Tumor Biol. (2014) 35:11781–11789 DOI 10.1007/s13277-014-2304-3
Inducing apoptosis effect of caffeic acid 3,4-dihydroxy-phenethyl ester on the breast cancer cells Jiaoyuan Jia & Mei Yang & Yu Chen & Hongyan Yuan & Jinghe Li & Xueling Cui & Zhonghui Liu
Received: 5 May 2014 / Accepted: 1 July 2014 / Published online: 5 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract To explore the antitumor effect of caffeic acid 3,4dihydroxy-phenethyl ester (CADPE) on the breast cancer cell lines and illuminate the related mechanism. After treatment with different concentrations of CADPE for 24, 48, and 72 h, cell proliferation ability of the breast cancer cell lines MDAMB-231 and MDA-MB-435 was analyzed by the MTT. Changes of the cell cycles were evaluated by PI staining. Cell apoptosis was examined by flow cytometry after Annexin V/7AAD double staining. Nuclear morphologic changes were observed under the inverted fluorescence microscope after staining with Hoechst 33342. Mitochondrial membrane potential and reactive oxygen species (ROS) level were estimated by JC-1 and DCFH-DA staining. In addition, the expression level of mitochondrial signaling pathway proteins Bcl-2, Bax, and caspase-3 were evaluated by Western blot. CADPE has the distinct cytotoxic effect to the breast cancer cells, and J. Jia : Z. Liu (*) Department of Immunology, College of Basic Medical Sciences, Jilin University, Changchun, Jilin 130041, China e-mail: [email protected]
Y. Chen Department of Obstetrics and Gynaecology, Changhai Hospital, The Second Military Medical University, Shanghai 200433, China H. Yuan Department of Immunology, College of Basic Medical Sciences, Jilin University, Changchun, Jilin 130041, China M. Yang Department of Pathology, The Second Hospital, Jilin University, Changchun, Jilin 130041, China J. Li Department of Oncology and Hematology, The Second Hospital, Jilin University, Changchun, Jilin 130041, China X. Cui Department of Genetics, College of Basic Medical Sciences of Jilin University, Changchun, Jilin 130041, China
the effect is dose dependent. It did not change the cell cycles but induced the cell apoptosis of the breast cancer cells. At the same time, after CADPE treatment, the expression levels of caspase-3 and Bax in the breast cancer cells were upregulated and Bcl-2 expression was declined. The ROS level in the breast cancer cells was enhanced, and mitochondrial membrane potential of the cells was downregulated. CADPE has the antitumor functions. It can induce the cell apoptosis through downregulating Bcl-2 expression, enhancing Bax and caspase-3 expression levels, upregulating ROS level and reducing the mitochondrial membrane potential of the breast cancer cells to trigger the mitochondrial signal pathway.
Keywords Caffeic acid 3,4-dihydroxy-phenethyl ester . Breast cancer . Apoptosis . ROS
Breast cancer is the most prevalent tumor and a major cause of morbidity and mortality among women . Each year, more than one million new cases appeared all over the world and brought serious harm to women health . Surgery together with chemotherapy is the main method for breast cancer treatment; the chemotherapy cannot only relieve or cure part of the tumor but it can also bring serious side effects to the patients, so finding a new therapeutic schedule with high efficiency and low toxicity has always been the target of the medical research. Caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE), a compound originally isolated from medicinal plants Sarcandra glabra and Teucrium pilosum , has attracted more interests due to its proven pharmacologic safety and its many biologic activities, such as suppression of hepatocellular carcinoma growth  and gastric carcinoma cell migration . In the present study, we investigated the effects of CADPE on the breast cancer cell lines MDA-MB-231 and MDA-MB435 and explored the related mechanism.
