| Ovarian cancer is the most common female reproductive system malignancy.The onset of the disease is concealed,and most patients have been accompanied by metastases in the pelvic and abdominal cavity at the time of diagnosis.Although cytoreductive surgery and postoperative platinum plus taxane combination chemotherapy can greatly improve the short-term efficacy of ovarian cancer,70%of patients with advanced ovarian cancer will still relapse,and the 5-year survival rate has not been obvious improved.Therefore,to explore the mechanism of recurrence and metastasis of ovarian cancer,to find new therapeutic targets and methods may provide new ideas for the clinical treatment of ovarian cancer.In recent years,many studies have shown that the occurrence and development of tumors are not only closely related to the tumor cells themselves,but also closely related to their microenvironment.Tumor-Associated Macrophages(TAMs)are the most abundant types of immune cells in the tumor microenvironment.Studies have shown that TAMs in ovarian cancer tissues are mostly M2 type,and there is a clear relationship between the infiltration density of M2-TAMs and poor prognosis.Curcumin is a fat-soluble polyphenolic compound extracted from the rhizome of traditional Chinese medicines such as turmeric and genus(such as turmeric,medlar,and calamus).Studies have shown that curcumin can effectively inhibit the malignant behavior of ovarian cancer cells and has broad application prospects in the treatment of ovarian cancer.At the same time,curcumin has many functions such as anti-oxidation,anti-bacterial,anti-inflammatory,analgesic and wound healing,and has demonstrated its immunomodulatory potential in many inflammatory disease models.Therefore,this subject mainly studies the regulation of curcumin on the polarization of TAMs and its relationship with the malignant behavior of ovarian cancer,and preliminary discussion on related mechanisms.Part I:Regulation of Curcumin on Polarization of TAMsObjective:To determine the regulation of curcumin on TAMs by different concentrations of curcumin on TAMsMethods:(1)Primary TAMs were obtained from the ascites of ovarian cancer patients by immunomagnetic bead sorting for subsequent culture.(2)The effects of different concentrations of curcumin on the proliferation of human ovarian cancer cell lines SKOV3 and OVCAR-3,as well as primary TAMs,were determined by CCK-8 method.(3)Flow cytometry was used to detect the changes of TAMs phenotype after 48 hours of different concentrations of curcumin,and the optimal concentration was determined.(4)Detection of gene and protein expression of phenotype-associated cytokines by TAMs by real-time PCR and ELISA.Results:Higher concentrations of curcumin significantly inhibited the proliferation of human ovarian cancer cells.SKOV3 was more sensitive to curcumin than the OVCAR-3cell line.The concentration of 20?M and below had no significant effect on the proliferative capacity of the three cells,and was a suitable concentration range.The TAMs in the ascites of patients with ovarian cancer were mostly M2,and the proportion of CD206~+cells was 54.89±2.92(%).After 48 hours of different concentrations of curcumin,there was no significant difference in the proportion of CD86~+cells;the proportion of CD206~+cells decreased significantly,and the concentration of curcumin 20?M decreased most obviously.The secretion levels of IL-10 and IL-12 in the supernatant of the culture supernatant were detected by ELISA.The results showed that the IL-10 level in the supernatant of TAMs without the intervention of curcumin was as high as 111.74±27.37ng/L,and the intervention of 20?M curcumin for 48 h.IL-10 levels decreased significantly to 14.97±2.30 ng/L;IL-12 secretion levels increased significantly from 5.01±4.37 ng/L to 25.19±9.83 ng/L.The results of real-time PCR showed that the relative expression of M1-related cytokines IL-1,IL-12 and TNF-?mRNA increased;the relative expression of M2-related cytokines,chemokines TGF-?,CCL23 and CXCR2 mRNA decreased significantly.Conclusion:(1)Higher concentrations of curcumin can effectively inhibit the proliferation of ovarian cancer cells.