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Experimental Therapeutics |
Departments of Radiation Oncology [E. E., L. G., H. O., D. E. H.] and Radiology [E. D.], Vanderbilt University School of Medicine, and Department of Biomedical Engineering [D. E. H.], Vanderbilt University, Nashville, Tennessee 37232
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PI3K catalyzes the addition of a phosphate group to the inositol ring of phosphoinositides (6) . One target of these products is the serine/threonine Akt (protein kinase B). Akt subsequently phosphorylates several downstream targets, including the Bcl-2 family member Bad and caspase-9, thus inhibiting their proapoptotic functions (7 , 8) . Akt has also been shown to phosphorylate the forkhead transcription factor FKHR (9) . In addition, many other members of the apoptotic machinery as well as transcription factors contain the Akt consensus phosphorylation site (5) , further suggesting that Akt plays a prominent role in inhibiting apoptosis.
In the present study, we analyzed the role of the PI3K/Akt signal transduction pathway during the response of the vascular endothelium to radiation. We found that radiation alone is sufficient to induce Akt phosphorylation through a PI3K-dependent mechanism. We studied the effects of PI3K antagonists on the viability of irradiated endothelial cells. Wortmannin is an extremely potent inhibitor of PI3K, with an IC50 of 1.9 nM (10) . A second compound, LY294002, has been shown to inhibit PI3K with an IC50 of 1.4 µM (11) . We observed that wortmannin and LY294002 enhanced apoptosis in irradiated endothelial cells at 4 nM and 2 mM, respectively, suggesting that PI3K contributes to endothelial cell viability. Furthermore, PI3K antagonists enhanced radiation-induced obliteration of tumor vasculature, resulting in enhanced tumor growth delay.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wortmannin (Sigma Chemical Co., St. Louis, MO) was stored in the dark at 4°C and dissolved in DMSO. LY294002 (Biomol, Plymouth Meeting, PA) was stored in DMSO at -20°C. Wortmannin was diluted in medium immediately before administration to cell cultures. Both drugs were administered to cells 30 min before radiation. An Eldorado 8 Teletherapy 60Co Unit (Atomic Energy of Canada Limited) was used to irradiate endothelial cell cultures at a dose rate of 0.84 Gy/min. Delivered dose was verified by use of thermoluminescence detectors.
Western Immunoblots.
HUVECs in culture were treated with VEGF or radiation, 3 Gy, as described above. Cells were counted and then washed with iced-cold PBS twice before the addition of lysis buffer (20 nM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium PPI, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin). Protein concentration was quantified by the Bio-Rad method. Equal amounts of protein were loaded into each well and separated by 8% SDS-PAGE gel, followed by transfer onto 0.45 µm nitrocellulose membranes. Membranes were blocked by use of 10% nonfat dry milk in PBST for 2 h at room temperature. The blots were then incubated with the rabbit-antihuman [Phospho-AKT (Ser-473) or AKT, 1:1000; Cell Signaling] antibodies overnight at 4°C. Donkey antirabbit IgG secondary antibody (1:1000; Amersham) was incubated for 1 h at room temperature. Immunoblots were developed by using the enhanced chemiluminescence (ECL) detection system (Amersham) according to the manufacturer’s protocol and autoradiography.
Viability Assays.
The number of cells undergoing apoptosis was quantified by microscopic analysis of apoptotic nuclei. Cells were then fixed and stained with H&E at 24 h after treatment with radiation and/or PI3K antagonists. Cells were then examined by light microscopy. For each treatment group, four high power fields (x40 objective) were examined, and the number of apoptotic and total cells was determined. From these numbers, the percentage of apoptotic cells for each group was determined.
The DNA Laddering Assay was performed as follows. After treatment with radiation and/or PI3K antagonists, HUVECs were placed in the incubator for 24 h. The medium was collected in centrifuge tubes to retain any floating, apoptotic cells. The remaining cells were trypsinized and added to the collected medium. The tubes were spun down at 2500 rpm for 10 min at 4°C. To the cell pellets, 500 µl of DNA lysis buffer [5 mM Tris-HCl (pH 7.4), 20 mM EDTA, and 0.5% Triton X-100] were added, along with 25 µl of 20 mg/ml proteinase K and 60 µl of 10% SDS. This mixture was incubated at 50°C for 1 h. Six hundred µl of chloroform were then added. The tubes were shaken and spun at 14,000 rpm for 10 min. The aqueous layer was extracted and combined with two volumes of cold 95% ethanol. The DNA was allowed to precipitate for 1 h, followed by centrifugation for 10 min at 14,000 rpm for 10 min. The DNA pellet was then resuspended in 10 nM Tris and 1 mM EDTA. Ten µg of DNA were run on a 2.0% agarose gel stained with ethidium bromide. The gel was then photographed under UV light.
