Effect Of Anti-human IgM Antibody On The Growth, Apoptosis And Cell Cycle Of Human Laryngeal Squamous Cell Carcinoma Hep-2 Cells

Abstract Objective:Immunoglobulin (Ig) is very important immune molecule in the body's immune system, and its main function is mediating humoral immunity. According to the traditional immunological theory, Ig is the specific product of B lymphocytes and other cells don't produce Ig. However, in recent years, researchers have found that Ig is ectopicly expressed in multiple epitheliogenic malignant tumor tissues and carcinoma cell lines and has a role as growth factor. Our research group has reported the expression of IgM in epitheliogenic malignant tumor tissues and carcinoma cell lines. In this study, we investigated the expression of IgM protein in laryngeal squamous cell carcinoma Hep-2 cells, and conducted a preliminary study on the biological activity of IgM protein ectopic expressing in Hep-2 cell. Methods:The immunocytochemical technique (strept avidin-biotin complex method, SABC method), Western Blot and flow cytometry (FCM) were utilized to examine the expression of IgM protein in cultured human laryngeal squamous cell carcinoma Hep-2 cells. Through the cell proliferation inhibition assay (MTS method), setting the time and concentration gradient, to investigate the effect of anti-human IgM antibody on Hep-2 cell growth and proliferation activity, and cell morphology were observed. The Hep-2 cell proliferation ability was also checked by soft agar colony forming assay with different concentrations of anti-human IgM antibody. By flow cytometry (Annexin V/PI staining) and Hoechst staining method to detect apoptosis and related morphological changes of Hep-2 cell after anti-human IgM antibody administration. Finally, flow cytometry with PI staining was utilized to analyze the effect of antihuman IgM antibody on cell cycle of Hep-2 cell. Results: The results of immunocytochemical technique (SABC method) indicated that there was IgM protein expression in human laryngeal squamous cell carcinoma Hep-2 cell and the mainly location of IgM was in cytoplasma. The positive signal was detected by Western Blot in the Hep-2 cell protein extracts, which molecular weight was equal to 75KD of IgMμchain. Application of indirect immunofluorescence flow cytometric analysis, the fluorescence intensity of Hep-2 cell was 12.4±2.55, which was much lower than 17.2±2.30 in positive control Raji cell and 2.29±0.50 in negative control(P<0.05), indicating the expression of IgM protein in Hep-2 cells. MTS results showed that the inhibition rate increased with the increase of interaction time and this inhibitory effect presented significantly on the third day. The inhibition rate also increased with the increase of concentration, which became obvious from the concentration of 50μg/ml. It indicated that the anti-human IgM antibody significantly inhibit Hep-2 cell growth in a dose and time-dependent manner (P<0.05) and alter the morphology of Hep-2 cell. Soft agar colony formation assay showed anti-human IgM antibody inhibit the proliferation of Hep-2 cell with a concentration-dependent manner, which was significantly different to the control groups (P<0.05). The antihuman IgM antibody significantly increased apoptosis of Hep-2 cells with typical apoptotic morphological changes. The apoptosis rate of Hep-2 cells examined by flow cytometry with Annexin V/PI staining and Hoechst staining were (20.33±1.93)% and (20.45±3.45)% respectively. These apoptosis rates were significantly higher in comparison with controls (P<0.05). Flow cytometry showed anti-human IgM antibody could also affect the cell cycle of Hep-2 cell, decreasing the percentage of G1 phase cell and increasing the S phase cells so that the cell cycle arrested at S phase (P<0.05). Conclusions:There is IgM protein expression in human laryngeal squamous cell carcinoma Hep-2 cell and the expression of IgM is localized mainly in cytoplasma. The administration of anti-human IgM antibody suppresses the growth and viability of Hep-2 cells and increase apoptosis. Furthermore, the anti-human IgM antibody causes cell cycle blockage. These results support a role of IgM as a growth factor in laryngeal squamous cell carcinoma, which may provide a new target for laryngeal squamous cell carcinoma treatment in the future.