(2)Sorting TAMs from ascites to construct a co-culture model of primary TAMs and ovarian cancer is a feasible solution to better reflect disease status.(3)TAMs in ascites of patients with advanced ovarian cancer showed M2 type.(4)Curcumin can regulate the polarization of TAMs and decrease the expression of M2-TAMs.The effect of 20?M curcumin is most obvious.Part II:The effect of TAMs on the malignant behavior of ovarian cancer and the intervention of curcuminObjective:To investigate the effects of TAMs on the malignant behavior of ovarian cancer and the intervention of curcumin,and to explore its related mechanisms.Methods:The culture supernatant obtained in the first part of the experiment was co-cultured with ovarian cancer OVCAR-3 and SKOV3.Considering the problem of curcumin metabolism in culture supernatant and the anti-tumor effect of curcumin itself,five groups were established in this study to further clarify that curcumin can affect the malignant behavior of ovarian cancer by regulating the polarization of TAMs.They were:blank cell group:cultured in 1640 medium;Curcumin20?M group:cultured in 1640 medium containing 20?M curcumin;TAMs group:TAMs culture supernatant collected in the first part of the experiment;TAMs(Curcumin 20?M)group:Using the first part of the experiment,the TAMs culture supernatant was treated with 20?M curcumin;TAMs+Curcumin 20?M group:The TAMs culture supernatant collected in the first part of the experiment was used,and 20?M curcumin was added.According to the above grouping,the effects of TAMs on the malignant behavior of ovarian cancer and the intervention of curcumin were investigated by CCK-8 test,single layer wound healing test and Transwell invasion test.The possible mechanisms of epithelial mesenchymal transition(EMT)related marker gene and protein expression levels and ELISA determination of cytokine levels were investigated by real-time PCR and Western blot.Results:(1)CCK-8 results showed that TAMs can significantly promote the proliferation of two ovarian cancer cells(P<0.001).Compared with TAMs group,TAMs(Curcumin 20?M)group significantly inhibited the proliferation of cells(P<0.05).There was no significant difference between the TAMs+Curcumin 20?M group and the TAMs group.In cell scratch assay and Transwell invasion assay,TAMs can significantly promote the migration and invasion of ovarian cancer cells.Compared with TAMs group,TAMs (Curcumin)group and TAMs+Curcumin group could reduce cell migration and invasion ability,and TAMs(Curcumin)group inhibited ovarian cancer migration and invasion ability.(2)In the cell scratch test,we observed that the morphology of ovarian cancer cells changed in TAMs group,TAMs(Curcumin)group and TAMs+Curcumin group compared with the blank group,and the cell morphology of TAMs group changed.Most obvious,the cell morphology is slender,extending out of the tentacles,and it has a long fusiform shape with obvious stromal cell-like changes.The morphology of SKOV3 cells was more pronounced than that of OVCAR-3 cells.The results of real-time PCR and Western blot showed that the expression of E-cadherin mRNA and protein were down-regulated in TAMs group,while the expression of N-cadherin and Vimentin mRNA and protein were up-regulated,further confirming ovarian cancer cells(SKOV3,OVCAR-3)and EMT occurred after co-culture of TAMs supernatant.The TAMs(Curcumin)group inhibited ovarian cancer cell EMT compared to the TAMs group.The results of ELISA showed that the content of TGF-?1 in the supernatant of TAMs group was as high as 4204.48+1130.17ng/L,and the content of TGF-?1 in the supernatant of TAMs(Curcumin 20?M)group decreased to 2601.79±275.02 ng/L(P<0.05).Conclusion:TAMs culture supernatant can significantly promote the proliferation,migration and invasion of ovarian cancer cells.The mechanism may be related to the secretion of a large amount of TGF-?1 by M2-TAMs,which in turn activates the TGF-?pathway to induce EMT.Low-dose curcumin(20?M)inhibits the migration and invasion of ovarian cancer cells by regulating the polarization of TAMs and inhibiting EMT in ovarian cancer cells. |
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