| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Cancer Therapy: Preclinical |
Authors' Affiliations: 1 Division of Radiotherapy, Department of Oncology, Transplants and Advanced Technologies in Medicine and 2 Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Pisa, Italy; 3 Division of Medical Oncology, Department of Experimental and Clinical Medicine and Surgery "F. Magrassi and A. Lanzara," Second University of Napoli; 4 Department of Endocrinology and Molecular and Clinical Oncology, Federico II University of Napoli, Naples, Italy; and 5 Departments of Radiation Oncology and Medicine, University of Colorado Health Sciences Center, Denver, Colorado
Requests for reprints: Romano Danesi, Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Via Roma 55, 56126 Pisa, Italy. Phone: 39-050-830148; Fax: 39-050-562020; E-mail: r.danesi{at}med.unipi.it.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Experimental Design: ZD6474, ionizing radiation, and gemcitabine, alone or in combination, were given in vitro to MIA PaCa-2, PANC-1, and Capan-1 cells and in vivo to MIA PaCa-2 tumor xenografts. The effects of treatments were studied by the evaluation of cytotoxicity, apoptosis, cell cycle, EGFR and Akt phosphorylation, modulation of gene expression of enzymes related to gemcitabine activity (deoxycytidine kinase and ribonucleotide reductase), as well as vascular endothelial growth factor immunohistochemistry and microvessel count.
Results: In vitro, ZD6474 dose dependently inhibited cell growth, induced apoptosis, and synergistically enhanced the cytotoxic activity of gemcitabine and ionizing radiation. Moreover, ZD6474 inhibited phosphorylation of EGFR and Akt and triggered cell apoptosis. PCR analysis showed that ZD6474 increased the ratio between gene expression of deoxycytidine kinase and ribonucleotide reductase. In vivo, ZD6474 showed significant antitumor activity alone and in combination with radiotherapy and gemcitabine, and the combination of all three modalities enhanced MIA PaCA-2 tumor growth inhibition compared with gemcitabine alone.
Conclusions: ZD6474 decreases EGFR and Akt phosphorylation, enhances apoptosis, favorably modulates gene expression in cancer cells, and acts synergistically with gemcitabine and radiotherapy to inhibit tumor growth. These findings support the investigation of this combination in the clinical setting.
Several studies showed that pancreatic cancer is characterized by dysregulation of molecular mechanisms involved in cell proliferation, invasiveness, and angiogenesis (3). Epidermal growth factor receptor (EGFR) is a key driver of cell proliferation, and increased tumor expression of EGFR has been associated with more advanced disease, resistance to chemoradiotherapy, and poor clinical prognosis (4, 5). Furthermore, a recent study showed that patients with pancreatic cancer overexpressing EGFR had shorter survival (6). Similarly, overexpression of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) correlates with increased tumor growth rate, microvessel density, tumor metastatic potential, and poor prognosis in a variety of malignancies, including pancreatic cancer (7, 8).
The alterations of EGFR and VEGFR activity may confer a substantial growth advantage to pancreatic cancer cells. However, the signaling pathways triggered by these receptors can be blocked by antibodies binding to the external domain of the receptor or by small-molecule inhibitors of receptor-associated tyrosine kinase inhibitor.
A selective VEGFR-2 tyrosine kinase inhibitor, SU5416, produced a significant in vivo antitumor effect on MIA PaCa-2 xenografts. Furthermore, a combination of SU5416 and gemcitabine resulted in a pronounced suppression of tumor growth (9). Similarly, EGFR blockade with the C225 antibody (cetuximab) plus gemcitabine produced additive antitumor effects in an orthotopic pancreatic carcinoma model in nude mice (10). A phase II study in patients with positive immunohistochemical staining for EGFR in tumors has shown promising results (11). The tyrosine kinase inhibitors gefitinib (ZD1839) and erlotinib (OSI-774) have been combined with gemcitabine, and a recent report in 569 patients with advanced pancreatic cancer showed a median survival of 6.4 months in the erlotinib/gemcitabine group compared with 5.9 months in the gemcitabine alone group (12). In that study, patients responded equally well to treatment with erlotinib regardless of the expression level of EGFR. However, the criteria for optimal clinical development of target-specific anticancer agents alone or in combination with conventional cytotoxic drugs are still to be firmly established.
