| Evasion of the immune system（or immune tolerance）is crucial for the cancer development,progression and resistance to treatment.Under physiolo-gical conditions,the PD-1/PD-L1 interaction facilitates the development of immune tolerance,preventing excessive immune cell activity that may lead to tissue destruction and autoimmunity.At the same time,PD-L1 expression is also an immune evasion mechanism used by various malignancies and is usually associated with a poor prognosis of tumor patients.The role of PD-L1in tumors is mainly to cause central and peripheral immune tolerance,immune failure of tissue cells,and regulate the anti-cancer immune responses.PD-L1 expression is also considered as a predictive biomarker against PD-1/PD-L1 treatment responses.About 20%to 30%of non-small cell lung cancer（NSCLC）expresses PD-L1 in their sampled tumors and infiltrating immune cells.The difference of PD-L1 expressions between intracellular and intercellular suggesting that sampling of tumor tissue may affect the results of PD-L1 detection.The regulation of PD-L1 expression in tumor cells is influenced by many factors,including both intracellular and extracellular tumor microenvironmental factors.In addition,it is also subject to epigenetic regulation,and the regulation process is extremely complex and even contradictory.Therefore,the control of PD-L1 expression in tumor cells should be considered from multiple perspectives.Hydrogen sulfide（H2S）,a new type of gas signal molecule,has a bidirectional“bell-shaped pharmaco-logical effect”of promoting cancer and suppressing cancer,that is,lower concentrations of H2S tend to promote cancer cell proliferation,while higher concentrations of H2S tend to inhibit cancer cell proliferation.The biosynth-esis of H2S in vivo can affect the transmission effect of cell signaling pathways,and can also influence the survival state of cells through the tumor microenvironment.Fewer reports on the relationship between H2S and lung cancer growth or lung cancer cell proliferation were found.In this study,NSCLC was used,and the PD-1/PD-Ls expression characteristics of clinical NSCLC patients were investigated.The effects of exogenous H2S（NaHS）on the proliferation of NSCLC cells and the regulation of PD-L1 expression of these cells were studied in vitro and its mechanism was explored.Based on the results of this study,the following scientific hypothesis was confirmed:H2S at low concentrations can promote NSCLC cell proliferation,and the promoting effect is related to PD-L1 up-regulation in NSCLC cells.3 parts are in the current research.Part one Study on the expression of PD-1 and its ligands in non-small cell lung cancerObjective:To investigate the positive expression rate of PD-1 and its ligands in NSCLC patients,analyze the correlation between the expression of PD-1/PD-Ls and clinicopathological features of patients.Method:A total of 48 patients with NSCLC were enrolled in this study.The lung cancer tissues of each patient were made into paraffin sections.The expression of PD-1,PD-L1 and PD-L2 was observed by immune-histochemical staining.And analyze the relationship between the expression of PD-1,PD-L1 and PD-L2 and the clinicopathological features of patients.The subjects were divided into three groups:PD-1 group,PD-L1 group,and PD-L2 group.The observations and evaluation contents of each group are the same.The results were comprehensively evaluated in terms of the patient’s general condition,the expression rate of PD-1 and its ligand,and the pathological features of the tumor.Results:1.The immunohistochemical staining test showed that PD-1 was expre-ssed in most immune cells in lung adenocarcinoma and squamous cell carcinoma,and PD-L1 and PD-L2 were in lung adenocarcinoma and squamous cell carcinoma.The positive rate of PD-1 was 35.4%,64.6%for PD-L1 and 45.8%for PD-L2.The expression of PD-1,PD-L1,and PD-L2 are independent of each other.2.The expressions of PD-1,PD-L1 and PD-L2 were not related to gender and age of the patients（P>0.05）,and were not significantly related to the survival time of lung cancer patients（P>0.05）.There was no significant difference of survival time between the patients with positive and negative expression of PD-1,PD-L1 and PD-L2（P>0.05）.3.The expression of PD-1 and PD-L2 has not related to the tumor tissue type,tumor differentiation,tumor stage or lymph node metastasis;PD-L1expression has not correlated to tumor histological type,tumor differentiation or lymph node metastasis,however,the PD-L1 expression in stage III was significantly higher than