Clonogenic survival analysis was performed as we have described (2) . Briefly, three HUVEC culture plates were treated at each radiation dose level. After treatment with radiation and/or antagonists, cells were trypsinized, counted by hemocytometer, and subcultured into fresh medium. After 14 days, the cells were fixed with cold methanol and stained with 1% methylene blue. Colonies with >50 cells were counted, and the surviving fraction was determined.
Tumor Vascular Window Model.
Life Technologies, Inc. penicillin-streptomycin solution (200 µl) was injected into the hind limb of the mouse before the procedure. The dorsal skinfold window is a 3-g plastic frame applied to the skin of the mouse and remains attached for the duration of the study. The chamber was screwed together, while the epidermis was incised and remained open with a plastic covering. The midline was found along the back, and a clip was placed to hold the skin in position. A template, equivalent to the outer diameter of the chamber, was traced, producing the outline of the incision. A circular cut was made tracing the perimeter (7-mm diameter) of the outline followed by a crisscross cut, thus producing four skin flaps. The epidermis of the four flaps was then removed using a scalpel with an effort to follow the hypodermis superior to the fascia. The area was then trimmed with fine forceps and iris scissors. The template was removed, and the top piece of the chamber was fixed with screws. During surgery, the area was kept moist by applying moist drops of PBS with 1% penicillin/streptomycin solution. The bottom portion of the chamber was put in place, and the top was carefully positioned on the cut side so that the window and the circular incision were fitted. Antibiotic ointment was applied at this time. The three screws that hold the chamber together were then positioned into the chamber holes and tightened so that the skin was not pinched, thus avoiding diminished circulation. Tumor blood vessels developed in the window within 1 week.
We studied the time- and dose-dependent response of tumor blood vessels to radiation using the window model. Vascular windows were treated with 3 Gy of superficial X-rays using 80 kVp (Pantak X-ray Generator). Five mice were studied in each of the treatment groups. LY294002 (3 mg/kg) was injected i.p. 15 min before irradiation. The window frame was marked with coordinates, which were used to photograph the same microscopic field each day. Vascular windows were photographed using x4 objective to obtain a x40 total magnification. Color photographs were used to catalogue the appearance of blood vessels on days 0–7. Photographs were scanned into Photoshop software, and vascular center lines were positioned by ImagePro Software and verified by an observer blinded to the treatment groups. Tumor blood vessels were quantified by the use of ImagePro software, which quantifies the vascular length density of blood vessel within the microscopic field. Center lines were verified before summation of the vascular length density. The mean and 95% confidence intervals of vascular length density for each treatment group were calculated, and variance was analyzed by the General Linear Models and Bonferroni t test.
Tumor Growth Delay.
C57BL/6 mice received injections s.c. in the right thigh with 106 viable cells of a murine glioblastoma (GL261) suspended in 0.2 ml of a 0.6% solution of agarose. Each set of six mice was stratified into four groups on day 1 (radiation, LY294002, LY294002 + radiation, and control) to control for mean tumor volume. An equal number of large- and intermediate-sized tumors were present in each group. Mouse tumors were stratified into groups so that the mean tumor volume of each group was comparable. Irradiated mice were immobilized in Lucite chambers, and the entire body was shielded with lead except for the tumor-bearing hind limb. Radiation was administered within 15 min of LY294002 (3 mg/kg) injection. A total dose of 24 Gy was administered in eight fractionated doses over 10 days. The first dose was administered on day 1. The third group received LY294002 administered by i.p. injection. The fourth group received LY294002 on days 1, 3, 5, and 7 of radiation therapy. The radiation therapy was administered in the same manner as the radiation-only group.
Twice weekly tumor volumes were measured using skin calipers as described previously (12, 13, 14)
. Tumor volumes were calculated from a formula (a x b x c/2) that was derived from the formula for an ellipsoid (
d3/6). Data were calculated as the percentage of original (day 0) tumor volume and graphed as fractional tumor volume ± SD for each treatment group.
