Study On Mechanism Of Effective And Synergistic Anti-NSCLC Interaction Of Monomers Under Zhou’s KJY Prescription

Zhou’s prescription, referring to the anti-lung cancer effective prescription summarized by the TCM master Zhou Zhong-ying according to the clinical experience, is comprised of drugs with four effects such as resolving hard lump, eliminating dampness and phlegm as well as nourishing yin and benefiting qi. According to the preliminary study of the research group, over20-target monomeric compounds with effective cytotoxicity components are gained through high throughput screening. However, it still remains necessary to carry out in-depth research on molecular mechanism inhibiting non-small cell lung cancer (NSCLC) of monomeric compound.OBJECTIVE:To prepare effective monomeric compounds-related drugs with four effects, including Bibenzyl, Ophiopogonin B,(-)-epigallocatechin gallate, Isoliquiritigenin, Quercetin as well as20-(S)Ginsenoside Rg3(hereinafter referred to as Bb,OpB, EGCG, ISL, QC and Rg3); to conduct study on in vitro anti-tumor effect of lung cancer A549and H460cells; to carry out research on synergistic antitumor mechanism of monomeric combination and compatibility, to determine the effective matching of monomer combination prescription.METHOD:Determine the contributions of growth and acumulated value of monomeric inhibitory lung cancer A549and H460cells through measurement of half maximal inhibitory concentration IC50; determine the reservation component of monomer combination prescription through L16(45) orthogonal experimental design; analyze the impacts on cell proliferation cycle through orthogonal experiment L9(34) and by selecting the effective matching dose of the monomer combination prescription for lung cancer A549and H460cells from the inhibiting role of drug in cell growth and its impacts on the formation of SubG1; study the synergistic role of monomer combination prescription and single agent in apoptosis of A549and H460cells through Flow Cytometry (FCM) analysis based on cell membrane redistribution and high content screening (HCS); observe the corresponding morphological changes in cell apoptosis of submicroscopic structure through Transmission Electron Microscope (TEM); discuss the molecular mechanism of monomer combination prescription in regulating A549and H460cell apoptosis and death receptor-relevant protein expression through Western Blot testing.RESULT:According to the result of IC50, it indicates that monomer Bb has no apparent role in inhibiting the growth of A549and H460cells, thus monomer Bb is abandoned; monomers OpB, EGCG and QC have good dose-dependent relationship while monomers ISL and Rg3manifest negative inhibiting reaction. Through inhibition experiment of L16(45) orthogonal experiment in combination with EGCG-OpB-Rg3-QC-ISL on cell growth, the reservation component and dose of monomer combination prescription is determined. The concentration screening value of monomer EGCG is50μM. the one of OpB is10μM, that of Rg3is25μM while QC is50μM respectively. Thus, the monomer I with the negative inhibiting role is abandoned.According to L9(3) orthogonal experimental design, the effective matching ratio of OpB/EGCG/QC/Rg3in the preliminary monomer combination prescription is OpB:EGCG:QC:Rg3=7.23:22.92:3.02:3.93(μg/ml). The result of IC50shows that IC50value of A549cell in24h and48h is17.70and5.69μg/ml respectively and that of H460cell in24h and48h is11.19and2.68μg/ml respectively. This matching prescription OpB/EGCG/QC/Rg3has good dose-and time-dependent relationship on growth inhibition of the two cells.Through the influences of drug on SubGl of cell proliferation cycle, the combination of EGCG/OpB/QC/Rg3is determined. As for A549cell, the effective matching of the Prescription EGCG/OpB/QC/Rg3-A549is EGCG/OpB/QC/Rg3=50:10:10/5(μM) and37.10μg/ml. as for H460cell, that of the Prescription EGCG/OpB/QC/Rg3-H460is EGCG/OpB/QC/Rg3=50:2:10/5(μM) and31.31μg/ml. The orthogonal experiment shows that monomer EGCG is a major factor inducing the apoptosis of lung caner A549andH460cells at late stage through the analysis of the formation of SubG1. Monomer OpB is the synergistic factor for A549cell, but plays an antagonistic role in H460cell. Monomers QC and Rg3assist EGCG factor in playing a role in arrest other links of the cell cycle.According to the analysis of the impacts on DNA ploidy of cell cycle, monomer ISL mainly induces the reduction in SubGl for A549and H460cells; monomer QC can induce cell arrest at phase S; monomer Rg3can induce A549cell arrest at G2phase and H460cell at G1phase.According to the cell membrane phospholipids redistribution analysis on induction of cell apoptosis, single agent EGCG can induce the early-stage apoptosis of cells for the response increase in Annexin V (+) fluorescence value of the two cells. The response of monomer combination Prescription EGCG/OpB/QC/Rg3in early-stage apoptosis of A549cell is remarkably larger than that of single agent and doublet ones. Additionally, the doublet EGCG/OpB is larger than the single agent EGCG. it is thus indicated that it has synergistic effect. However, the response in H460cell is not remarkable, indicating it has antagonistic role. Through the response analysis of Annexin V (+)/PI (+) fluorescence intensity, the Prescription EGCG/OpB/QC/Rg3can induce the late-stage apoptosis and necrocytosis of the two cells and its reaction on A549cell is larger than that on H460cell. Through HCS multi-channel fluorescence scanning and