ONCOLOGY REPORTS 26: 1287-1294, 2011 Induction of apoptosis by casticin in cervical cancer cells through reactive oxygen species-mediated mitochondrial signaling pathways DAN CHEN, JIANGUO CAO, LI TIAN, FEI LIU and XIFENG SHENG Medical College, Hunan Normal University, Changsha 410013, P.R. China Received May 13, 2011; Accepted June 20, 2011 DOI: 10.3892/or.2011.1367 Abstract. Casticin, one of the main components from Fructus cancer cases are diagnosed every year, with a five-year survival Viticis, has been reported to inhibit the growth of various cancer rate of about only 52% (3,4). Currently, we generally utilize cells, including the human cervical cancer cell line HeLa. The radiotherapy and/or chemotherapy as a strategy in the clinical purpose of this study was to examine the apoptotic activity and treatment of cervical cancer, thereby inducing apoptosis in molecular mechanism of casticin action on human cervical tumor cells (5-8). However, conventional therapeutic drugs cancer cells. The apoptotic activity of casticin on human cervical not only trigger apoptosis but also exert lethality on normal cancer HeLa, CasKi, SiHa and peripheral blood mononuclear cells, resulting in serious toxicity (5-8). Therefore, clinically cells (PBMCs) was measured using a histone/DNA ELISA speaking, it is important for us to develop effective, new treat- assay, flow cytometry with propidium iodide (PI) staining and ments for human cervical cancer that can induce apoptosis DNA agarose gel electrophoresis. The mitochondrial membrane with higher selectivity and lower toxicity. potential and reactive oxygen species (ROS) production were Casticin is one of the main components from Fructus evaluated by flow cytometry analysis. Caspase activities were Viticis (Manjingzi is the Chinese name), which is a traditional assayed using a caspase colorimetric activity assay kit. Protein Chinese medicine prepared from the fruit of Vitex trifolia L. expression levels of cytochrome c, Bax, Bcl-2, Bcl-xL and XIAP (family Verbenaceae) that is also used as a folk medicine, were analyzed by Western blotting. Casticin caused accumula- is known to be an anti-inflammatory agent, and is used to tion of the Sub-G1 cells and increased reactive oxygen species treat certain cancers in China (9). The chemical structure of (ROS) production in HeLa, CasKi, SiHa cell lines, but not in casticin is shown in Fig. 1. It has been shown that the C-3' and PBMCs. Apoptosis of HeLa cells was induced by casticin via C-5 hydroxyl as well as the C-3 and C-4' methoxyl groups are mitochondrial release of cytochrome c due to the reduction of required for the significant antiproliferative activity of flavone mitochondrial trans membrane potential, activation of caspase-3 and that casticin (5,3'-dihydroxy-3,6,7,4'-tetramethoxyl- and -9, and the production of reactive oxygen species. The pan flavone, also called vitexicarpin) also contains these groups caspase inhibitor zVAD-FMK, the caspase-9 inhibitor zLEHD- (10). Casticin has been reported to inhibit the proliferation and fmk and N-acetylcysteine suppressed casticin-induced apoptosis. growth of a variety of cancer cells, including human cervical Bax was upregulated, while expression levels of Bcl-xL and cancer cells (HeLa) (10-14). Therefore, it is worth investigating XIAP were downregulated. However, there was no change in the whether casticin can induce apoptosis in HeLa cells and the expression of Bcl-2 under the same treatment. Our results indicate molecular mechanisms by which casticin causes apoptosis. that casticin-induced apoptosis of cervical cancer cells is medi- Work from our laboratory and others previously demon- ated by ROS generation and mitochondrial signaling pathways. strated that similar flavones, such as apigenin (15), chrysin (16), luteolin (17) and quercetin (18), cause a higher level of Introduction reactive oxygen species (ROS) in cancer cells than in normal cells, resulting in cytotoxicity. In the present study, we exam- Cervical cancer is one of the most common malignancies in ined the effects of casticin on the apoptosis of human cervical the world among women (1,2). About 500,000 new cervical cancer cell lines. We show for the first time that casticin causes mitochondrial membrane potential collapse, cytochrome c release, caspase-3 and -9 activation and apoptotic cell death triggered by ROS generation in human cervical cancer cells. Correspondence to: Dr Jianguo Cao, Medical College, Hunan Normal University, Changsha 410013, P.R. China Materials and methods E-mail: [email protected] Drugs and chemical reagents. Casticin was purchased from Key words: human cervical cancer, casticin, apoptosis, reactive Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). oxygen species, mitochondria Casticin has a molecular weight of 374.3 ku, appears as yellow crystals and has a purity of 98.0%. Casticin was prepared in 1288 CHEN et al: CASTICIN INDUCES APOPTOSIS BY ROS AND MITOCHONDRIAL SIGNALING PATHWAYS dimethyl-sulfoxide (DMSO) at a concentration of 10 mmol/l, and