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Experimental Therapeutics, Preclinical Pharmacology |
-PE38
Departments of 1 Otolaryngology, 2 Pharmacology, 3 Radiation Oncology, and 4 Biostatistics, University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania; and 5 Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland
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ABSTRACT |
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(TGF-) fused to Pseudomonas exotoxin (TGF-–PE38)-abrogated head and neck squamous cell carcinoma (HNSCC) tumor growth in vitro and in vivo.
Experimental Design: In vitro cytotoxicity assays were carried out to determine the sensitivity of HNSCC cells to TGF-–PE38. TGF-–PE38-treated HNSCC cells were examined by immunoblotting for cleaved poly(ADP-ribose) polymerase to evaluate apoptosis. Nude mice bearing established HNSCC xenografts were treated with several doses of TGF-–PE38 to evaluate the antitumor efficacy in vivo. Tumor sections were stained with terminal deoxynucleotidyl transferase-mediated nick end labeling for apoptosis. To determine the effect of oral administration of TGF-–PE38, gavage injections of TGF-–PE38 were administered, and the esophagus and surrounding soft tissue were then stained for apoptotic cells.
Results: HNSCC cell lines examined were sensitive to low doses of TGF-–PE38 (EC50 in the range of 1.6 to 10 ng/mL). HNSCC cells treated with TGF-–PE38 undergo apoptosis. Antitumor effects were observed using 0.1 and 0.03 µg of TGF-–PE38 administered intratumorally. At these doses, the treatment was well tolerated. Tumors treated with the toxin had a higher number of apoptotic cells compared with the control tumors. No apoptotic cells were observed in the pharyngoesophageal tissues of the mice after gavage administration of the toxin suggesting that the toxin could be orally administered without toxicity.
Conclusions: These results indicate that topical or intratumoral administration of low doses of TGF-–PE38 may demonstrate antitumor effects in HNSCC without associated systemic toxicity.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TGF-), has been shown to inhibit tumor growth in vitro and in vivo (3)
. Several strategies have been developed to target EGFR, including small molecule tyrosine kinase-specific inhibitors, monoclonal antibodies, and antisense approaches (4)
. These EGFR-targeting strategies are currently in clinical trials or have been approved for clinical administration by the Food and Drug Administration (e.g., Iressa/ZD1839). Another strategy that has proven effective in the treatment of malignant brain tumors is the use of EGFR ligand fused to a cytotoxin (5
, 6)
.
Pseudomonas exotoxin, derived from the bacteria Pseudomonas aeruginosa, exerts its toxic effects by inactivating protein synthesis in mammalian cells. The exotoxin has three functional domains, including a cell binding domain, a domain responsible for cytosol translocation, and a cytotoxic domain. The exotoxin has been engineered to remove the cell-binding domain, and site-specific mutations have been introduced to increase its chemical stability (7)
. The active cytosolic form of Pseudomonas exotoxin A (PE38) has been fused to the EGFR ligand TGF- to generate a targeted toxin known as TGF-–PE38 (8)
. Thus, only cells that express EGFR will internalize the fusion protein with minimal toxicity to normal cells that don’t express or demonstrate low levels of EGFR. TGF-–PE38 is currently under clinical investigation for the treatment of malignant brain tumors.
In the present study, we tested the antitumor efficacy of TGF-–PE38 in HNSCC preclinical models. Four HNSCC cell lines tested in vitro for their sensitivity to TGF-–PE38 and were demonstrated to undergo apoptotic cell death. The in vivo antitumor efficacy of TGF-–PE38 was tested in HNSCC tumors expressing high levels of EGFR. Antitumor effects without apparent toxicity were demonstrated with intratumoral administration of TGF-–PE38. These results suggest that TGF-–PE38 may be used to prevent or treat HNSCC.
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MATERIALS AND METHODS |
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In vitro Cytotoxicity Assay.
HNSCC cells were plated at a density of 5 x 104 cells/mL in a 24-well plate. The next day cells were treated with increasing doses of TGF-–PE38 (0 to 10 ng/mL) in serum containing media. After 24 hours, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out to measure the number of metabolically active cells. The IC50, at which 50% of the cells were dead at 24 hours, was calculated from the dose-response curve generated using GraphPad PRISM software (version 3).
Immunoblotting.
