Inhibitory Effect Of LSD1 Inhibitor Combined With Paclitaxel On Drug-Resistant Gastric Cancer Cells And Its Associated Mechanism

Part I: The inhibitory effect of GSK-LSD1 combined with PTX on MGC803/PTX cellsObjective: Use the combined index(CI)to judge the effect of drug combination on MGC803/PTX cells,and to compare the ability of combinated drug and single drug to inhibit cell survival.Methods: MTT assay was used to determine the inhibitory rate of the single drug group.Select concentrations of the inhibition rate between 5% and 30% of to screen the combined concentration and determine the combinated time.After determining the PTX concentration,determine the concentration of GSK-LSD1 and calculate the CI value.Results: Determine successfully the optimal time for the combination of GSK-LSD1 and PTX was 72 h and the optimal concentration was 100 μM and 10 n M,respectively.The CI value was 0.334 and the inhibition rate was 68.09%.Part II: Study on the anti-tumor effect and mechanism of the combined groupObjective: To study the anti-tumor effects of the combined group on MGC803/PTX cells and related mechanisms.1.Drug combination induced apoptosis of MGC803/PTX cellsMethods: The morphological changes of cells and nuclei were observed under fluorescence microscope.The colony formation ability was detected by plate cloning.Annexin V/PI staining was used to detect the apoptosis rate;Total-PI3 K,P-PI3 K,Total-AKT,P-AKT(Ser473),Bax,BCl-2,Total-caspase3,Cleaved-caspase3 were detected by Western blot.,Total-PARP,Cleaved-PARP and other protein expression changes.Results: The combination group had the ability to break up cell morphology and nucleus significantly.Compared with the single-use group,the combination group had lower colony formation rate and higher apoptosis rate.The combination group down-regulated the expression of P-PI3 K,P-AKT(Ser473)and BCl-2,and up-regulated the expression of Bax,Cleaved-caspase3 and Cleaved-PARP.2.Drug combination inhibited the metastasis of MGC803/PTX cellsMethods: Cell scratch test and Tanswell test were used to test the cell transfer ability.Western blot was used to detect the expression of metastasis-associated proteins ZO-1 and Vimentin.Results: The width of the scratches formed in the combination group was wider.The number of cells in the subcompartmental membrane of the Tanswell experiment group was less than that of the single-use group;the combination group up-regulated the expression of ZO-1 and down-regulated the expression of Vimentin.3.Drug combination promoted the autophagy of MGC803/PTX cellsMethods: DCFH-DA staining was used to detect intracellular reactive oxygen species.Immunofluorescence experiments and transmission electron microscopy were used to observe the production of autophagosomes.Western blot was used to detect the expression of LC3 B,P62 and other proteins.In the combination group,autophagy inhibitor 3-MA was used to detect the reversal of the expression of LC3 B and P62 protein.Results: DCFH-DA staining detected a significant increase in intracellular reactive oxygen species in the combination group;immunofluorescence experiments and transmission electron microscopy revealed that the combination group produced more autophagosomes.The combination group up-regulated the ratio of LCII/LCI and down-regulated the expression of P62;the combination group added 3-MA and then down-regulated the ratio of LCII/LCI and up-regulated the expression of P62.In conclusion:1.LSD1 and tubulin are two targets of antitumor drugs.The combination of two target drugs in drug-resistant cells is a new drug combination method.This study determined that the optimal time for the combination of GSK-LSD1 and PTX was 72 h,the optimal concentrations were 100 μM and 10 n M,the inhibition rate was 68.09%,and the CI value was 0.334.According to Soriano et al.,0.2 ≤ CI ≤ 0.4 was used.Strong synergy;2.The combination of MGC803/PTX cells and cell nucleus morphology,colony formation rate,apoptotic rate,etc.,have significant changes;the combined group down-regulated the expression of P-PI3 K,P-AKT(Ser473),BCl-2;The expression of Bax,Cleaved-caspase3 and Cleaved-PARP were up-regulated,indicating that the combination group induced apoptosis through PI3K/AKT-mediated apoptosis pathway;3.The combination group significantly inhibited the MGC803/PTX metastasis compared with the single use group,while up-regulated the expression of ZO-1 and down-regulated the expression of Vimentin,indicating that the combination group can inhibit cell proliferation by inhibiting metastasis;4.Compared with the single-use group,the combination group significantly increased intracellular reactive oxygen species and autophagosome content,and up-regulated the ratio of LCII/LCI and down-regulated the expression of P62,indicating that the combined group promoted autophagy.