| As the leading cause of death among the patients diagnosed with gynecological malignancies worldwide,ovarian cancer has received increasing attention.The 5-year survival rate for advanced ovarian cancer patients is less than 30%.Surgery combined with chemotherapy is the first line of treatment.From traditional chemotherapy drugs such as platinum to molecular targeted drugs and immunotherapy,the treatment of ovarian cancer has been continuously optimized,but the desired effect has not been achieved in patients,so we urgently need to develop new anti-tumor drugs.Enzymeinstructed self-assembly(EISA)is an emerging anti-tumor therapy.Different from traditional drugs dependent of receptor-ligand interactions,which play anti-tumor roles by interfering with a specific cell function,the anti-tumor effect of EISA is often multi-step and multi-sided,with the characteristics of selectivity,high efficiency,and high biological safety.A tumor cannot develop without its nest---the tumor microenvironment.Tumor-associated macrophages(TAMs)are the largest number of immune cells in the tumor microenvironment,and their functional characteristics tend to be M2-type macrophages.With the high plasticity of macrophages,macrophages can be reprogrammed from M2-type to M1-type,which can enhance its anti-tumor effect.We prepared a small molecule compound--phospho-tyrosine cholesterol(PTC),which can initiate EISA with the action of alkaline phosphatase.In this study,we combined in vitro and in vivo experiments to study the inhibitory effect on ovarian cancer cells,the reprogramming effect on macrophages and the combined anti-tumor effect of PTC' EISA in ovarian cancer.This topic is divided into the following three parts:(1)the inhibition effect of PTC on ovarian cancer cells and its mechanism;(2)the reprogramming effect of PTC on macrophages and its mechanism;(3)combined anti-tumor effect of PTC on ovarian cancer.Objective 1.To investigate the inhibition effect of PTC on ovarian cancer cells and its mechanism;2.To investigate the reprogramming effect of PTC on macrophages and its mechanism;3.To investigate the combined anti-tumor effect of PTC on ovarian cancer.Methods 1.Prepare and characterize PTC.The morphology and particle size of PTC before and after EISA were detected by transmission electron microscopy.Expression of ALP in ovarian cancer tissues were retrieved using TCGA database.The ALP assay kit was used to detect the activity of ALP in ovarian cancer cells and normal epithelial ovarian cell.CCK8 kit was used to detect the inhibitory effect of PTC on ovarian cancer cells,and to compare the inhibitory effect of PTC with cisplatin on ovarian cancer cells.The effects of PTC on apoptosis of ovarian cancer cells were detected by flow cytometry.Western blot was used to detect the effects of PTC on expression of cleaved caspase-3,the steroid receptor activator SRC and its downstream AKT signaling pathway in ovarian cancer cells.2.To induce differentiation into macrophages,THP-1 cells were treated with PMA to generate THP-1 macrophages.To obtain THP-1 derived M2 macrophages,macrophages were then cultured with IL-4 and IL-13.To obtain RAW264.7 derived M2 macrophages,RAW264.7 cells were treated with IL-4.q RT-PCR was used to detect the expression of CD206 and IL-10.The ALP assay kit was used to detect the activity of ALP in THP-1 and RAW264.7 derived M2 macrophages.CCK8 kit was used to detect the inhibitory effect of PTC on M2 macrophages.q RT-PCR was used to detect the expression of CD206,IL-10,TNF-? and i NOS in M2 macrophages after PTC treatment.Changes in the secretion levels of IL-10 and TNF-? in the cell supernatants were detected by ELISA.Ascites macrophages were extracted from 6 patients with ovarian cancer.After 48 h of PTC treatment,the expression of CD206 and CD86 in ascites macrophages were detected by flow cytometry.The ROS detection kit was used to detect the effect of PTC on the ROS levels of THP-1 and RAW264.7 derived M2 macrophages by fluorescence microscope,flow cytometry and microplate reader.Cytoskeletons were labeled with rhodamine phalloidin,and the effect of PTC on the distribution of cytoskeletons of THP-1 and RAW264.7 derived M2 macrophages were measured by immunofluorescence and elongation factors.3.The transwell device was used to construct the co-culture system of both human and mouse ovarian cancer cells and M2 macrophages in vitro.The effect of PTC on the apoptosis of ovarian cancer cells and the expression of CD206 and CD86 in macrophages in the co-culture system were detected by flow cytometry.The secretion levels of IL-10 and TNF-in the supernatant of the co-culture system were detected by ELISA.The subcutaneous tumor model of ovarian cancer in C57BL/6 immunologically sound mice were established to observe the effect of PTC on the growth of subcutaneous transplanted tumor.Immunohistochemistry was used to detect the expression of cleaved caspase-3 in tumor tissues.The single-cell suspension of mouse tumor tissues were prepared and the expression of CD206 and CD86 in macrophages were detected by flow cytometry.Immunofluorescence