SU11248 (SUNITINIB) SENSITIZES PANCREATIC CANCER TO THE CYTOTOXIC EFFECTS OF IONIZING RADIATION
KYLE C. CUNEO, M.D.,* LING GENG, M.D.,y ALLIE FU, B.S.,y DARREN ORTON, PH.D.,z DENNIS E. HALLAHAN, M.D.,xyk AND ANURADHA BAPSI CHAKRAVARTHY, M.D.y
* Vanderbilt University School of Medicine, Nashville, TN; y Department of Radiation Oncology, Vanderbilt University Medical Center, Nashville, TN; z Institute of Chemical Biology, Vanderbilt University, Nashville, TN; Departments of xCancer Biology and yRadiation Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center; and k Department of Biomedical Engineering, Vanderbilt University, Nashville, TN
Purpose: SU11248 (sunitinib) is a small-molecule tyrosine kinase inhibitor which targets VEGFR and PDGFR iso- forms. In the present study, the effects of SU11248 and ionizing radiation on pancreatic cancer were studied.
Methods and Materials: For in vitro studies human pancreatic adenocarcinoma cells lines were treated with 1 mM SU11248 1 h before irradiation. Western blot analysis was used to determine the effect of SU11248 on radiation- induced signal transduction. To determine if SU11248 sensitized pancreatic cancer to the cytotoxic effects of ion-
izing radiation, a clonogenic survival assay was performed using 0–6 Gy. For in vivo assays, CAPAN-1 cells were injected into the hind limb of nude mice for tumor volume and proliferation studies.
Results: SU11248 attenuated radiation-induced phosphorylation of Akt and ERK at 0, 5, 15, and 30 min. Further- more, SU11248 significantly reduced clonogenic survival after treatment with radiation (p < 0.05). In vivo studies revealed that SU11248 and radiation delayed tumor growth by 6 and 10 days, respectively, whereas combined treatment delayed tumor growth by 30 days. Combined treatment with SU11248 and radiation further attenuated Brdu incorporation by 75% (p = 0.001) compared to control.
Conclusions: SU11248 (sunitinib) sensitized pancreatic cancer to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in pancreatic cancer. © 2008 Elsevier Inc.
Pancreatic cancer, Sunitinib, SU11248, Radiosensitizer, VEGFR, PDGFR.
It is estimated that 32,300 people will die of pancreatic cancer this year, making it the fourth leading cause of cancer related death in US men and women (1). Pancreatic adenocarcinoma is one of the most aggressive neoplasms. Even with the advances made in oncology, this disease continues to have a dismal prognosis. More than 80% of patients are surgically unresectable at the time of presentation. Patients with resect- able disease have a median survival of 15–19 months and long-term survival of less than 20% (2).
Concurrent chemoradiation has a controversial role in the treatment of pancreatic cancer. Results reported by the Gas- trointestinal Tumor Study Group in 1985 showed a twofold increase in survival with adjuvant chemoradiation (3). How- ever, recent data from a European Organization for Research and Treatment of Cancer trial showed only a small benefit from adjuvant therapy (4). In contrast, the European Study Group for Pancreatic Cancer (ESPAC)-1 trial suggested that
chemoradiation may worsen outcome (5). It should be noted that this trial has been widely criticized (6, 7). For unre- sectable locally advanced pancreatic cancer, chemoradiation has been shown to result in a moderate increase in survival (8). Currently, 5-FU based chemoradiation is recommended by the National Comprehensive Cancer Network (NCCN) as adjuvant therapy for resectable tumors and as definitive ther- apy for locally advanced disease. SU11248 (sunitinib malate, Sutent) is a multitargeted tyrosine kinase inhibitor and is a po- tent inhibitor of VEGFR1, VEGFR2, VEGFR3, PDGFRa, and PDGFRb (9–13). Preclinical studies suggest that this compound affects the growth of both the tumor vasculature and the tumor cells themselves (9–13). Sunitinib malate is approved for use in advanced-stage renal cell carcinoma and in imatinib-resistant gastrointestinal stromal tumors. Re- sults from a Phase II clinical trial in advanced-stage renal cell carcinoma showed a 40% partial response rate, with 27% of patients having stable disease at 3 months (14). Additionally,
Reprint requests to: Bapsi Chakravarthy, M.D., Vanderbilt Uni- versity Medical Center, B-1003, The Vanderbilt Clinic (TVC), Nashville, TN 37232-5671. Tel: (615) 322-2555; Fax: (615) 343-
1061; E-mail: [email protected]
Supported by NIH grants RO1-CA112385, RO1-CA70937, RO1-CA88076, RO1-CA89888, P50-CA90949, Vanderbilt-Ingram
Cancer Center, CCSG P30-CA6848. K.C. Cuneo is supported by the Vanderbilt University Medical Scholars Program.
