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Abstract
Introduction
Epidermal growth factor receptor (EGFR) is expressed in human epithelial tumors including salivary cancers, and known to be correlated with tumor progression and poor clinical courses in some epithelial tumors. In this study, we determined the therapeutic effect of the anti-EGFR monoclonal antibody Erbitux (C225, cetuximab) in combination with the DNA topoisomerase I inhibitor irinotecan (CPT-11) on human salivary adenoid cystic carcinoma (SACC) cells growing in nude mice.
Materials and Methods
At first, immunocytochemical analysis for the expression of EGFR and phosphorylated EGFR (pEGFR) on a human salivary ACC cell line (ACC3). To determine the in vivo effects of Erbitux and CPT-11, nude mice with orthotopic parotid tumors were randomized to receive intraperitoneal Erbitux (1 mg) two times per week, intraperitoneal Irinotecan (50 mg/kg) once per week, Erbitux plus CPT-11, or placebo. (control) Tumor volume and weight were measured. And mechanisms of in vivo activity of Erbitux and/or CPT-11 were determined by immunohisto-chemical/immunofluorescent analyses.
Results
Immunocytochemical staining of ACC3 demonstrated that EGFR was expressed and phosphorylated. CPT-11 inhibited ACC tumor growth in nude mice. Tumors of mice treated with CPT-11 and CPT-11 plus Erbitux exhibited increased tumor cell apoptosis and decreased microvessel density, which correlated with a decrease in the tumor volume in nude mice. But, CPT-11 seems not to be synergistic with Erbitux in our ACC3 model system.
References
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Fig. 1.
Immunocytochemical analysis of the chamber slides in salivary adenoid cystic carcinoma cells. ACC3 cells were immunostained for the expression of EGFR and phosphorylated EGFR. Representative results are shown.(original magnification ×200) (ACC3: adenoid cystic carcinoma cell line, EGFR: epidermal growth factor receptor)
Fig. 2.
A. CPT-11 inhibits the growth of salivary ACC xenografts in nude mice. ACC cells (5×105) were injected into the parotid gland of nude mice. Seven days after the injection of the tumor cell suspension, the mice (n=10) were treated with intraperitoneal injection of Erbitux at 1 mg/injection twice weekly, CPT-11 given via intraperitoneal injection at 50 mg/kg once a week, or both Erbitux and CPT-11. Ponits, mean tumor volume measured twice per week and expressed once per week.(bars: SE) B. Tumor weights of each group at the time of sacrifice. Results are means±SE. (ACC: adenoid cystic carcinoma)
Fig. 3.
Representative gross features of the tumors of each group at the time of sacrifice. A. Control B. Erbitux 1 mg/injection, twice weekly. C. CPT-11 50 mg/kg, once a week. D. CPT-11+Erbitux.
Fig. 4.
Histological (H&E staining), immunohistochemical/immunofluorescent and TUNEL analyses for degree of EGFR tyrosine kinase phosphorylation, cell proliferation, and apoptosis. After 5 weeks of treatment with Erbitux, CPT-11, or Erbitux+CPT11, ACC3 orthotopic tumors were sectioned and immunostained for EGFR, pEGFR, PCNA, and stained with TUNEL. Treatment with Erbitux alone or in combination with CPT-11, inhibited the degree of phosphorylation of EGFR. Note that all the treatment group showed decreased number of PCNA-positive tumor cells. And Some of CPT11-treated tumor cells demonstrated apoptosis. Repersentative features are shown.(original magnification ×100, in panel of TUNEL ×200) (EGFR: epidermal growth factor receptor, pEGFR: phosphorylated EGFR, ACC3: adenoid cystic carcinoma cell line, PCNA: proliferating cell nuclear antigen, TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)
Fig. 5.
Immunohistochemical and immunofluorescent double staining for tumor-associated endothelial cells in Erbitux/CPT-11-treated orthotopic tumors of ACC3. Microvessel density was decreased in CPT-11-treated tumors, but there was no apoptosis of the tumor-associated endothelial cells.(original magnification ×100, in panel of CD31 ×200, in panel of CD31/TUNEL ×200) (ACC3: adenoid cystic carcinoma cell line)
Table 1.
Quantitative immunohistochemical analysis of ACC3 tumors in parotid glands of nude mice
(1: ACC3 cells (5×105) were injected into the parotid glands of nude mice. Seven days later, mice were randomized for treatment with twice-weekly intraperitoneal injections of Erbitux (1 mg/mouse), once-weekly intraperitoneal injections of CPT-11 (50 mg/kg), a combination of both drugs, or placebo as a control. Specimens were processed for immunohistochemical analyses 5 weeks after initiation of therapy, 2: PCNA and TUNEL positivity was quantitated as the ratio of positively stained cells/total cells ×100 per field in 10 random 0.159-mm2 fields at ×100 magnification and 0.039-mm2 fields at ×200 magnification, 3: P<0.05 as compared with controls (Wilcoxon rank-sum test), 4: Microvessel density was determined by measuring the number of completely stained blood vessels in 10 random 0.159-mm2 fields at ×100 magnification, 5: CD31/TUNEL positivity was quantitated by measuring the number of CD31/TUNEL-positive cells in each of 10 random 0.039-mm2 fields at by measuring the number ×200 magnification)
(ACC3: adenoid cystic carcinoma cell line, PCNA: proliferating cell nuclear antigen, TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)
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