Anti-Cancer effects of 131i-nimotuzumab on immune deficiency mouse bearing human larynx cancer

JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 144 ANTI-CANCER EFFECTS OF 131I-NIMOTUZUMAB ON IMMUNE DEFICIENCY MOUSE BEARING HUMAN LARYNX CANCER Nguyen Thi Kim Huong*; Ho Anh Son**; Nguyen Thi Thu*** Vo Thi Cam Hoa***; Pham Huy Quyen*; Nguyen Linh Toan** SUMMARY Objectives: To assess the anti-cancer effects of 131 I-nimotuzumab on immune deficient mouse bearing human laryngeal cancer. Subjects and methods: Three groups of mice bearing human laryngeal cancers were injected via the tail vein by 131 I-nimotuzumab, nimotuzumab or 0.9% NaCl, separately. Tumor volume was measured every week. The average survival time and survival rate was calculated for mice in each group. Results: 131 I-nimotuzumab could reducing the tumor volume with extending the average life span and increase the survival rate for the mice. Conclusions: 131 I-nimotuzumab had the highly anti-cancer effects on immune deficient mice bearing human laryngeal cancer. * Keywords: Larynx cancer: 131 I-nimotuzumab; Nude mouse. INTRODUCTION Laryngeal cancer is the second common malignancy in head and neck cancer. Larynx cancer accounts for 2% of all cancer cases [2]. In Vietnam, larynx cancer ranks common second after nasopharyngeal cancer in head and neck cancer and ranks fourth of all types of cancers. This disease is common in men (80%), usually in ages 40 - 60 years, but the current trend is that more young people are affected with growing burden on society and health system [1]. Epidermal growth factor receptor (EGFR) is often overexpressed in larynx cancer with over 90% in head and neck cancer. EGFR signaling plays an important role in the development, progression, invasion, metastasis and angiogenesis in head and neck cancer. In addition, overexpression of EGFR has a poor prognosis of overall survival in patients in Eastern and Western countries. In particular, EGFR expression level is considered to be a predictor of laryngeal squamous cell carcinoma [3]. Currently treatment for head neck cancer and larynx cancer is the still the major challenge due to its inadequate response to conventional treatments such as chemotherapy or radiotherapy. Targeted therapy is one of the promising cancer treatments and has superior advantages over conventional method. This method however is that focuses only destroying cancer cells, and in addition, targeted therapies have limited adverse effects on * Haiphong Medical-Pharmaceutical University ** Vietnam Military Medical University *** Nuclear Research Institute Corresponding author: Ho Anh Son (hoanhson@vmmu.edu.vn) Date received: 03/10/2017 Date accepted: 20/11/2017 JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 145 healthy organs and on the overall condition of the cancer patients. Using radiolabeled antibody, or radioimmunotherapy is an emerging method which attracting research attention during the past half century. There have been some successes in this field that have been applied to the clinical treatment of cancer patients. However, this is a new approach that has not yet been investigated in Vietnam. In this study, we used EGFR-specific monoclonal antibody, nimotuzumab in combination with 131I to form 131I-nimotuzumab complex, and evaluated the antitumor activity of this complex in immunocompromised mice xenografted with the human laryngeal cancer line Hep2. This is the initial basis for the development of this complex application for laryngeal cancer treatment in the future. SUBJECTS AND METHODS 1. Subjects. The immune-deficient BALB/c nude mice are imported from Charlie-River Company, USA. The mouse was bred in clean room conditions with the air is filtered and have positive pressure. Room temperature is maintained at 25 ± 20C, humidity 55 ± 5%, light is automatically turned on at 7:00 am, off at 7:00 pm. Chaw (Zeigler, USA) and drinking water be sterilized before use. Each mouse cage is mounted on a self- ventilated rack and filters through the membrane ensuring good isolation from pathogens. 2. Materials. * 131I-nimotuzumab: Nimotuzumab monoclonal antibody combines with 131I to form 131I-nimotuzumab complex. The technical procedure was carried out at Vietnam Nuclear Research Institute (NRI), with radiation concentrations of 100 - 200 mCi/mL. * Human laryngeal cancer line: Human laryngeal cancer cell line Hep2 were purchased from ATCC (American Type Culture Collection, P.O. Box 1549, Manassas, VA 20108, USA). 