Effects And Molecular Mechanism Of Eugenol On Myeloid-derived Suppressor Cells

Background:Cancer-associated fibroblasts(CAFs)are one of the main stromal cells in the tumor microenvironment(TME).CAFs play a crucial role in the tumorigenesis and progression of tumors,such as promoting the formation of tumor-related blood vessels,exert the function of immune suppression,promote the recruitment of Treg,and inhibit the recruitment and chemotaxis of CD8+T cells.Currently,the treatment strategies targeting CAFs mainly include the following aspects.First,the number of CAFs can be directly reduced or eliminated.The second is to reverse the function of CAFs and further reduce the tumor-promoting activity of CAFs.The third is to induce the normalization of CAFs or block the related signaling pathways of CAFs activation.Some natural small molecules have significant inhibitory effects on tumor growth.Studies have shown that curcumin can inhibit the growth of various tumor cells,but whether curcumin has any effect on the growth and activity of CAFs,which is the most important cells in TME,is still unclear.This study mainly explored the effect of curcumin on Prostate cancer associated fibroblast(CAF)and its related mechanism through in vitro experiments.Our results show that curcumin can further induce apoptosis and cell cycle arrest of CAF cells through ROS-mediated endoplasmic reticulum stress.Objective:This study mainly studied the effect of curcumin on CAFs and the specific molecular mechanism,hoping to further explore the potential application of curcumin in tumor immunotherapy and potential target cells,and our study complements the molecular mechanism of curcumin acting on CAF.Methods:The effect of curcumin on CAF,PC-3,NAF and the proliferation of spleen cells were determined by MTT assay.Flow cytometry was used to detect the pro-apoptotic effect of curcumin on CAF,cell cycle arrest,intracellular ROS,ΔΨm and T cell proliferation.The long-term effects of curcumin on the proliferative activity of CAF were examined by colony cloning.The expression of Bax,Bcl-2,Bcl-XL,caspase9,caspase8,cleaved caspase3,cleaved PARP,p-PERK,p-eIF2a,ATF4,CHOP,p-p53,cyclinD1 in CAF after curcumin treatment were determined by Western Blot.Quantitative real-time PCR(qRT-PCR)was used to detect the mRNA expression of ATF4,CHOP,Bim,PUMA,Bax,bcl-2,etc.The caspase 3 activity assay was used to detect the changes of caspase 3 activity in CAF.Results:In this study,we found that curcumin can selectively inhibit the proliferative activity of CAF.Further,colony cloning experiments showed that curcumin had a long-term inhibitory effect on CAF proliferation,that is,CAF cells treated with curcumin formed fewer colonies.Then,the apoptosis experiments showed that curcumin could induce CAF apoptosis significantly,and showed a significant dose-dependent effect.Western Blot showed that the cleaved caspase 3,cleaved PARP,Bax,and other pro-apoptotic proteins in CAF treated with curcumin were observably up-regulated,while the expression levels of Bcl-2 were decreased.In addition,the caspase 8,a marker protein of exogenous apoptosis,was barely detected.These results suggested that the inhibition effect of curcumin on CAF was mainly caused by inducing non-exogenous apoptosis.Cell cycle arrest will also affect the cell apoptosis and proliferation activity,according to the results of cell cycle,curcumin can significantly increase the proportion of CAF in G2-M phase.The results of Western Blot showed that the expression level of p-p53 increase,which suggests that curcumin may increase further induced phosphorylation of p53 to cause the cell cycle arrest of CAF,resulting in cell apoptosis and other events.The results of Flow cytometry showed that after curcumin treatment,the intracellular ROS levels of CAF increased significantly.The results of Western Blot showed that p-PERK,p-eIF2a,ATF4 and CHOP were up-regulated observably.However,after NAC pretreatment,a ROS remover reagent,the expression of these proteins are no longer increases.These results suggest that curcumin can be up-regulated the levels of intracellular ROS in CAF,and further lead to endoplasmic reticulum stress.In addition,after NAC pretreatment,the proportion of CAF apoptosis was notably reduced,and the proportion of CAF cells in G2-M phase was also markedly reduced.The colony cloning experiments also showed that NAC pretreatment could restore the proliferative activity of CAF.Moreover,the results of Western Bolt also showed that after pretreatment with NAC,the cleaved PARP,cleaved caspase-3 and p-p53 were decreased notably,the activity of caspase-3 was also observably decreased,and the ΔΨm was also recovered.These results suggested that curcumin induced CAF cell apoptosis and G2-M phase arrest was associated with ROS up-regulation,and the mechanism was associated with the ROS mediated endoplasmic reticulum stress,which via the PERK-eIF2α-ATF4 axis.In addition,we also explored the effect of low concentration of curcumin on CAF,and found that low concentration of curcumin had no significant effect on the proliferation activity of CAF,and then use CAF supernatant,which was used the curcumin pretreated,to culture the T cells.The results showed that the inhibitory effect of CAF on T cells was weakened after curcumin treatment.These results indicated that curcumin could reverse the immunosuppressive effect of CAF on T cells.Conclusion:This study mainly explored the effect of curcumin on CAF,and the results showed that curcumin can selectively inhibit the proliferative activity of CAF,and has a long-term inhibitory effect on the proliferative activity of CAF.In addition,the inhibitory effect of curcumin on CAF was mainly related to the non-exogenous apoptosis and the cell cycle arrest at G2-M phase of CAF induced by curcumin.The pathway is associated with the ROS mediated PERK-eIF2α-ATF4 axis in endoplasmic reticulum stress.Moreover,low concentrations of curcumin can reverse the inhibition of CAF on T cells.The decrease in the number of CAF and the release of immunosuppressive effect provided a favorable direction for the immunotherapy of tumor cells,which also suggested the new application of curcumin in tumor therapy.