Effect Of ?-Elemene On Human Breast Cancer MCF-7 Cell Line In Vitro And In Vivo And Its Mechanism

Breast cancer is the most common malignancy in women,with more than one million new cases worldwide each year.The disease ranks fifth after lung cancer,liver cancer,gastric cancer,and colorectal cancer,and its incidence increases with age.According to tumor size,histological tumor type,grade,lymph node status or hormone receptor status,corresponding treatment strategies are performed,such as surgery,targeted therapy,hormone therapy,chemotherapy,radiation therapy or a combination of both.In fact,these treatments can cause harmful damage to healthy tissue.In addition,many factors can lead to treatment failure,such as cancer cells remaining after surgery,resistance to chemotherapy,and physiological barriers to treatment,such as the blood-brain barrier and the cell barrier that prevents access to treatment.Therefore,we need natural chemical therapeutic agents with small side effects and low toxicity.?-elemene is a semi-terpenoid active compound extracted from Curcuma zedoaria.?-elemene has been approved by the State Food and Drug Administration for anti-cancer treatment,including brain cancer,breast cancer,prostate cancer,ovarian cancer and lung cancer.?-elemene has been shown to have a wide range of antitumor activities,which can inhibit cell proliferation,induce apoptosis,inhibit neovascularization,and inhibit tumor invasion and metastasis ?-elemene is a natural plant-derived ingredient,known for its efficacy,safety,and fewer adverse reactions,making it an effective drug for cancer treatment.Although ?-elemene can induce the anti-tumor effect of cancer cells from a variety of sources,the effect of ?-elemene on breast cancer is less studied,and its mechanism of action is unclear.In this study,we explored the therapeutic application of ?-elemene in breast cancer,and the molecular mechanism was further discussed.ObjectiveTo explore the inhibitory mechanism of ?-elemene on breast cancer cell MCF-7 invitro and in vivo and its related molecular mechanism.Methods:1 In vivo experiments: Female SPF grade BALB / C nude mice(28-35 days),weight 17-20 g,after constructing human breast cancer MCF-7 nude mice xenograft models,they were randomly divided into model groups,50 mg / kg ?-elemene group(low dose group),100 mg / kg ?-elemene group(high dose group)and 25 mg / kg 5-FU group(positive control group),six in each group.The ?-elemene administration group and the positive control 5-FU group were administered intraperitoneally once every other day,In model group,the same volume of 0.9% Na Cl solution was administered.The nude mice of each group were weighed daily,and the tumor size of each group of nude mice was measured using a vernier caliper every 3 days during the experiment.After 21 days,the nude mice were killed by anesthesia,the tumors were separated and weighed,and fixed in a 4% paraformaldehyde solution.The tumor mass and transplantation tumor inhibition rate of each group were calculated.And we observed the differences in transplanted tumors in nude mice with HE staining.TUNEL staining was used to evaluate the effect of ?-elemene on tumor cell apoptosis.Ki-67,LOX and Cyclin D1 protein levels were detected,and the expression level of p-m TOR protein were detected by immunofluorescence.2 In vitro experiments: Human breast cancer cells MCF-7 were selected for in vitro experiments,and ?-elemene was added to MCF-7 cell culture solution at different concentrations.MTS method,colony formation assay and carboxyfluorescein acetoacetate(CFDA-SE)labeling method were used to detect the effect of ?-elemene on cell proliferation.The cell cycle of breast cancer was detected on Beckman Coulter.The apoptosis morphology and fluorescence intensity of MCF-7 cells were detected by Hoechst 33258 staining,the mitochondrial membrane potential changes of cells were detected by JC-1 staining,and Annexin V / PI double staining to detect the change of apoptosis rate of breast cancer cells by ?-elemene.Western blot was used to detect Cleaved Caspase-9,Cleaved Caspase-7,Cleaved Caspase-3,Caspase-9,Caspase-7,Caspase-3,Bax,Bcl-2,Cyclin D1,Cyclin D3,CDK2,CD K4,CDK6,p18,p21,p27,LOX,p-m TOR and m TOR protein expression levels.Result:1 Effect of ?-elemene on breast cancer xenografts in nude mice1.1 Effects of ?-elemene on MCF-7 nude mice xenograft volume,weight and tumor inhibition rateThe weight of nude mice in the model group increased first and then decreased,while that in the administration group increased all the time.There was no significant difference in body weight between the administration group and the model group,indicating that ?-elemene had little effect on the body weight of nude mice;the tumor volume of the administration group was smaller than that of the model group,and the difference was statistically significant(p < 0.05).The results showed that compared with the model group,the inhibitory rate of ?