| Objective Select the effective monomer of Jinfukang Oral Liquid and commonly used traditional Chinese medicine for lung cancer treatment.”Declined Healthy Qi and Hidden Posion” is the core etiology and pathogenesis of lung cancer,and circulating tumor cells are an important part of ”Hidden Posion”.Reveal the molecular mechanism of apoptosis in human lung cancer circulating tumor cells(CTC-TJH-01)induced by the important component of Jinfukang component Polyphyllin?.Explored initially the effect of Polyphyllin? combined with paclitaxel on the proliferation of CTC-TJH-01.Methods 1.Using CCK-8 cell proliferation assay to screen the monomer compounds contained in clinical anti-tumor traditional Chinese medicines: Polyphyllin?,Ursolic acid,Bufalin,Curcumin,and Astragaloside ?.Detect the effect of five monomers on the proliferation of CTC-TJH-01,H1975 and 16 HBE cell lines,and evaluate the toxicity of five monomers on tumor cell lines,and select out the best monomer compounds.2.Flow cytometry and ?-Galactosidase Staining were used to detect the effect of the best herbal monomer compound saponin VII on cell cycle,senescence and apoptosis of CTC-TJH-01 and H1975 cell lines.Explore the action pathway of Polyphyllin? to interfere with CTC-TJH-01 and H1975 cell.3.Flow cytometry was used to detect variance of the mitochondrial membrane potential and intracellular caspase 3/8/9 levels of CTC-TJH-01 and H1975 cell.Effects of Polyphyllin? on nuclear DNA damage was observed by laser confocal microscopy.Western Blot was used to examine the effects on the changes of death-related proteins in CTC-TJH-01 and H1975 cell lines induced by Polyphyllin?.Explore the underlying molecular mechanisms in this process.4.The CCK-8 cell proliferation assay was used to detect the cytotoxicity of different concentrations of Polyphyllin? combined with paclitaxel on CTC-TJH-01.Evaluated the synergistic effect of Polyphyllin? combined with paclitaxel on CTC-TJH-01.Results 1.Polyphyllin ?,Ursolic acid,Bufalin and Curcumin inhibit the proliferation of CTC-TJH-01 and H1975 cells(P<0.05).With prolonged action time and drug concentration,the effect is gradually enhanced.Astragaloside IV had no significant effect on the proliferation of CTC-TJH-01 and H1975 cell lines.2.Polyphyllin? can arrest CTC-TJH-01 and H1975 cells in G1 phase(P<0.05)and induce apoptosis(P<0.001),Polyphyllin? can induce CTC-TJH-01 and The mitochondrial membrane potential of H1975 cells was decreased(P<0.05).Polyphyllin? had no significant effect on CTC-TJH-01 and H1975 cell senescence.Polyphyllin? can induce nuclear shrinkage of CTC-TJH-01 cells and DNA damage in the nucleus,whereas this phenomenon was not observed in H1975.3.Polyphyllin? significantly up-regulated the expression of caspase3 / 8 / 9 in CTC-TJH-01 and H1975 cells(P<0.05).Polyphyllin? significantly up-regulated the expression of PARP1,?-H2 AX,TNF-R1 and DR5 proteins in CTC-TJH-01 cells(P<0.05),and down-regulated the expression of XIAP and survivin(P<0.05).Polyphyllin? significantly up-regulated the expression of TNF-R1,DR5 and Bax in H1975 cells(P<0.05),and significantly down-regulated the expression of Bcl-2,XIAP and survivin(P<0.05).4.When Polyphyllin? and paclitaxel are combined in different ratios,the toxic effects on the proliferation on CTC-TJH-01 cells are different.When the molar concentration ratio is:(Resaponin VII: paclitaxel)=(2:1),they have synergistic effect on the inhibition of CTC-TJH-01 proliferation.Conclusions 1.Polyphyllin?,compound of JINFUKANG,inhabited proliferation of CTC-TJH-01 and H1975,blocked the cell cycle of them in G1 phase and induced apoptosis.2.Polyphyllin? induces apoptosis in CTC-TJH-01 cells mainly through DNA damage pathway mediated by the death receptor,whereas H1975 cells apoptosis are induced by mitochondrial pathway mediated by death receptor.3.When combined with the molar concentration ratio of Polyphyllin?: paclitaxel =2:1,the inhibition on CTC-TJH-01 cells proliferation was synergistic. |
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