Study On The Mechanism Of Triptolide Inhibition On Cisplatin Resistant Epithelial Ovarian Cancer Based On The Polarization Of TAMs

Research Background and Objective:The treatment of cisplatin resistant human epithelial ovarian cancer and advanced ovarian cancer has become a difficult problem for doctors at present.In our previous work,tripterygium lactone（TP）was found to have an inhibitory effect on cisplatin resistant ovarian cancer,and the relevant inhibitory mechanism was invest-ig-ated.Tumor-related macrophages（TAMs）can induce the growth of malignant tumors,and the polarization of TAMs plays an important role in the occurrence and development of malignant tumors,while the PI3K/Akt/Nκ-B signaling pathway plays an important role in the polarization of tumor-related macrophages.Our previous work also found that TP can inhibit the growth of ovarian cancer by blocking the PI3K/AKT signaling pathway.It was hypothesized that TP could promote the polarization of TAMs to M1-type macrophages and inhibit its polarization to M2-type macrophages by inhibiting the PI3K/AKT/NF-κB signaling pathway.Therefore,this study observed the effects of TP on ovarian tumors at the molecular,cellular,tissue and animal levels,and observed the polarization of TAMs and the changes of proteins related to the PI3K/AKT/NF-κB signaling pathway,so as to verify that TP plays an anti-tumor role by regulating the change of tumor microenvironment and regulating the PI3K/AKT/NF-κB signaling pathway.This new perspective provides the experimental and theoretical basis for the treatment of ovarian cancer and advanced ovarian cancer patients with TP as cisplatin resistant chemotherapy drugs.Methods:1.In vitro experiments1.1 Establishment of TAMs in vitro model1.1.1 THP-1 cells differentiation:the logarithmic phase of THP-1 cell lines,with containing 10%fetal bovine serum,1%glutamine,1%double resistance（penicillin and streptomycin）make up the 1640 medium THP-1 cells,adjust cell concentration for the 6*10⁵/hole vaccination in 6 orifice,adding different concentrations of PMA,PMA concentration of（0、100、200）ng/ml,finally added to the above 1640 medium 2 ml,with 0 ng/ml Bore PMA as a negative control,24 h and 48 h after observing the hole cell wall and morphological changes,to determine the optimization of PMA induced differentiation of THP-1 concentration and action time.1.2 Establishment of stable M1 and M2 macrophages:With 100 ng/ml PMA induced THP-1 cells after 24 h,When the cells are in a good state of adherence,respectively,with 100μg/ml IL-4 and 10 ng/ml LPS stimulation THP-1 cell line induced by PMA（can be respectively M2 TAMs and M1TAMs）,After cell growth was observed and centrifuged,the supernatant of each group was collected by centrifugation after 48 hours.The OD values of the supernatant IL-10 and IL-12 were detected by ELISA,and the induction of macrophages in vitro model was identified.1.3 CCK-8 method was used to detect the effect of TP on the proliferation of A2780/DDP cells:the A2780/DDP cells in the logarithmic growth period were taken as the research objects and treated with different concentrations of TP.The experiment was divided into 6 groups:（0、6.25、12.5、25、50、100）ng/ml TP,and the light absorption value（OD value）of each pore was detected by CCK-8 after the cells were treated for 24h respectively,and the IC50 value of TP on A2780/DDP cells was calculated for 24h.1.4 Observe the effects of TP on the proliferation,invasion and metastasis of A2780/DDP cells1.4..1 Influence of EDU detection of TP on proliferation of A2780/DDP cells:According to the operation method of EDU kit,the cells were divided into:（1）blank group:only medium（2）6.25ng/ml TP group（3）12.5ng/ml TP group（4）25ng/ml TP group（5）50ng/ml TP group（6）co-culture group:cells plus induced M2 macrophage supernatant.1.4.2 Cell scratch test,extracellular matrix adhesion test and Transwell test were used to detect the invasion and metastasis capacity of A2780/DDP cells after intervention with different concentrations of TP.After the tumor cells were treated with different concentrations of TP,the changes in the invasion and metastasis ability of the cells before and after the TP treatment were observed by the above three different experimental methods.2.In vivo experiments2.1 Establish subcutaneous tumor modelA2780/DDP cells were taken in the logarithmic growth period and the cell density was adjusted to 2*10⁷/ml.The mice were inoculated subcutaneously（under the armpit）,and when the tumor size was about 100mm³,they were randomly divided into five groups:negative control group,blank control group,DDP group,TP group,and combined（DDP+TP）group,with 6 nude mice in each group.2.1.1 Methods of administration for each group:（1）negative group:normal saline 50 ml/kg/d,intraperitoneal injection,administration once a day for 14 days.