Blood flow within these tumors was quantified by Power Doppler after the third fraction of irradiation. Tumor blood flow was imaged with a 10–5 MHz linear Entos probe attached to an HDI 5000 (probe and HDI 5000 from ATL/Philips, Bothell, WA) as we have described previously (15) . Power Doppler sonography images were obtained with the power gain set to 82%. A 20-frame cineloop sweep of the entire tumor was obtained with the probe perpendicular to the long axis of the lower extremity along the entire length of the tumor. Color area was quantified using HDI-lab software (ATL/Philips). This software allows direct evaluation of power Doppler cineloop raw. The color area was recorded for the entire tumor. Five mice were entered into each treatment group. Values for color area were averaged for each tumor set, and treated groups were compared with controls with the unpaired Student t test.
Statistical Analysis.
We used the General Linear Model (logistic regression analysis) to test for associations between the numbers of apoptotic cells present in culture, clonogenic survival, tumor blood flow, and tumor volumes. We applied the Bonferroni method to adjust the overall significant level equals to 5% for the multiple comparisons in this study. All statistical tests were two-sided, and differences were considered statistically significant for P < 0.05. SAS software version 8.1 (SAS Institute, Inc., Cary, NC) was used for all statistical analyses.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To determine whether PI3K participates in Akt phosphorylation in response to growth factors or irradiation, two distinct PI3K antagonists, 4 nM wortmannin or 2 µM LY294002 was added 30 min before treatment. Fig. 2
shows that either LY294002 or wortmannin inhibited Akt phosphorylation after treatment of HUVECs with 3 Gy. These PI3K antagonists had no effect on total Akt levels during the 1 h studied in this experiment.
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. LY294002 or wortmannin alone produced 2 and 3% of apoptotic cells with no statistically significant (P = 0.8) change in the number of apoptotic nuclei as compared with untreated control HUVECs, respectively. Treatment with radiation alone increased the percentage of apoptotic cells to 5 and 6%, which was statistically significantly increased as compared with untreated control HUVECs (P < 0.05, Bonferroni t test). HUVECs pretreated with PI3K antagonists before irradiation showed an increase in apoptotic nuclei to 16 and 17% of cells, respectively (P < 0.001). To verify the presence of apoptosis, DNA fragmentation was studied by analysis of DNA laddering as shown in Fig. 4
. Cells treated with LY294002 and radiation showed laddering of DNA, whereas cells treated with radiation alone or LY294002 alone did not demonstrate DNA laddering.
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shows the results of colony-forming assays of HUVECs treated with radiation. HUVECs treated with LY294002 alone for 30 min reduced plating efficiency to 70% as compared with the plating efficiency of untreated control cells. LY294002 enhanced radiation-induced cytotoxicity as compared with HUVECs treated with radiation alone (P < 0.05, Bonferroni t test).
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shows the vasculature within the dorsal skinfold windows before and 96 h after treatment with radiation alone, LY294002 alone, or LY294002 and radiation. Fig. 6D
shows the mean vascular length density from three windows. Tumor blood vessels receiving no treatment continue to increase in length to 111% as compared with vessels at the 0-h time point. Radiation alone reduced vascular length density at 96 h to 63% that of vessels before treatment (P = 0.13). LY294002 alone produced minimal vascular regression, which was 85% as compared with vessels before treatment (P = 0.41). Treatment with LY294002 before irradiation resulted in a marked reduction in vascular length density by 96 h, which was 5% of that of the same tumor windows before treatment. The vascular destruction achieved by LY294002, together with radiation, was significantly statistically greater as compared with radiation alone (P = 0.043).
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shows tumor volume measurements in groups of mice treated with each of these conditions. Untreated control tumors reached 1 cm3 at 7 days as compared with 9 days after treatment with radiation or LY294002 alone. Tumors in mice treated with LY294002 before irradiation demonstrated growth delay that extended beyond 15 days (Fig. 7)
. Growth delay after combined treatment was statistically significantly less than that of tumors treated with radiation alone or LY294002 alone (P = 0.04). LY294002 did not cause mortality in mice studied during the 19 days of study.
