| Introduction Programmed death-1(PD-1)interacts with programmed death ligand-1(PD-L1)to form an immunosuppressive microenvironment and play a key role in tumor immune escape.Multiple monoclonal antibody drugs based on blocking PD-1/PD-L1 binding have been approved for the treatment of malignant tumors such as melanoma,non-small cell lung cancer and kidney cancer.However,these immune checkpoint inhibitors can cause immune-related adverse events(ir AEs)widely during the application process,which affects various organs and tissues and can be life-threatening when the disease is severe.Currently,glucocorticoids are mainly used to prevent and relieve the occurrence of ir AEs.Glucocorticoids have a suppressive effect on the immune system.Therefore,whether glucocorticoids have a negative effect on the therapeutic effect of immune checkpoint inhibitors is a clinical issue with great concern.A clinical retrospective analysis has shown that glucocorticoids attenuated the therapeutic effect of PD-1/PD-L1 monoclonal antibodies on non-small cell lung cancer,while glucocorticoids had no significant effect on immunotherapy in melanoma.The formation of an immunosuppressive microenvironment is a prerequisite for the efficacy of PD-1/PD-L1 monoclonal antibodies.Therefore,the expression of PD-L1 in tumor cells is another key factor that determines its therapeutic effect.Nowadays,the effect of glucocorticoids on PD-L1 expression in tumor cells has not been reported in the literature.The purpose of this study was to investigate the role of glucocorticoids in regulating PDL1 expression in tumor cells and to explore the potential effects of glucocorticoids on immunotherapy effects from the perspective of tumor cells.Methods Human non-small cell lung cancer cells NCI-H292 and A549,human melanoma A2058 cells and A375 cells,human kidney cancer 786-O cells and 769-P cells were used as cell models to evaluate the effect of glucocorticoids on PD-L1 expression in different tumor cells.1)The effects of dexamethasone on PD-L1 protein expression in different tumor cells were investigated by Western Blot in the basic conditions and IFNγ-induced PD-L1 up-regulation models.2)Western blot method was used to detect the timedependent regulation of PD-L1 protein expression with dexamethasone on human nonsmall cell lung cancer cells NCI-H292.3)The concentration-dependent effects of dexamethasone on PD-L1 protein levels on non-small cell lung cancer cells NCI-H292 and A549 were examined by Western Blot in the IFNγ-inducted model.4)The effect of dexamethasone on non-small cell lung cancer cells’ surface protein CD274 was investigated by flow cytometry.5)NCI-H292,A2058 and 786-O cell lines were treated with glucocorticoids in the basic condition and IFNγ-induced models,and the PD-L1 expression were examined by Western Blot.6)Blocking protein synthesis with cycloheximide(CHX),the protein degradation rate of PD-L1 in the basic condition or IFNγ-induced model was determined with Western Blot.7)Proteasome inhibitor MG132 was further used to prevent PD-L1 protein ubiquitin-dependent degradation,while lysosomal inhibitor CQ was used to inhibit PD-L1 protein lysosomal-dependent degradation,and the PD-L1 protein levels was detected by Western Blot after treated with dexamethasone in the basic condition or IFNγ-induced model.8)Glucocorticoids were objected to NCI-H292 cells,and the expression changes of PD-L1 were detected by qRT-PCR.9)Treated with dexamethasone with/without IFNγ on A549 and NCI-H292 cell lines,the m RNA levels of PD-L1 were detected by q-RT-PCR.Human non-small cell lung cancer NCI-H292 cells,human melanoma A2058 cells,and human kidney cancer 786-O cells were employed to further explore the different regulatory mechanisms of glucocorticoids on PD-L1 expression on different tumor cells.1)Collect different tumor cell lysates and the protein levels of glucocorticoid receptors were observed by Western Blot.2)After treated with dexamethasone,amcinonide and betamethasone,the expression changes of target genes of GR on human non-small cell lung cancer cells NCI-H292 were detected by q-PCR.3)Human melanoma A2058 cell lines and human kidney cancer 786-O cell lines were treated with dexamethasone and used to detect the activation changes of target genes of GR by q-RTPCR.4)The total protein and phosphorylation level of Stat1 and IRF1(the upstream transcription factors of PD-L1)were determined with Western Blot in the basic