Investigation On The Inhibition And Mechanism Of Three Lactone Compounds On Breast Cancer Cell MCF-7

Inula helenium L.is a kind genus inula flower of the compositae herbaceous,the report shows that it contains more than 40 different components,and the most important compositions are terpenoidsandinulin.Alantolactone and Isoalantolactone are sesquiterpene lactones extractions from Inula helenium L.Aucklandia lappa.is a kind compositae herbaceous,which root has many kinds of pharmacological functions.Costunolide is sesquiterpene lactones extracted from Aucklandia lappa.The studies of three kinds of lactone compounds are focusing on its anti-inflammatory,anti-oxidate,antibacterial and antitumor activity,especially the effect of antitumor is particularly prominent.Many reports have pointed out that alantolactone,isoalantolactone and costunolide can inhibit gastric cancer,liver cancer and colon cancer,but there are few reports about the inhibitory activity and action of mechanism in breast cancer.Therefore,in this study,three kinds of lactones compounds,which including alantolactone,isoalantolactone and costunolide,were used to explore the inhibition of proliferation,metastasis and the possible mechanism on breast cancer cell.In the study,the breast cancer cell MCF-7 have been selected as the research object.The cell morphology were tested after treated with alantolactone(10,20,30 and 40 μM),isoalantolactone(100,200,300 and 400 μM)and costunolide(10,20,30 and 40 μM)under the microscope.MTT assay was used to detect the inhibitory effect of alantolactone,isoalantolactone and costunolide on MCF-7 cells at different treatment time(24 h,48 h)and different concentrations.Hoechst33258 assay and Annexin V-FITC/PI staining were used to detect the inhibition of breast cancer cell apoptotic rate under different concentrations of alantolactone(10,20 and 30 μM),isoalantolactone(100,200 and 300 μM)and costunolide(20,30 and 40 μM).Wound healing assay and colony formation assay were used to measure the invasion and colony forming abilities of the breast caner cell after treated with different concentrations of three kinds of lactone compounds.The expression level of apoptosis related proteins Bcl-2,Bax,p53,caspase3,caspase12 and metastasis associated protein MMP2,MMP7,MMP9 were measured by Western blot assay,and the expression of p38,NF-κB and Nrf2 signal pathway were also tested.The experimental results show that: In the microscopic observation,different concentrations of alantolactone,isoalantolactone and costunolide could change the shape of breast cancer cell,the cell morphology was changed from diamond to circle,and the number of the cells decreased significantly with increase of the drug concentration and time,it indicated that three kinds of lactone compounds not only change the cell morphology,but also inhibit the proliferation ability of breast cancer cells.The alantolactone,isoalantolactone and costunolide could significantly inhibit the viability and growth activity of breast cancer cell with dose-dependent and time-dependent in MTT assay.The results of Hoechst33258 assay and flow cytometry indicated that three kinds of lactone compounds with different concentrations could effectively induce the apoptosis of breast cancer cells.The experimental results of wound healing assay showed that the migration ability of the breast cancer cell treated with the different concentrations of alantolactone,isoalantolactone and costunolide was inhibited,which compared with the control group,and the breast cancer cells almost never migrate with the increase of drugs concentration and treating time,indicating that the three lactone compounds could effectively inhibit migration ability of breast cancer cell.The results of colony experiments revealed that the number of colony formation was significantly reduced after the treatment of alantolactone,isoalantolactone and costunolide,which indicated that the colony formation ability of the cells was inhibited.The Western Blot assay results showed that alantolactone,isoalantolactone and costunolide could activate p38 MAPK and induce its phosphorylation and increase the expression of pro-apoptotic protein Bax,cancer suppressor gene p53,and apoptosis related cleaved protein of Caspase-3 and Caspase-12,and down regulate of the expression of Bcl-2 protein,so the breast cancer cell was induced to apoptosis.The three kinds of lactones compounds could inhibit the activation of NF-κB(p65)signaling pathway through prevent the nuclear transfer of p65,inhibit the phosphorylation of IκB in the upstream signal pathway of NF-κB(p65),led to the decrease of IκB phosphorylation level,and reduced the expression of the metastasis related proteins induced by NF-κB,the proliferation and metastasis of cancer cells were inhibited,so the apoptosis of breast cancer cells was further induced.Alantolactone,isoalantolactone and costunolide could also induce cell apoptosis by activating Nrf2 into the nucleus.The above experimental results showed that alantolactone,isoalantolactone and costunolide could inhibit the growth,proliferation and metastasis of breast cancer cells,which indicated that it had the potential to be an anticancer drug and also provided some certain basis for the clinical application of breast cancer treatment.