Apoptosis Induced By Cetuximab In Conjunction With Hyperthermia And Radiotherapy In Human NPC CNE Cell Line

Part One: The experimental study on apoptosis of human nasopharyngeal carcinoma CNE cell line induced by cetuximab in vitro Objective: To investigate the apoptosis mechanism of CNE cell line induced by cetuximab in vitro. Methods: The working concentration of cetuximab was defined as its IC(20～30) at 48 hours action against CNE which was determined by MTT assay. Cells apoptosis was assayed by transmission electron microscopy(TEM), fluorescent Hocehst 33258 staining and flow cytometric analysis(FCM). The expression of Bcl- 2,Bax proteins were determined by Western Blot. Results: A working concentration of Cetuximab at 10μg/ml was obtained and subsequently used in the following experiments. Characteristical changes of apoptosis in CNE cells were observed and the apoptosis rate of 24, 48h was 14.30±0.78%, 15.30±0.95% respectively(P<0.05 compared with control group). In addition, Western Blot analysis indicated the level of expression of the proteins Bax significantly increased while Bcl-2 decreased by the treatment. Conclusion: Cetuximab could significantly induce the apoptosis of CNE cells, possibly through the Bax up-regulation and Bcl-2 down-regulation. Part Two : The experimental study on apoptosis of human nasopharyngeal carcinoma CNE cell line induced by cetuximab combined with hyperthermia in vitroObjective: To investigate the apoptosis mechanism of CNE cell line induced by cetuximab combined with hyperthermia in vitro. Methods: Apoptosis was assayed by fluorescent Hochest 33258 staining and flow cytometry(FCM) in three experimental groups: cetuximab(10ug/ml) or hyperthermia(43℃, 30min) alone, cetuximab combined with hyperthermia. The expression of Bax, Bcl-2, EGFR proteins were determined by Western Blot. Results: Characteristical changes of apoptosis in CNE cells were observed in all experimental groups, especially in cetuximab combined with hyperthermia group. FCM also showed more prominent apoptosis induced by cetuximab combined with hyperthermia group compared to cetuximab or hyperthermia alone groups. Western Blot analysis indicated that the ratio of Bax/Bcl-2 significantly increased while the expression of the protein EGFR decreased by the treatment,the level of expression of the protein EGFR decreased gradually after the treatment of hyperthermia. Conclusion: Cetuximab combined with hyperthermia synergistically increases the apoptosis in CNE cells, possibly by further down-regulating expression of EGFR to up-regulate Bax/Bcl-2 ratio.Part Three : The experimental study on apoptosis of human nasopharyngeal carcinoma CNE cell line induced by cetuximab in conjunction with radiotherapy and hyperthermia in vitroObjective: To investigate the apoptosis mechanism of CNE cell line induced by Cetuximab in conjunction with radiotherapy and hyperthermia in vitro. Methods:Apoptosis was assayed by fluorescent Hocehst 33258 staining and flow cytometry (FCM) in five experimental groups: control with no treatment (C), radiotherapy alone(2,4,8,12Gy)(R), Cetuximab with radiotherapy(TR), Cetuximab with hyperthermia(43℃, 30min)(TH), and Cetuximab with radiotherapy plus hyperthermia(TRH). The expression of Bcl-2, Bax proteins were determined by Western Blot. Results: Characteristical changes of apoptosis in cells treated with cetuximab were observed in all the experimental groups, especially in TRH. FCM also showed more prominent apoptosis induced by TRH group compared to the control, R, TR and RH groups. In addition, Western Blot analysis indicated that the ratio of the proteins Bax/Bcl-2 by the treatment. Conclusion: Cetuximab in conjunction with radiotherapy and hyperthermia synergistically increases the apoptosis in CNE cells, possibly through further up-regulation of the ratio Bax/Bcl-2.