Reversal Of A549 Resistance By Hedyotis Diffusa Active Compound Combined With Paclitaxel

Objective:To study the effect of combination of active fraction of Hedyotis diffusa and paclitaxel on reversing multidrug resistance of cancer cells and explore its mechanism.Method:(1)Screening of active fractions of Hedyotis diffusa:95%ethanol was heated and refluxed to extract Hedyotis diffusa.Different effective fractions were extracted by petroleum ether,dichloromethane and ethyl acetate:HDW-A of petroleum ether,HDW’B of dichloromethane Ⅰ,HDW-C of ethyl acetate Ⅰ,HDW-D of dichloromethane Ⅱ,HDW-E of ethyl acetate Ⅱ,HDW-F of ethyl acetate Ⅱ,and HDW-F of ethyl acetate Ⅱ.MTT assay was used to determine the inhibitory effect of different extracts on the growth of human lung cancer cell A549 and human lung cancer cell A549/T resistant to taxol,and to screen the best active site;(2)LC-MS qualitative analysis of the active parts of Hedyotis diffusa:qualitative analysis of the chemical constituents in HDW-E and HDW-F by liquid chromatography-mass spectrometry;(3)Investigation of the combination ratio of HDW-E and HDW-F with paclitaxel PTX respectively,and MTT method was used to determine the active parts of Hedyotis diffusa diffusa.Combination index(Cl)was used as an index to optimize the optimal combination ratio;(4)Study on the effect and mechanism of combination of two drugs in reversing multidrug resistance:detection of cell apoptosis,cell cycle,cell proliferation and expression of AKT-mTOR-p70 signaling pathway protein,observation of taxol accumulation in A549/T cells by confocal laser microscopy and intracellular spindles after administration Morphological changes;(5)Evaluation of anti-tumor activity in vivo:A nude mice model of A549/T cells was established.Negative control group(saline),paclitaxel group(10mg/kg),HDW-F group(0.1 g/kg)and combination group(10mg/kg+0.1g/kg)were set up to investigate the effect of combination of two drugs on the inhibition rate.Result:(1)Screening of active fraction of Hedyotis diffusa:HDW－E was the best active fraction of Hedyotis diffusa selected by MTT test.HDW-E was purified and anthraquinone component HDW-F was obtained.The purified HDW-F could inhibit the growth of A549 cells and A549/T cells;(2)LC-MS qualitative analysis of the active part of Hedyotis diffusa:LC-MS analysis showed that the main chemical constituents in HDW-E were iridoid terpenoids,anthraquinones and flavonoids,while anthraquinones were the main compounds in HDW-F;(3)Investigation of the combination ratio of the two drugs:HDW-E concentration was 17.5 μg/mL,HDW-F concentration was 21.5 μg/mL and 1.58 μM PTX were administered in combination.At this time,the optimal combination ratio was reached,and the combined index Cl values were 0.787 and 0.756,respectively;(4)Study on the effect and mechanism of reversing multidrug resistance by combination of HDW-E and HDW-F:Compared with paclitaxel,HDW-E and HDW-F could significantly inhibit cell proliferation,induce apoptosis and arrest cell cycle in G2/M phase,respectively.Down-regulation of phosphorylated AKT-mTOR-p70 signaling pathway protein;laser confocal microscopy showed that HDW-E and HDW-F partially reversed the low accumulation of paclitaxel in drug-resistant cells.Compared with the control group and the two drug-treated groups,they could induce nuclear polynucleus;(5)In vivo animal experiments,paclitaxel combined with HDW-F can significantly inhibit the growth of tumors.Conclusion:The results showed that HDW-E and HDW-F had synergistic effects with paclitaxel respectively.They could inhibit cell proliferation,arrest cell cycle,induce cell apoptosis,down-regulate the expression of AKT-mTOR-p70 signaling pathway protein and inhibit the growth of tumors.Study on the mechanism of drug resistance showed that HDW-E and HDW-F could reverse drug resistance by increasing the accumulation of paclitaxel in A549/T cells.However,the target of reversing paclitaxel resistance by HDW-E and HDW-F remains to be further studied.LC-MS analysis showed that anthraquinones were the main active components of HDW-F purified from HDW－E,and HDW-F also had a good reversal effect on paclitaxel resistance,these results suggest that anthraquinones from Hedyotis diffusa can reverse the resistance to taxol.HDW-E is superior to HDW-F in inhibiting cell growth.It is speculated that the iridoid terpenoids and flavonoids contained in HDW-E also have certain anti-tumor activity.The specific mechanism needs further study.This study provides a basis for the study of the anti-tumor effect of Hedyotis diffusa and its reversal of multidrug resistance,and provides a reference for clinical drug use.