HNSCC cells were plated at a density of 5 x 104 cells/mL in 100-mm dishes. The next day, cells were treated with TGF-–PE38 (0 to 8 ng/mL) in serum-containing media. To demonstrate specificity of TGF-–PE38 to EGFR, cells were incubated with EGFR-specific antibody C225 (ImClone Systems, Inc., New York, NY) at a concentration of 6 µg/mL for 2 hours before TGF-–PE38 treatment. Cisplatin (80 µmol/L; Bedford Laboratories, Bedford, OH) was used to induce apoptosis in HNSCC cells as a positive control for poly(ADP-ribose) polymerase (PARP) cleavage. After 24 hours, cells were harvested as described previously (11)
. Protein quantitation was performed on the supernatant using Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA). Forty micrograms of protein were loaded on an 8% PAGE gel and electrophoresed along with 10 µL of prestained broad range protein marker (Cell Signaling Technology, Beverly, MA). After electrophoresis, proteins were transferred onto a Protran membrane in a semi-dry transfer apparatus (Bio-Rad Laboratories) and subjected to immunoblotting for EGFR (Transduction Laboratories, Lexington, KY), PARP (Santa Cruz Biotechnology, Santa Cruz, CA), or ß-Actin (Oncogene Research Products, Boston, MA) to demonstrate equal loading.
In vivo Studies.
HNSCC 1483 cells was trypsinized, washed with 1x HBSS, counted using a hemocytometer, and 1 x 106 cells were injected s.c. in female nude mice 5 to 6 months of age obtained from Harlan-Sprague Dawley (Indianapolis, IN). There were five mice per group. When tumors reached a diameter of 2 to 3 mm, they were injected once every alternate day for a week with 0.625, 0.1, or 0.03 µg of TGF-–PE38 in 50 µL of 0.2% BSA for a total of three injections. Control mice received injections of 0.2% BSA in saline. Tumors were measured three times a week with a vernier caliper in three dimensions. Tumor volumes were determined using the formula 4/3 
(d1 x d2 x d3), where d1, d2, and d3 are the diameters in three dimensions (12)
. Mice were sacrificed when control tumors reached a volume of 1 cm3. Blood and serum from mice treated with 0.1 and 0.03 µg of TGF-–PE38 were collected for analysis via cardiac puncture. Blood was collected in 0.5 mol/L EDTA. Serum was collected from 0.5 mL of the blood. Hematologic and serum chemistry analysis was conducted by Antech Diagnostics (Farmingdale, NY). Tumors were snap frozen, sectioned, and fixed in 4% paraformaldehyde solution for 20 minutes, followed by staining for apoptotic cells using TUNEL apoptotic cell detection kit (Roche, Penzberg, Germany) using the manufacturer’s protocol.
To asses the toxicity of TGF-–PE38 to the esophagus, male C3H/HeNsd (Harlan-Sprague Dawley) mice were administered 200 µL of water followed by 100 µL of water containing 0.4 mg of TGF-–PE38 by placing a feeding tube at the mouth of the esophageal inlet and injecting the toxin. The mice were allowed to swallow the toxin. Subgroups of mice were sacrificed 24 or 48 hours after injection, and the remaining mice were followed over a 30-day period for survival. The esophagus was removed from the mice sacrificed at 24 or 48 hours, frozen in ornithine carbamyl transferase, sectioned, and stained for apoptotic cells using a Promega Apoptosis Detection kit (Promega, Madison, WI). The slides were fixed in 4% methanol-free formaldehyde solution in PBS for 15 minutes at room temperature, washed three times in PBS for 5 minutes at room temperature, and treated with proteinase K (20 µg/mL) solution for 8 to 10 minutes at room temperature. The slides were rinsed in PBS, fixed in 4% methanol-free formaldehyde solution for 5 minutes at room temperature, rinsed, and placed in equilibration buffer for 5 minutes. Fifty microliters of a reaction mixture containing equilibration buffer, terminal deoxynucleotidyltransferase enzymes, and a nucleotide mix was added followed by incubation at 37°C for 60 minutes. The sections were washed three times in 2x SSC for 15 minutes at room temperature, followed by addition of propidium iodide (1 µg/mL) for 15 minutes at room temperature, washed three times in PBS at room temperature, mounted in antifade, and observed under a fluorescent microscope. Animal use and care was in strict compliance with institutional guidelines established by the University of Pittsburgh, Institutional Animal Care and Use Committee.
