Study On Inhibitory Effects Of Icotinib On The Human Tongue Carcinoma Cell Line Tca8113

Carcinoma of tongue is one of the most common malignant tumor of the oral and maxillofacial region. It has rather complicated biological characters, with fast progress, high postoperative recurrence and poor prognosis. The keys to improving curative effects for tongue carcinoma are prevention of recurrence as well as choice of treatment. The rapid development of molecular biology makes the bio-therapy to be the fourth kind of treatment mode following prior three kinds of methods, ie, surgery, radiotherapy and chemotherapy. In the meantime, molecular targeting medicine is active and vigorous in clinical application. Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) was reported recently and had active effects on carcinoma. Icotinib is one kind of EGFR tyrosine kinase inhibitor, experiments had confirmed that Icotinib could inhibite the proliferation of carcinoma of A431 and BGC-823. That has not been reported about its inhibitory effects on carcinoma of tongue. In present study, Tca8113 cells were exposed to different concentration of Icotinib and continued incubating for different time. Then they were detected about the proliferation, distribution of cell cycle, apoptosis and cell protine (cyclinD₁, P₂₇, matrix metalloproteinase-9, E-cadherin). This study will provide experiment evidence for further exploring the mechanism of EGFR tyrosine kinase inhibitor and for EGFR tyrosine kinase inhibitor to be used to treat carcinoma of tongue.ObjectiveTo investigate the effects and machanisms of Icotinib on human carcinoma of tongue Tca8113 cell line.Materials and Methods1. Human sequamous cell carcinoma of tongue Tca8113 cell line was provided by The college of stomatology, Shanghai JiaoTong University.2. Icotinib was provided by Zhejiang Beida Medicine Company.3. MTT method: Incubated Tca8113 cells were treated with Icotinib at different concentration (0μmol/L, 5μmol/L, 10μmol/L, 20μmol/L, 40μmol/L, 80μmol/L ) and continued incubating for 12h, 24h, 48h, 72h, 96h respectively. The inhibitory rate of cell proliferation was evaluated by MTT. At the same time, morphology of cells were observed with microscope.4. Distribution of cell cycle and apoptosis were detected by flow cytometer (FCM).5. Immunocytochemical method, indirectly immunfluorescene and FCM were used to detect the expression of the cell cycle protine cyclinD₁, P₂₇, matrix metalloproteinase -9 (MMP-9), E-cadherin (E-cad) after Tca8113 cells were treated with Icotinib.6. The datas were analyzed by SPSS 13.0 software. Analysis of variance(ANOVA) was used to analyze the datas. Statistically significant leveal was considered as "alpha equals 0.05" .Results1. Growth inhibitory effect of Icotinib on Tca8113 cells: Following treatment with Icotinib at different concentration (5μmol/L, 10μmol/L, 20μmol/L, 40μmol/L, 80μmol/L), the proliferation of Tca8113 cells was inhibited significantly (P < 0.01). The inhibitory rate of cells proliferation at the same conceration of Icotinib has statistical significance in different time (12h, 24h, 48h, 72h, 96h) (P<0.01). The results demonstated that Icotinib could inhibit the Tca8113 cells growth by time- and dose-dependment manners.2. Cell cycle kinetic analysis: FCM showed that Icotinib induced a remarkable cell cycle arrest, accompanied by a decreasing in the percentage of cells in the S phase. And percentage of apoptotic cells was gradually increased with the increasing of Icotinib concentration and/or increasing of treating time. The statistical analysis showed changes of the cell cycle and percentage of apoptotic cells were positive correlated to Icotinib concentration and treated time.3. Immunocytochemical, indirectly immunfluorescene and FCM results indicated that Icotinib could decrease the expression level of the cyclinD₁, MMP-9 and increase the expression level of the P₂₇, E-cad of Tca8113 cells.Conclusions1. Icotinib can inhibit the proliferation of Tca8113 cells in a time- and dose -dependment manners.2. Icotinib can change distribution of Tca8113 cell cycle. It has evident effect that makes cells arrest at G₀/G₁ phase. It can induce apoptosis of Tca8113 cell.3. Icotinib can decrease the expression level of the cyclinD₁ and increase the expression level of the P27.4. Icotinib can decrease the expression level of the MMP-9 and increase the expression level of the E-cad. This further demonstrate that Icotinib can inhibite infiltration and metastasis of malignant tumor.