Conflict of interest: none.
Received Aug 28, 2007, and in revised form Feb 5, 2008.
Accepted for publication Feb 13, 2008.
a Phase III trial in imatinib-intolerant gastrointestinal stromal tumors showed an improvement of time to progression in pa- tients treated with sunitinib vs. placebo (15, 16). SU11248 has also shown promise in a number of other diseases includ- ing acute myelogenous leukemia (AML), head and neck can- cers, neuroendocrine tumors, and breast cancer (17).
A previous report showed that SU11248-sensitized endo- thelial cells and tumor vasculature to the cytotoxic effects of ionizing radiation (11). In this study, the effect was attrib- uted to attenuating signal transduction through radiation- induced survival pathways including PI3k/Akt signaling. In the current study, we looked at the ability of SU11248 to sensitize human pancreatic adenocarcinoma cell lines to the cytotoxic effects of ionizing radiation. This study serves as a preclinical model for the use of sunitinib malate with concurrent chemoradiation in pancreatic cancer and other gastrointestinal malignancies.
METHODS AND MATERIALS
Cell lines MiaPaCa2, Panc-1, and CAPAN-1 (human, pancreatic carcinoma) were obtained from American Type Culture Collection (Manassas, VA) and maintained in high-glucose (4.5 g) DMEM supplemented with 10% fetal bovine serum and 1% penicillin-strep- tomycin. All cell lines were kept at 37◦C in a 5% CO2 incubator.
SU11248 was synthesized in its non-salt form by Dr. Darren Orton at the Institute of Chemical Biology at Vanderbilt University and stored in the dark at 4◦C. SU11248 was diluted with DMSO to a stock concentration of 1 mM. To be consistent with prior reports, the compound was administered to cells 60 min before irradiation at a con- centration of 1 mM. A Mark-1 Irradiator 137Cs (JL Shepard and Asso- ciates) was used to irradiate cultures at a dose rate of 1.897 Gy/min.
Cell lysis and immunoblot analysis
Cells lines were treated with or without 1 mM SU11248 for 1 h, then irradiated and processed at the indicated times. During process- ing, cells were washed twice with phosphate-buffered saline fol- lowed by 150 mL lysis buffer (20 nM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM Na-PP, 1 mM PMS fluoride, and 1 mg/mL leupeptin). Protein concentration was quantified by the BioRad method (Hercules, CA). A total of 40 mg of total protein was loaded into each well of a 10% sodium dodecyl sulfate– polyacrylamide gel electrophoresis gel and separated. Protein was transferred onto a polyvinylidene fluoride membrane (Millipore; Billerica, MA) and probed with antibodies for phospho-Akt (S473/T308), Akt, phospho-ERK (T202/Y204), ERK, PDGFR-
beta(958), VEGFR1, VEGFR2 (Cell Signaling Technology, Dan- vers, MA), and actin (Sigma-Aldrich) in a 5% milk TBST solution. Membranes were then washed and probed with HRP labeled mouse anti-rabbit secondary antibodies (Sigma-Aldrich). Membranes were developed and scanned into PhotoShop software. Experiments were performed at least three times to insure reproducibility.
Cell lines were grown to 70–80% confluency. Cells were washed with phosphate-buffered saline, suspended with trypsin, and ad- justed to specific densities for each condition. The cells were then plated and allowed to attach for 4 h. Cells were treated with 1 mM SU11248 at a 1:1,000 dilution in DMSO or control vehicle followed 60 min later by 0, 2, 4, or 6 Gy. Media were changed after irradiation.
Fourteen days later, plates were fixed with 70% ethanol and stained with 1% methylene blue. Colonies larger than 50 cells were counted with a dissection microscope. Plating efficiency was calculated as (number of colonies/number of cells plated)/(number of colonies for corresponding control/number of cells plated). The mean and standard error were calculated for each treatment condition.
