| Objective:Monoclonal antibodies targeting PD-1/PD-L1 have achieved remarkable results in the treatment of tumors and have been the breakthrough in the field of cancer immunotherapy.However,with these immune checkpoint inhibitors being more widely used in the clinic,their limitations have gradually become apparent.Apart from the effective treatment outcome in melanoma and non-small cell lung cancer,the overall response rate of monoclonal PD-1/PD-L1 antibodies in most solid tumor treatment does not exceed 20%,and some studies suggest that this is partly due to the difficulty of penetrating into tumor tissues owing to large molecular weight of antibodies.Furthermore,the non-specific activation of the immune system caused by the antibodies can affect almost all human organs and tissues,and causes a wide range of adverse events thus severely limiting the clinical use of PD-1/PD-L1 monoclonal antibodies.Compared with these antibodies,small molecule drugs usually have the advantages of good permeability of organs or tumors,feasibility of oral administration and little stimulation to immune system.Therefore,it has become a heat to explore small molecule drugs directly modulating PD-L1 expression.Based on the regulatory mechanisms of PD-L1 in transcription,translation and cell membrane expression,it provided potential treatment targets for the development of small molecule inhibitors.Hence,our study applied non-small cell lung cancer cells highly expressing PD-L1 as cell models to select drug candidates that are able to regulate PD-L1 from a compound library,and finally one compound,butein,was found.Butein is a natural flavonoid derived from the bark of Rhus verniciflua Stokes,which has been reported to exert antitumor effects by promoting tumor cells apoptosis,inhibiting angiogenesis in tumor tissues and preventing tumor cell migration.However,it is unclear whether butein can exert an antitumor immune effect based on its ability to regulate PD-L1 expression.Therefore,this study aimed to explore the key mechanisms of PD-L1 regulation by butein.In addition,this study will examine whether the regulation of PD-L1 by butein can influence the killing effect of T cells on tumor cells through in vitro T cell coculture assay.At the same time,the immunosuppressive(PD-1 combined with PD-L1)mouse model(CT26 mouse model)was used to further clarify whether butein can exert significant antitumor effects in vivo by reducing the expression of PD-L1.In conclusion,this study aims to elucidate the mechanism of antitumor effect of butein in downregulating PD-L1 and provides a candidate compound for the development of small molecule inhibitors that can modulate PD-L1 expression.Methods:1.Different human-derived non-small cell lung cancer cell lines NCI-H292,NCI-H460,NCI-HCC827 as well as patient-derived human primary lung cancer cells were used as cellular models to evaluate the regulatory effects of butein on PD-L1expression in tumor cells.It is known that when cytotoxic T cells infiltrate in tumor tissues,the secreted IFNγcan further mediate the expression of PD-L1 on the surface of tumor cells.To this end,this study mimics a tumor immunosuppressive microenvironment by supplementing the medium with additive IFNγ.The NCI-H292cell line highly expressing PD-L1 was selected as representative cell line for basal expression level of PD-L1,while NCI-H460 and NCI-HCC827 cells with relatively low PD-L1 expression represented IFNγ-induced cell lines.(1)Firstly,the effects of butein on basal expression and the level of PD-L1 in IFNγ-induced expression cell lines were investigated by Western blotting,and the effect of butein in regulating PD-L1 under two conditions was further confirmed in non-small cell lung cancer cell lines and primary cells.(2)Flow cytometry was used to investigate the ability of butein to modulate PD-L1 expressed on the membrane surface of NCI-HCC827.(3)The effect of butein in NCI-H292 and NCI-HCC827 on PD-L1 m RNA production in tumor cells was determined by q RT-PCR.(4)To examine the alteration of PD-L1 degradation rate in NCI-H292 cells by butein,a protein synthesis inhibitor actinomycin(CHX)was used to block protein synthesis and to determine whether butein regulated the protein synthesis process of PD-L1.(5)Finally,the proteasome inhibitor MG132 and lysosomal pathway inhibitor CQ were used to investigate whether the efficacy of butein remained after the natural degradation of PD-L1 was blocked,and then to explore whether butein could affect the degradation of PD-L1.2.Cell lines under both basal level or IFNγ-induced conditions were selected to explore the effect of butein on STAT12,a key transcription factor of PD-L1.Since butein had an effect of PD-L1 regulation under IFNγ-induced conditions,which was mainly associated with the IFNγ-JAK-STAT1 signaling pathway,therefore,this study examined the effect of butein on STAT1.(1)The changes in total protein levels and phosphorylation levels of STAT1 in cells after administration were analyzed by Western blotting to clarify the regulatory role of butein on STAT1.