Among these new biological agents, ZD6474 may have a role in the treatment of tumors characterized by dysregulation of growth factor–dependent pathways controlling cell cycle, proliferation, apoptosis, and invasiveness, such as pancreatic cancer, because of its ability to inhibit both VEGF flk-1/KDR receptor (VEGFR-2) and EGFR (13). By targeting VEGFR-2-dependent tumor angiogenesis and EGFR-dependent cancer cell proliferation, ZD6474 offers the potential advantages of inhibiting two key pathways of tumor growth.
Therefore, the present study was aimed at investigating the ability of ZD6474 to synergistically interact with gemcitabine and ionizing radiation in preclinical models of pancreatic cancer as well as the cellular and molecular features of such a combination.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Cell cultures. The MIA PaCa-2, PANC-1, and Capan-1 cell lines (American Type Culture Collection, Manassas, VA), which have been previously characterized for their ability to produce VEGF and EGF (7, 9, 14), were grown in DMEM with 10% FBS and 2.5% horse serum (MIA PaCa-2), DMEM with 10% FBS (PANC-1), and RPMI 1640 with 20% FBS (Capan-1), glutamine, and penicillin-streptomycin. Cells were cultivated in 75 cm2 flasks (Costar, Cambridge, MA) at 37°C in 5% CO2 and 95% air and harvested with trypsin-EDTA when they were in logarithmic growth.
Treatments and assay of cytotoxicity. Exponentially growing cells were plated in 100-mm tissue culture dishes (Costar), allowed to attach for 24 hours, and irradiated by using a 6 MV photon linear accelerator (General Electric, Buckinghamshire, United Kingdom). After irradiation, cells were harvested, and 5 x 104 cells per well were plated in six-well sterile plastic plates (Costar) and allowed to grow for 72 hours. Cytotoxicity assays included cells treated with (a) gemcitabine (0.001-10 µmol/L) for 24 or 72 hours, (b) ZD6474 (0.001-10 µmol/L) for 72 hours, (c) gemcitabine (0.001-10 nmol/L) and ZD6474 (0.001-10 nmol/L) for 72 hours, (d) gemcitabine (0.001-10 nmol/L) for 24 hours followed by a 24-hour washout in drug-free medium and then ZD6474 (0.001-10 nmol/L) for 72 hours, and (e) the reverse sequence. After all treatments were completed, cytotoxicity was expressed as the percentage of surviving cells relative to untreated controls, and the IC50s of cell growth were calculated by nonlinear least squares curve fitting (GraphPad Prism, Intuitive Software for Science, GraphPad Software, San Diego, CA).
Interaction between ZD6474, gemcitabine, and ionizing radiation was assessed using the combination index (CI), where CI < 1, CI = 1, and CI > 1 indicated synergistic, additive, and antagonistic effects, respectively. Data analysis was done by the Calcusyn software (Biosoft, Oxford, United Kingdom).
Cell cycle analysis. Cells were plated at 1 x 106 in 100-mm plastic dishes (Costar) and allowed to attach for 24 hours. After treatments with ionizing radiation, gemcitabine (24 hour), ZD6474 (72 hours), and ZD6474-gemcitabine combinations at their IC50 levels, cells were harvested with trypsin-EDTA and washed twice with PBS. DNA was stained with a solution containing propidium iodide (25 µg/mL), RNase (1 mg/mL), and NP40 (0.1%), and samples were kept on ice for 30 minutes. Cytofluorimetry was done using a FACScan (Becton Dickinson, San Jose, CA), and data analysis was carried out with CellQuest software, whereas cell cycle distribution was determined using Modfit software (Verity Software, Topsham, ME).
To evaluate the effect of treatments on synchronized cells, additional experiments were done using cells cultivated in medium without FBS for 24 hours. After confirmation of the synchronization of serum-starved cells by fluorescence-activated cell sorting, cells were allowed to grow in fresh medium with FBS for 72 hours and then treated with gemcitabine, ZD6474, and their combinations as described above.