aliquots were stored at -80˚C. Stock solutions were diluted to the desired final concentrations with growth medium just prior to use. The following were purchased from Hunan Clonetimes Biotech Co., Ltd. (Changsha, China): Dulbecco's modified Eagle's medium (DMEM; Invitrogen), RPMI-1640 medium (Invitrogen), fetal bovine serum (Invitrogen), Cell Apoptosis ELISA detection kit (Roche), Caspase-3 Activity detection kit (Millipore, Bedford, MA), Caspase-9 colo- rimetric Activity assay kit (Millipore), propidium iodide (PI; Sigma), ethidium bromide (EB; Sigma), Apoptotic Figure 1. The chemical constitution of casticin. DNA Ladder detection kit (Bodataike Company, Beijing), 2',7'-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes Inc, Eugene, OR), CBZ-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk: Livermore), CBZ-Leu-Glu(OMe)-His- test/control agents or vehicle with 10% fetal bovine serum for Asp(OMe)-fluoromethyl ketone (zLEHD-fmk: Livermore), 24 h. This assay was performed as previously described (16). mouse anti-human Bcl-2, Bax, XIAP, Bcl-XL, cytochrome c and β-actin antibodies (Santa Cruz Biotechnology), and horse- Determination of reactive oxygen species. Intracellular ROS radish peroxidase-conjugated anti-mouse secondary antibody accumulation was measured by FCM using the fluorescent (Cell Signaling Technology). probe DCHF-DA as previously described (16). Cells (2x106) were collected after treatment with various concentrations of Cell lines and cell culture. Human cervical cancer cell test agents and washed with serum-free medium and incubated lines (HeLa, CasKi and SiHa) were purchased from the with 1 ml DCHF-DA for 30 min at 37˚C in the dark. After China Centre for Type Culture Collection (CCTCC; Wuhan, incubation, the cells were washed with serum-free medium China), were maintained in DMEM supplemented with 10% three times and analyzed within 30 min by FCM (American fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin BD Company, FACS420) at an excitation wavelength of and 100 µg/ml streptomycin, and were incubated at 37˚C 488 nm and an emission wavelength of 525 nm. in a humidified atmosphere of 5% CO2. Normal human peripheral blood mononuclear cells (PBMCs) were isolated Measurement of mitochondrial transmembrane potential. by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) Mitochondrial membrane potential was measured by flow density-gradient centrifugation and were cultured in RPMI- cytometry using the cationic lipophilic green fluorochrome 1640 medium supplemented with 20% FBS. PBMCs were rhodamine-123 (Rh123) (Molecular Probes). Disruption acquired from a healthy volunteer after obtaining informed of ΔΨm is associated with a lack of Rh123 retention and a consent. decrease in fluorescence. Briefly, the cells were washed twice with PBS and incubated with 1 µg/ml Rh123 at 37˚C Histone/DNA ELISA for detecting apoptosis. The cell apop- for 30 min. Then, the cells were washed twice with PBS, and tosis ELISA detection kit (Roche) was used to detect apoptosis Rh123 intensity was determined by flow cytometry. Cells with in cells treated with casticin according to the manufacturer's reduced fluorescence (less Rh123) were counted as having lost protocol. Briefly, cells were seeded in a 96-well plate at a density some of their mitochondrial membrane potential. of 1x104 cells/well for 24 h, and testing agents were then added to culture medium containing 10% fetal bovine serum. After Analysis of caspase-3 and -9 activities. Analysis of caspase-3, 24 h, we transferred the cytoplasm of the control and treatment and -9 activities was performed using the Caspase apoptosis groups to the 96-well plate pre-coated with streptavidin and detection kit according to the manufacturer's instructions. In previously incubated with the biotinylated histone antibody brief, cell lysates were prepared after the respective treatment and the peroxidase-tagged mouse anti-human DNA for 2 h at with tested agents. Assays were performed in 96-well plates room temperature. Absorbance was measured at 405 nm using by incubating 20 µg cell lysates in 100 µl reaction buffer (1% the EXL-800-type enzyme-linked immunosorbent apparatus NP-40, 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 10% glycerol) (Bio-Tek, Shanghai). containing 5 µM of the caspase-3 substrate, Ac-DEVD-pNA, or the caspase-9 substrate, Ac-LEHD-pNA. Lysates were then Flow cytometry Analysis using PI staining. Cells were seeded incubated at 37˚C for 2 h. Next, absorbance was measured at at a density of 4x106 cells/well in 100 ml culture flasks for 405 nm with an enzyme-labeling instrument (ELX-800 type, 24 h followed by treatment with medium containing various Bio-Tek). In the caspase inhibitors assay, cells were pretreated concentrations of casticin with 10% fetal bovine serum for with a caspase inhibitor (20 µM of zVAD-fmk, zDEVD-fmk, 24 h or 4.0 µM casticin for the indicated time. Propidium zIETD-fmk or zLEHD-fmk) for 1 h prior to the addition of iodide staining to analyze DNA content was performed as test agents.