| Objective: To investigate the growth inhibition effect on human adenocarcinoma A549 cell line and Rad51 changes in different sequential and synchronous applications of gefitinib and cisplatin. The curative effect and mechanisms of EGFR-TKI combined with chemotherapy in vitro were analyzed so as to obtain experimental data for clinical purpose in the future.Methods(:1)A549 cells were treated with gefitinib (final concentration 5μmol/L) and cisplatin at different concentrations titres (0.25,0.5,1.0,2.0,4.0, 8.0,16.0μg/ml) in different combination methods. In the first part of study, A549 cells were divided into three groups. Group A(P-G): Cells were treated initially for 24h to cisplatin at different concentrations, followed by a washout and treated with gefitinib for 48h; GroupB(PG): Cells were treated initial for 24h with cisplatin at different concentration titres and gefitinib synchronously, followed by a washout and additional treatment with gefitinib for 24h; Group C(G-P): Cells were initially exposed to gefitinib for 48h, followed by a washout and additional treatment with cisplatin at different concentrations for another 24h. The inhibitory rate and IC50 were calculated with the MTT assay and growth curve was also drawn(.2)In part two of the study, cells were treated with gefitinib (final concentration 5μmol/L) and cisplatin(final concentration 1μg/ml)in the same way as in part one. In addition to group A, B and C, a fourth blank control group (Group D) was added in which no gefitinib and cisplatin were added. Cell cycle distribution and apoptosis status were then analyzed by flow cytometry. (3)In the third part of the study, A549 cells were divided into four groups and treated as in part two of the study. The Rsd51 level was analyzed by western blot analysis.Results:(1)Compared with IC50 of group A(1.21μg/ml), the IC50 of group B (3.62μg/ml) and group C (4.43μg/ml) was raised with statistical significant difference(P <0.05). Right shifted growth curve were observed in group B and C compared to group A.(2)The percentage of A549 cells in phase G0-G1 and phase S in the group A, B, C and D were (5.95±0.14,51.51±1.34)%, (7.52±0.17,86.51±1.91)%, (30.89±1.01,22.19±0.71)% and(34.55±0.84,52.01±2.44)% respectively. The percentage of cells in phase G0-G1 in the group A and B was significantly increased compared to group C(P <0.05). The percentage of cells in phase S in group C was significantly increased compared to group A and B(P <0.05). The apoptotic ratio in group A, B, C and D of A549 cells wer(e34.74±0.94)%,(6.09±0.22)%,(2.14±0.09)% and (1.28±0.17)% respectively. The apoptotic ratio in group A and B was significantly higher than group C(P <0.05). Apoptotic ratio in group A was higher in group B(P <0.05), but no difference were found between group C and group D(P>0.05).(3)The DNA repair protein Rad51 expression level(indicated as gray-scale value)of group A,B,C and D were 20.01±2. 73,40.63±4.26,42.83±3.63 and 13.11±2.65 respectively. The Rad51 expression in all gefitinib and cisplatin treated study groups were increased to different extent compared to the blank control group D(P <0.05). Rad51 expression was more prominent in group B and C than in group A(P <0.05)but no difference was observed between group B and group C.Conclusion(1)P-G sequential mode creates a stronger growth inhibition effect than PG synchronous mode and G-P sequential mode on A549 cells in vitro.(2)The apoptotic ratio of A549 cells treated in G-P sequential mode did not increased significantly whereas treatment in P-G sequential mode and PG synchronous mode could effectively enhance the apoptotic ratio of A549 cell. Among them, treatment in P-G sequential mode showed greater apoptosis-inducing effect.(3)Compared with other synchronous and sequential mode, P-G sequential treatment method could effectively reduced the DNA repair protein Rad51 expression in A549 cell induced by cisplatin, enhancing apoptotic ratio, growth inhibition and cytotoxicity of chemotherapy.
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