Analysis of apoptosis. Cells were treated with ionizing radiation, gemcitabine (24 hours), ZD6474 (72 hours), and their combinations at IC50 levels and, at the end of the 72-hour incubation period, washed twice with PBS and fixed in 4% buffered paraformaldehyde for 15 minutes. Cells were resuspended and incubated for further 15 minutes in a solution containing 8 µg/mL bisbenzimide HCl. Cells were spotted on glass slides and examined by fluorescence microscopy (Leica, Wetzlar, Germany). A total of 200 cells from randomly chosen microscopic fields was counted, and the percentage of cells displaying chromatin condensation and nuclear fragmentation relative to the total number of counted cells (apoptotic index) were calculated.
Assays of EGFR phosphorylation. The phosphorylation of tyrosine residue at position 1173 of EGFR (pY1173EGFR) was analyzed using a pY1173-specific ELISA (BioSource International, Camarillo, CA) and normalized to total EGFR content. Cells were treated as described above for apoptosis analysis; at the end of incubation, cells were scraped, washed twice with PBS, centrifuged (1,000 x g for 5 minutes at 4°C), and resuspended in 25 µL of extraction buffer for 30 minutes on ice, with vortexing as per protocol directions. Cell extract (5 µL) was diluted to 100 µL of NaN3 (15 mmol/L), centrifuged at 15,000 x g for 10 minutes at 4°C, and transferred to microtiter wells coated with a monoclonal antibody specific for human EGFR. A standard curve was run with each assay using 100, 50, 25, 12.5, 6.25, 3.12, and 1.6 units/mL of human pY1173EGFR and 10, 5, 2.5, 1.25, 0.625, 0.312, and 0.16 ng/mL of human full-length EGFR. After overnight incubation at 4°C, the solution was removed from wells and 100 µL of rabbit anti-pY1173EGFR and rabbit anti-EGFR were added into each well of pY1173EGFR and full-length EGFR ELISA, respectively. Plates were incubated at room temperature for 1 hour and washed four times, and 100 µL of a solution containing horseradish peroxidase–labeled anti-rabbit IgG were added to each well. After 30 minutes, wells were drained and a chromogen solution was added; 30 minutes later, reactions were ended by 100 µL of a stop solution and the absorbance was read at 450 nm after 20 minutes. To calculate pY1173EGFR and full-length EGFR concentrations, a standard curve method was used. Values of pY1173EGFR calculated from the standard curve were then normalized for total EGFR, and protein content was measured by the Lowry reagent (Sigma).
The phosphorylation of tyrosine residue 992 of the cytoplasmatic domain of EGFR in control and treated cells was analyzed with a pY992EGFR-specific ELISA and normalized to the total EGFR content following the manufacturer's instructions (Active Motif, Carlsbad, CA).
Assay of Akt phosphorylation. To evaluate if ionizing radiation, gemcitabine, ZD6474, and ZD6474-gemcitabine treatments, at their IC50 levels, could affect the activation of intracellular proteins involved in survival signaling, Akt protein activation by phosphorylation at serine residue 473 (pS473Akt) was assayed with ELISA (BioSource International) and normalized to the total Akt content as described previously (15).
PCR analysis. Total RNA was extracted from cells treated as described above in "cell cycle analysis" using the TRI Reagent LS. RNA was dissolved in RNase-free water containing 10 mmol/L DTT and 200 units/mL of RNase inhibitor and measured at 
abs 260 nm. RNA (1 µg) was reverse transcribed at 37°C for 1 hour in 100 µL reaction volume containing 0.8 mmol/L deoxynucleotide triphosphate, 200 units of Moloney murine leukemia virus reverse transcriptase, 40 units of RNase inhibitor, and 0.05 µg/mL of random primers. The cDNA was amplified by quantitative PCR with the Applied Biosystems 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Reactions were done in triplicate using 5 µL cDNA, 12.5 µL of Taqman Universal PCR Master Mix, 2.5 µL of probe, and 2.5 µL of forward and reverse primers specific for deoxycytidine kinase (dCK) and ribonucleotide reductase (RR) regulatory (RRM1) and catalytic (RRM2) subunits in a final volume of 25 µL. Samples were amplified by an initial incubation at 50°C for 5 minutes followed by incubation at 95°C for 10 minutes and 40 cycles of denaturation at 95°C for 15 seconds followed by annealing and extension at 60°C for 1 minute. Target gene quantitation was done as described previously (16). Finally, because dCK and RR are involved in gemcitabine chemosensitivity, the dCK/(RRM1 x RRM2) expression ratio was calculated (16).
