the PI3K/Akt pathway is the mammalian target of rapamycin (mTOR). mTOR controls translation of specific mRNA transcripts that encode proteins for cell cycle progression and cell proliferation (7, 19). In our present study, we show that induction of mTOR inhibition 2 days before the beginning of fractionated radiotherapy resulted in improved tumor growth control in vivo. mTOR inhibitor RAD001 possesses a dose-modifying effect on radiotherapy. In vitro, proliferation of HUVEC seemed to be most sensitive to a combination of mTOR inhibition and radiotherapy, whereas tumor cells showed a cell line – specific resistance. Hence, improved tumor growth control by combination of mTOR inhibition and radiotherapy might be based on damage to established tumor blood vessels (antivascular effect) and growth inhibition of new blood vessels in tumors (antiangiogenic effect).

Materials and Methods Cell lines and cell culture. HUVECs were purchased from PromoCell and were maintained in polystyrene flasks (Falcon, Becton Dickinson) with growth factor – supplemented (Supplement Pack; PromoCell) endothelial-cell basal medium (PromoCell) containing 2% fetal bovine serum (FBS; Life Technologies) as detailed by the manufacturer. The murine cancer cell line CT-26 colon cancer derived from BALB/c mice was cultured in RPMI 1640 (PAN Systems) containing 10% FBS, gentamicin, sodium pyruvate, and HEPES buffer. The human pancreatic cancer cell line L3.6pl was maintained in DMEM supplemented with 10% FBS, sodium pyruvate, nonessential amino acids, L-glutamine, vitamin solution, and penicillin-streptomycin mixture. In vitro cell proliferation assay. HUVECs and tumor cells were cultured in 96-well microtiter plates (500-700 cells per well) in respective


Clin Cancer Res 2008;14(3) February 1, 2008

CancerTherapy: Preclinical diametershort2  diameterlong. The volume of necrosis was subtracted from the entire tumor volume. Experimental protocol for in vivo experiments. Fourteen to 16 days after tumor cell implantation, animals were anesthetized by inhalation of isoflurane-N2O [FiO2 0.35, 0.015 L/L isoflurane (Forene); Abbott GmbH] and fractionated radiotherapy was applied on 5 consecutive days in doses of 2 Gy (5  2 Gy) or 4 Gy (5  4 Gy; Philips RT100 250 kV, 8 mA). In the orthotopic pancreatic tumor model, animals were positioned in the right lateral position and tumors were palpated in the abdomen. The radiation beam was focused on the intra-abdominal tumor through a plexiglass tube of 1.5 cm in diameter. The maximal depth dose of radiation was delivered to the center of the pancreatic tumor, which was estimated to be located 4 mm beneath the skin in the animals. Mice bearing subcutaneous tumors were positioned in prone position. The radiation beam was again focused through the plexiglass tube and the maximal depth dose of radiation was delivered to the center of the tumors, which was estimated to be located 3 mm beneath the skin in the animals. Radiotherapy of tumors was done with or without mTOR inhibition by RAD001. The mTOR inhibitor RAD001 was administered by daily i.p. injection of 1.5 mg/kg body weight. RAD001 treatment was introduced 2 days before the beginning of fractionated radiotherapy, the day after the last fraction of radiotherapy, or as single therapy on day 12 (L3.6pl) or 14 (CT-26) after tumor cell implantation. Administration of RAD001 was continued until the end of experiments. Animals were euthanized when abnormalities in behavior and ingestion occurred because of tumor burden. Immunohistochemical determination of CD31. Frozen tissues of L3.6pl tumors were sectioned (10 Am), mounted on SuperFrost Ultra Plus slides (Menzel GmbH), and air dried for 30 min. The cryosections were fixed in cold ethanol (10 min) and washed with PBS. The sections were incubated with the primary goat – anti-mouse CD31/platelet/ endothelial cell adhesion molecule 1 antibody (1:250; Santa Cruz Biotechnology) overnight at 4jC and rinsed with PBS. Sections were then incubated with the biotinylated secondary donkey – anti-goat antibody (1:200; Santa Cruz Biotechnology) for 1 h at ambient temperature. Positive reactions were visualized by incubating the slides with avidin-biotin for 1 h, followed by incubation with 3-amino9-ethylcarbazole for an additional 30 min. The immunostained sections were counterstained with hemalaun, rinsed with distilled water, and mounted with Ultra Mount (Dako). Statistical analysis. All results are given as mean F SE. Data analysis was done with a statistical software package (SigmaStat for Windows; SPSS Science). One-way ANOVA test adjusted by least square difference test was used for the estimation of stochastic probability in intergroup comparison. P values

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