Radiosensitization Effect Of Dihydroartemisinin On Hypopharyngeal Carcinoma Fadu Cell Xenograft Tumors In Nude Mice

Objective: The purpose of the present investigation is to observe the inhibition and radiosensitization effects of dihydrartemisinin(DHA)on human hypopharyngeal carcinoma Fadu cell xenografts in nude mice,and to provide the basis of its tumor inhibition and radiosensitization in vivo;try to find out the mechanism of the inhibition and radiosensitization effects,to explore the roles of signal transducer and activator of transcription 3(STAT3)in the process;to provide experimental basis for the development of new anti-tumor drugs and to supply a new strategy for prevention and therapy of hypopharyngeal carcinoma.Methods: Human hypopharyngeal carcinoma Fadu cells growing at logarithmic growth phase were inoculated subcutaneouly in nude mice on the right flank(0.2ml,2~6�107 cells).Fifteen days later,the grown tumors were big enough to be planted into other nude mice on the right flank under strict disinfection condition till they grew well in vivo.After two weeks,20 mice were randomly divided into 4 groups: blank control group,DHA group,simple irradiation group,DHA plus irradiation group(n=5).Control group and simple irradiation group were intraperitoneally injected with 10% DMSO each day;DHA group and DHA plus irradiation group were injected with DHA dissolved in 10% DMSO each day.The injection concentration of DHA is50mg/kg;each group was treated for 28 days.Irradiation therapy was applied5 hours after DHA injection.One Gy irradiation dose was used simple irradiation group and DHA plus irradiation group three times a week.The total radiation dose is 12 Gy.We observed the general condition of the mice per day.Twenty-eighty days later,the tumors were removed.The tumor size and weight were measured before and after treatment.The results of HE stainingof tumors were observed under the microscope.Flow cytometry was used to observe cell cycle distribution and apoptosis.Tumors samples were taken to do Western blot to detect the expression of STAT3,p-STAT3,cyclin D1 and apoptosis-related proteins including Cyclin D1,Bcl-2,Bax.Results:1 Xenograft mice model grown with human hypopharyngeal carcinoma was successfully established.2 The mice in blank control group,DHA group,irradiation group and irradiation plus DHA group were generally in good conditions,with no significant difference among different groups.3 In comparison of control with group,the inhibition rates were statistically increased in DHA group,irradiation group and DHA plus irradiation group.The inhibition rates of DHA group,irradiation group,and combination of irradiation with DHA group were 40.74%,61.08%,80.09%,respectively;the three groups of growth inhibition rates were 33.08%,53.06%and 68.68%.4 HE staining of tissue sections showed that application of DHA resulted in morphology changes of the tumor cells,but not as effective as the irradiation;irradiation plus DHA had significant synergistic effect.5 Flow cytometric analysis of cell cycle showed that irradiation induced G 2/M arrest.Combination DHA with irradiation significantly shortened G2/M phase,and mainly arrested in G0/G1 phase and S phase(compared with irradiation group,P < 0.05).DHA plus irradiation induced tumor cell apoptosis more efficiently compared with the control group(P < 0.05)and with radiotherapy alone(P < 0.05).6 Western-blot showed that total STAT3 expression did not change.However,the expression of p-STAT3 decreased when DHA was applied.It was interesting to note that p-STAT3 expression in radiation group was remarkably increased in comparison with control group,application of DHA significantly decreased the expression of p-STAT3.DHA,radiotherapy reduced the expression of CyclinD1,Bcl-2.Expression of these proteins inDHA plus irradiation group also decreased significantly,except that the expression of Bax was increased.Conclusions:1 DHA has inhibitory effect on hypopharyngeal carcinoma xenograft mice models generated from Fadu cells.2 The irradiation significantly inhibited tumor growth in mice bearing xenografted hypopharyngeal carcinomas.3 DHA and irradiation have synergistic effect on tumor inhibition.The apoptosis rate of hypopharyngeal carcinoma Fadu cells caused by irradiation was effectively increased by DHA in nude mice.DHA significantly shortened the G2/M arrest caused by irradiation.The synergistic effects of DHA combined with irradiation may be related to the blocking of STAT3 activation pathway by DHA,along with the regulation of its downstream proteins,which play important roles in the radiosensitization by DHA.