Synergistic Effects And Mechanisms Of EGFR-blockade By Cetuximab Combined With Chemotherapeutic And/or Radiotherapy In Laryngeal Squamous Carcinoma Cells Hep-2in Vitro

Objective Cetuximab is a mouse-human chimeric IgG monoclonal antibody,specific binding with the cell surface of epidermal growth factor receptor （EGFR）,competitively blocking the epidermal growthfactor and its ligand binding, thusblocking the proliferation of tumor cells, metastasis, invasion and angiogenesis, andother biological effects. Cetuximab and some chemotherapeutic drugs or radiationhaving a synergistic killing effect on different types of tumor cells, reducing theresistance of different types of tumor cells to Cetuximab, improve the sensitivity ofright Cetuximab. This study by observing the cetuximab plus cisplatin andradiation-induced laryngeal squamous carcinoma cell line Hep-2proliferation,apoptosis and cell cycle impact explore cetuximab with cisplatin and radiationcombined application of Hep-2cell-killing effect and its preliminary study of theregulatory mechanism.Methods1. In vitro cultured human laryngeal squamous cell carcinoma cell lineHep-2, take the logarithmic growth phase cells tested.2. Cisplatin and radiation, respectively, in combination with cetuximab growthinhibition rate of Hep-2cells was significantly higher than that of cisplatin andradiation alone or in combination （P <0.001）, to have a synergistic killing effect. 3. Experimental group were given cetuximab1036μg/ml, cisplatin3μg/ml, radiation4Gy, cisplatin3μg/ml+radiation4Gy, the cetuximab1036μg/ml+cisplatin3μg/ml,cetuximab monoclonal antibody1036μg/ml+radiation4Gy, cetuximab1036μg/ml+of cisplatin3μg/ml+radiation4Gy, while the establishment of a negative controlgroup （without adding any drug）, respectively. role in human laryngeal carcinomaHep-2cell lines24h inverted microscope observationcell morphology, CCK-8kit todetect cell growth inhibition rate of different intervention programs on Hep-2apoptosis rate and cell cycle distribution by flow cytometry.Results1. Different doses of cetuximab, cisplatin, and radiation of Hep-2cellswas inhibited by time-a dose-dependent, and in a certain concentration range was24h half maximal inhibitory concentration（IC50） were the cetuximab1036.84μg/Lcisplatin3.08μg/ml, radiation4.18Gy; of Hep-2cells are more sensitive to the growthinhibitory effects of cetuximab.2. Cisplatin and radiation, respectively, in combination with cetuximab growthinhibition rate of Hep-2cells was significantly higher than that of cisplatin andradiation alone or in combination （P <0.001）, to have a synergistic killing effect.3. Cetuximab and radiation, cetuximab in combination with cisplatin for24h in thestage of early apoptotic Hep-2cells were （21.92±3.00）%,（18.44±2.57）%,significantly higher than thenegative control group with radiation, cisplatin alone orin combinatioμgroup （P <0.001）, prompting cetuximab to cisplatin or radiation hassignificant synergy in vitro differentiation of human laryngeal carcinoma Hep-2celllines apoptosis.4. Single-drug Cistercian infliximab group, the proportion of cells in S phasecompared to the proportion of negative control group was significantly higher （P<0.001）; cetuximab+radiatioμgroup （P=0.005）, cetuximab+cisplatin+radiationgroup （P=0.002）, the proportion of G₀/G₁phase cells was significantly higher than the radiatioμgroup.Conclusions1. Laryngeal squamous cell Hep-2cell lines are sensitive tocetuximab-induced apoptosis in a time-and dose-dependent manner, in a certainconcentration range.2. Cisplatin and/or radiation and Cistercian infliximab combined with significantgrowth inhibition of Hep-2cells, a synergistic inhibitory effect on the proliferation ofHep-2cells.3. Cetuximab and cisplatin or radiation associated with significantly increasedapoptosis rate of Hep-2, suggesting that cetuximab to cisplatin or radiation in vitrodifferentiation of human laryngeal carcinoma Hep-2cell lines apoptosissignificantlysynergies.4. Cetuximab by Hep-2cells during cell division arrest in S phase to prevent theuncontrolled proliferation inhibition of cell division. Cetuximab and radiationassociated with can also be people of Hep-2cell lines arrest in the G₀/G₁phase of theradiation-sensitive in vitro can significantly induce apoptosis, which radiation has astrong synergy. This may be one of the mechanisms of the combination of cisplatinand radiation with cetuximab synergy on Hep-2cells.