| Background and objective:Biologically active immunoglobulins such as IgG and IgA have been shown to exist in epithelial malignant tumor tissue and carcinoma cell lines. However, the expression and function of immunoglobulin M(IgM) in nasopharyngeal carcinoma(NPC) are largely unknown. Our preliminary studies using serological proteomics have identified the expression of membrane-bound IgMs in NPC cell line CNE-1, as well as several other epitheliogenic malignant tumor tissues and carcinoma cell lines.The objectives of this study are:1) To further characterize the expression patterns of IgM in nasopharyngeal carcinoma and its cell lines(HNE-1 and CNE-1);2) To examine the relationship between IgM expression levels and clinical progress and prognosis of nasopharyngeal carcinoma patients;3) To investigate the pathophysiologic mechanisms of IgM in nasopharyngeal carcinoma cells.Methods:1. The immunocytochemistry SABC method, SDS-PAGE, Western-blotting and flow cytometric indirect immunofluorescent techniques were utilized to examine the expression of IgM in cultured HNE-1 and CNE-1 cells.2. To examine the expression of IgM in paraffin imbedded human nasopharyngeal carcinoma tissue, the immunohistochemistry SABC method was utilized. Meanwhile, the IgM expression levels in nasopharyngitis tissue were measured and were used as control. Furthermore, a COX regression model was utilized to analyze the relationship between IgM expression levels and multiple clinical manifestations of nasopharyngeal carcinoma including tumor recurrence, metastasis, and survival rate.3. To assess the in vitro anti-cancer effect of antihuman IgM antibodies, multiple cellular parameters were investigated following administration of antihuman IgM antibodies to the culture of HNE-1 cells. First, the proliferation and related morphology of HNE-1 cells were examined by MTS method. Second, flow cytometry with PI staining, Hoechst and TUNEL staining were utilized to detect apoptosis and related morphological changes of HNE-1 cells. Finally, flow cytometry was utilized to analyze the effect of antihuman IgM antibodies on HNE-1 cell cycles.Results:1. There was IgM expression in nasopharyngeal carcinoma cell lines, with 91.56％+1.72％of HNE-1 cells and 91.00％+1.79％of CNE-1 cells expressing IgM. The IgM expression was predominantly found in cytoplasm, though some IgM was expressed on cell membrane of CNE-1 cells. Furthermore, an immunoglobulin-like protein was found in the protein extract of nasopharyngeal carcinoma cells by using SDS-PAGE and Western-blot analysis. This protein reacted specifically with immunoglobulin μchain and has a molecular weight(75KD) analogous to that ofμchain. The mean fluorescence intensity of IgM in HNE-1 and CNE-1 was 10.50+0.80 and 6.99±0.43 respectively, higher than nasopharyngitis control tissue (P＜0.05).2. The follow-up rate was 94.85％and the overall median survival period was 63.50 months. The recurrence rate was 21.74％and the metastasis rate was 41.30％during the follow-up period. The overall survival rate was 47.83％, recurrence-free survival rate 45.65％and metastasis-free survival rate 37.00％. The 1, 3 and 5 year actuarial survivals were 93.48％, 71.74％and 58.66％, respectively.3,Overall, IgM was expressed in 68.48％of nasopharyngeal carcinoma whereas only 24.00％control nasopharyngitis tissue expressed IgM(P＜0.05). The IgM expression was mainly localized in cytoplasm in poorly differentiated nasopharyngeal carcinoma. However, in moderately and well-differentiated nasopharyngeal carcinoma or in control tissue, the IgM was expressed on the plasma membrane(P＜0.05). The IgM expression in nasopharyngeal carcinoma was negatively correlated with both clinical stage and T-stage(P＜0.05). There was no significant correlation between IgM expression and age, sex, duration, pathological type, degree of differentiation, or N-stage. There was no significant correlation between IgM expression and local carcinoma recurrence, cervical carcinoma recurrence, or distant metastasis. The IgM expression was correlated with overall survival rate(P＜0.05). But there was no significant correlation between IgM expression and disease-free survival rate. Furthermore, neither overall survival rate nor disease-free survival rate was related to the intensity of IgM expression. A multivariate anaiysis using the COX regression model indicated that both recurrence and metastasis were related to a poor prognosis for nasopharyngeal carcinoma(P＜0.05) whereas there was no correlation between IgM expression and prognosis.4. The increase of IgM concentration or increase of interaction time of IgM and cell culture caused an increase of OD value and inhibition rate of HNE-1 cells. This inhibitory effect presented immediately upon adding IgM with a concentration higher or equal to 50μg/ml. This inhibitory effect could also present three days after adding IgM at a lower concentration. Thus, the antihuman IgM antibody significantly inhibited HNE-1 cell growth in a dose and time-dependent manner(P＜0.05). In addition, the IgM appeared to alter the morphology of HNE-1 cells.The antihuman IgM antibody significantly increased apoptosis of HNE-1 cells with typical apoptotic morphological changes. The apoptosis rate of HNE-1 cells examined by flow cytometry with PI staining, Hoechst and TUNEL staining were 31.1％+2.81％, 27.26％+1.69％and 26.19％+2.17％respectively. These apoptosis rates were significantly higher in comparison with control(P＜0.05). Furthermore, the antihuman IgM antibody decreased the percentage of G1 phase cells and increased S phase cells(P＜0.05).Conclusion:1. There is IgM expression in nasopharyngeal carcinoma cell lines(HNE-1 and CNE-1). The expression of IgM is localized mainly in cytoplasma of nasopharyngeal carcinoma cells, and on cytolemma in a subpopulation of CNE-1 cells; 2. IgM is highly expressed in nasopharyngeal carcinoma and its expression localization is strongly correlated with pathological stages. Further studies are needed to determine whether the localization difference of IgM expression is related to its functional difference;3. The expression of IgM may be an early event in nosogenesis of nasopharyngeal carcinoma. The detection of IgM expression may provide predictive information on nasopharyngeal carcinoma prognosis;4. The administration of antihuman IgM antibody suppresses the growth and viability of HNE-1 cells and increase programmed cell death. Furthermore, the antihuman IgM antibody causes cell cycle blockage. These results support a role of IgM as a growth factor in nasopharyngeal carcinogenesis.
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