Effect Of Pulsatilla Saponins A On Radiosensitivity Of Lung Cancer Cells And Its Mechanism

Purpose:In order to identify the biological effects of herbal extracts Pulsatilla saponin A on the proliferation and radiosensitivity of human non-small cell lung cancer cell lines, and to preliminarily explore the potential mechanisms.Method:The normal human bronchial epithelial cell lines Beas-2B cells, and human non-small cell lung cancer cell lines A549, H1975 were utilized as cell model.(1) MTT viability assay was performed to detect the impact of Pulsatilla saponin A on cell proliferation, and to identify the appropriate drug concentrations which were used in the following experiments;(2) Colonogenic assay was used to determine the effect of Pulsatilla saponin A on the radiosensitivity of non-small cell lung cancer cell lines;(3) Flow cytometry was applied to analyze cell cycle progression of Pulsatilla saponin A on non-small cell lung cancer cells after irradiation;(4) Annexin V-FITC cell apoptosis assay was used to determine apoptosis of Pulsatilla saponin A on non-small cell lung cancer cells after irradiation;(5) Phosphorylation ?-H2 AX immunofluorescence staining and comet assay were performed to identify the effect of Pulsatilla saponin A on non-small cell lung cancer cells DNA damage after irradiation;(6) Western blot assay was utilized to determine the changes of protein expression for DNA damage repair、cycle、apoptosis of Pulsatilla saponin A on non-small cell lung cancer cells after irradiation;(7) Statistical analysis.Result:(1) Pulsatilla saponin A obviously inhibited the viability of non-small cell lung cancer cell lines A549 and H1975 cells, and the IC50(50% inhibitory concentration) were 13.12±0.79μg/ml and 10.56±0.68μg/ml respectively, while the IC50 of Pulsatilla saponin A on normal human bronchial epithelial cell line Beas-2B was much lower(19.84±1.12μg/ml). The drug concentration(about 2μg/ml) of cell survival 80% on NSCLC cells was used in the following experiments;(2) Compared to NSCLC cells exposed to ionizing irradiation(IR), Pulsatilla saponin A combined with IR obviously enhanced the radiosensitivity of non-small cell lung cancer cell lines A549 and H1975, and the SERs were 1.17(P<0.05) and 1.28(P<0.05) respectively;(3) Pulsatilla saponin A significantly increased the IR induced G2/M cell cycle arrest of A549 and H1975 cells: compared with IR treatment alone, Pulsatilla saponin A combined with IR raised the G2/M cell ratio from 33.16±1.45 to 46.05±1.76 in A549 cell(P<0.05),and from 7.62±1.32 to 63.68±1.31 in H1975 cell(P<0.05);(4) Pulsatilla saponin A enhanced the early apoptosis of A549 cells after irradiated. After the combination of Pulsatilla saponin A and IR, the early apoptotic portion was increased from 26.81±1.70 to 58.36±2.18 in A5409 cells, compared with IR treatment alone(P<0.05). In H1975 cells, late apoptotic portion induced by IR alone was 24.31±1.94, and significantly increased to 46.69±2.42 when treated with Pulsatilla saponin A combined with IR(P<0.05);(5) Pulsatilla saponin A enhanced IR induced DNA damage of non-small cell lung cancer cell lines A549 and H1975. IR induced phosphorylated γH2AX foci formation was(6.38±2.47)/cell in A549 cell,(12.08±5.28)/cell for Pulsatilla saponin A treatment,(21.84±4.87)/cell in A549 after single dose IR, and(30.17±7.85)/cell in A549 after treatment with IR and Pulsatilla saponin A together(P<0.05). In H1975 cell,(10.08±3.87)/cell γH2AX foci were determined in control cells, while(24.18±3.52) foci/cell were identified after Pulsatilla saponin A treatment; the number of foci was(16.06±2.77)/cell in H1975 exposed to IR alone, and increased to(34.66±10.06)/cell in H1975 treated with Pulsatilla saponin A and IR(P<0.05). In single cell gel electrophoresis experiment, relative length of comet tail(Tail DNA%) in parental A549 cells was(2.24±0.09)%/cell, while Tail DNA% was increased to(4.38±0.89)%/cell、(5.33±0.41)%/cell and(6.04±1.48)%/cellin A549 treated with Pulsatilla saponin A, IR, and Pulsatilla saponin A combind with IR, respectively(P<0.05). In H1975 cells, Tail DNA% was(1.12±0.41)%/cell 、(4.91±1.35)%/cell、(2.87±0.91)%/cell and(6.92±1.35)%/cell when treated with mock therapy, Pulsatilla saponin A, IR alone, and Pulsatilla saponin A combind IR, respectively(P<0.05);(6) Western blot analysis suggested that, compared with IR group, the expression of DNA damage repair associated protein Mre11、DNA-PKcs 、Ku80, cell cycle related protein Cyclin E 、Cyclin B1 and anti-apoptotic protein Bcl-2 were inhibited in A549 and H1975 cells after exposed to Pulsatilla saponin A plus IR.Conclusion:(1) Pulsatilla saponin A not only inhibited the cell viability in vitro, but also enhanced IR induced DNA damage in human non-small cell lung cancer cell line A549 and H1975.(2) The potential mechanism of Pulsatilla saponin A affected the sensitivity of non-small cell lung cancer cells might be related to its regulation of cell cycle G2/M phase arrest, apoptosis induction and DNA damage repair inhibition after IR.