| Objective:Laryngeal squamous cell carcinoma is a common human malignancy, currently the main treatment is surgery and radiation therapy. Chemotherapy drugs commonly used in treatment of laryngeal squamous cell carcinoma are not satisfied. People have been exploring new chemotherapy and chemotherapy drugs diligently. Meanwhile, the expression of immunoglobulin (Ig) in epitheliogenic malignant tumor has caused researchers'great concern. Further and systemic research on the phenomenon of Ig ectopic expression in a variety of malignant tumors, including laryngeal cancer, has become a hot and difficult spot, and provided new ideas for investigating the occurrence and development of cancer. In the current study, we investigated the effect of anti-human IgM antibody on the growth of laryngeal squamous cell carcinoma Hep-2 cell transplanted tumor established in nude mice, and the expression of IgM, gp96 protein and apoptosis of tumor cells, so as to explore the anti-tumor roles and possible mechanism of anti-human IgM antibody, and provide experimental evidence for using anti-human IgM antibody in the prevention and treatment of laryngeal squamous cell carcinoma. Methods:Human laryngeal squamous cell carcinoma Hep-2 cell was cultured and then subcutaneously inoculated into the right flank of Balb/c-nu/nu nude mice to establish the transplanted mode of human laryngeal squamous cell carcinoma, then the 15 nude mice were randomly divided into 3 groups:experimental group(IgM group, intratumoral injection anti-human IgM antibody 1.5mg/tumor for five times with a 3-day interval), control group of IgG (treated with normal goat IgG) and control group of PBS (treated with normal PBS). The weight of nude mice and the shortest and longest diameter of the tumor were measured with slide gaud at 3-day interval, and tumor volume (mm3) was calculated using the following standard formula:(the longest diameter)×(the shortest diameter)2/2, and the side effect of anti-human IgM antibody was observed during treatment. The mice were sacrificed on day3 after the last treatment, the tumor and main organs were also dissected, stained with hematoxylin-eosin for histological examination. IgM and gp96 protein were assessed immunohistochemically, and the expression of them were observed microscopically. By image analysis system, the average optical density values of IgM and gp96 protein in xenografts were analyzed. The expression of apoptotic cells was measured with TUNEL-AP method, and the percentage of TUNEL-positive cells was calculated by image analysis system. Results:(1) The model of laryngeal squamous cell carcinoma xenografts in nude mice was successfully established, the tumorigenic rate in nude mice injected with Hep-2 cell was 100%. Three groups of mice all appeared the growth of implanted tumors in situ. The tumor tissues were proved to be laryngeal squamous cell carcinoma by pathological section examination. Three groups of animals had been all survive during the experimental phase. (2) Food and drink of nude mice was normal, and mental state of nude mice was good, without dying and the action was free in three groups. The weight and pathological section examination of heart, liver, spleen, lung, kidney had no obvious difference. (3) The tumor volume of the experimental group was (64.58±26.74)mm3, significantly lower than that of the control group of IgG (204.89±110.55)mm3 and the control group of PBS (258.36±71.07)mm3 (P<0.05).No significant difference was found in body weight of the nude mice in experimental group (22.40±1.92)g, the control group of IgG (25.62±4.00) g and the control group of PBS (22.42±1.93)g (P>0.05). The weight of tumor in the experimental group was (0.07±0.05)g, significantly lower than that of the control group of IgG (0.26±0.20)g, and the control group of PBS (0.25±0.09)g (P<0.05).The tumor growth inhibition ratio 1 (comparison with the control group of IgG) was 73.63%, and the tumor growth inhibition ratio 2 (comparison with the control group of PBS) was 73.28%. (4)The results of immunohistochemical technique to determine the expression of IgM and gp96 protein in implanted tumor of mice showed that the expression of IgM protein in experimental group (0.04±0.03) dropped dramatically, the difference between experimental group and control group of IgG (0.10±0.03) and control group of PBS (0.11±0.04) was statistically significant (P<0.05). The expression of gp96 protein in experimental group (0.04±0.01) dropped dramatically, the difference between experimental group and control group of IgG (0.08±0.02) and control group of PBS (0.10±0.03) was statistically significant (P<0.05). (5) The percentage of TUNEL-positive cells was measured with TUNEL method, which was (2.32±1.03)% in experimental group, (0.47±0.23)% in control group of IgG and (0.38±0.23)% in control group of PBS, respectively. The difference was significant (P<0.05). Conclusions:(1) Anti-human IgM antibody had obvious inhibitory effect on the growth of human laryngeal squamous cell carcinoma Hep-2 cell xenografts in nude mice. (2) Anti-human IgM antibody can inhibit the expression of IgM protein. It was suggested that anti-human IgM antibody may be binding with IgM antigen produced by laryngeal squamous cell carcinoma and inhibit tumor growth. (3) Anti-human IgM antibodies can inhibit the expression of gp96 protein, which may be related to the inhibition of IgM protein expression and promoting tumor cell apoptosis. (4) Anti-human IgM antibody can promote apoptosis of tumor cell in laryngeal squamous cell carcinoma xenografts, which may be related to the inhibition of IgM and gp96 protein expression. (5) Anti-human IgM antibody had no obvious toxicity and side effect. (6) Anti-human IgM antibody may become one part of combined therapy of human laryngeal squamous cell carcinoma in the future. Further studying on IgM ectopic expression will provide original broad prospect for preventing and treating human laryngeal squamous cell carcinoma. |
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