Statistical Methods.
To determine whether blood parameter values of the toxin-treated mice differed from the control group and whether any differences from the control group were associated with the delivered dose, three exact permutation tests were performed. We first determined if there was a dose-response effect, i.e., a linear trend. The Jonckhere-Terpstra test was used to assess a linear trend among treatment groups. If this test was found to be nonsignificant, the Kruskal-Wallis test was used to determine whether lab values among the treatment groups differed from each other. If the Kruskal-Wallis test was significant, a combination of the Wilcoxon rank-sum test and plots was used to determine which treatment groups differed.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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–PE38 Is Cytotoxic to HNSCC Cells.
. HNSCC cell line PCI-15b had the highest level and PCI-37a had the lowest level of EGFR among the four cell lines tested. To determine the antitumor efficacy of TGF-–PE38 in vitro, the four HNSCC cell lines were treated with increasing concentrations of TGF-–PE38 for 24 hours. The number of metabolically active cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and a dose-response curve was generated. 1483 and OSC-19 were sensitive to TGF-–PE38 with IC50 of 3 ng/mL and IC50 of 4.78 ng/mL, respectively (Fig. 2A)
. HNSCC line PCI-15b (containing the highest level of EGFR expression) was the most sensitive to TGF-–PE38 with an IC50 of 1.6 ng/mL, and PCI-37a (expressing the lowest EGFR levels) was the least sensitive (IC50 of 10 ng/mL; Fig. 2B
).
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–PE38 is cytotoxic to HNSCC cells in vitro, we investigated the mechanism whereby it causes tumor cell death. HNSCC cell lines 1483 and OSC-19 were treated with TGF-–PE38 (4 or 8 ng/mL) for 24 hours. Both adherent as well as cells in the supernatant were lysed and analyzed by immunoblotting for cleaved PARP. Treatment of HNSCC cells with TGF-–PE38 induced cleavage of PARP at both doses of TGF-–PE38, indicating that the cells were undergoing apoptosis (Fig. 3, A and B)
. Cisplatin treatment was used as a positive control for apoptosis. These data suggest that HNSCC cells treated with TGF-–PE38 undergo apoptotic cell death after 24 hours of treatment.
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–PE38 Specifically Targets Cells Expressing EGFR.–PE38 in targeting HNSCC cells via EGFR, HNSCC cells (1483 and OSC-19) were pretreated with the EGFR-specific monoclonal antibody C225 for 2 hours. C225 binds to the extracellular domain of EGFR preventing ligand binding to the receptor. Subsequent treatment of C225 pretreated cells with TGF-–PE38 resulted in an abrogation of TGF-–PE38-induced PARP cleavage (Fig. 4)
. Cells treated with TGF-–PE38 (4 ng/mL) alone exhibited PARP cleavage, although those treated with either vehicle control, C225 alone, or with C225 did not. These results indicate that TGF-–PE38 exerts its effects on cells by specifically binding to EGFR followed by internalization into the cytosol.
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–PE38 Inhibits HNSCC Tumor Growth In vivo.–PE38 for a total of three injections. Inhibition of tumor growth was seen at all doses of TGF-–PE38 (Fig. 5, A and B)
. Although five mice treated with 0.625 µg of TGF-–PE38 had a dramatic reduction in tumor burden (Fig. 5A)
, this dose was associated with systemic toxicity as demonstrated by cachexia and mortality in some of the treated animals. The lower doses of TGF-–PE38 (0.1 and 0.03 µg) were well tolerated (the experiment was repeated twice with similar results). The lower doses (0.1 and 0.03 µg) are comparable with those used in mouse models of glioma where TGF-–PE38 was inoculated into the caudate nuclei of mice (14)
. TGF-–PE38-treated HNSCC tumors demonstrated lower growth rates compared with control tumors (Fig. 4B)
, indicating that TGF-–PE38 could effectively be used to treat HNSCC tumors in vivo.
