| Cancer development involves dynamic interactions between tumor cells and the immune system,and a hallmark of tumors is their ability to evade immune surveillance.Despite the infiltration of tumor-specific effector T cells at tumor site,these immune cells are largely ineffective in suppressing tumor growth.A common mechanism of immune evasion for most tumors is the establishment of an immunosuppressive microenvironment,composed of infiltrating T cells and innate immune cells in addition to the proliferating tumor cells and tumor stroma.The tumor microenvironment is the primary location in which tumor cells and the host immune system interact.Characterization of the nature of immune responses in the human cancer microenvironment holds the key to understanding protective tumor immunity and improving and empowering current cancer immunotherapy.Accumulating evidence has revealed that the interaction between tumor cells and the host immune system fosters tumor immune evasion and ultimately results in tumor dissemination,relapse,and metastasis.Immune checkpoints are molecules involved in the maintenance of immunologic homeostasis and therefore help to maintain peripheral tolerance to self-molecules.Immune tolerance is critical in preventing excessive autoimmunity throughout life.The programmed cell death 1(PD1)is a prominent checkpoint receptor.Upon engagement by its ligands,PD-L1/PD-L2,in the tumor microenvironment,PD1 dampens T effector functions,thus protecting cancer cells from immune-mediated rejection.The PD1/PD-L1 signaling also has cell-intrinsic functions in certain types of mouse and human tumors through modulating downstream effectors.As it boosts cancer growth and promotes tumorigenesis,a number of antibody-based therapeutics targeting the PD1/PD-L1 axis have entered clinical trial.Despite significant progress in cancer therapy by conventional approaches such as surgery,chemotherapy and radiation,the overall survival rates of cancer patients have not been substantially improved in the last decade.Chemotherapy has been widely used to inhibit cancer cell proliferation and induce cancer cell apoptosis.However,resistance of cancer cells to chemotherapy remains a big challenge.Complementary strategies such as immunotherapy could be helpful toeradicate cancer cells when combined with chemotherapy.Nevertheless,chemopreventive agents may not only directly cause immune cell death due to their cytotoxicity,but also alter tumor-reactive immune responses.Thus,a better understanding of how chemotherapy may affect the immune responses of cancer cells,in particular,the anti-tumor immunity or immune evasion,is necessary for the improvement of the efficacy of chemotherapy combining with immune remedy.Interferon-? is a cytokine that is critical for innate and adaptive immunity.Once antigen-specific immunity develops,IFN-? is secreted by activated effector T cells.Interferon-g upregulates MHC class I and class II molecules and promotes antigen presentation on tumour cells.By these functions,IFN-? was expected to work as an antitumour agent.Nevertheless,in a clinical trial,tumour progression was promoted upon administration of IFN-? to ovarian cancer patients.The mechanisms of tumour promotion by IFN-? remain unknown.IFN-? is also known to upregulate PD-L1 expression on tumour cells.the present study examined the significance of PD1 expression in regulating the chemoresistant ability of gastric cancer against 5-FU.Furthermore,the relationship between IFN-? status and PD-L1 is assessed.Then,Our findings shed light on the relationship between IFN-?/PD-L1 expression and tumour microenvironment,and may provide a new therapeutic strategies to combat drug resistence cancer.Methods:1)The 5-FU resistant variant SGC7901/5-FU and BEL-7402/5-FU was obtained from the parent cell line by step by step exposure to increasing concentrations of5-FU.MTT assay was used to evaluate the cell viability.PD-L1 expression was analyzed by western-blot.2)To investigate the association between PD-L1 and 5-FU resistance in SGC7901/5-FU and BEL-7402/5-FU cells,PD-L1 si RNA or PD1-expression vector was transfected into cells.MTT assay was used to evaluate the cell viability.PD-L1,ABCC1 and Bcl-2 expression was analyzed by western-blot.3)BEL-7402/5-FU cells were treated with a series concentration of IFN-?