（2）blank group:normal saline 50 ml/kg/d,intraperitoneal injection was given once a day for 14 days.（3）DDP group:cisplatin 4 mg/kg/d,intraperitoneal injection,1,8days,2 days.（4）in the TP group,the final volume was diluted to 0.2ml with normal saline of 0.15mg/kg/d tripterygium lactone alcohol,and the drug was given intraperitoneally,once a day,for 14 days.（5）（DDP+TP）group:TP was given by intraperitoneal injection at 0.15mg/kg/d once a day for 14 days.DDP was given by intraperitoneal injection at 4mg/kg/d for 2 days.The nude mice in each group were sacrificed by bloodletting 24 hours after the administration,and peripheral blood and tumor graft specimens were collected for later use.2.2 Detection of IL-12 and IL-10 levels in peripheral blood of nude mice:the levels of IL-12 and IL-10 in peripheral blood of nude mice were detected by ELISA.First,the peripheral blood of nude mice was centrifuged,and the supernatant was retained.2.3 Measurement of weight and volume of xenograft tumor in nude mice:after the death of nude mice,the tumor was subcutomized,and the wet weight of the tumor was measured.The longest diameter（a）and the shortest diameter（b）of the tumor were measured with verisimulus caliper,and the tumor volume was calculated according to the formula V=ab²/2.2.4 TUNEL method was used to detect the apoptosis of subcutaneous transplanted tumor cells in nude mice of the TP group,and immunohist-ochemistry was used to detect the expression of CD16/32 and CD206 proteins in the transplanted tumor tissues of each experimental group.2.5 Western blotting was used to analyze the expression changes of P13K/AKT/NF-NF-κB signaling pathway related factors AKT,P-AKT,P65,P-P65 and the changes of MMP-2,MMP-9 and VEGF proteins in each group.Image J analysis software was used to calculate the average optical density of various proteins in different groups of images.Results:1.In vitro experiments1.1 Establishment of TAMs in vitro model1.1.1 THP-1 cell lines were induced by 100ng/ml PMA for 24 hours,and THP-1 cells gradually became adherent from a single circular suspension cell,with irregular morphology and obvious pseudopodia protrusions,transparent cytoplasm and good refractive power,and the number of adherent cells increased significantly.The cells grow best.Therefore,100ng/ml PMA induced thp-1 cells for 24h were selected as the induction dose and action time of macrophages.1.1.2 By ELISA,the IL-10 OD values of the M2-type macrophages before and after cell stimulation were（0.203±0.059）and（0.610±0.048）（P<0.05）,and the IL-12 OD values of the M1-type macrophages before and after cell stimulation were（0.203±0.068）and（0.613±0.057）（P<0.05）,respectively.It indicates that the induction of macrophages in vitro is feasible.1.2 Effects of TP at different concentrations on the proliferation of A2780/DDP cells:TP inhibited A2780/DDP cells,and the inhibition rate of6.25ng/ml TP on cells was（6.63±1.21）%,12.5ng/ml TP on cells was（39.43±2.46）%,and 25ng/ml TP on cells was（70.91±2.59）%.The inhibition rates of 50 and 100ng/ml TP were 100%,and the cell survival rate was 124.24%in the co-culture group.Therefore,the IC50 of A2780/DDP cells after TP treatment for24h was 12.5ng/ml.1.3 Effects of TP on proliferation,invasion and metastasis of A2780/DDP cells:1.3.1 EDU results suggested that a large number of red stained cells were found in the co-culture group,and the number of red stained cells was significantly increased compared with the negative control group.However,the number of cells in the TP group was less than that in the co-culture group,and with the increase of TP concentration,the number of new proliferating cells decreased,indicating that after the intervention of TP,the number of Edu and new proliferating cells in the DNA replication period of cells infiltrated into the cells was less,reflecting the poor cell proliferation,and the percentage of cells entering the S phase was less.1.3.2 After 24h co-culture of A2780/DDP cells treated by M2 macrophages,it was found that the distance between the cells was shortened,the cell morphology was changed,and the cell morphology was elongated into a long spindle.Co-culture of M2 macrophages with A2780/DDP cells led to active growth of tumor cells.The cell scratch experiment confirmed that the kinetic energy of tumor cells was significantly enhanced after co-culture of M2 macrophages.The kinetic energy of cells in the TP group was weaker than that in the co-culture group,and the cells in the scratch area were less than that in the co-culture group,and the number of cells in the scratch area decreased with the increase of TP concentration.Similarly,cell adhesion experiments and Transwell experiments confirmed that after co-culture of A2780/DDP cells with M2 macrophages,more tumor cells showed adhesion and crossed the compartment.The number of tumors adhering to and passing