Tumor volume study
Approximately 1 million CAPAN-1 cells were suspended in 0.1 mL of cell medium then injected into the hind limb of nude mice. After tumors were visible (approximately 1 week) the mice were then di- vided into four groups: control, radiation therapy (XRT), SU11248, and SU11248 + XRT. Each mouse received daily treatment with either 40 mg/kg SU11248 or control vehicle via intraperitoneal injec- tion for a total of five treatments. Mice receiving XRT were irradiated 1 h after drug administration with 2 Gy fractions via an X-ray gener- ator. Tumor volume measurements were taken every 2–3 days for the duration of the study and the average and standard error were calcu- lated (n = 5). The Institutional Animal Care and Use Committees guidelines were followed during all aspects of treatment.
BrdU incorporation study
Approximately one million CAPAN-1 cells were suspended in 0.1 ml of cell medium then injected into the hind limb of nude mice. One week later the mice were divided into four groups: control, XRT, SU11248, and SU11248 + XRT. Mice received a total of three treat- ments with 40 mg/kg SU11248 or three 2-Gy fractions as stated pre- viously. Twelve hours after the third treatment, the mice were injected with bromodeoxyuridine (BrdU) labeling reagent (Zymed Laboratories, South San Francisco, CA). Six hours after injection, mice were sacrificed. The tumors were harvested, fixed in paraffin, and sectioned. Slides were stained with anti-BrdU primary antibody (mouse immunoglobulin G) followed by rhodamine red labeled goat anti-mouse secondary antibody (Invitrogen Molecular Probes, Carlsbad, CA). Sections were counterstained with the nucleophilic dye 4’,6-diamidino-2-phenylindole. Photographs were obtained, scanned into PhotoShop software, and quantified. The mean and standard error of BrdU-incorporating cells were determined (n = 4).
SU11248 attenuates radiation induced activation of Akt and ERK
To determine whether SU11248 attenuates cell viability signaling pathways, we studied Akt and ERK signal trans- duction in human pancreatic carcinoma cells. MiaPaCa2 and Panc-1 (human, pancreatic adenocarcinoma) cells were grown to 70–80% confluency on culture plates. Cells were then treated with 1 mM SU11248 or control vehicle and irradiated 1 h later with 3 Gy. At 0, 5, 15, and 30 min after radiation, plates were harvested and whole cell lysates were obtained. Figure 1 shows the results from Western blot anal- ysis using antibodies to phospho-Akt (S473 and T308), total Akt, phospho-ERK (T202 and Y204), total ERK, and actin for the MiaPaCa2 and PANC-1 cell lines. After radiation, control cells showed an initial decrease in phospho-Akt followed by an increase at 15 and 30 min. Treatment with SU11248 before irradiation attenuated this response at all time points, indicating that this compound affects the ac- tivation of this pathway after irradiation. A similar response
Fig. 1. Effect of SU11248 on radiation-induced signal transduction. MiaPaCa2 and PANC-1 (human, pancreatic adeno- carcinoma) cells were grown to 80% confluency treated with 1 mM SU11248 for 1 h, then irradiated with 3 Gy. At the indicated time points, protein was extracted, quantified, and separated in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel by electrophoresis. In a separate experiment, cell lysates from the above cell lines and CAPAN- 1 were tested for PDGFR and VEGFR1/2 expression. Shown are the resulting Western immunoblots using antibodies to P-Akt, total Akt, P-ERK, total ERK, VEGFR1, VEGFR2, and PDGFR and actin.
was seen with ERK phosphorylation. Radiation-induced phosphorylation of ERK at 15 and 30 min. This response was attenuated by treating the cells with SU11248 before ir- radiation. To confirm that PDGFR and VEGFR are expressed in the cell lines that were tested, Western blot analysis was performed. Figure 1c shows that VEGFR1, VEGFR2, and PDGFR are expressed at different levels in all three cell lines. The PANC-1 cell line expressed the highest levels of PDGFR; however, it had less VEGFR1 expression than the
CAPAN-1 and MiaPaCa2 cell lines. All three pancreatic cancer cell lines had substantially less expression than the control cell lines, 3T3 (fibroblast, PDGFR) and HUVEC (vascular endothelial cell, VEGF).