3. Methods. * Xenograft experiments: Hep2 cells (1 x 106) were subcutaneously injected in a volume of 100 µL into the left and right thigh of thirty immune-deficient BALB/c nude mice. All animals were housed in individually ventilated cages, after the tumor appears, and has a diameter of approximately 30 mm, then the mice were randomly divided into three groups: - 131I-nimotuzumab treatment group (n = 10): mice were intravenous ịnjected a single dose of 1 mCi/ml of 31I-nimotuzumab through tail veins. - Nimotuzumab treatment group (n = 10): mice were intravenous injected a single dose of 1 mCi/ml of nimotuzumab through tail veins. - Control group (n = 10): mice were intravenous ịnjected with 0.9% NaCl through tail veins. The sizes of the tumors were measured once per week using microcaliper from week 1 to week 8 after injection. In addition, JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 146 mortality of mice was monitored daily and the number of days was recorded for each mouse in the treatment and control groups throughout the follow-up period. Life tables were used to calculate the survival rates and average survival time. * Ethics statement: All animal care and experimental protocols were approved by the animal ethics committee of the Vietnam Military Medical Universtiy under the guidelines of the Animals (Scientific Procedures) Act 1986. * Statistics: Data was analyzed using the ANOVA tests or chi-square test where appropriate. Values are expressed as means ± SD. The statistical package for the Social Sciences version 21.0 (SPSS, Chicago, IL) was used for all the analyses. p values of < 0.05 were considered statistically significant. RESULTS 1. Treatment with131I-nimotuzumab reduces the volume of tumors. To investigage the effect of 131I-nimotuzumab in vivo, we measured tumor size in xenograft mice model barering human laryngeal cancer cell line Hep2 after treatment with 131I-nimotuzumab (nimotuzumab and NaCl 0.9% as control) at different times after injection. Figure 1: Volume of the tumor after one week of treatment. A) The table compares the average volume of tumors of mice of different treatment groups; there was no statistically significant difference between the groups of mice. B) Histogram showing the average tumor volume of the study groups. Results showed that at one week after treatment, the volume of tumors of groups of mice was similar. JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 147 Figure 2: Volume of the tumor after two weeks of treatment. A) The table compares the average volume of tumors of mice of different treatment groups; in that group of mice treated with 131I-nimotuzumab had significantly smaller tumor volume than control group, with * = p < 0.05, 131I-nimotuzumab vs. control; and no significant difference between the other groups. B) Histogram shows the average tumor volume of the study groups. However, by the second week after injection, the tumor volume in the 131I-nimotuzumab treated mice group was significantly lower than the control group (p < 0.05). While the tumor volume of the group treated with nimotuzumab was smaller than the control group, but there was no significant difference between the two groups, also no difference between the groups of 131I-nimotuzumab and nimotuzumab treatment. Figure 3: Volume of the tumor after eight weeks of treatment. A) The table compares the average volume of tumors of mice in different treatment groups; the group of mice treated with 131I-nimotuzumab had significantly smaller tumor * * JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 148 volume than control groups, with * = p < 0.05, 131I-nimotuzumab vs. control; and no significant difference between the other groups. B) Histogram shows the average tumor volume of the study groups. Figure 4: Volume of tumors in different groups of mice during eight weeks of treatment. The mice treated with 131I-nimotuzumab had a significantly smaller tumor volume than the control group at most of the time of the survey, from the 2nd week of treatment, with * = p < 0.05 ; ** = p < 0.01, 131I-nimotuzumab vs. control; and no significant difference between the other groups. Follow-up at subsequent times showed that the 131I-nimotuzumab treatment group had significantly smaller tumor volumes than the control group at eight weeks (with p < 0.05 - 0.01). While there was no statistically significant difference between the other groups at that time. The results show that 131I-nimotuzumab complex had a significant effect in inhibiting the development of human larynx cancer when compared to the control group, in addition the effect of 131I-nimotuzumab is stronger than nimotuzumab when compared with the control group. Thus, the efficacy of anti-cancer increased with the combination of 131I with nimotuzumab versus using nimotuzumab alone. * * * ** ** ** ** JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 149 2. Treatment with 131I-nimotuzumab increased average survival time and the survival rates of mice. Figure 5: The average survival time and the survival rates of mice treated with 131I-nimotuzumab, nimotuzumab and control group. A) The average survival time analysis of the three groups of mice, in which the 131I-nimotuzumab treatment group had a significantly higher survival time than the control group, with p < 0.05; and there was no difference in which between the treatment group with nimotuzumab. Use the chi square test. B) Histogram showing everage survival time and survival rates of mice treated with 131I-nimotuzumab, nimotuzumab, and control group. The effect of 131I-nimotuzumab was