(A)Cell proliferation was determined by assessing the mitochondrial reduction of MTT. Bars indicated means ± standard deviation of three independent experiments performed in triplicate (n =

9). Compared with untreated control cells, P > 0.05 were found in all of the treated groups. (B)Known numbers of single cells were plated into culture dishes in RPMI1640 BAY 63-2521 clinical trial containing 10% FBS and treated with gefitinib in several doses. Cells were then harvested by trypsinization and counted by a hemocytometer with trypan blue dye. Data points mean of triplicate samples. Data were expressed as means ± SE for three experiments. P > 0.05 vs. control group by Student’s t-test was found in every treated Adavosertib ic50 group. Expression of PTEN in H-157 cells after irradiation treatment After different dosage radiation (0, 1, 2, 4, 6, 8, and 10 Gy), the PTEN expression increased in a time-dependent manner. The highest expression were observed in H-157 cells treated with 4~6 Gy irradiation. At the same time, we also measured that PTEN expression increased at 3 h and returned to baseline at 12 h after irradiation (Figure 3). Based on this, we concluded that 6 Gy was the best dosage for improving PTEN expression and the same time as treatment with irradiation was the optimal time for addition

of gefitinib. Figure 3 Expression of PTEN in H-157 cells after irradiation treatment. Vactosertib (A) The H-157 cells which exposed to 1, 2, 4, 6, 8, and 10 Gy of X-rays were analyzed as shown in right panel. After irradiation, the cells were incubated for 6 h, and then were examined. (B) After incubation of X-irradiated (6 Gy) cells for 3, 6, 9 and 12 h, the PTEN protein was examined by Western blotting. Irradiation Treatment was shown to increase PTEN levels in H-157 cell lines tested, and H-157 cells learn more exposing to 4~6 Gy expressed major amounts of PTEN. Survival curve and cell growth curve of gefitinib-treated H-157 cells after irradiation

The cloning efficiency of H-157 was between 60% and 90%. The survival curve of control and gefitinib-treated H-157 cells after irradiation was shown in Figure 4. The radiobiological parameters of H-157 cells treated with irradiation and gefitinib were D0 = 1.14, Dq = 0.22, N = 1.57, while those of irradiation-treated H-157 cells were D0 = 1.51, Dq = 0.88, N = 3.84. In the present study, SER (sensitive enhancement ratio) = D0 (irradiation+gefitinib group)/D0 (irradiation group) = 1.51/1.14 = 1.32. The SER in gefitinib-treated cells indicated that treatment with gefitinib significantly improved the biological effect of irradiation following PTEN high expressed. At the same time, the cell growth curve was also down-regulated by gefitinib after irradiation (Figure 4). The data presented herein suggested the resistance for gefitinib was reversed by irradiation in H-157cells. Figure 4 Irradiation reversed the resistance of H-157 cells to gefitinib.