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, blood flow was measured in these same tumors by use of amplitude-modulated (power) Doppler sonography. Fig. 8
shows the signal intensity of blood flow in tumors in the hind limb of mice after treatment with radiation, LY294002, or LY294002 and radiation. Blood flow is indicated by the bright signal shown on these Doppler images. Doppler sonography showed that tumor blood flow was obliterated in response to treatment with LY294002 before irradiation. Blood flow was unchanged after treatment with radiation alone or LY294002 alone. Thus, tumor vascular destruction was associated with growth delay in tumors treated with LY294002 before irradiation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our finding that Akt is phosphorylated within 5 min of irradiation indicates that PI3K activation is a very early step in the initiation of radiation-induced signal transduction in the vascular endothelium. Although Akt phosphorylation may be downstream of any of these pathways, PI3K-specific concentrations of two structurally dissimilar PI3K antagonists (LY294002 and wortmannin) implicate PI3K in Akt activation after irradiation. Ionizing radiation has been shown previously to rapidly activate kinases. For example, the phosphotransferase activity of PKC increases within 1 min after doses in the range of 10 Gy (18)
. PKC substrates are subsequently phosphorylated within 5–10 min of irradiation, which is comparable with the rate of phosphorylation of Akt in irradiated HUVECs. Alternatively, radiation induces activation of nuclear kinases in response to DNA strand breaks. The nuclear kinases activated by DNA strand breaks include DNA-PK, ATM, and ATR (19, 20, 21, 22, 23)
. Other protein kinases that are rapidly activated in response to ionizing radiation include PKC
, which activates MAPP kinase kinase-7 and c-Jun NH2-terminal kinase signal transduction (24)
, and epidermal growth factor receptor, which in turn activates the extracellular signal-regulated kinase/mitogen-activated protein kinase kinase pathway (25)
. We propose that the mechanism of radiation-induced activation Akt phosphorylation in the vascular endothelium could involve these signaling pathways.
PI3K antagonists have been shown previously to enhance the cytotoxic effects of radiation (26, 27, 28) . However, these studies have used relatively high concentrations of kinase inhibitors, which also inhibit enzymes involved in DNA repair such as DNA-PK and ATM. The low concentrations of wortmannin (4 nM) and LY24002 (2 µM) used in the present study are relatively specific for PI3K. Concentrations of wortmannin required to inhibit DNA-PK and ATM are manyfold greater than those used in the present study. For example, a wortmannin concentration of 20 µM was used to inhibit DNA-PK and inhibit DNA double strand break repair (14 , 29 , 30) . Wortmannin has been shown to inhibit DNA-PK and ATM, with IC50s of 16 and 150 nM, respectively (26, 27, 28) . Similarly, the concentration of LY294002 required to inhibit DNA repair is 6 µM (31) . Moreover, we have found that these PI3K antagonists also enhance the effects of radiation on blood vessels in SCID mice that are deficient in DNA-PK activity.4 Interestingly, cells deficient in DNA-PK (SCID cells) or ATM (AT cells) also demonstrated partial radiation sensitization by PI3K antagonists (32) . Furthermore, radiation-induced p21 stabilization is inhibited by LY294002 or wortmannin in SCID and ATM cells (33) . These authors propose that enzymes other than DNA-PK and ATM contribute the radiosensitizing effect of LY294002 and wortmannin.
The mechanisms by which PI3K antagonists enhance the cytotoxic effects of radiotherapy are demonstrated by the enhancement of apoptosis in irradiated endothelial cells in the presence of PI3K antagonists. Akt has been shown to phosphorylate the Bcl-2 family member Bad and caspase-9, thus inhibiting their proapoptotic functions (7 , 8 , 34) . Moreover, Akt activation has been shown to prevent apoptosis by inhibition of cytochrome c release (35 , 36) . PI3K antagonists may prevent Akt activation by growth factors and radiation and thereby allow apoptosis to proceed. This is supported by our findings that PI3K antagonists added before irradiation enhance radiation-induced apoptosis in the vascular endothelium.
We used the dorsal skinfold window model and Power Doppler analysis of hind limb tumors to determine whether radiosensitization of tumors by PI3K antagonist involves destruction of the microvasculature. These assays allow for the longitudinal assessment of tumor vascular response to therapy (2 , 37) . Vascular length density allows the quantification of tumor vascularity within the window (2) . The sum of the lengths of all vessels before treatment is compared with that in the same window after therapy. Likewise, Power Doppler provides the measure of tumor blood flow before treatment, which is compared with that in the same tumor after therapy. These assays have been used previously to assess tumor vascular response to radiation (37, 38, 39, 40) . Both assays showed that PI3K antagonist LY294002 enhanced radiation-induced tumor vascular destruction. These findings support the in vitro studies showing attenuation of viability in the vascular endothelium by pretreatment with PI3K antagonists.
