Effect Of Caffeic Acid3,4-dihydroxy-phenethyl Ester On Apoptosis Of Human Breast Cancer Cells

Breast cancer is one of the most prevalent tumors and does serious harm towomen’s health. The abnormal activation of growth factors and their receptors playsan important role in breast cancer. Extracting active ingredients from phytomedicineswith low toxicity against growth factors has always been the focus of modern medicalresearch. Caffeic acid3,4-dihydroxy-phenethyl ester (CADPE), a compoundoriginally isolated from medicinal plants Sarcandra glabra and Teucrium pilosum, hasattracted more interests due to its proven pharmacologic safety and its wideanti-tumor biologic activities. In the present study, we investigated the effects ofCADPE on the breast cancer cells, and explored the related mechanism as follows.Results in the study suggest that CADPE could inhibit breast cancer cell proliferationand induce apoptosis, and it is expected to become effective antagonists of growthfactors.Methods1. Assay of Cell proliferationProliferation of MCF-7, MCF-7ADR, MDA-MB-231and MDA-MB-435cellswas assayed by cell proliferation kit.2. Examination of cells apoptosisThe changes of nucleic morphologies were observed under the fluorescencemicroscopy by Hochest-33342staining. The apoptosis rate was assayed by flowcytometry detected after Annexin V-PE and7-AAD staining.3. Analysis of mitochondrial apoptosis pathwayAfter the cells were cultured in RPMI1640culture medium containing10%fetalcalf serum and different concentrations of CADPE, the reactive oxygen species (ROS)levels were examined with ROS fluorescence probes2’,7’-dichlorodihydrofluoresceindiacetate (DCFH-DA), and the mitochondrial membrane potential was detected byJC-1staining. The expressions of Bcl-2, Bax and caspase-3proteins were measured by Western blotting.4. Assay of Growth factor receptors and respective phosphorylationThe serum-starved MDA-MB-231and MDA-MB-435cells were treated withCADPE for24h and subsequently stimulated by VEGF, EGF, PDGF and HGFrespectively for15min. The cells were collected and subjected to western blot analysisto test the activity of VEGFR, EGFR, Src and C-Met phosphorylation.5. Western blotting for assay of expressions of Her-2, c-myc and maz expressionsMDA-MB-231and MDA-MB-435were treated with CADPE and then wereanalyzed Her-2, c-myc and maz expression by Western blotting.6. The growth of breast cancer MDA-MB-231cells in the Nude MiceWhen the tumors appeared, the nude mice were administered with a single doseof2.5mg/kg CADPE twice a week and normal saline as control. The tumor size andbody weight of each mouse were observed at least twice a week.Results1. Changes of proliferation of breast cancer cells treated by CADPEThe results showed that CADPE suppressed the proliferation of breast cancerMCF-7, MCF-7ADR, MDA-MB-231and MDA-MB-435cells.2. Effect of CADPE on apoptosis of breast cancer cellsAfter MDA-MB-231and MDA-MB-435cells were treated with CADPE for24h, the Hoechst staining results showed that the CADPE treated cells had typicalmorphologic characteristics of cell apoptosis. Annexin V-PE/7-AAD double stainingand flow cytometric analyses showed that the number of apoptotic cells increased inall these CADPE treated groups of MDA-MB-231and MDA-MB-435cells,compared with the control group. The apoptosis rates in20μmol/L of CADPE treatedgroups for48h were significantly higher than the0μmol/L CADPE treated groups(p<0.01).3. ROS production in breast cancer cells induced by CADPEWhen the MDA-MB-231and MDA-MB-435cells were treated with0μmol/L,10μmol/L,20μmol/L,40μmol/L and80μmol/L of CADPE for48h, the ROS level ofcells were up-regulated significantly.4. Disruption of mitochondrial membrane potential in breast cancer cells treatedby CADPETo determine the changes of mitochondrial membrane potential in MDA-MB-231 and MDA-MB-435cells after CADPE treatment, JC-1staining was carried out. Thefluorescence microscope observation showed progressive loss of red JC-aggregatefluorescence and appearance of green monomer fluorescence in the cytoplasm,thatmeaned depletion of mitochondrial membrane potential occurring.This decreaseoccurred in a dose-dependent manner.5. Effects of CADPE on expression of caspase-3, Bax and Bcl-2in breast cancercellsThe results of Western Blot showed that CADPE treatment increased theexpression of caspase-3and Bax, but decreased Bcl-2expression in a dose-dependentmanner.6. Inhibition of phosphorylation of VEGFR, EGFR, C-Met in breast cancer cellstreated by CADPEAfter MDA-MB-231cells and MDA-MB-435cells were treated with VEGF,EGF, PDGF and HGF respectively, corresponding phosphorylation level of VEGFR,EGFR, Src and C-Met were significantly increased,while the phosphorylation level ofVEGFR, EGFR and C-Met changed relatively less in the groups which previouslyincubated with CADPE.7. Effects of CADPE on phosphorylation of EGFR in breast cancer cells in doseand time-dependent mannerThe expression of p-EGFR was decreased with the prolonged time of incubationwith CADPE,or with the CADPE dose increasing.8. Expression of Her-2, c-myc and maz in breast cancer cells treated by CADPEMDA-MB-231and MDA-MB-435cells were treated with CADPE, theexpression of Her-2, c-myc and maz reduced compared to untreated group.9. Effect of CADPE on growth of human breast cancer xenografts in nude miceThe tumor volumes of CADPE group were significantly smaller than that of thecontrol group after38、42days (P=0.046,0.025), The tumor weights of two groupswere different statistically (P=0.017). But mice weight of the two groups had nosignificant difference.Conclusions1. CADPE represented a significant cytotoxicity in breast cancer cell linesMCF-7, MCF-7ADR, MDA-MB-231and MDA-MB-435cells. It could suppress the proliferation in dose-dependent and time-dependent mode. CADPE could alsosuppress the growth of MDA-MB-231human breast cancer Xenografts in Nude Mice2. CADPE elevated the expression of caspase-3, Bax and delevated theexpression of Bcl-2. CADPE could down regulate the Mitochondrial MembranePotential and up-regulated the ROS level of these cells. The mitochondrial signalpathway took part in the process of CADPE inducing apoptosis in breast cancer celllines.3. CAPDE could suppress the activity of VEGFR, EGFR and C-Met whichmaybe the part mechanism responsible for inhibitting cell growth and inducingapoptosis.4. CADPE could suppress Her-2, c-myc and maz protein expression in breastcancer cells indicating which have a negative regulatory role in these landmarkoncogenes in breast cancer.In conclusion, CADPE can inhibit breast cells proliferation activity and induceapoptosis through mitochondrial apoptosis pathway.CADPE could inhibit tyrosinekinase receptor activity including EGFR,VEGFR and C-Met and inhibit oncogeneexpression of Her-2, c-myc and maz.It maybe the part mechanism responsible forinhibitting cell growth and inducing apoptosis. Thus, these results suggest thatCADPE maybe could be utilized as anticancer drug against breast cancer.