was used to detect the expression of CD206,CD86,CD8 and Foxp3 in tumor tissues.The biosafety of PTC in vivo was further evaluated by the body weight changes and HE staining of heart,liver,spleen,lung and kidney.Results 1.PTC was evenly distributed and uniform in size under electron microscope.After adding ALP,the shape was round with good dispersion and uniform in size.The average particle size of PTC was 7.4±1.2 nm and 17.3±2.4 nm before and after EISA.The TCGA database showed that ALP was highly expressed in ovarian cancer.The ALP activity of ovarian cancer cell lines was higher than that of normal ovarian epithelial cell.The IC50 of SKOV3 and ID8 of PTC were 5.48±0.082?g/ml,2.15±0.075?g/ml,respectively.EISA of PTC can effectively inhibit the growth of ovarian cancer cells in a dose-dependent manner.The IC50 of SKOV3 and ID8 of DDP were 4.34±0.066?g/ml,3.41±0.066?g/ml,respectively.The inhibition efficiency of PTC was equal to or better than cisplatin.PTC could up-regulate the expression of cleaved caspase-3 in ovarian cancer cells,and the apoptosis rate of ovarian cancer cells were significantly increased.PTC could down-regulate the phosphorylation of steroid receptor activator SRC and inhibit the activation of its downstream AKT pathway.2.ALP was active in THP-1 and RAW264.7 derived M2 macrophages.The IC50 of THP-1 and RAW264.7 derived M2 macrophages were 60.95±0.296?g/ml,76.79±0.502?g/ml,respectively.EISA of PTC could down-regulate the expression of CD206,IL-10 and up-regulate the expression of TNF-?,i NOS in THP-1 and RAW264.7 derived M2 macrophages,accompanied by a decreased secretion of IL-10 and increased secretion of TNF-?.EISA of PTC could downregulate the proportion of CD68+CD206+ in ascites macrophages of ovarian cancer patients,and reduce the mean fluorescence intensity(MFI)of CD206.Although the change of CD68+CD86+ was not statistically significant,the MFI of CD86 increased.The ROS kit was used to detect the ROS levels of THP-1 and RAW264.7 derived M2 macrophages after PTC treatment.The fluorescence intensity of cells were significantly increased by the fluorescence microscopy,the number of positive cells and the MFI were increased by flow cytometry,and the OD value of cells were significantly increased by the microplate reader.Immunofluorescence of cytoskeletons labeled with rhodamine phalloidin demonstrated that THP-1 and RAW264.7 derived M2 macrophages showed a tendency of cell morphology from slender to round,with a significant reduction in elongation after PTC treatment.3.In the human co-culture system(SKOV3 and THP-1 derived M2 macrophages),the apoptosis rate of SKOV3 cells was increased,the proportion of CD68+CD206+ in THP-1 derived M2 macrophages was decreased and the proportion of CD68+CD86+ was increased,accompanied by a decreased secretion of IL-10 and increased secretion of TNF-? after PTC treatment.Similar results were observed in the mouse co-culture system(ID8 and RAW264.7 derived M2 macrophages).In vivo experiments,subcutaneous tumor growth of PTC treated mice slowed down and the tumor size was smaller than the control group.Immunohistochemistry showed a significantly higher level of cleaved caspase-3 expression in tumor tissues after PTC treatment.Flow cytometry showed that the proportion of F4/80+CD206+ in tumor tissues of PTC treatment group decreased,the MFI of CD206 decreased,and the proportion of F4/80+CD86+ increased.Immunofluorescence showed a decreased expression of CD206 and Foxp3 and an increased expression of CD86 and CD8 in the PTC treatment group.There was no significant weight loss in the PTC treatment group,and the change trend was similar to that in the control group.HE staining sections of heart,liver,spleen,lung and kidney of mice in the PTC treatment group showed no significant pathological changes.Conclusions 1.The particle size of PTC is appropriate and evenly dispersed.EISA of PTC effectively inhibited the growth of ovarian cancer cells in a dose-dependent manner,and its cancer inhibition efficiency was equal to or better than cisplatin.EISA of PTC plays a role in cancer inhibition through multiple ways: EISA of PTC not only activated caspase cascade pathway and promoted apoptosis of ovarian cancer cells,but also inhibited activation of steroid receptor activator SRC and its downstream AKT pathway,thus inhibiting the growth of ovarian cancer cells.2.EISA of PTC could not only reprogramme THP-1 and RAW264.7 derived M2 macrophages from M2 to M1,but also remodeled the ascites macrophages of ovarian cancer patients toward M1.On one hand,EISA of PTC could improve the level of ROS in macrophages,on the other hand,it could reprogramme the macrophages by regulating the distribution of cytoskeleton.3.EISA of PTC could not only inhibit the tumor growth,but also reprogrammed the macrophages from M2 to M1.Therefore EISA of PTC could play a combined anti-tumor role of "killing two birds with one stone" at both cellular and animal levels in ovarian cancer.EISA of PTC also weakened the immunosuppressive state of ovarian tumor microenvironment. |
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