Although the irradiated hind limb does not show injury after treatment with LY294002 and radiation, normal vascular endothelium may be sensitized by PI3K antagonists. We found no difference in the apoptosis induced in endothelial cells from normal blood vessels (HUVECs) as compared with transformed vascular endothelium (human microvascular endothelial cells). Brachytherapy is one strategy to circumvent the potential toxicities associated with radiosensitization of normal endothelium.
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FOOTNOTES |
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1 Supported in part by NIH Grants R01-CA58508, R01-CA70937, R01-CA89674, R21-CA89888, P30-CA68485, and P50-CA90949 and the Vanderbilt University Department of Radiation Oncology. 
2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Vanderbilt University, 1301 22nd Avenue South, B-902, The Vanderbilt Clinic, Nashville, TN 37232-5671. Phone: (615) 343-9244; Fax: (615) 343-3075; E-mail: dennis.Hallahan{at}mcmail.vanderbilt.edu
3 The abbreviations used are: RTK, receptor tyrosine kinase; PI3K, phosphatidylinositol 3-kinase; HUVEC, human umbilical vein endothelial cell; VEGF, vascular endothelial growth factor; PKC, protein kinase C; DNA-PK, DNA-dependent protein kinase;ATM, ataxia telangiectasia mutated; SCID, severe combined immunodeficient.
4 D. E. Hallahan, unpublished observation.
Received 2/25/02. Accepted 6/20/02.
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B. Lu, Y. Mu, C. Cao, F. Zeng, S. Schneider, J. Tan, J. Price, J. Chen, M. Freeman, and D. E. Hallahan Survivin As a Therapeutic Target for Radiation Sensitization in Lung Cancer Cancer Res., April 15, 2004; 64(8): 2840 - 2845. [Abstract] [Full Text] [PDF]
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J. K. Park, Y. M. Chung, B.-G. Kim, Y.-A Yoo, B.-S. Yang, J. S. Kim, and Y. D. Yoo N'-(phenyl-pyridin-2-yl-methylene)-hydrazine carbodithioic acid methyl ester enhances radiation-induced cell death by targeting Bcl-2 against human lung carcinoma cells Mol. Cancer Ther., April 1, 2004; 3(4): 403 - 407. [Abstract] [Full Text] [PDF]
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C. d. L. Davies, L. M. Lundstrom, J. Frengen, L. Eikenes, O. S. Bruland, O. Kaalhus, M. H. B. Hjelstuen, and C. Brekken Radiation Improves the Distribution and Uptake of Liposomal Doxorubicin (Caelyx) in Human Osteosarcoma Xenografts Cancer Res., January 15, 2004; 64(2): 547 - 553. [Abstract] [Full Text] [PDF]
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J. Tan and D. E. Hallahan Growth Factor-Independent Activation of Protein Kinase B Contributes to the Inherent Resistance of Vascular Endothelium to Radiation-Induced Apoptotic Response Cancer Res., November 15, 2003; 63(22): 7663 - 7667. [Abstract] [Full Text] [PDF]
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T. M. Grana, C. I. Sartor, and A. D. Cox Epidermal Growth Factor Receptor Autocrine Signaling in RIE-1 Cells Transformed by the Ras Oncogene Enhances Radiation Resistance Cancer Res., November 15, 2003; 63(22): 7807 - 7814. [Abstract] [Full Text] [PDF]
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A. J. Schueneman, E. Himmelfarb, L. Geng, J. Tan, E. Donnelly, D. Mendel, G. McMahon, and D. E. Hallahan SU11248 Maintenance Therapy Prevents Tumor Regrowth after Fractionated Irradiation of Murine Tumor Models Cancer Res., July 15, 2003; 63(14): 4009 - 4016. [Abstract] [Full Text] [PDF]
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P. Sonveaux, A. Brouet, X. Havaux, V. Gregoire, C. Dessy, J.-L. Balligand, and O. Feron Irradiation-induced Angiogenesis through the Up-Regulation of the Nitric Oxide Pathway: Implications for Tumor Radiotherapy Cancer Res., March 1, 2003; 63(5): 1012 - 1019. [Abstract] [Full Text] [PDF]
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