through the cell decreased after TP treatment,and this phenomenon was more obvious with the increase of TP concentration.The results showed that the migration and invasion ability of A2780/DDP cells could be enhanced after co-culture with M2macrophages,and the invasion and metastasis ability of tumor cells decreased after TP intervention.2.In vivo experiments2.1 After tumor formation in nude mice,the tumor volume and weight of each group were different,and the tumor volume of each group was:the control group（466.463±52.493）mm³,the DDP group（309.843±52.595）mm3,the TP group（223.870±22.585）mm³,and the combined group（149.460±33.670）mm³.The tumor volume of DDP group,TP group and combined group was significantly smaller than that of control group（P<0.05）.The tumor volume of TP group and DDP group was greater than that of the combined group（P<0.05）.Tumor weight of each group:control group（0.851±0.126）g,DDP group（0.541±0.055）g,TP group（0.394±0.034）g,combined group（0.244±0.047）g.The tumor weight of TP group,DDP group and combined group was significantly lower than that of control group（P<0.05）.The tumor weight of TP group and DDP group was greater than that of the combined group（P<0.05）.2.2 Detection of serum levels of IL-12 and IL-10 in peripheral blood of nude mice:（1）detection of serum IL-10 in the blank control group（104.50±5.91）pg/ml,the negative control group（39.84±4.12）pg/ml,the DDP group（66.89±12.33）pg/ml,the TP group（63.15±11.82）pg/ml,and the combined group（34.05±7.60）pg/ml.Serum IL-10 in the negative control group,DDP group,TP group and combined group was all smaller than that in the blank control group（P<0.05）.Serum IL-10 in both DDP group and TP group was higher than that in the combined group（P<0.05）.（2）detection of serum IL-12:the blank control group（37.16±4.23）pg/ml,the negative control group（167.59±8.04）pg/ml,the DDP group（80.81±6.82）pg/ml,the TP group（79.17±6.05）pg/ml,and the combined group（139.48±6.13）pg/ml.Serum IL-12 in the negative control group,DDP group,TP group and combined group was all greater than that in the blank control group（P<0.05）.Serum IL-12 in both DDP group and TP group was lower than that in the combined group（P<0.05）.2.3 HE staining of tumor tissues showed cell atypia,the nuclei were stained blue-purple,the cytoplasm was stained red,the nucleus-to-plasma ratio was increased,and megakaryocytes or multinucleates were changed.TUNEL showed that the apoptosis of the TP group was significantly higher than that of the blank control group（8.83±0.75）and TP group（18.83±1.17）under the microscope（P<0.01）.2.4 Immunohistochemical detection of the expression of CD16/32 and CD206 proteins in tumor tissues of nude tumor-forming mice in each experimental group:compared with the blank control group,CD16/32 proteins in DDP group,TP group and（TP+DDP）group were higher than that in the blank control group,indicating that the cytoplasm was stained to brown-yellow,especially in the combination group,CD16/32 proteins were strongly positive.The cytoplasm of the blank control group was flaxen and CD16/32 was weakly positive.Compared with the blank control group,CD206 protein expression in DDP group,TP group and（TP+DDP）group was lower than that in the blank control group,and part of the cytoplasm was stained to light yellow,especially in the combination group,CD206protein was weakly positive.The cytoplasm of the blank control group was stained dark yellow and CD206 protein was strongly expressed.2.5 Western-blot analysis of protein expression in each group:The results of western-blot indicated that,compared with the blank control group,the proteins of VEGF,MMP-2 and MMP-9 in the DDP group,TP group and（DDP+TP）group were lower than that in the blank control group（P<0.05）,while the expression of（DDP+TP）histones was the lowest.There was no difference in non-phosphorylated p65 and AKT in each group（P>0.05）.The expression levels of phosphorylated p65 and phosphorylated AKT in each group were lower than those in the blank control group（P<0.05）,and the expression levels of phosphorylated proteins in the combined group were the lowest.Conclusion:1.In vitro induction of TAMs is feasible.2.TP inhibited the growth of A2780/DDP cells and promoted apoptosis in a time-dose dependent manner.3.TP can induce apoptosis of human Cisplatin-resistant ovarian cancer A2780/DDP cells in collaboration with DDP,and increase the sensitivity of A2780/DDP to Cisplatin.4.The mechanism of tumor cell apoptosis induced by TP may be related to the inhibition of the PI3K/AKT/NF-κB survival signaling pathway,the down-regulation of phosphorylated AKT and phosphorylated NF-κB,and the inhibition of the expression of MMP-2,MMP-9 and VEGF proteins,thus inhibiting the polarization of TAMs to M₂-type macrophages,promoting the polarization of TAMs to M₁-type macrophages and promoting the apoptosis of tumor cells.