Evaluation of in vivo activity of treatments in nude mice xenografts. Athymic nude mice (4- to 6-week-old females) were obtained from the National Cancer Institute (Bethesda, MD). All animal procedures and maintenance were conducted in accordance with the institutional guidelines of the University of Colorado Health Sciences Center (Denver, CO). Mice were injected s.c. with 107 MIA PaCa-2 cells resuspended in 200 µL Matrigel (Collaborative Biomedical Products, Bedford, MA). After 7 days, when tumors reached a volume of 0.1 cm3, mice were randomized to receive i.p. administration of 30 mg/kg/d ZD6474 on days 1 to 5 of each week for 4 weeks, 120 mg/kg/d gemcitabine on days 1 and 3 of each week for 4 weeks, and/or radiotherapy at 2 Gy/dose daily on days 2 and 4 of each week for 4 weeks. Radiation was given using a linear accelerator with customized blocking to treat only the flank containing the pancreatic xenograft. For each experiment, treatment groups comprised eight mice. Tumor volume was measured using the following formula: (
/ 6) x larger diameter x (smaller diameter)2.
Immunohistochemical analysis. Immunohistochemistry was done on formalin-fixed, paraffin-embedded tissue sections (5 µm) of MIA PaCa-2 xenografts as reported previously (17). After overnight incubation at 4°C with the appropriate primary antibody, sections were washed and treated with a secondary biotinylated goat antibody (1:200 dilution; Vectastain avidin-biotin complex method kit, Vector Laboratories, Burlingame, CA), washed again, exposed to avidin-conjugated horseradish peroxidase H complex, and incubated in diaminobenzidine and hydrogen peroxide. The following antibodies were used: anti-Ki67 monoclonal antibody (1:100 dilution, clone MIB1; DBA, Milan, Italy), a rabbit anti-human/mouse VEGF polyclonal antibody (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit anti-human/mouse VEGFR-2 antibody (1:50 dilution; Acris Gmbh, Hiddenhausen, Germany), a rabbit anti-human/mouse pVEGFR-2 antibody (1:100 dilution; Acris), a rabbit anti-human/mouse polyclonal EGFR antibody (1:100 dilution; Acris), and a rabbit anti-human/mouse pY1173EGFR antibody (1:100 dilution; Acris). The slides were then rinsed in distilled water, counterstained with hematoxylin, and mounted. To determine the percentage of positive cells, at least 1,000 cells per slide were counted and scored. Blood vessels were detected using an anti-factor VIII–related antigen monoclonal antibody (1:50 dilution; DAKO, Milan, Italy) and stained with a standard immunoperoxidase method (Vectastain avidin-biotin complex method kit). Each slide was scanned at low power (x10 to x100 magnification), and the area with the highest number of vessels (hotspot) was identified. This region was then scanned with a microscope at x250 magnification (0.37 mm2). Five fields were analyzed, and for each field, the number of stained blood vessels was counted. Microvessel count was scored in individual tumors as the average of five field counts.
Statistical analysis. All experiments were done in triplicate and repeated at least twice. Data were expressed as mean ± SE and analyzed by Student's t test or ANOVA followed by the Tukey's multiple comparison; the level of significance was P < 0.05.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
gemcitabine and the reverse schedule, respectively. Similarly, simultaneous exposure to gemcitabine and ZD6474 showed synergism, and both drugs synergistically interacted with ionizing radiation (Fig. 1). Finally, the combination of ZD6474, gemcitabine, and ionizing radiation displayed a strong synergistic effect against all pancreatic cancer cells (Fig. 1).