also assessed through the average survival time and survival rate when compared on the mice treated with nimotuzumab and control group. Results showed that the mice treated with 131I-nimotuzumab, the average survival time was 15.829 weeks, significantly higher than of control group, with average survival time was 14.222 weeks (p = 0.033), and trend to higher than the mice treated with nimotuzumab had aveage survival time was 15.657 even the difference was not statistic significant. At the same time, the survival rate of the treated group by 131I-nimotuzumab was 0.9, which was higher than the control group with the survival rate was 0.5, and was the same with the mice treated with nimotuzumab. The results showed that 131I-nimotuzumab treament increased average survival time and the survival rates of mice bearing laryngeal cancer compared to control group. DISCUSSION In this study we investiaged the combination of 131I with nimotuzumab, an anti-EGFR monoclonal antibody and assessed the anti-cancer effects of this complex in immunodeficiency mice bearing human laryngeal cancer. Results showed JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 150 that 131I-nimotuzumab complex was higher effective against larynx cancer than nimotuzumab alone when compared to control group. Treatment with 131I- nimotuzumab reduced the volume of tumors, also increased the average survival time and the survival rate of mice compared to the using nimotuzumab group alone and control group. Thus, our findings suggest that the combination of 131I and nimotuzumab increases the effective treatment of human larynx cancer on animal model. 131I and nimotuzumab itself has certain effects on the treatment of head and neck cancer and larynx cancer in particular, but the effect is still limited [4] and usually is combined with other treatment methods to improve the efficiency [5]. In our study, although the mice treated with nimotuzumab had a smaller tumor volume than the control group at most of the time of study, however, the difference was not significant. This suggests that using nimotuzumab alone is not so effective in treating laryngeal cancer, which is consistent with some previous studies. In this study we conducted a combination of nimotuzumab with 131I to exploit the advantages of these substances. Among EGFR-specific monoclonal antibodies (cetuximab, gefitinib, erlotinib, panitumumab and nimotuzumab), the nimotuzumab had the distinct properties and advantages than the other antibody types. Nimotuzumab binds sustainable and stable to the receptor via double bonds so it can bind to cells with moderate to high levels of EGFR expression. Due to nimotuzumab has a low affinity with the healthy cells with low EGFR expression, so it avoids dose-dependent toxicity limitation. Unlike other anti-EGFR antibodies with typical skin toxicity, in clinical trials with nimotuzumab, 90% of patients showed no evidence of severe skin toxicity [6, 7]. Among the radioactive isotopes used for radiotherapy, 131I has outstanding advantages such as: the half-life of 131I is eight days, which is in accordance with the half-life of the antibody, thus, it will prolong the duration of treatment. In addition, 131I emission  ray, which allows imaging to monitor the absorption, distribution, metabolism and excretion of radioactive medications. It also emisses the shorter  for effectively treat tumors even small ones. Easy renal clearance of 131I helps to limit the accumulation of iodine in the bone [8]. Moreover, accumulation of clinical trials have demonstrated the efficacy of 131I in the treatment of hematopoietic and solid tumors. 131I also has an advantages such as: inexpensive, can be used to locate the tumor and use it for therapeutics, and has a long history of successfully treating malignant tumors. In addition, 131I emits the powerful -rays as an additional element that supports the emission of  particles in radioimmunoassay, as  particles generate large amounts of energy efficiency to destroy the cancer cells [9, 10]. Accordingly, the superior efficacy of the 131I-nimotuzumab complex in the treatment of larynx cancer on animal model is probably due to the combination of the advantages of each constituent. The combination of 131I and nimotuzumab has proven to be the primary efficacy in treatment of laryngeal cancer. Thiscombination combination is also JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 151 innovative point in this study and opens new prospects for the treatment of laryngeal cancer and other tumors that overexpress the EGFR. The results of this study are an important basis for further research into the application of 131I-nimotuzumab complex in the treatment of larynx cancer in clinical trials. CONCLUSIONS 131I-nimotuzumab complex is effective in treatment of immune deficiency mouse model bearing human larynx cancer. 131I-nimotuzumab treatment animal showed the reduction the tumor volume. It increased survival time and survival rates of mice. 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