The Effect Of GDF15 On Curcumin-Induced Ferroptosis In Triple Negative Breast Cancer Cells

BackgroundTriple-negative breast cancer(TNBC)accounts for about 15%-20% of all breast cancer cases.Due to the lack of effective therapeutic targets,there is no effective treatment method other than chemotherapy in systemic therapy,so the recurrence and metastasis rate is high and the prognosis is poor.Ferroptosis is a programmed death mode first discovered and proposed by Dixon in 2012.It is an iron-dependent form of cell death caused by excessive lipid peroxidation.It can be induced by drugs like erastin,sorafenib,artemisinin and their derivatives.Curcumin,a polyphenol extracted from turmeric,is a safe active ingredient in traditional Chinese medicine and has strong anti-tumor effects.Studies have shown that it can also induce ferroptosis in tumor cells.Growth differentiation factor-15(GDF15)is highly expressed in various malignant tumors,and high levels of serum GDF15 in patients often predict poor prognosis.Studies have shown that GDF15 is involved in the regulation of iron metabolism-related genes,so it may also play a regulatory role in the process of ferroptosis.It is hoped that this study can provide new means for triple-negative breast cancer treatment.Chapter I Curcumin induces ferroptosis in TNBC cells ObjectiveTo determine whether curcumin induces ferroptosis in human TNBC cell lines MDA-MB-231 and BT549.MethodsTreating human TNBC cell lines MDA-MB-231 and BT549 with curcumin/erastin and ferrostatin-1 to confirm whether curcumin induces ferroptosis in these two cell lines.The cell viability was detected by CCK-8 assay.Treating MDA-MB-231 cells with curcumin,and the intracellular iron ion content,glutathione(GSH)content and lipid peroxide(Lipid ROS)accumulation level were detected.Results1.After curcumin treatment of MDA-MB-231 and BT549 cells,the cell viability decreased with the increase of concentration,and the inhibitory effect of curcumin was about 80% of the level of erastin.2.Ferrostatin-1 rescued the curcumin-induced decreased cell viability of MDA-MB-231 but it failed to rescue that in BT549 cells.3.Curcumin induced the increase of Iron Ion level and lipid ROS accumulation in cells,and induced the decrease of glutathione in cells(p<0.01).ConclusionCurcumin induces ferroptosis in TNBC cell line MDA-MB-231,and cause ferroptotic characteristic metabolic changes.Chapter II Effects of GDF15 on curcumin-induced ferroptosis in TNBC ObjectiveTo study the role and mechanism of GDF15 in curcumin-induced ferroptosis in TNBC cells through in vitro cell experiments and in vivo animal xenograft experiments.Methods1.Treating MDA-MB-231 with curcumin,m RNA and protein levels of GDF15 were detected by real-time quantitative PCR and Western Blotting.2.MDA-MB-231 cells were transfected with GDF15 specific si RNA.Groups were set up in the form of control group,GDF15 knockdown group,control+curcumin group,GDF15 knockdown+curcumin group.Cell activity,intracellular iron ion content,GSH content,and lipid ROS were detected in each group.m RNA and protein levels of GDF15,SLC7A11,GPX4,FTH1,and FTL were detected.3.After knocking down the GDF15 by CRISPR/Cas9,MDA-MB-231 cells were transplanted into BALB/c mice.After tumor formation,the mice were intraperitoneal injected with curcumin and the tumor size was measured,recorded and pictured.Results1.After curcumin treatment of MDA-MB-231,the m RNA and protein levels of GDF15 decreased with increasing curcumin concentration(p<0.001).2.Knockdown of GDF15 expression promoted curcumin-induced cytotoxicity,increase in cellular iron content and lipid ROS,and decrease in GSH content(P<0.01).It also promoted curcumin-induced protein levels decrease of GPX4,SLC7A11,FTL and FTH1(P<0.05).3.After curcumin treatment,the tumor volume and weight of nude mice in GDF15 knockdown group were significantly lower than those in control group(P<0.05).ConclusionKnockdown of GDF15 inhibits the expression of SLC7A11,GPX4,FTH1 and FTL,thereby affecting the function of system Xc-and the content of intracellular iron ions,and promoting curcumin-induced ferroptosis in MDA-MB-231 cells.In vivo experiments further verified that down-regulation of GDF15 promoted the antitumor activity of curcumin on subcutaneously transplanted tumors in nude mice.Chapter III Expression of GDF15 before and after neoadjuvant chemotherapy in breast cancer and its relationship with the efficacy of neoadjuvant chemotherapy ObjectiveThrough the expression changes of GDF15 before and after neoadjuvant chemotherapy(NAC)in breast cancer and its relationship with the efficacy of NAC,to explore whether it can be a new therapeutic target in breast cancer treatment or a predictor of neoadjuvant chemotherapy.MethodsThe online bioinformatics tool UALCAN was used to analyze the expression of GDF15 in breast cancer tissue and normal breast tissue,and KM plotter was used to analyze the relationship between m RNA level of GDF15 expression and the prognosis of breast cancer patients;79 breast cancer patients who received NAC were enrolled in this study.Core needle biopsy specimens before NAC and tumor resection specimens after NAC were collected to detect the expression of GDF15,x CT,and GPX4 protein levels in each sample by immunohistochemistry and immune response score(IRS)standards.The correlation of clinicopathological factors,the correlation of GDF15 expression with x CT and GPX4 expression,and the effect of GDF15 expression on the curative effect of patients.Conclusion1.GDF15 is highly expressed in breast cancer compared with normal breast tissue.Breast cancer patients with high GDF15 expression are faced with lower recurrence-free survival and overall survival low GDF15 expression patients(p<0.05).2.The high expression of GDF15 in breast cancer patients is correlated with ER-negative and triple-negative subtype(p=0.041,0.038,respectively).3.The expression of GDF15 in breast cancer patients is positively correlated with the expression of ferroptosis-related proteins x CT and GPX4.(x CT: r=0.495,p<0.01;GPX4: r=0.478,p<0.01).4.The expression of GDF15 was significantly decreased after NAC(p<0.001).In the subgroup analysis,the above changes still existed in the population with good curative effect(CR+PR),while in the population with poor curative effect(SD+PD),there was no significant difference in the expression of GDF15 before and after NAC(P> 0.05).5.The degree of decline of GDF15 expression is positively correlated with the efficacy.(r=0.477,p<0.01)ConclusionGDF15 is highly expressed in breast cancer tissues,and high expression of GDF15 in breast cancer patients predicts poor prognosis.The expression of GDF15 in breast cancer patients is positively correlated with the expression of ferroptosis-related genes;NAC reduces the expression of GDF15 in breast cancer tissue,and this change is more obvious in patients with good curative effect.The change of its expression level is related to curative effect.