(2)si RNA of STAT1 was applied to test whether the pharmacological efficacy of butein was abolished to demonstrate butein acted in a STAT1-dependent manner.(3)The m RNA levels of STAT1 in different cell lines were compared by q RT-PCR after the administration of butein to explore the effect of butein on the m RNA level of STAT1.(4)CHX was used to block STAT1 protein synthesis,and changes in protein half-life of STAT1 before and after administration of butein was analyzed to investigate the role of butein on STAT protein stability.3.A T-cell coculture assay was applied to evaluate T cell killing ability on tumor cells after PD-L1 was suppressed by butein.(1)PBMCs were isolated from fresh blood samples of healthy volunteers and activated into T cells by stimulation with anti-CD3/CD28/CD2 antibodies.(2)T cells were then cocultured with tumor cells and PD-1 monoclonal antibody was set as a positive control.(3)At the end of the incubation,different samples were fixed and stained with crystal violet,and then were washed and photographed with a microscope(washed until there existed no floating color).4.In this study,a mouse tumor model of immunosuppressive microenvironment(PD-1 interacts with PD-L1)was used to investigate the inhibitory effect of butein on tumor growth by modulating PD-L1 expression.CT26 mouse model is known to be an immunogenic mouse model of colon cancer that expresses PD-L1 and can be induced by cytokine IFNγ.In addition,this model well responds to PD-1/PD-L1 monoclonal antibodies and has been widely applied in cancer immunotherapy.Thus,this study chose this model to investigate the antitumor effect of butein.(1)PD-L1 expressions before and after butein treatment were examined by Western blotting in CT26 cells in vitro.(2)CT26 tumor cells were injected in the flank of immunocompetent BALB/c mice and divided into two groups(control and butein)to investigate the antitumor effect and toxic side effects.(3)At the end of the mouse administration experiment,tumor tissues were obtained to determine membrane PD-L1 expression and infiltrated CD8~+T cells ratio by flow cytometry.(4)m RNA levels of PD-L1 and STAT1 in tumor tissues were analyzed by q RT-PCR.(5)Total protein expressions of PD-L1 and STAT1 were examined by Western blotting.(6)CT26 tumor cells were also injected in the flank of immunodeficient BALB/c mice to investigate the antitumor effects of butein.Results:1.Butein significantly downregulated PD-L1 expression and was associated with inhibition of its m RNA level.In both basal and IFNγ-induced PD-L1 expressing cell lines,total PD-L1 protein levels were examined after being treated with different concentrations of butein.The results showed that butein significantly inhibited PD-L1 expressed in NCI-H292 and IFNγ-treated NCI-H460 and NCI-HCC827 cell lines in a concentration-dependent manner.To confirm the effect of butein in downregulating PD-L1,butein(20μM)was administered to other NSCLC cell lines and patient-derived lung cancer primary cells for 24 h under basal level and IFNγ-induced conditions.The experimental results continued to show a significant downregulatory effect of butein on PD-L1.As PD-L1expressed on the cell surface can bind to PD-1 and exert an inhibitory effect of immune cells.Thus,we examined the effect of butein on membrane PD-L1 expression by flow cytometry.The results showed that butein inhibited PD-L1 levels on the surface of tumor cell membranes and produced a significant effect under IFNγ-induced conditions.To further explore the mechanism of PD-L1 inhibition by butein,we used NCI-H292 and NCI-H460 cell lines to investigate the effect of butein on PD-L1expression in a time-dependent manner.It turned out that butein downregulated PD-L1in a relatively short period after butein treatment,suggesting that the regulatory effect of butein on PD-L1 might occur at an earlier transcriptional process.Therefore,we examined the effect of butein on m RNA levels of PD-L1 as well as protein stability.The results showed that(1)the relative m RNA level of PD-L1 in NCI-H292 cells decreased from 1.00±0.00 to 0.37±0.07(p=0.0006)after 24 h of butein action;In addition,in IFNγ-induced NCI-HCC827 cells,butein decreased the relative m RNA levels of PD-L1from 1.00±0.00 to 0.70±0.09(p=0.0286)and 17.15±1.39 to 7.27±0.86(p=0.0037),respectively;(2)After administration of CHX to inhibit protein synthesis of PD-L1,we found that the protein half-life of PD-L1 was not affected by butein;(3)After respectively blocking the ubiquitin-proteasome degradation pathway and the lysosomal degradation pathway of PD-L1 with MG132 and CQ,butein continued to downregulate the total protein level of PD-L1,indicating that butein did not affect the degradation process of PD-L1.In summary,butein significantly downregulated PD-L1expression and was associated with the inhibition of the m RNA level of PD-L1.2.Butein affected the m RNA level of PD-L1 by inhibiting STAT1 m RNA.Butein was able to downregulate PD-L1 under IFNγ-induced conditions,and the IFNγ-JAK-STAT1 signaling pathway plays an important part in this condition.Therefore,this study examined the effect of butein on STAT1.