SU11248 sensitizes pancreatic cancer to the cytotoxic effects of ionizing radiation
To determine whether SU11248 sensitizes pancreatic can- cer cells to the cytotoxic effects of radiation, we studied
clonogenic survival. MiaPaCa2 and PANC-1 cell lines were subcultured onto plates at specific densities, treated with 1 mM SU11248, and irradiated 1 h later with 0, 2, 4, or 6 Gy. Two weeks later, cells were fixed with 70% ethanol and stained with methylene blue. Colonies larger than 50 cells were counted for each treatment condition. Figure 2 shows the dose response curves after treatment with SU11248 or control vehicle. Treatment with SU11248 shifted the dose–response curve downward and significantly reduced plating efficiency at 2, 4, and 6 Gy (p < 0.05). These data indicate that SU11248 sensitizes pancreatic cancer cells to the cytotoxic effects of radiation in vitro. Treatment with SU11248 alone for 1 h had no significant effect (p > 0.10) on plating efficiency compared with control in either cell line used. Synergy between SU11248 and radiation is implied because there was no significant difference between the con- trol and drug alone groups, but there was a significant difference between the drug plus radiation vs. radiation alone groups.
Effects of SU11248 and radiation on growth of pancreatic cancer xenografts
To determine whether SU11248 enhances the therapeutic efficacy of radiation in pancreatic cancer xenografts, we
studied the CAPAN-1 cell line in mouse models. CAPAN- 1 was used instead of the other two cell lines because of in- creased tumorgenicity. CAPAN-1 cells were grown in the hind limb of nude mice. After tumors were visible, the mice received five treatments with 40 mg/kg SU11248 or five 2-Gy fractions over 7 days. Figure 3a shows the growth curves for each treatment condition. After treatments were complete (Day 7) tumors in mice receiving the control vehi- cle, SU11248 alone, and XRT alone grew rapidly. In the combined treatment group, the tumor growth curve re- mained flat up to Day 21. Figure 3b shows the mean (n = 5) tumor growth delay for each treatment condition using a fivefold increase in volume as reference. Treatment with SU11248 alone or radiation alone delayed tumor growth by 6 and 10 days, respectively, compared with controls. Combined treatment with SU11248 and radiation delayed tumor growth by 30 days. This delay was greater than what would be predicted by an additive effect. A simple test of synergy performed by multiplying the growth fractions for the radiation and drug alone groups at Day 9 predicts that a simple additive effect would result in a tumor
1.25 times the original volume. Because the tumor volume for the drug plus radiation group at this time point was unchanged, there was a synergistic interaction between radiation and SU11248 in this study.
0 2 4 6
Fold Volume Increase
SU + IR
0 2 4 6
1 5 9
13 17 21 25 29 33 37 41 45
Gy SU11248 IR
Fig. 2. SU11248 decreases clonogenic survival in irradiated pan- creatic cancer cells. MiaPaCa2 and PANC-1 cells were plated at spe- cific densities. After attached cells were treated with 1 mM SU11248 for 1 h, then irradiated at 0, 2, 4, or 6 Gy. Two weeks later, plates were fixed and stained. The surviving colonies were counted and plating efficiency was determined. Shown are the dose–response curves for each treatment condition with mean and standard error (n = 4). There is a statistically significant difference between each treatment condition (p < 0.05).
Fig. 3. SU11248 and fractionated radiation synergistically delay tu- mor growth. CAPAN-1 (human, pancreatic adenocarcinoma) cells were injected into the hind limb of nude mice. After tumors were visible, the mice were treated five times with 40 mg/kg SU11248 or irradiated with 2 Gy over the first 7 days. (a) Mean tumor volume and standard error (n = 5) for each treatment condition during and after the course of treatment. (b) The tumor growth delay compared with control was determined using a fivefold increase in tumor vol- ume as reference.
Fig. 4. Effects of SU11248 and radiation on tumor cell proliferation. CAPAN-1 cells were injected into the hind limb of nude mice. After tumors were visible, the mice were treated three times with 40 mg/kg SU11248 or 2 Gy. Twelve hours after the last treatment, mice were injected with BrdU labeling reagent, then sacrificed 6 h later. Immunohistochemistry was performed using anti-Brdu antibody (red) and cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (blue).
(a) Representative photographs for each treatment condition. (b) The mean number of Brdu incorporating cells and stan- dard error (n = 4) for each treatment condition were quantified. *p < 0.05.
Effects of SU11248 and radiation on BrdU incorporation
To determine whether SU11248 alters the proliferation of pancreatic cancer, we studied tumor cells for BrdU incorpo- ration. CAPAN-1 xenografts were grown in the hind limb of nude mice. The mice were then treated with three 40-mg/kg doses of SU11248 or irradiated with 2-Gy fractions. Twelve hours after the final treatment mice were injected with BrdU. Tumors were harvested 6 h later. Figure 4a shows the images obtained using immunohistochemistry staining for BrdU (red) and the 4’,6-diamidino-2-phenylindole nuclear counter- stain (blue). The mean number of BrdU positive cells and standard error (n = 4) are shown in Fig. 4b. Treatment with radiation alone had no significant change in BrdU incorpora- tion. Treatment with SU11248 alone significantly reduced BrdU incorporation by 40% (p = 0.012) and combined treat-
ment significantly reduced BrdU incorporation by 75% (p = 0.001) compared with controls.
