| Background and objectives:Epithelial Ovarian Cancer is one of the common malignant tumors in the female reproductive system and the mortality rate is the first in gynecologic malignancies.At present,the main treatment of EOC is cytoreductive surgery and postoperative adjuvant paclitaxel plus platinum chemotherapy for 6-8 courses.Despite continuous attempts to improve surgical skills,increase the proportion of satisfactory tumor cell cytoreductive surgery,use new chemotherapeutic drugs,and combine different treatment options,80%of patients still relapse and metastasize after initial treatment,resulting in the 5-year overall survival rate has not been fundamentally improved.The study found that ovarian cancer is an immunogenic tumor and its occurrence and progression are closely related to the inflammatory microenvironment.Cytokines are secreted in the microenvironment of ovarian cancer,and exert immunosuppressive effects,which promotes immune escape in ovarian cancer.Many studies at home and abroad have found that the increase in the levels of interleukin-6(IL-6),IL-17A,and transforming growth factor beta 1(TGF-?1)is associated with poor prognosis of ovarian cancer,and TGF-?1 can induce Tregs,which is important for the maintenance of CD4~+CD25~+Treg function status,further inhibiting immunity.Sulforaphrane(SFN)is extracted from cruciferous plants.A large number of studies have shown that sulforaphrane has anti-oxidation,anti-inflammatory and induces apoptosis of tumor cells;it can interrupt the binding of LPS to TLR4/MD2 complex by directly binding cysteine 133 in myeloid differentiation protein-2.Thereby inhibiting the activation of the downstream signaling pathway.The aim of this study was to investigate the effect of SFN on the secretion of IL-6,IL-17A and TGF-?1 expressed by human ovarian cancer cells,and the effect of supernatant cultured on peripheral blood T lymphocytes on the distribution of Treg in order to improve the immunosuppressive state of ovarian cancer and improve the prognosis of patients with ovarian cancer.Methods:1.Enzyme-linked immunosorbent assay(ELISA)was used to determine the concentration of IL-6,IL-17A,TGF-?1 secretion of human ovarian cancer cells OVCAR3(MyD88~+)and A2780(MyD88~-)induced by SFN,paclitaxel(PTX),lipopolysaccharide(LPS)and SFN combined with PTX or LPS after 48 hours;2.Collecting the supernatant of human ovarian cancer cells OVCAR-3 and A2780 induced by SFN,PTX,LPS and SFN combined with PTX or LPS,co-cultivation with peripheral blood T lymphocytes for 24 hours,the distribution of Treg in each group was measured by flow cytometry.Results:1.OVCAR-3 cell can secrete IL-6,IL-17A and TGF-?1,their contents are(109.37±4.03)pg/ml,(76.01±0.18)pg/ml and(1212.54±5.97)pg/ml;A2780 cells almost no secrete IL-17A,IL-6,secrete a certain amount of TGF-?1,the content of which are(9.63±3.85)pg/ml,(12.81±1.45)pg/ml,(1033.27±41.42)pg/ml.2.SFN can reduce IL-6,IL-17A,TGF-?1 secreted by OVCAR-3 cells(P<0.05),but has no effect on A2780 cells(P>0.05),lipopolysaccharide and paclitaxel can induce IL-6,IL-17A and TGF-?1 secretion in OVCAR-3 cells,but have no effect on A2780 cells(P>0.05).SFN can reduce the secretion of IL-6,IL-17A and TGF-?1 in OVCAR-3 cells induced by lipopolysaccharide and paclitaxel(P<0.05),but has no effect on A2780 cells(P>0.05).3.The expression of CD4~+CD25~+Foxp3~+in the OVCAR-3 cell supernatant treated T cells group was significantly higher than that in the normal T cell group(7.43±0.12),and the difference was statistically significant(P<0.05).The CD4~+CD25~+Foxp3~+value of SFN-induced OVCAR-3 cell supernatant treated T cell group(4.43±0.21)was lower than that of the OVCAR-3 cell supernatant treated T cell group(10.40±0.10),and the difference was statistically significant(P<0.05);The CD4~+CD25~+Foxp3~+value of LPS-induced OVCAR-3 cell supernatant treatment T cell group(11.40±0.56)was higher than that of OVCAR-3 cell supernatant treatment T cell group(10.40±0.10),the difference was statistically significant(P<0.05);The CD4~+CD25~+Foxp3~+value of PTX-induced OVCAR-3 cell supernatant treatment T cell group(11.13±0.32)was higher than that of OVCAR-3 cell supernatant treatment T cell group(10.40±0.10),the difference was statistically significant(P<0.05);4.The CD4~+CD25~+Foxp3~+value of the T cell group induced by SFN combined with LPS-induced OVCAR-3 cell supernatant(9.23±0.31)was lower than that of LPS-induced OVCAR-3 cell supernatant treated T cell group(11.40±0.56),statistically significant(P<0.05);The CD4~+CD25~+Foxp3~+value of the T cell group induced by SFN combined PTX-induced OVCAR-3 cell supernatant(8.87±0.89)was lower than that of the PTX-induced OVCAR-3 cell supernatant treated T cell group(11.13±0.32),statistically significant(P<0.05);the expression of CD4~+CD25~+Foxp3~+in the T cell group of A2780 cell supernatant(8.87±0.35)was significantly higher than that in the normal T cell group(7.43±0.12),and the difference was statistically significant(P<0.05),there was no significant difference in the expression of CD4~+CD25~+Foxp3~+in the T cell groups treated by supernatant of A2780 cells induced by different drugs(P>0.05).Conclusion:1.Sulforaphrane can down-regulate the secretion of IL-6,IL-17A and TGF-?1by human ovarian cancer OVCAR-3 cells by blocking the TLR4/MyD88 pathway,but not for human ovarian cancer A2780 cells.2.There are a large number of pro-inflammatory and immunosuppressive factors in MyD88~+human ovarian cancer OVCAR-3 cell culture supernatant,and it can promote the expression of CD4~+CD25~+Foxp3~+Treg cells when co-culture of peripheral blood T lymphocytes,so that the body is in immunosuppressive state.Moreover,PTX and LPS can promote immunosuppressive state;while SFN can inhibit the expression of CD4~+CD25~+Foxp3~+Treg cells by blocking the TLR4/MyD88pathway and down-regulating the immunosuppressive factors secreted by MyD88human ovarian cancer OVCAR-3 cells to decrease its distribution percentage,alleviate the body's immunosuppressive state,and finally down-regulate the immunosuppressive effect. |
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