|
gemcitabine combination in MIA PaCa-2 (+14.8% and +10.2%), PANC-1 cells (+8.9% and +7.5%), and Capan-1 cells (+12.0% and +8.3%), respectively (Table 1
). On the other hand, minimal perturbations were observed after gemcitabineZD6474 combination, whereas ZD6474 treatment caused an accumulation in the G1 phase of all cell lines (Table 1). Gemcitabine arrested serum-starved cells in S phase (+16.5%, 10.3%, and 17.1% in MIA PaCa-2, PANC-1, and Capan-1 cells, respectively), whereas ZD6474, in the same conditions, induced cells to accumulate in the G1 phase. Moreover, ionizing radiation and drug treatments induced apoptosis as shown by the presence of a hypodiploid region in the fluorescence-activated cell sorting scan, which was confirmed by fluorescence microscopy after bisbenzimide staining. Indeed, cells exposed to ionizing radiation, gemcitabine, ZD6474, and their combinations, at the IC50s, presented typical apoptotic morphology with cell shrinkage, nuclear condensation and fragmentation, and rupture of cells into debris. Gemcitabine increased apoptotic cell death in MIA PaCa-2, PANC-1, and Capan-1 cells up to 19.7%, 17.7%, and 18.8%, respectively (Fig. 2
). Fluorescence microscopy also revealed markedly higher levels of apoptosis in the ZD6474-treated and ionizing radiation–treated cells when compared with controls. Furthermore, the combination of the two drugs and ionizing radiation significantly increased the apoptotic index compared with gemcitabine-treated cells (Fig. 2).
|
|
ZD6474 and increased up to 112% to 128% by the reverse sequence, whereas p992EGFR levels were not affected by both combinations.
|
ZD6474 exposure) to –87.8% (MIA PaCa-2 cells after ZD6474gemcitabine treatment).
Modulation of gene expression. Ionizing radiation significantly enhanced dCK gene expression in all cell lines, whereas RRM1 and M2 levels were minimally increased; as a consequence, a 4.3-, 2.4-, and 4.6-fold increase in dCK/(RRM1 x RRM2) in MIA PaCa-2, PANC-1, and Capan-1 cells, respectively, was observed (P < 0.05). A significant increase in dCK/(RRM1 x RRM2) was also obtained after gemcitabine treatment in all cell lines (Table 1). Finally, ZD6474 significantly modulated this ratio through an increase in dCK and a reduction in RRM1 and RRM2 expression (Table 1), whereas the sequential ZD6474gemcitabine and gemcitabineZD6474 treatments determined only a 1.5- to 1.7-fold increase of the dCK/(RRM1 x RRM2) ratio in cancer cells.
Inhibition of xenograft growth in nude mice. The mean volume of tumor nodules in the control group was 511.9 ± 29.3 mm3 by day 46 (Fig. 4 ), whereas tumor volumes in mice treated with ZD6474, gemcitabine, or fractionated radiotherapy alone were significantly lower than controls (P < 0.05, Student's t test) at the same time point.
|
On the contrary, the combination of all three modalities, ZD6474, gemcitabine, and radiation, produced a substantial growth delay at the end of treatment in all mice (mean tumor volume of 66.94 ± 16.0 mm3) compared with both untreated mice (P = 0.003) and single-agent gemcitabine-treated mice (P = 0.02). Moreover, all treatments were well tolerated and no difference in animal behavior or body weight was found between groups (data not shown).
Finally, immunohistochemistry of excised tumors revealed a marked inhibition of Ki67 and VEGF staining in animals treated with the combination of ZD6474, gemcitabine, and radiotherapy (Table 2 ). Similarly, immunostaining done in four untreated and ZD6474-treated mice showed that both tumor cells and endothelial cells of tumor stroma from the control group expressed EGFR, VEGFR, pEGFR, and pVEGFR-2, and although tumor cells showed only a weak membrane staining of VEGFR-2 and pVEGFR-2, immunohistochemical analysis showed that treatment with ZD6474 significantly decreased the phosphorylation of EGFR and VEGFR in both cell types. Finally, quantification of tumor-induced vascularization in the areas of most intense neovascularization revealed that ZD6474 treatment reduced the number of microvessels (from 25 to 10 microvessels per field compared with control mice), whereas ionizing radiation and gemcitabine produced a slight reduction (Table 2). An almost complete suppression of tumor microvessels was observed after combined treatment with ZD6474, gemcitabine, and ionizing radiation (Table 2).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Taken together, these studies indicate a potentially important role for EGFR and VEGFR signaling in pancreatic cancer progression and suggest that blocking these receptors may result in significant disease control. Indeed, recent experimental approaches with an anti-VEGFR-2 antibody (24) or small molecules targeting the VEGFR tyrosine kinase (25, 26), alone or in combination with chemotherapeutic agents (24, 25), have shown promising results in vivo. Moreover, gemcitabine in combination with the anti-VEGF antibody bevacizumab produced a 21% partial response rate and 45% disease stabilization rate in patients with stage IV pancreatic cancer (27). Similarly, the anti-EGFR antibody cetuximab resulted in an additive effect with gemcitabine in an orthotopic model of pancreatic carcinoma in nude mice (10) and produced 12% partial responses and 63% stable diseases in advanced pancreatic cancer patients (11). Finally, a recent study showed that AEE788, an EGFR/ErbB2 and VEGFR tyrosine kinase inhibitor, in combination with gemcitabine, produced apoptosis and significantly suppressed human pancreatic cancer in nude mice (28).