The MAPK and Akt signaling cascades play an important role in tumor cell proliferation. A mutation at codon 12 in the c-K-ras oncogene occurs in more than 85% of pancreatic cancers and results in constitutively active MAPK signaling (2, 18–20). Additionally, the activation of PI3k/Akt signaling has been implicated to play a role in the survival of pancreatic cancer cell lines (19, 21). Low-dose ionizing radiation has been shown to transiently activate MAPK/ERK and PI3K/Akt sig- naling; compounds that target these pathways are effective radiosensitizers (22, 23). Therefore targeting these pathways
has the potential to affect pancreatic cancer cell growth by both blocking constitutively active pro-survival pathways and by enhancing the cytotoxic effects of ionizing radiation.
In the current study, we found that both Akt and ERK are transiently activated after treatment with low-dose ionizing radiation in pancreatic cancer cell lines. Western blot analysis showed that treatment with SU11248 before irradiation atten- uated the phosphorylation of ERK and Akt, indicating that SU11248 blocks signal transduction through these pathways. Additionally, treatment with SU11248 sensitized pancreatic cancer cell lines to the cytotoxic effects of ionizing radiation in in vitro clonogenic survival assays. SU11248 is a potent in- hibitor of tyrosine kinases including VEGFR and PDGFR. Western blot analysis from this report suggests that both VEGFR and PDGFR are expressed in pancreatic cancer cell lines. Previous reports have shown that both Akt and ERK are downstream of these receptors (24). The radiosensitizing effect that was seen in the clonogenic survival assays is likely related to the effect of SU11248 on Akt and ERK signaling. To confirm the in vitro effects of SU11248 on pancreatic cancer, we used a hind limb xenograft tumor model. A previ- ous report from our laboratory showed that SU11248 sensi- tizes endothelial cells to the cytotoxic effects of ionizing radiation (11). As mentioned earlier, SU11248 also directly affects the growth of tumor cells themselves. A therapy that targets both tumor vasculature and tumor cells has the poten- tial to effectively enhance the effects of ionizing radiation on tumor growth. In our model, combined treatment with SU11248 and radiation significantly delayed tumor growth. Similar to the in vitro data, the effect seen in the in vivo assays indicated a synergistic interaction between SU11248 and ra- diation. This synergy was confirmed using the method char- acterized by Zaider to describe the interaction of two agents (25). The effect seen is likely related to enhanced destruction of tumor vasculature in addition to enhanced killing of
pancreatic cancer cells.
We administered SU11248 60 min before irradiation with standard 2-Gy fractions. According to pharmacodynamic data, SU11248 reaches an effective plasma concentration within 30 min, peaks by 6 h, and remains at an effective level for 12 h after a single dose of 40 mg/kg (13). Therefore, at the time of irradiation, the mice theoretically had pharmacologi- cally effective concentrations of SU11248; these concentra- tions lasted for at least 12 h. It is interesting that the tumor growth curve of the combined group remained flat for 2 weeks after treatment. These data suggest that only a small percentage of viable tumor cells remained after combined treatment or new tumor vasculature formation was substan- tially delayed.
We previously showed that SU11248 synergistically enhanced the effects of radiation on tumor vasculature in vivo (11). In the current study, we used BrdU incorporation to study in vivo tumor cell proliferation and found that SU11248 and radiation synergistically attenuated Brdu in- corporation in tumor cells. In this model, radiation produced a minimal effect by itself, whereas SU11248 alone signifi- cantly reduced BrdU incorporation. Comparatively, in the tumor growth delay assay, radiation alone was more effective than SU11248 alone.
The development of specific small-molecule inhibitors is an important contribution to the field of oncology. By target- ing specific molecules that contribute to tumor proliferation and function, we can theoretically develop therapies with fewer side effects. In the current study, a small molecule inhibitor was used to inhibit both constitutively active and ra- diation-induced signal transduction. The use of small mole- cule inhibitors as radiosensitizers represents advancement in radiation oncology. SU11248 and other targeted therapies have the ability to effectively enhance the therapeutic ratio of radiation therapy. SU11248 is a promising drug for future clinical trials with chemoradiation in the treatment of pancre- atic cancer.
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