-elemene(50,100 mg / kg)on the transplanted tumor of MCF-7 nude mice was 76.17%,83.17%,and that of the positive control group was 71.58%.1.2 Effect of ?-elemene on proliferation of MCF-7 xenografts in nude miceCompared with the model group,He staining results showed that ?-elemene(50,100 mg / kg)tumor tissue necrosis increased significantly,cytoplasmic light staining,in which ?-elemene(100mg / kg)effect was the most significant,followed by ?-elemene(50mg / kg)in the positive control group.The results of Ki-67 showed that compared with the model group,the expression of Ki-67 in tumor tissue of the positive control group and ?-elemene(50,100 mg / kg)group was significantly reduced,and the number of positive cells was decreased.1.3 Effect of ?-elemene on apoptosis of human breast cancer cell transplantation tumorsCompared with the model group,TUNEL staining results showed that 50 mg /kg and 100 mg / kg ?-elemene could increase the number of green fluorescent cells,the fluorescence intensity was getting stronger,and breast cancer cells showed an apoptotic morphology.These results showed that ?-elemene could induce apoptosis of xenografts of MCF-7 in nude mice.1.4 Effect of ?-elemene on the expression of LOX,cyclin D1 and p-m TOR in MCF-7 cellsImmunohistochemistry analysis showed that compared with the model group,?-elemene(50,100 mg / kg)significantly reduced the expression of LOX and cyclin D1 protein.Immunofluorescence staining showed that ?-elemene reduced the level of p-m TOR protein in tumor cells.These results indicated that ?-elemene could inhibit the expression of LOX and cyclin D1,and inhibit the phosphorylation of m TOR.2 Effect of ?-elemene on human breast cancer cell MCF-72.1?-elemene inhibited MCF-7 cell proliferationHuman breast cancer cells MCF-7 were treated with ?-elemene(50,100,200,300,400 ?mol / L)at different concentrations and applied to MTS experiments 24,48,and 72 hours later.The results showed that ?-elemene inhibited MCF-7 cell viability in a dose and time-dependent manner.The ?-elemene concentration was 400 ?mol / L,and the inhibition was most significant at 72 h.The results of the colony formation experiments showed that ?-elemene significantly inhibited the formation of human breast cancer cell MCF-7 clones with increasing ?-elemene(100,200,300 ?mol / L)concentration(p <0.05).CFDA-SE staining showed that compared with the blank control,as the ?-elemene(100,200,300 ?mol / L)concentration increased,the fluorescence intensity also gradually increased.2.2 ?-elemene promoted MCF-7 cell apoptosisCompared with the blank control group,?-elemene(100,200,300 ?mol / L)was applied to MCF-7 cells.As the concentration increased,the nuclei of some MCF-7cells became broken,nuclear chromatin was concentrated,and ?-elemene promoted the eversion of phosphatidylserine in the cell membrane of MCF-7 cells,reduced the mitochondrial membrane potential.Western blot showed that ?-elemene upregulated the expression of cleaved caspase-9,cleaved caspase-7,cleaved caspase-3 and Bax,reduced the expression of apoptosis-related proteins caspase-9,caspase-7,caspase-3and Bcl-2 proteins,and these results indicated that ?-elemene could induce apoptosis in human breast cancer cells.2.3 Effect of ?-elemene on the MCF-7 cycle of human breast cancer cellsCompared with the blank control group,with the increase of ?-elemene concentration(100,200,300 ?mol / L),?-elemene significantly increased MCF-7cells in the G2 / M phase in a concentration-dependent manner.The ratio indicated that ?-elemene blocked breast cancer cell MCF-7 cell cycle at G2 / M phase.Compared with the blank control group,?-elemene(100,200,300 ?mol / L)decreased the expression levels of the cycle-related proteins CDK6,Cyclin D1,Cyclin D3,p18,p21,and p27,while CDK4 protein expression increased.However,CDK2 protein levels did not change significantly.These results demonstrated that ?-elemene could block the MCF-7 cycle of breast cancer cells in the G2 / M phase.2.4 Effect of ?-elemene on the expression of LOX,p-m TOR and m TOR proteins in MCF-7 cellsCompared with the blank control group,with the increase of ?-elemene(100,200,300 ? mol / L),the expression level of p-m TOR and LOX protein decreased,but the total protein of m TOR did not change significantly.These results showed that?-elemene could down-regulate LOX protein levels and inhibit m TOR protein phosphorylation expression.Conclusion:?-elemene can significantly inhibit the growth of breast cancer xenografts in nude mice.Its antitumor effect may be related to the inhibition of proliferation and induction of apoptosis.?-elemene can also inhibit the proliferation of human breast cancer cell line MCF-7 in vitro,and induce apoptosis and cycle arrest of breast cancer cells,In vitro and in vivo experiments demonstrate that the therapeutic effect of ?-elemene on breast cancer is related to the decrease of LOX and cyclin D1 protein levels and the inhibition of m TOR phosphorylation.