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–PE38 were analyzed for apoptotic cells by terminal deoxynucleotidyl transferase-mediated nick end labeling. The treated tumors had four times the number of apoptotic cells compared with the control cells as evidenced by the nuclear staining of fragmented DNA (Fig. 6)
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–PE38.–PE38-treated mice for evidence of systemic toxicity. Blood parameters of mice treated with 0.1 and 0.03 µg of TGF-–PE38 were examined for levels of hepatic enzymes aspartate aminotransferase and alkaline phosphatase. In addition, we examined other hematologic indices, including hemoglobin, hematocrit, creatinine, albumin, absolute lymphocytes, and absolute polymorphonuclear leukocytes, because the information is required for future clinical applications. Blood parameters from the treated and control mice were compared (Table 1)
. The Jonckhere-Terpstra test was nonsignificant for all of the laboratory end points. There was no dose-response effect for any of the laboratory values. The Kruskal-Wallis test was significant for hemoglobin values of mice treated with 0.03 µg of TGF-–PE38 compared with the untreated control mice and mice treated with the higher dose of TGF-–PE38 (0.1 µg; P = 0.0041; Fig. 7A
). However, the lack of an apparent dose-response association suggests that the lower hemoglobin values detected in the group receiving 0.03 µg of TGF-–PE38 were not due to TGF-–PE38 therapy. There was no significant difference in hematocrit levels across groups, thereby providing additional evidence that the changes in hemoglobin levels in the 0.03 µg group are not due to TGF-–PE38 (Fig. 7B)
. No differences between the control animals and the TGF-–PE38 treatment groups were found for the other laboratory end points. In summary, there was no indication that intratumoral administration of TGF-–PE38 caused abnormal hematology or serum chemistry laboratory values when compared with untreated control mice at the doses tested.
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–PE38 in immunocompetent mice. C3H/HeNsd mice were treated with a single dose of TGF-–PE38 (0.4 mg) via oral gavage administration. All mice appeared healthy with no deaths, weight loss, decreased activity, or difficulty in swallowing. Examination of the mice sacrificed 24 or 48 hours (Fig. 8)
after injection for the appearance of apoptotic cells in the esophagus revealed no apoptotic cells. The esophagus not only demonstrated lack of apoptotic cells but also showed no inflammatory pathological changes after gavage administration of the toxin. Five mice administered either saline or TGF-–PE38 (0.4 mg) and followed for a period of 30 days demonstrated no indications of toxicity as manifest by stable weight and activity levels.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Pseudomonas exotoxin A is a protein secreted by the bacteria Pseudomonas aeruginosa. Over the last decade, the protein has been engineered to minimize its size while retaining its potency at ADP ribosylation of elongation factor 2, which arrests protein synthesis (17
, 18)
. To efficiently enter cells it requires a targeting moiety and then can translocate to the cytosol where it exerts its cytotoxicity. PE38 has been fused to several ligands such as the Fv portion of erb B2 antibodies (18)
and cytokines, including interleukin 4 (13)
and interleukin 13 (19)
. PE38 has also been fused to the EGFR ligand TGF- (14)
, enabling its use in the treatment of tumors expressing high levels of EGFR. HNSCC cells demonstrate elevated EGFR levels compared with normal epithelia (1)
. To date, there are no reports demonstrating the efficacy of TGF-–PE38 in the treatment of HNSCC.
In the present study, HNSCC cell lines were sensitive to TGF-–PE38 at IC50 values ranging from 1.6 to 10 ng/mL. TGF-–PE38 has been previously reported to be cytotoxic to human glioma cell lines with a reported ID50 ranging from 0.1 to 35 ng/mL (14)
. To determine whether HNSCC cells treated with TGF-–PE38 undergo apoptosis, we analyzed levels of cleaved PARP. Cleavage of PARP is one of the last steps in the apoptotic pathway downstream of the mitochondria. Cells undergoing apoptosis demonstrate increased levels of cleaved PARP. Although there are reports on the type of apoptotic cell death observed after TGF-–PE38 treatment in HNSCC, our results corroborate findings in a previous study where HNSCC cells treated with interleukin 13 fused to PE38 underwent apoptotic cell death with cleaved PARP and caspases 3, 8, and 9 accumulation (20)
. To investigate the antitumor efficacy of TGF-–PE38 in vivo, we treated nude mice bearing s.c. HNSCC tumors with the agent.