Materials and methods Materials Culture medium RPMI-1640, trypsin-EDTA, penicillin/ streptomycin, and fetal calf serum were bought from Gibco Company (Grand Island, NY, USA). MDA-MB-231 and MDA-MB-435 cell lines were obtained from American Type of Culture Collection (ATCC; Manassas, VA, USA). Hoechst 33342, Ribonuclease A solution, and proteinase K were the products of Sigma-Aldrich (St. Louis, MO, USA). Primary and secondary antibodies of caspase-3, β-actin, Bax, and Bcl2 for Western blot analysis were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Other chemicals and reagents used in this study were of analytical grade. Cell culture and treatment The MDA-MB-231 and MDA-MB-435 cells were cultured in RPMI-1640 culture medium supplemented with 10 % FBS and antibiotics under a fully humidified atmosphere and 37 °C and 5 % CO2 condition. Cells in the log phase were treated with various concentrations of CADPE synthesized in our lab as described before . Cell proliferation assay to investigate the effect of CADPE on cell proliferation of MDA-MB-231 and MDA-MB-435 cells with Cell proliferation was tested by Cell titer 96 ® AQueous nonradioactive cell proliferation assay. Briefly, log phase MDAMB-231 and MDA-MB-435 cells were treated with 0.25 % trypsin to make single-cell suspension with RPMI-1640; the cell concentration was adjusted to 5×107/L, and 100 μl of this single-cell suspension was planted into a 96-well plate and incubated at 37 °C with 5 % CO2 for 24 h. The medium was replaced with 100 μl RPMI-1640 medium containing different concentrations of CADPE (0, 10, 20, 40, and 80 μmol/l) and 10 % fetal calf serum, and cells were cultured at 37 °C and 5 % CO2 condition for 0, 24, 48, and 72 h. Then, 20 μl Cell titer 96 ® AQueous nonradioactive cell proliferation assay reagent (Promega, USA) was added to each well for 4 h of additional incubation. The absorption values at 490 nm were determined with an automatic enzyme-linked immunosorbent assay plate reader (BMG POLARstar Omega, BMG Labtech, Germany). Cell cycle analysis After treated with 0, 20, and 40 μmol/l CADPE for 48 h, MDA-MB-231 and MDA-MB-435 cells were collected and washed twice with phosphate-buffered saline (PBS). DNA of the washed cells (1×106) was subjected to incubation with
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600 μl PI solution (Triton X-100, 0.1 %; PI, 20 μg/ml; RNase, 200 μg/ml) for 20 min, the cell cycles were determined with flow cytometry and analyzed utilizing CellQuest software (BD company, USA). Hoechst 33342 staining After treatment with series concentrations of CADPE for 24 h, breast cancer cells MDA-MB-231 and MDA-MB-435 were fixed with 4 % formaldehyde solution and washed with PBS for three times. After stained with 10 μmol/l of Hoechst 33342 for 10 min, the cells were washed twice with PBS again, and the changes of nucleic morphologies were observed under the fluorescence microscopy (MPS60, Leica Microsystems Welzlar GmbH, Bensheim, Germany). Flow cytometry analysis after Annexin V-PE and 7-AAD double staining Apoptosis rates of MDA-MB-231 and MDA-MB-435 cells were measured after Annexin V-PE/7-AAD (BD Pharmingen, San Diego, CA, USA) double staining according to the instruction of the kit. Briefly, MDA-MB-231 and MDA-MB435 cells were cultured with RPMI-1640 culture medium containing 10 % fetal calf serum and different concentrations of CADPE in 6-well plates, collected and washed twice with PBS solution, and then rewashed with binding buffer (0.01 M HEPES/NaOH (pH 7.4), 0.14 M NaCl, and 2.5 mM CaCl2). Subsequently, cells were stained with Annexin V-PE/7-AAD at room temperature and dark condition for 15 min. The cells were washed with cold PBS for three times and detected with flow cytometry (BD Company, USA). The cells marked as Annexin V− and 7-AAD− were considered viable cells, Annexin V+ and 7-AAD− were counted as early apoptotic cells, Annexin V+ and 7-AAD+ cells were late apoptotic cells, and Annexin V− and 7-AAD+ were necrotic cells. Mitochondrial membrane potential measurement after JC-1 staining The mitochondrial membrane potential was determined using the mitochondria-specific lipophilic cationic fluorescence dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) detection kit according to the manufacturer’s instructions (Abnova Corporation, Walnut, CA, USA). In brief, MDA-MB-231 and MDA-MB-435 cells were treated with 10 or 20 μmol/l CADPE, co-cultured with 100 μl JC-1 for 30 min at 37 °C and dark condition, washed twice with PBS followed by measurement of the fluorescence intensity by fluorescent microscopy (Carl Zeiss MicroImaging GmbH, Goettingen, Germany). JC-1 can enter mitochondria selectively. In healthy cells with high mitochondrial membrane potential, the dye reagent aggregates and emits red
