The Study On Gigantol Inhibiting The Stemness Of Gastric Cancer Cells By Regulating The Polarization Of Macrophages

Objectives: In our present study,we aimed to explicit the effect of gigantol on the biological behavior of gastric cancer cells,and to explore the mechanism of gigantol inhibiting the stemness of gastric cancer cells by regulating the polarization of macrophages.Methods: First,human gastric cancer cell line NCI-N87 was stimulated with gigantol in vitro,and the proliferation of NCI-N87 was detected by CCK-8 method.Transwell assay was used to observe the effect of different concentrations of gigantol on the migration ability of NCI-N87.The protein levels of apoptosis-related proteins,CD133,and key transcription factors were detected by Western blot.Secondly,the Kaplan-Meier Plotter online survival analysis website was used to analyze the correlation between M2 polarization-related markers（CD163,Arg-1,Stat6）and gastric cancer patients’ survival.Then,mouse macrophages Raw264.7 were induced to polarize to M1/M2 and treated with gigantol,the groups are as follows: M0 group,M1（LPS+IFN-?）group,M2（IL-4）group,M0G（M0+gigantol）group,and M2G（M2+gigantol）,q RT-PCR and Western blot assays were used to detect the protein and m RNA levels of M1/M2 polarization related markers.Finally,the co-culture model of gastric cancer cells and macrophages was constructed by collecting conditioned media of different groups and divided into GC（gastric cancer cells）+M0 group,GC+M1group,GC+M2 group,GC+M0G group,GC+M2G group.In the co-culture model,transwell assay was used to compare the migration ability of NCI-N87 cells in different groups.Spheroid formation assay was applied to depict the stemness of NCI-N87 of different groups.The m RNA levels of key transcription factors were detected by the q RT-PCR method,and the protein expression of stemness marker CD133 and key transcription factors were detected by Western blot.Results: Firstly,the CCK-8 results showed that gigantol could effectively inhibit the proliferation of NCI-N87 cells,and after different concentrations of gigantol（0,2.5,5,10,20,40,80,160,320 μM）treated on gastric cancer cells for 24 h,48 h and 72 h,the proliferation ability of gastric cancer cells decreased in a dose-and time-dependent manner.IC50（48 h）value was 168.1 μM.Transwell assay results showed that gigantol significantly inhibited the migration ability of NCI-N87 cells.Western blot results showed that the ratio of the anti-apoptotic protein Bcl-2 to the pro-apoptotic protein Bax and the expression of the anti-apoptotic protein Bcl-XL decreased in a concentration-dependent manner after different concentrations of gigantol treated on NCI-N87 cells,and the levels of stemness marker CD44 and pluripotency stemness factors Klf4,Sox2,and Nanog decreased（P<0.05）.Secondly,online KM-plotter analysis revealed that the expression of CD163,Arg-1,Stat6 were negatively correlated with the survival time of gastric cancer patients after diagnosis（P<0.001）.Patients with lower levels of CD163,Arg-1,Stat6 exhibited a longer survival time.Then,when Raw264.7 was intervened with gigantol,the q RT-PCR and Western blot results showed that the expression of M1 and M2 polarization-related markers CD206,CD163,and Arg-1 were significantly decreased in the M2 G group.Finally,after NCI-N87 cells were treated with condition-medium of different groups,transwell assay results showed that the migration ability of NCI-N87 cells in the GC+M0G group and GC+M2G group was significantly inhibited.In sphere formation assay,we observed an increased volume and a higher number of mammosphere in the GC+M2 group while the additional gigantol could significantly rescue this trend.The results of q RT-PCR and Western blot assays showed the GC+M0G group and GC+M2G group significantly reduced the levels of stemness marker CD133 and pluripotency stemness factors Oct4,Klf4,Sox2,and Nanog of NCI-N87 cells compared with the GC+M0 group and GC+M2G group,respectively.Conclusions: Gigantol could inhibit the proliferation and migration of human gastric cancer cell NCI-N87,promote its apoptosis,and inhibit its stemness.The mechanism of gigantol’s inhibitory effect on the malignant behavior and stemness of gastric cancer cells NCI-N87 may be through the inhibition of M2 polarization of macrophages.