| ObjectiveMedullary thyroid carcinoma (MTC), as a malignant tumor originates from thyroid follicular paracrine cells. Thyroid follicular paracrine cells do not absorb iodine,and is not sensitive to radiation,so the radiation effect is not good. Thyroid folicular paracrine cells, which has no affinity to iodine and has no sensitive to radiation, therefore the radiation therapy does not work well. To investigate the effects of Janus kinase inhibitor AG490 in JAK-STAT signal pathways and in radio-sensitivity, we cultivated the TT medullary thyroid gland carcinoma cells in AG490 in vitro in order to provide theoretical basis for the effective treatment of MTC in clinic.Methods1. To cultivate the TT medullary thyroid gland carcinoma cells in the concentrations of 0,25,50,100 umol/L with AG490, cell proliferation was detected by MTT assay, absorbance value was observed on ld,2d,3d,4d and 5d in order to measure the proliferation index.2. To cultivate the TT medullary thyroid gland carcinoma cells in the concentrations of 0,25,50,100 umol/L withAG490 for 48h to observe the apoptosis rate.3. To cultivate the subculture TT medullary thyroid gland carcinoma cells in the concentrations of 0,25,50,100 umol/L with AG490 for 24h.The protein expression of JAK2, p-STAT3, Bcl-2 and Bax were evaluated by Western blotting.4. Experimental group:TT medullary thyroid gland carcinoma cells was cultivated in the concentration of 50 umol/L with AG490.Control group: TT medullary thyroid gland carcinoma cells was cultivated without AG490. The adherent cells was irradiated with increased dose-(0,2,4,6,8,10 Gy), and counted the cells by microscope after 24h, then calculated the planting efficiency(PE) and survival fraction(SF),.Using GraphPad Prism 5 software to draw the cell survival curve. To calculate SF, mean lethal dose(D0), dose quasithreshold (Dq), extrapolation number(N), sensitization enhancement ratio at D0(SER DO), and sensitization enhancement ratio at Dq(SER Dq) when the radiobiological parameters ray was 2Gy.5. To Irradiate TT medullary thyroid gland carcinoma cells that cultivated in the concentrations of 0,25,50,100umol/L with AG490 with 4 Gyto measure the protein expression of JAK2 and p-Stat3 of each group.ResultsCompared with the concentration of 0 umol/L of AG490, the proliferation of thyroid TT cells were decreased in the concentrations of 25, 50,100 umol/L of AG490, the inhibition on thyroid TT cell was gradually increased along with the increased of the concentration of AG490.The apoptosis rate of TT thyroid cell were (4.86±0.79)%, (15.24±1.00)%, (33.11±2.40)% and (64.47±2.29)% respectively in the concentrations of 0, 25,50,100 umol/L of AG490, and the apoptosis rate was gradually increased with the increased of the concentration of AG490. Compared with 0 umol/L of AG490, the protein expression of JAK2,P-Stat3 and Bcl-2 were gradually reduced with the increased of the concentration, protein expression of Bax was increased with the increased of concentration. The Poisson correlation analysis showed that JAK2 and p-Stat3 were positively correlated(r=0.67); JAK2 and Bcl-2 were positively correlated(r=0.79); p-Stat3 and Bcl-2 were positively correlated(r=0.70); JAK2, p-Stat3, Bcl-2 and Bax were negatively correlated(r=-0.44, r=-0.42, r=-0.40).SF of experimental group and control group were reduced along with the increased of the irradiation dose, and the medicated group was significantly lower. Comparison between 6 kinds of radiobiological parameters:with 4 Gy dose of irradiation, JAK2 in the concentrations of 0,25,50,100 umol/L of AG490 were 0.94±0.09,0.78±0.02,0.31±0.01, 0.15±0.03 respectively, p-Stat3 were 0.94±0.83,0.85±0.0.13,0.43±0.03,0.23±0.02 respectively. Compared with irradiation group, the protein expression of JAK2 and p-Stat3 were reduced, and when the concentration of AG490 was 50 umol/L or more, the differences was more significant(P<0.05).ConclusionThe inhibition on thyroid TT cell is gradually increased along with the increased of the concentration of AG490, and the apoptosis rate is gradually increased, which further clarifies the proliferation of thyroid TT cells is significantly decreased. SF of two groups are reduced along with the increased of the irradiation dose, and the medicated group is lower, and radiobiological parameters also change accordingly. AG490 could increase the radiosensitivity of human medullary thyroid carcinoma cells. The protein expression of JAK2, p p-Stat3 and Bcl-2 are gradually reduced with the increased of the concentration, protein expression of Bax is increased with the increased of concentration, which suggests that AG490 can regulate expression of apoptosis protein(Bcl-2 and Bax). the sensitization effect of AG490 and mechanism of apoptosis protein regulation is further confirmed. |
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