The Study Of A Natural Compound,Ilamycin E Against Triple-Negative Breast Cancer

BackgroundBreast cancer is the most common malignant cancer among women world-wild.It's the leading cause of cancer death in women.Triple-negative breast cancer(TNBC),which accounts for 15%-20%of breast cancers,lacks expression of estrogen receptor(ER)?progesterone receptor(PR)and human epidermal growth factor receptor-2(HER-2).Because of the high metastasis and relapse risks,TNBC is the most malignant subtype of breast cancers without efficient cancer therapies.The cytotoxicities of the Ilamycins have not been reported previously.Thus further study is still needed to explain the potential anti-tumor activity of Ilamycins.Methods1.SRB assayIn order to find out the potential anti-tumor activity of Ilamycins,SRB assays were performed using breast cancer cell lines HCC 1806,HCC 1937,MDA-MB-468,MDA-MB-231,T47D,MCF7,SKBR3,BT474 and MCF10A cell line.The cell viability at different drug dosages were plotted in EXCEL and IC50 values were obtained from the graphs.2.DNA synthesis assayDNA synthesis of HCC1937 and MDA-MB-468 were tested with the Click-iT EdU microplate assay kit(Invitrogen,Carlsbad,CA)according to the manufacturer's protocols.Finally,3 fields were randomly captured,and EdU positive cells and total cell number were counted for each sample.The EdU positive cell were divided by total cell number as results for analysis.3.Cell cycle analysisAfter treated with Ilamycin E for 24hr,Adherent and detached HCC1937 and MDA-MB-468cells were digested,harvested,and fixed in 75%ethanol at 4 ?.1X106 cells were resuspended in 100ul 0.6%NP-40(2mg/ml RNaseA,O.lmg/ml propidium iodide)incubated for 30 min at 37 ? in dark.The cells were then analyzed on an Accuri C6 flow cytometer(BD).4.Cell apoptosis analysisHCC1937 and MDA-MB-468 cells were treated with Ilamycin E for 24-36hr,cells were harvested and stained with anti-Annexin V antibody(eBioscience)and propidium iodide,and then analyzed by an Accuri C6 flow cytometer(BD)5.Western blotting analysisAfter incubating with Ilamycin E for 0-48hr,discard the culture media,cells were collected using lysis buffer(with 1%P8340),and then the cell lysate was centrifuged and the resulting supernatant was collected.Then mixed with the loading buffer and subjected to SDS-PAGE and transferred onto poly(vinylidene fluoride)(PVDF)membranes.The membranes were incubated with diluted primary antibodies horseradish peroxidase(HRP)conjugated secondary antibodies.After Western lighting chemiluminescence reagent plus added,viewed on an ImageQuant LAS4000 Biocompound imager to visualize the expression levels of specific proteins.6.Statistical analysisAll of the results were shown as Mean±SD,and statistical analysis were performed by Student's t-test using the SPSS program(version 12.0)(SPSS,Chicago,IL,USA).P<0.05 was defined to be statistically significant(*,P<0.05,**,P<0.01,***,P<0.001).Results1.Ilamycin E inhibits breast cancer cell viability.Ilamycin E,but not Ilamycin F exhibits potential inhibition of breast cancer cell lines HCC 1806,HCC 1937,MDA-MB-468,MDA-MB-231,T47D,MCF7,SKBR3,BT474 and MCF10A cell line.And HCC1937 was the most sensitive cell line with the lowest IC50 14.24?M.2.Ilamycin E significantly inhibits TNBC cells' DNA synthesisEdU assay shows Ilamycin E blocked DNA synthesis in TNBC cell lines HCC1937 and MDA-MB-468.3.Ilamycin E leads to TNBC cells G1/S phase arrest.When treated with Ilamycin E(0,10,20,30 ?M)for 24hr,the G0/G1 phase of HCC1937 and MDA-MB-468 cells were significant increased,whereas the S and G2/M phase of cells were decreased.It shows that Ilamycin E can significantly leads to G1/S phase arrest of HCC1937 and MDA-MB-468 cell lines.4.Ilamycin E induces cell apoptosis of TNBC cell lines.After treatment with Ilamycin E for 24-36hr,Annexin V positive cells were significantly increased,which shows a dose-dependent manner in both HCC1937 and MDA-MB-468 cell lines.5.Ilamycin E regulates the expression of cell cycle,apoptosis related genes in TNBC cell lines.Western blot results show that Ilamycin E can dramatically reduce the protein levels of cyclin D1,and increase expression of p21 and p27.Consistent with the result of cell apoptosis in flow cytometry,it turns out an increase of apoptotic proteins cleaved PARP,cleaved caspase 3,7,9 and a deregulation of anti-apoptotic proteins Bcl-2 and Survivin.Further study shows that Ilamycin E inhibits KLF5 but induces EGR1 transcription and activates ER-Stress pathway.ConclusionIn this study,Ilamycin E,a natural compound extracted from marine microorganism,showed potent anti-cancer activity in triple-negative breast cancer cell lines.Ilamycin E significantly reduced the expression of Cyclin D1 and increased that of p21 and p27,and led to cell cycle G0/G1 phase arrest.Ilamycin also effectively induced apoptosis likely via inducing ER stress-intrinsic apoptosis pathway in both HCC1937 and MDA-MB-468 breast cancer cell lines.Furthermore,Ilamycin E significantly decreased the expression of KLF5,and induced the expression of tumor suppressor EGR1.These results suggest that Ilamycin E may inhibit triple-negative breast cancer and provide a new strategy for breast cancer therapy.