(0 ?g/L,5?g/L,10 ?g/L,20 ?g/L)and 5-FU(20 ?g/m L).MTT assay was used to evaluate the cell viability.PD-L1,JAK,p-JAK,STAT3 and p-STAT3 expression was analyzed by western-blot.In addition,PD-L1 si RNA was transfected into BEL-7402/5-FU cells and followed the treatment with IFN-?(10 ?g/L)and 5-FU(20 ?g/m L),then MTT assay was used to evaluate the cell viability.Results:1)To detect the sensitivity of the SGC7901/5-FU,BEL-7402/5-FU cell line and its parental cell line to 5-FU,an MTT assay was performed.The results demonstrated that compared with the SGC7901 cell line(IC50 =2.2 ?g/m L),the SGC7901/5-FU cells(IC50 =8.16 ?g/m L)exhibited obvious resistance to 5-FU.2)The proliferation of the two cell lines were also measured with MTT method.The result showed that the proliferation of SGC7901/5-FU and BEL-7402/5-FU cells were higher than those of parental cells.3)The results indicated that PD-L1 expression was up-regulated in SGC7901/5-FU and BEL-7402/5-FU cells,suggesting that PD-L1 may contribute to 5-FU drug resistance in cancer cells,4)To investigate the association between PD-L1 and 5-FU resistance in SGC7901/5-FU and BEL-7402/5-FU cells,PD-L1 si RNA or negative control was transfected into SGC7901/5-FU and BEL-7402/5-FU cells.We examined the expression of PD-L1 in cells by IFA and western-blot,to detect the efficacy of transfection.After confirming the successful downregulation of PD-L1 in cells,MTT assay was used to evaluate the cell viability and proliferation in 96-well plate.After 24 hour of transfection,5-FU was added and incubated with cells for another 48 h.The proliferation of PD-L1 si RNA cells decreased obviously,when compared with negative si RNA cells.Moreover,we observed that SGC7901/5-FU and BEL-7402/5-FU promoted apoptosis and decreased resistance to 5-FU,when compared with negative si RNA cells,suggesting that downregulation of PD-L1 decreased the chemoresistant ability of SGC7901/5-FU and BEL-7402/5-FU.5)To further determine the effects of PD-L1 expression on chemoresistance of cancer cells,PD-L1 expression vector or the control vector was transfected into SGC7901/5-FU and BEL-7402/5-FU cells.RT-PCR analysis confirmed that the PD-L1 vector was able to increase PD-L1 expression in SGC7901/5-FU and BEL-7402/5-FU cells compared with that in the control group.The proliferation of PD-L1 expression cells(both in SGC7901/5-FU and BEL-7402/5-FU cells)increased obviously,when compared with empty vector control.The SGC7901/5-FU and BEL-7402/5-FU cells which were transfected with the PD-L1 vector exhibited a significantly higher survival rate than the cells in the empty vector control group following 5-FU exposure.Moreover,the apoptosis of PD-L1expression cells decreased obviously,suggesting that the expression of PD-L1 may promote resistance of the SGC7901/5-FU and BEL-7402/5-FU cells to 5-FU.6)BEL-7402/5-FU cells were treated with a series concentration of IFN-?(0 ?g/L,5?g/L,10 ?g/L,20 ?g/L)and 5-FU(20 ?g/m L).The result showed that the proliferation of BEL-7402/5-FU cells with IFN-? treatment were lower than those of control cells.7)The results indicated that PD-L1 expression was up-regulated in BEL-7402/5-FU cells followed the treatment with IFN-?,8)Upregulated p-JAK and p-STAT3 were observed in BEL-7402/5-FU cells followed the treatment with IFN-?.9)PD-L1 si RNA or negative control was transfected into BEL-7402/5-FU cells and followed the treatment with IFN-?.The proliferation of BEL-7402/5-FU PD-L1 si RNA cells with IFN-? treatment were lower than those of control cells.Conclusion:PD1 signaling in cancer cells could participate in the chemoresistant ability of cells in vitro,possibly through enhanced proliferation,inhibitory apoptosis and expression of MDR transporters.Moreover,IFN-? could induce PD-L1 expression and promotes tumour proliferation via JAK-STAT-3 pathway in durg resistence cell lines. |
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