testing, it shows that A549cell Hoechst33528channel under the monomer combination Prescription EGCG/OpB/QC/Rg3, the cell apoptosis manifests positive correlation between dose and time. The change in H460cell is not remarkable. According to the result of A549cell Annexin V channel under the Prescription EGCG/OpB/QC/Rg3, it shows that the early-stage cell apoptosis manifests dose-and time-dependence and the role of apoptosis induction is larger than the positive drug4.0μg/ml cisplatin. As for H460cell, it has obvious role if given the medium and high dose. According to the result of PI channel of A549and H460cells under the Prescription EGCG/OpB/QC/Rg3, it shows that the growing rate in late-and early-stage apoptosis is synchronic.Transwell experiment shows that the inhibition of monomer Prescription EGCG/OpB/QC/Rg3in A549cell invasion capacity manifests positive correlation dose dependency. The inhibition of single agent in A549cell invasion capacity has synergistic role in compatibility of Prescription EGCG/OpB/QC/Rg3. Within8h of drug action, each prescription does not good response in transwell invasion capacity of H460cell. It shows that the invasion capacity of A549cell is larger than that of H460cell.Through TEM, the influences of the monomer and their combination on submicroscopic structure of cell can be observed. According to the result of A549cell, it indicates that the single agent EGCG can induce the reduction in volume of A549cell, karyopyknosis and apoptosis morphology of chromatin condensation. The single agent OpB mainly induces vacuolar degeneration or autophagy of A549cell organelle. There are no obvious changes in single agent QC. The single agent Rg3mainly induces swelling degeneration of organelle and cell nucleus, damages of karyotheca and cell degeneration and necrosis. Under the monomer combination Prescription EGCG/OpB/QC/Rg3, the early-and late-stage apoptosis morphology of A549cell can be observed. Meanwhile, swelling of organelle&cell nucleus, damages on cell membrane and disintegration of cell can also be observed. It is thus indicated that the interaction characteristic of the drug can be synergistically manifested in the mirror of one cell.For H460cell, the single agent OpB can induce vacuolar degeneration of organelle or autophagy. Single agent QC can induce fatty or atheromatous degeneration of endoplasmic reticulum or organelle of the cell. There are no obvious changes in single agents EGCG and Rg3. Monomer combination Prescription EGCG/OpB/QC/Rg3can induce the formation of early-stage apoptosis morphology of H460cell. It can be found that the cell apoptosis is accompanied with vacuolar degeneration or autophagy, swelling of organelle and cell nucleus, thus manifesting the common characteristic of drug matching function.According to Western Blot testing result, the monomer Prescription EGCG/OpB/QC/Rg3 and single agent E can upregulate pro-apoptotic protein bax and caspase series and downregulate anti-apoptosis protein bcl-2and induce the apoptosis of A549and H460cells. Cascade reaction of caspase8and caspase is activated through signal response of exogenous pathway of cell apoptosis. Besides, the rise in caspase-9and bax/bcl-2through endogenous pathway is caused and the cell apoptosis is induced. Single agent OpB plays a synergistic role in assisting single agent EGCG in inducing the regulation of A549cell apoptosis protein. It has obvious negative regulatory role in apoptosis protein of H460cell. Therefore, single agent compatibility prescription shall adopt different-dose matching scheme for NSCLC of different clone sources.For monomer combination Prescription EGCG/OpB/QC/Rg3and single agent EGCG, the protein expression in death receptors of A549and H460cells including DcR3, DR5, Fas, TNFR1and TNFR2. Then the apoptosis of cell can be induced through activating caspase8and cascade reaction of caspase. Under the role of doublet system EGCG/OpB, the expression of decoy death receptor DcR3of A549cell can be reduced and that of DR5, Fas, TNFR1and TNFR2proteins can be increased. Nevertheless, EGCG/OpB can increase the expression of decoy death receptor DcR3protein of H460cell. Therefore, the combination of other functional receptors and ligands can be inhibited competitively.Conclusion:The monomer combination Prescription EGCG/OpB/QC/Rg3of Zhou’s KJY prescription-relevant monomeric compounds EGCG, OpB, QC and Rg3compatibility could effectively inhibit growth and proliferation of in vitro tumor of lung cancer A549and H460cells in vitro. It also could form arrest of lung cancer A549and H460cells proliferation cycle and induce cell apoptosis. Monomer OpB plays a synergistic role in assisting monomer EGCG in inhibiting the growth of A549cell and promoting cell apoptosis and also has antagonistic role in H460cell. The monomer combination Prescription EGCG/OpB/QC/Rg3plays a positive regulatory role in expression of apoptosis-related proteins of exogenous and endogenous pathways of A549and H460cells. Monomer OpB can upregulate the expression of decoy receptor DcR3protein of H460cell. Thus the combination of functional receptor and ligand is inhibited competitively, which may be one of reasons for having antagonistic role in cell apoptosis induced by monomer EGCG. Therefore, different-dose monomeric matching schemes should be adopted for cell systems of NSCLC with different clone sources, so that more effective regulatory and inhibiting roles can be played as it is in line with the principle of individualized TCM therapy.