(1)Different tumor cell lysates were collected after being treated with butein and the protein levels of STAT1and its phosphorylated form were determined by Western blotting.The results showed that butein downregulated the total protein level of STAT1 in a concentration-dependent and time-dependent manner.(2)To further validate the STAT1-dependent effect of butein,the efficacy of butein was investigated when si RNA was used to knockdown STAT1,and the results showed that the efficacy of butein was abolished when STAT1was knockdown.Therefore,this study further investigated whether STAT1 m RNA levels or the protein half-life were affected by butein.(3)STAT1 m RNA was significantly inhibited in NCI-H292,NCI-HCC827 and NCI-H460 cell lines after 24 h of butein.(4)When protein synthesis was inhibited,the protein half-life of STAT1 was not affected by butein.These results suggested that butein inhibited STAT1 m RNA level and affected the transcriptional process of PD-L1.3.Butein increased the killing effect of T cells by downregulating PD-L1 on tumor cells.To examine whether downregulation of PD-L1 by butein could increase the killing effect of T cells,we cocultured T cells with two tumor cell lines,NCI-H292 and NCI-H460,and PD-1 monoclonal antibody was set as a positive control.The results showed that butein enhanced the killing effect of T cells on tumor cells in a concentration-dependent manner,as evidenced by a decrease in surviving tumor cells.It showed a similar result in both NCI-H292 and NCI-H460 cell line.Futrthermore,the result exhibited that the downregulation of PD-L1 caused by butein exerted a therapeutic effect similar to that of monoclonal antibodies and enhanced the antitumor killing effect of T cells.4.Butein exerted antitumor effect by downregulating PD-L1 expression.To determine the antitumor effect of butein,we applied CT26 tumor cell to explore the role of butein in PD-L1 regulation in vivo and in vitro.(1)Butein could downregulate PD-L1 in CT26 cells in vitro in a dose-dependent manner.(2)In immunocompetent BALB/c mice,the tumor volume of the control group grew from54.64±4.75 mm~3 to 1452.97±91.46 mm~3,while the tumor volume was from 55.29±6.99 mm~3 to 715.20±74.47 mm~3 in butein-treated group,which exhibited a tumor inhibition rate of 50.78%,and these indicated that butein significantly inhibited tumor growth in vivo.(3)No significant body weight loss was observed both in the control and butein-treated group,indicating that butein had no effect on their normal growth.(4)After continuous administration of butein,the mice were sacrificed and the tumor tissues were obtained to determine the cell surface expression of PD-L1 and infiltrated CD8~+T cells ratio.The results showed that butein could significantly downregulate cell membrane PD-L1 expression and increased the infiltration of CD8~+T cells in tumor tissues.(5)By comparing the total protein expression and m RNA levels of PD-L1 and STAT1 in the tumor tissues from both groups,we found that butein significantly downregulated the total protein levels of PD-L1 and STAT1,and significantly inhibited the relative m RNA levels of PD-L1 and STAT1.(6)When CT26 cells were injected in the flank of immunodeficient BALB/c-nude mice,there was no significant difference in tumor growth between the control group as well as the butein-administered group.In summary,butein exerted antitumor immune effects through PD-L1 downregulation and this effect was dependent on the immune system.Conclusions:By using a cell-based model to select drugs that are able to modulate PD-L1expression,butein was found to have a strong capacity of PD-L1 regulation,which was further confirmed to play a same role in different NSCLC cell lines both under basal and IFNγ-induced conditions.In addition,this study found that butein inhibited the m RAN level of PD-L1 without affecting the protein stability of PD-L1.It was shown that butein significantly downregulated both STAT1 expression and its phosphorylation levels under both basal and IFNγ-induced conditions,which was associated with inhibition of STAT1 m RNA.In addition,the results of T cell coculture assay with tumor cells showed an enhanced killing effect of T cells.Meanwhile,butein inhibited tumor growth in an immunosuppressive(PD-1 bounds to PD-L1)CT26 mouse model,but this ability was abolished in BALB/c-nude immunodeficient mice,which suggested that butein exerted antitumor immunomodulatory effects through downregulation of PD-L1and was dependent on the immune system.This study elucidated the mechanism of butein in downregulating PD-L1 in tumor cells,and provided a theoretical basis for the further development of butein in being a small molecule inhibitor to modulate PD-L1expression. |
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