condition and IFNγ-induced model.Various murine tumor cells were used in vitro to evaluate the regulation of PD-L1 by glucocorticoids,and the correlation between glucocorticoid receptors and PD-L1 expression was initially investigated by in vivo experiments.1)Treated with different concentration dexamethasone on murine lung cancer LLC cells,murine melanoma B16 cells and murine colon cancer MC38 cells under the basic condition and IFNγ-induced model,cell extract was objected to Western blot to evaluate the m PD-L1 protein levels.2)Glucocorticoids were objected to murine colon cancer cell MC38 and the expression changes of PD-L1 were detected by Western blot.3)The MC38 xenograft model and LLC xenograft model were used to investigate the changes of GR or PD-L1 expression levels in tumor cells before/after immune surveillance.Results Glucocorticoids can significantly inhibit the expression of PD-L1 in non-small cell lung cancer cells,but do not affect the PD-L1 protein levels in renal cancer cells and melanoma cells.Western Blot results showed that administration of dexamethasone on NCI-H292 cells down-regulated the protein level of PD-L1.In the IFNγ-induced model of non-small cell lung cancer cells A549 and NCI-H292,dexamethasone significantly inhibited IFNγ induced up-regulation of PD-L1 protein.In order to further clarify the inhibitory effect of dexamethasone on PD-L1 protein expression,we use concentration gradient and different action time of dexamethasone in NSCLC.The results showed that the protein levels of PD-L1 decreased gradually in a time-dependent manner under the basic condition and in a concentration-dependent manner in the IFNγ-induced model.Since PD-L1 protein mainly plays a role on the surface of tumor cell membranes,we detected the inhibitory effect of dexamethasone on the surface of tumor cell membrane PD-L1 protein named CD274 by flow cytometry.The results showed that treated with dexamethasone in A549 under the basic condition,compared with the control group(1.27 ± 0.13%),the percentage of CD274 in the administration group(dexamethasone)decreased to 0.83 ± 0.28%(p = 0.0488).IFNγ could increased the expression level of CD274 significantly to 7.70 ± 2.36%(p = 0.0027),which proved that the induction model was successfully constructed.After simultaneously administration of dexamethasone and IFNγ,the CD274 expression levels decreased from 7.70 ± 2.36% to 0.99 ± 0.70%(p = 0.0059).Treated with various glucocorticoids in H292,which commonly used in clinical therapy,the Western Blot results showed that dexamethasone,prednisone,prednisolone,amcinonide and betamethasone could down-regulate PD-L1 protein levels.At the same time,we also investigated the regulation of PD-L1 by glucocorticoids in other tumor cells.The results showed that glucocorticoids did not affect the expression level of PD-L1,either on melanoma A2058 cells and A375 cells,or on renal cell carcinomas 786-O cells and 769-P cells.Next,we further investigated the regulation of PD-L1 expression by examining the stability and transcription level of PD-L1.Western Blot results showed that dexamethasone did not significantly affect the degradation rate of PD-L1 protein,either in the basic condition or in the IFNγ-induced model.It was shown that treaed with dexamethasone(1 μM),prednisolone(1 μM),amcinonide(1 μM)and betamethasone(1 μM)in H292 cell line,m RNA level of PD-L1 was down-regulated by 5.33 ± 0.04 times(p < 0.0001),5.27 ± 0.03 times(p < 0.0001),4.70 ± 0.02 times(p < 0.0001),8.36 ± 0.04 times(p < 0.0001).In the IFNγ-induced model,dexamethasone(1 μM)could downregulate PD-L1 transcription levels by 7.61 ± 0.12-fold(p = 0.0001)and 1.66 ± 2.40-fold(p = 0.0201)in A549 and H292 cells,respectively.Glucocorticoids could inhibit the level of phosphorylation of Stat1 Ser701 in NSCLC.Western Blot results showed that glucocorticoid receptors were commonly expressed on different tumor cells.The difference in GR expression was not consistent with the difference in the regulation of PD-L1 by glucocorticoids,indicating that the difference of GR expression in different tumor cells was not the reasons for the differences in the regulation of PD-L1 by glucocorticoids.Furthermore,we investigated the activation level of downstream target genes of GR during dexamethasone administration in different tumor cells by q-PCR.The results showed that dexamethasone(1 μM),amcinonide(1 μM)and betamethasone(1 μM)caused up-regulation of