that of stage I/II（P<0.05）.Part two Effect of exogenous H2S on proliferation and PD-L1 expres-sion of NCI-H1650 cellsObjective:To examine the effect of different concentrations of exogenous H2S on the proliferation of NSCLC cells（NCI-H1650）at different time and to evaluate the expression of PD-L1 on NCI-H1650 cells.Method:NCI-H1650 cell lines were used and were incubated with different concentrations of NaHS.According to the NaHS concentration,the cells were divided into four groups:0μM group,50μM group,100μM group,and 200μM group.Each group was incubated with three observation phases of 24h,48h and 72h.At the stage of logarithmic growth,the cells prolife-rations were detected by MTT assay and the PD-L1 expressions were detected by flow cytometry.Optical density（OD）values were used to reflect the proliferation of cells in each group and relative mean fluorescence intensity（MFI）was used to indicate the PD-L1 expression levels in each group.Results:1.In the same incubation period,each exogenous H2S with concentration of 50μM,100μM or 200μM could promote the proliferation of NCI-H1650cells,and with the increasing concentration of exogenous H2S,the prolifer-ation of cells was also increased obviously（P<0.05）.2.Under the intervention of same concentration of exogenous H2S,the proliferation of cells in four groups increased with the prolongation of incub-ation period,and the proliferation of NCI-H1650 cells at different incubation period in the same concentration was significantly different（P<0.05）.3.Flow cytometry results showed that during the same incubation period,exogenous H2S promoted the expression of PD-L1 in NCI-H1650 cells in a concentration-dependent manner,and the difference was significant among groups（P<0.05）.4.Under the exogenous H2S intervention with same concentration,the expression of PD-L1 in NCI-H1650 cells increased in a time-dependent manner（P<0.05）.Part three Study on the up-regulating mechanism of PD-L1 by exogen-ous H2S of NCI-H1650 cellsObjective:To investigate the up-regulating mechanism of PD-L1expression by HIF-1αof NCI-H1650 cells in condition of incubating with exogenous H2S.Method:Human NSCLC cell line NCI-H1650 cells were cultured to logarithmic growth phase and divided into four groups:control group,H2S group,YC-1[3-（5’-hydroxymethyl-2’-furyl）-1-benzylindazole]group,and H2S+YC-1 group.The expression of HIF-1αin each group was determined by immunofluorescence cytochemistry,and the PD-L1 expression in each group was detected by flow cytometry.Results:1.The expression of HIF-1αand PD-L1 in H2S group was significantly higher than that in control group（P<0.05）.2.The expression of HIF-1αand PD-L1 in YC-1 group was significantly lower than that in control group（P<0.05）.3.Compared with the control group,the expression of HIF-1αwas significantly decreased in H2S+YC-1 group,and the expression of PD-L1 was significantly increased（P<0.05）;In H2S+YC-1 group,the expression of HIF-1αwas significantly lower than that of H2S group（P<0.05）,and the expression of HIF-1αwas decreased even more（P<0.001）.The expression of PD-L1 in H2S+YC-1 group was higher than that in YC-1 group（P<0.05）.There was no significant difference in the expression of HIF-1αbetween H2S+YC-1 and YC-1 groups（P>0.05）.Conclusions:1.The expression of PD-L1 was not related to histological type,tumor differentiation or lymph node metastasis in NSCLC patients,but was related to tumor stage.The expression of PD-L1 in stage III NSCLC was significantly higher than that in stage I/II NSCLC.The expression of PD-1,PD-L1,and PD-L2 in NSCLC is independent of each other.The expressions of PD-1,PD-L1,and PD-L2 were not significantly associated with gender,age,and survival time of NSCLC patients.2.In a certain concentration and time range,exogenous H2S promoted the proliferation of NSCLC cells and the expression of PD-L1 in a concen-tration-and time-dependent manner.The mechanism of exogenous H2S promoting NSCLC cell proliferation is likely related to its up-regulation of PD-L1 expression.3.Exogenous H2S can up-regulate the expression of PD-L1 in NSCLC cells by up-regulating the expression of HIF-1α.The mechanism of exoge-nous H2S up-regulating PD-L1 expression in NSCLC cells is not completely clear.4.Reducing the production of H2S in the tumor or suppressing the expression of HIF-1αcan be used as an effective measure to inhibit the func-tion of PD-L1.The results of the current study can provide new ideas for the development of new anti-cancer drugs and cancer immunotherapy. |
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