Established tumors were treated intratumorally with varying concentrations of TGF-–PE38. Although the highest dose of TGF-–PE38 (0.625 µg) was associated with tumor eradication, there was also substantial toxicity as demonstrated by the death of two thirds of the mice. The maximum-tolerated dose of TGF-–PE38 in athymic nude mice has been reported to be at 0.3 µg when stereotactically injected into the caudate nuclei (14)
. In that study, 0.1 µg of TGF-–PE38 were safe and efficacious for the treatment of intracranial tumors in athymic nude mice (14)
. Our results corroborate this finding in that mice treated with lower doses of (0.1 and 0.03 µg) demonstrated tumor growth inhibition compared with the control. Furthermore, on staining the tumor sections with terminal deoxynucleotidyl transferase-mediated nick end labeling, we observed that the cells in the TGF-–PE38-treated tumors were undergoing apoptosis. TGF-–PE38 did not demonstrate any adverse effects in the mice at low doses. No significant difference was found between the blood parameters from treated and control mice, indicating that there was no drug-related toxicity. Although there are no reports of systemic administration of TGF-–PE38 in clinical trials, the enzymatically active domain of Pseudomonas exotoxin fused to EGF (DAB389,EGF) has been administered i.v. in cancer patients without reaching dose-limiting toxicities (21)
. Although no hematologic toxicities were observed, transaminase and creatinine elevations were reported. Our data corroborates the hematologic findings reported in the study; however, we did not see any significant elevations in the creatinine levels.
Intratumoral or topical administration of TGF--PE38 in patients suffering from easily accessible HNSCC tumors may result in exposure of normal esophageal tissue to the drug. Dose-limiting toxicity was observed at the highest dose of 100 ng/mL (4 µg) when injected stereotactically into glioblastoma tumors (14)
. This is the only dose-limiting toxicity reported in clinical trials with TGF-–PE38. To test the toxicity of TGF-–PE38 on normal esophageal tissue, we used a dose of 0.4 mg for oral gavage administration in immunocompetent mice. Higher doses may be toxic if administered intraesophageally. When tissues were examined for drug-related toxicity, there were no adverse effects observed in the TGF-–PE38-treated mice compared with the control animals, indicating that the drug can be injected into tumors of the oral cavity with little or no effect on normal regional tissues. The lack of toxicity may be due to low levels of EGFR expression in normal esophageal mucosa. Alternatively, the lack of toxicity could be attributed to low affinity of the human TGF- for the mouse EGFR. However, our in vitro data demonstrate that TGF-–PE38 is cytotoxic to murine fibroblasts expressing EGFR, implying that human TGF- can bind to murine EGFR (data not shown). To date, there are no reports of topical administration of TGF-–PE38. Most studies examining the antitumor effects of PE38 in vivo deliver the drug via intratumoral or intravenous administration (20)
. Although we did not specifically investigate the antitumor efficacy of an oral route of administration, our data indicate a lack of toxicity to normal epithelia and support additional investigations of the topical administration of the drug in patients with accessible oral tumors.
TGF- fused to Pseudomonas exotoxin has been previously demonstrated to have antitumor effects in several cancers, including brain (8)
, bladder (22)
, breast (23)
, and non–small-cell lung cancer (24)
. This is the first report demonstrating its effects in head and neck tumors. HNSCC cells expressing EGFR are susceptible to TGF-–PE38 at low concentrations and mice administered the drug do not suffer adverse effects. Because HNSCC tumors express high levels of EGFR compared with the normal adjacent mucosa, targeted therapy may prove efficacious in this tumor type. Increased EGFR expression in precancerous dysplastic lesions compared with normal adjacent mucosa may justify the use of TGF-–PE38 in prevention of tumor progression (25)
. Direct intratumoral injections of TGF-–PE38 are possible in patients with easily accessible head and neck tumors. The dose and treatment regimen of TGF-–PE38 could be extrapolated for clinical application from in vivo data presented in this study. These finding indicate that TGF-–PE38 may be used as a therapeutic agent for the treatment of HNSCC tumors.
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FOOTNOTES |
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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.
Requests for reprints: Jennifer Rubin Grandis, Department of Otolaryngology, The Eye and Ear Institute, Suite 500, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: (412) 647-5280; Fax: (412) 647-2080; E-mail: jgrandis{at}pitt.edu
Received 3/25/04; revised 6/24/04; accepted 7/14/04.
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REFERENCES |
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