the m RNA levels of KLF9 by 7.54 ± 1.11 times(p = 0.0005),9.24 ± 1.51 times(p = 0.0019)and 7.48 ± 3.19 times(p = 0.0244),respectively.Meanwhile,the transcription level of SNAI2 was increased by 3.51 ± 0.44 times(p = 0.0006),4.20 ± 0.62 times(p = 0.0009),and 3.13 ± 1.00 times(p = 0.0216)as well as the transcription level of MT2 A was increased by 3.43 ± 0.98 times(p = 0.0126),3.59 ± 1.18 times(p = 0.0189)and 3.60 ± 0.39 times(p = 0.0029),respectively.At the same time,after treated with dexamethasone(1 μM)on 786-O cell lines,KLF9,SNAI2,and MT2 A were up-regulated by 10.48 ± 1.20 times(p = 0.0008),4.90 ± 1.12 times(p = 0.0070)and 1.92 ± 0.55 times(p = 0.0498).These three target genes were also detected on A2580 cell lines.The results showed that KLF9,SNAI2,and MT2 A were upregulated by 1.30 ± 0.11 times(p = 0.0028),1.79 ± 0.15 times(p = 0.0009)and 6.69 ± 0.88 times(p = 0.0018).The above results indicated that glucocorticoid could activate GR target genes on different tumor cells.Next,we examined the levels of Stat1 and IRF1,which seems as the classic transcription factors of PD-L1 in tumor cells.Western Blot experiment results shows that treated with dexamethasone in the IFNγ-induced model of A549 cells,the phosphorylation level of Stat1 Tyr701 was significantly inhibited,while the phosphorylation level of Stat1 Ser727,the total protein level of Stat1,and the IRF1 protein level did not change significantly.Unlike the inhibition on A549,the phosphorylation levels of Stat1 Tyr701,Stat1 Ser727,and IRF1 on 786-O and A2058 were not suppressed by dexamethasone.Combining the results of the above experiments,we found that glucocorticoids can specifically down-regulate the phosphorylation level of Stat1 Tyr701 in NSCLC,thereby inhibiting the protein level and transcription level of PD-L1,and we did not observe phosphorylation level changes of Stat1 on 786-O and A2058.We initially believe that the difference in the regulation of PD-L1 protein on different tumor cells by glucocorticoids may be due to the specific inhibition of Stat1 Tyr701 phosphorylation by dexamethasone on A549 cells.Dexamethasone can inhibit PD-L1 expression on murine colon cancer MC38 cell and glucocorticoid receptors were negative related to PD-L1 expression.Different murine tumor cell models were used to investigate the regulation of PD-L1 by glucocorticoids in vitro.Western Blot showed that glucocorticoids can inhibit PD-L1 expression on murine colon cancer MC38 cells in IFNγ-induced model.On murine lung cancer LLC cells,dexamethasone could inhibit PD-L1 expression in a concentrationmanner.Glucocorticoids had no significant effect on PD-L1 expression on murine melanoma B16 cells.In MC38 xenograft model and LLC xenograft model,glucocorticoid receptor level was negatively correlated with its PD-L1 expression.Conclusion From the perspective of tumor cells,this study revealed the differences in the regulation of PD-L1 by glucocorticoids in different tumor cells.In human non-small cell lung cancer,glucocorticoids can significantly inhibit the expression of PD-L1 protein and also significantly inhibit the up-regulation of PD-L1 induced by IFNγ.PD-L1 expression levels do not changed significantly in melanoma and kidney cancer.In human non-small cell lung cancer,glucocorticoids can significantly inhibit the transcription level of PD-L1,but do not affect the stability of PD-L1 protein.IFNγ,as a classical cytokine to induce PD-L1 protein expression,mainly activates the transcription of PD-L1 by promoting the nuclear translocation of Stat1 and IRF1.In A549 cell line,we found that glucocorticoids could specifically down-regulate the phosphorylation level of Stat1 Tyr701 without affecting the phosphorylation level of Stat1 Ser727,the total protein level of Stat1 and the expression level of IRF1.It was found that glucocorticoids could inhibit the upregulation of PD-L1 caused by IFNγ in mouse colon cancer MC38 cells and murine lung cancer LLC cells.In MC38 xenograft model and LLC xenograft model,we initially determined that glucocorticoid receptor level was negatively correlated with its PD-L1 expression,which provided preliminary data supporting for our subsequent vivo experiments.Our findingis reveals the effects of glucocorticoids on PD-L1 expression on tumor cells and provides a theoretical basis for the potential impact of glucocorticoids on the efficacy of immunotherapy. |
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