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fluorescence (normal membrane potential). When the mitochondrial membrane potential collapses, JC-1 cannot accumulate in the mitochondria, the cells will show only green fluorescence (decline membrane potential). The examination of reactive oxygen species (ROS) level in breast cancer cell lines MDA-MB-231 and MDA-MB-435 The breast cancer MDA-MB-231 and MDA-MB-435 cells at logarithmic phase were planted into 24-well flat-bottom plates, 6×104 cell/well, in RPMI-1640 culture medium containing 10 % fetal calf serum and CADPE (the final concentrations of CADPE are 0, 10, 20, 40, and 80 μmol/l) under 37 °C and 5 % CO2 condition for 48 h. Then collected the cells and examined their ROS level with ROS fluorescence probes 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA; Invitrogen, USA). Briefly, discarded the supernatant of the cells, added 200 μl DCFH-DA (10 μmol/l) diluted with RPMI-1640 culture medium, and cultured the cells under 37 °C and 5 % CO2 and no-light condition for 1 h. Washed the cells with RPMI-1640 culture medium for three times, discarded the supernatant, and analyzed the fluorescence intensity with fluorescence microplate (exCitation wavelength w as 488 nm, emission wavelength was 525 nm). The examination was repeated three times. Expression of Bcl-2, Bax, and caspase-3 proteins by Western blot MDA-MB-231 and MDA-MB-435 cells were treated with series concentrations of CADPE (0, 10, and 20 μmol/l), collected and washed twice with cold PBS. Then cells were incubated with RIPA buffer (50 mmol/l Tris–HCl (pH 7.5), 150 mmol/l NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 2 mmol/l EDTA (pH 8.0), and 0.1 % SDS) at 4 °C for 30 min. After 13,000×g centrifugation at 4 °C for 15 min, protein concentrations were determined by the bicinchoninic acid assay (BCA) using bovine serum albumin as standard. Forty micrograms of total cellular proteins and 10 μl of 2× sample buffer were mixed together and subjected to heat treatment at 100 °C for 5 min to denature the proteins. The proteins were separated with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; 12 % for caspase-3, Bcl-2, and Bax), transferred onto a polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) using an electrotransfer system (0.8 mA/cm) (Ellard Inc., Seattle, WA, USA). The nitrocellulose membrane was blocked in the blocking buffer (5 % skim milk) at room temperature for 2 h, and incubated with the primary antibodies against Bcl, Bax,
β-actin, and caspase-3 proteins at room temperature for 2 h (antibodies were diluted to 1:500 in Tris-buffered saline (TBS-T) containing Tween-20 (20 mmol/l Tris– HCl, pH 7.6, 136 mmol/l NaCl, and 0.1 % Tween-20). The membranes were washed three times with TBS-T liquid, before incubated with antimouse IgG-conjugated HRP (1:5,000 dilution in TBS-T buffer) at room temperature for at least 1 h. Protein bands were visualized by enhanced chemilluminescence kit (Amersham, Buckinghamshire, UK) and L AS-4000 imaging system (FUJIFILM, Tokyo, Japan). Measurement of intracellular active oxygen Formation of intracellular active oxygen was determined using a fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA; Sigma Co.). DCF-DA, a nonfluorescent substance, changes into fluorescent green DCF in the presence of peroxides associated with intracellular hydrogen peroxides. CADPE was administered to MDA-MB-231 and MDAMB-435 cells and then cultured with 5 μmol/l DCF-DA at 37 °C for 30 min. These cells were harvested after washed with PBS (pH 7.4) three times and 1 % trypsin-EDTA treatment. Then cells were washed twice with PBS again and fluorescence was measured with flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA) and analyzed with the CellQuest software (BD, Franklin Lakes, NJ, USA). Statistical analysis Results were generated more than three times independently. Statistical analysis was done using SPSS17.0 software. p value less than 0.05 (p