Therefore, this study evaluated whether the combination of gemcitabine and radiotherapy with ZD6474, a potent inhibitor of both VEGFR and EGFR signaling, may represent a valuable therapeutic approach against experimental pancreatic cancer.
The present in vitro study showed that ZD6474 treatment caused a dose-dependent inhibition of cell growth in MIA PaCa-2, PANC-1, and Capan-1 pancreatic cancer cells, whose sensitivity fell within the range of IC50s previously reported for other tumor cell lines, including breast, colon, gastric, and ovarian cell lines, with functional EGFR but lacking VEGFR-2 (29). These effects occurred in a dose range that is comparable with that of the selective EGFR tyrosine kinase inhibitor gefitinib (17). Furthermore, ZD6474 significantly potentiated the apoptosis induced by the combined treatment with gemcitabine and ionizing radiation. These results agree with previous studies that reported enhanced apoptotic cell death after combined treatment of ZD6474 with paclitaxel, docetaxel, or oxaliplatin in several cell lines (28, 30). In contrast, in the esophageal cell line KYSE30, the sequence of ZD6474 for 48 hours followed by either docetaxel or oxaliplatin for 24 hours produced a marked arrest of cells in G0-G1 phase without induction of apoptosis (30). Therefore, we next determined whether ZD6474 could affect the activation of intracellular proteins that are involved in survival signaling. Indeed, EGF and VEGF are also known to be survival factors, rendering cells more resistant to apoptosis and chemotherapy through activation of Akt, which regulates antiapoptotic mechanisms by activating nuclear factor-
B and blocking proapoptotic proteins, including BAD (31, 32). It is likely that reduction of pS473Akt through EGFR inhibition plays an important role in the antitumor activity of ZD6474 in MIA PaCa-2, Capan-1, and PANC-1 cells. Previous in vitro and in vivo studies showed that the reduction of pS473Akt correlated with the enhancement of gemcitabine-induced apoptosis and antitumor activity, suggesting that the Akt pathway plays a significant role in mediating drug resistance in human pancreatic cancer cells (33, 34). The inhibition of antiapoptotic pathways could explain, at least in part, the synergistic interaction of ZD6474 and gemcitabine. Furthermore, gemcitabine decreased the amount of pS473Akt and significantly increased pY1173EGFR in all pancreatic cancer cells. This phosphorylation creates a major binding site for the protein tyrosine phosphatase SHP-1, which can dephosphorylate EGFR and could block EGFR-induced activation of the extracellular signal-regulated kinase signal transduction pathway (35) favoring ZD6474 activity. In Capan-1 cells, gemcitabine also significantly reduced pY992EGFR, which is involved in the direct binding of phospholipase C-
and stimulation of cell proliferation, thus providing a potential explanation of the synergistic interaction in the gemcitabineZD6474 sequence (36). However, in the other pancreatic cancer cells, gemcitabine caused only minimal effects at this phosphorylation site, and other factors, such as modulation of cell cycle, as previously detected with oxaliplatin (30), may explain this positive interaction. Indeed, cellular damage induced by chemotherapy or by ionizing radiations can convert EGFR ligands from growth factors into survival factors for cancer cells that express functional EGFR. In this context, the blockade of EGFR signaling in combination with cytotoxic drugs or radiotherapy could cause irreparable cell damage leading to apoptosis.
