Z-guggulsterone Modulates PD-L1 For Non-small Cell Lung Cancer In Combination With Antitumor Therap

Objective:To study the effective of Z-guggulsterone on the expression of PD-L1 in non-small cell lung cancer cells and the specific regulatory mechanism.Methods:In vitro,non-small cell lung cancer cell lines(A549 and H1975)and Lewis lung cancer cells in mice were treated with different concentrations and time gradients of Z-guggulsterone,and the effects of Z-guggulsterone on cell viability,apoptosis and cell cycle were detected,and the optimal drug concentration and treatment time were selected.The mRNA expression level of PD-L1 in A549,H1975 and Lewis cells was detected by real-time quantitative PCR assay.The expression level of PD-L1 on A549,H1975 and Lewis cells was determined by flow cytometry.PD-L1 promoter plasmid was constructed,and the effect of Z-guggulsterone on PD-L1 promoter in A549 and H1975 cells was detected by double luciferase assay.A549,H1975 and LLC cells were transfected with siRNA to inhibit the expression of FXR and A549 cells were transfected with lentiviral contracts to establish FXR-overexpressed stable cell lines.The expression level of PD-L1 mRNA and the surface of PD-L1 were tested by real-time quantitative PCR assay and flow cytometry,respectively.The expression level of FXR and its downstream molecules SHP were detected by western blot.Akt signaling pathway inhibitor MK2206 and Erk1/2 signaling pathway inhibitor PD325901 were added exogenically to inhibit Akt and Erk1/2 signaling pathway in A549 and H1975 cells,respectively.The expression level of PD-L1 in cells was detected by western blot and the expression level of surface PD-L1 was detected by flow cytometry.In vivo,Lewis lung cancer xenograft model in C57BL/6 mice were randomly divided into into 4 groups with 10 mice in each group:(1)Control group:intraperitoneal injection of normal saline+DMSO every 3 days for 15 days;(2)Low concentration group:10 mg/kg Z-guggulsterone was intraperitoneal injected every 3 days for 15 days;(3)The medium concentration group:20 mg/kg Z-guggulsterone was intraperitoneal injected every 3 days for 15 days;(4)The high concentration group:40 mg/kg Z-guggulsterone was intraperitoneal injected every 3 days for 15 days.The body wright and tumor volume of the transplanted mice were measured every 3 days.After the injection cycle was completed,the Lewis lung cancer transplanted tumor was removed from the mice,and the changes of PD-L1 expression in the transplanted tumor were detected by immunohistochemical stainingResults:1.Z-guggulsterone can inhibit the cell viability of A549,H1975 and LLC.The inhibition rates on A549,H1975 and LLC were 49.3%,24.9%and 44.2%at 40 μM and 48h for the treatment time and concentration of Z-guggulsterone.With the increase of drug concentration and prolonged treatment time,the proportion of cell cycle in G0/G1 phase of A549,H1975 and LLC gradually increased,while the proportion in S phase gradually decreased.Therefore,Z-guggulsterone can block the transformation of cell cycle from G0/G1 phase to S phase of A549,H1975 and LLC.However,we found that Z-guggulsterone had only a slight effect on apoptosis in A549,H1975 and LLC2.Within a certain rage,PD-L1 mRNA and surface PD-L1 expression level gradually increased with the concentration of Z-guggulsterone and the prolongation of the treatment time in A549,H175 and LLC.The activation of PD-L1 promoter in A549 and H1975 increased with the increasing concentration of Z-guggulsterone3.After inhibition of intracellular FXR by transfection with siRNA,PD-L1 mRNA level in A549,H1975 and LLC was significantly higher than that in cells without siRNA transfection,but lower than that in cells simultaneously treated with Z-guggulsterone.Similarly,the expression level of surface PD-L1 on A549,H1975 and LLC were altered.PD-L1 mRNA and surface PD-L1 expression level in transfection of FXR-overexpressed lentiviral were lower than those in mock.The expression level of PD-L1 mRNA and surface PD-L1 of A549 transfected with both FXR-overexpressed lentiviral and Z-guggulsterone was slightly higher than mock.4.A549 and H175 were simultaneous treated with Z-guggulsterone and Akt signaling pathway inhibitor MK2206 or Erk1/2 signaling pathway inhibitor PD325901 and the expression level of PD-L1 mRNA and surface PD-L1 were slightly higher than control,but were less than only treated by Z-guggulsterone cells.5.In the LLC model experiment in mice,the volume of the transplanted tumor gradually decreased with the increase of the concentration of Z-guggulsterone while the body weight of the mice showed no significantly difference.Immunohistochemical staining results showed that the expression level of PD-L1 on LLC surface of mouse transplanted tumor increased in a dose dependent manner.Conclusion:Z-guggulsterone has antitumor effect can regulate the expression of PD-L1 in non-small cell lung cancer partially through FXR,Akt and Erk1/2 signaling pathways.