The present study also showed that ZD6474, gemcitabine, and ionizing radiation increased the expression of dCK. This result agrees with previous studies, which indicated that stress stimuli and nucleoside analogues had a stimulatory effect on dCK (37). Because of the profound influence of dCK on tumor resistance to gemcitabine (38), the enhanced expression of dCK after ZD6474 exposure may explain the synergistic interaction observed in the ZD6474gemcitabine combination. Moreover, because dCK mRNA expression influenced radiosensitization by gemcitabine (39), we hypothesize that induction of dCK by ZD6474 may play a role in the synergistic interaction in the triple combination of ZD6474, gemcitabine, and ionizing radiation. However, the interaction between gemcitabine and ZD6474 with ionizing radiation may involve several other molecular mechanisms, including inhibition of RR activity (40), cell cycle modulation (41), promotion of apoptosis (42), and inhibition of tumor proliferation and angiogenesis (43).
Finally, because transcriptome analysis suggested that the synergistic interaction of gemcitabine with pemetrexed against MIA PaCa-2, PANC-1, and Capan-1 cells mainly relies on the increase of dCK/(RRM1 x RRM2) expression ratio (16), the significant enhancement of this ratio after ZD6474 and ionizing radiation could partly explain the positive interaction between these treatments and gemcitabine.
Tumor growth was also inhibited by ZD6474 in vivo; according to previous studies on solid tumor xenografts (13, 29, 44, 45), chronic administration of ZD6474 is well tolerated in athymic mice and produces significant inhibition of tumor growth, which may result from inhibition of both EGFR and VEGFR-2 activity. Furthermore, preclinical studies with cetuximab, gefitinib, and, more recently, ZD6474 showed that these agents potentiated the antitumor activity of standard cytotoxics and radiotherapy (17, 30, 44, 46). The present study confirmed these findings, showing a cooperative and long-lasting inhibition of tumor progression mostly after combined administration of ZD6474 and radiotherapy. Similar results were obtained with gemcitabine in combination with radiotherapy, thus confirming that doublet therapies were overall more active than single treatments, although the lack of statistical significance may reflect the inherent limitations of in vivo models to fully assess the efficacy of treatments. However, the triple combination therapy of ZD6474, gemcitabine, and ionizing radiation produced a significant tumor growth delay compared with gemcitabine alone.
In conclusion, the present study provides evidence of the feasibility of integrating three different treatment modalities, including target-specific and cytotoxic agents as well as radiotherapy, and provides a scientific rationale for translating the combination of ZD6474, gemcitabine, and radiotherapy in the clinical treatment of pancreatic cancer.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Note: C. Bianco and E. Giovannetti contributed equally to this work.
Received 4/ 4/06; revised 8/ 8/06; accepted 9/20/06.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
B/Rel activity in pancreatic cancer. Int J Cancer 2003;105:735–46.[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  A. Morabito, M. C. Piccirillo, F. Falasconi, G. De Feo, A. Del Giudice, J. Bryce, M. Di Maio, E. De Maio, N. Normanno, and F. Perrone Vandetanib (ZD6474), a Dual Inhibitor of Vascular Endothelial Growth Factor Receptor (VEGFR) and Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinases: Current Status and Future Directions Oncologist, April 1, 2009; 14(4): 378 - 390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Morgan, L. A. Parsels, J. Maybaum, and T. S. Lawrence Improving Gemcitabine-Mediated Radiosensitization Using Molecularly Targeted Therapy: A Review Clin. Cancer Res., November 1, 2008; 14(21): 6744 - 6750. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. L. Bradshaw-Pierce, C. A. Steinhauer, D. Raben, and D. L. Gustafson Pharmacokinetic-directed dosing of vandetanib and docetaxel in a mouse model of human squamous cell carcinoma Mol. Cancer Ther., September 1, 2008; 7(9): 3006 - 3017. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kleespies, I. Ischenko, M. E. Eichhorn, H. Seeliger, C. Amendt, O. Mantell, K.-W. Jauch, and C. J. Bruns Matuzumab Short-Term Therapy in Experimental Pancreatic Cancer: Prolonged Antitumor Activity in Combination with Gemcitabine Clin. Cancer Res., September 1, 2008; 14(17): 5426 - 5436. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Giovannetti, C. Lemos, C. Tekle, K. Smid, S. Nannizzi, J. A. Rodriguez, S. Ricciardi, R. Danesi, G. Giaccone, and G. J. Peters Molecular Mechanisms Underlying the Synergistic Interaction of Erlotinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, with the Multitargeted Antifolate Pemetrexed in Non-Small-Cell Lung Cancer Cells Mol. Pharmacol., April 1, 2008; 73(4): 1290 - 1300. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
