Sorbus Commixta Water Extract Induces Apoptotic Cell Death Via a ROS‑Dependent Pathway
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ONCOLOGY LETTERS 16: 4193-4200, 2018 Sorbus commixta water extract induces apoptotic cell death via a ROS‑dependent pathway SEONG-CHEOL MOON1,2*, HEE-JUNG CHOI1,2*, TAE-WOOK CHUNG1,2, JUNG-HEE LEE2, SYNG-OOK LEE3, MYEONG HO JUNG1, BYUNG JOO KIM1, JUN-YONG CHOI1,4 and KI-TAE HA1,2,4 1Department of Korean Medical Science, School of Korean Medicine and 2Korean Medical Research Center for Healthy Aging, Pusan National University, Yangsan, Gyeongnam 50612; 3Department of Food Science and Technology, Keimyung University, Daegu 42601; 4Korean Medical Clinical Research Center, Pusan National University Hospital, Yangsan, Gyeongnam 50612, Republic of Korea Received January 12, 2017; Accepted April 6, 2017 DOI: 10.3892/ol.2018.9217 Abstract. The stembark of Sorbus commixta Hedl. has been production was also increased by SCE treatment. However, the used for treating asthma, bronchitis, gastritis and edema. SCE-induced cytotoxic effects and the increased expression However, the anticancer and proapoptotic effects of the water of proapoptotic proteins, including p53 and p21, and reduced extract of the stembark of S. commixta (SCE) remain unknown. Bcl-2/Bax ratio, could be attenuated by N-acetyl cysteine, In the present study, it was shown that SCE inhibited the cell an ROS inhibitor. Taken together, these results indicate that viability of the hepatocellular carcinoma cell lines Hep3B and SCE is a potent proapoptotic herbal medicine, which exerts its HepG2, and of the colon carcinoma cell line HCT116. DNA effects via the ROS-mediated mitochondrial pathway. content analysis indicated that SCE increased the sub-G1 population of HCT116 cells. In addition, degradation of Introduction nuclear DNA and levels of proapoptotic cascade components, including caspase-9, caspase-3 and poly ADP-ribose poly- Apoptosis, a form of programmed cell death, is essential for merase, were augmented by SCE treatment. Mitochondrial maintaining fundamental physiological processes, including membrane potential and the ratio of B-cell lymphoma-2 embryonic development, tissue homeostasis, immune function, (Bcl-2)/Bcl-2-associated X protein (Bax) were also reduced. tumor suppression and infection resistance (1). Dysregulation Furthermore, SCE increased the expression of proapoptotic of apoptosis is considered to be one of the hallmarks of cancer, proteins, including p21, p27 and p53. Mouse double minute since it is associated with uncontrolled cell growth, tumori- 2 homology, a negative regulator of p53, was cleaved by genesis, cancer progression and resistance to therapy (2). The SCE treatment. Intracellular reactive oxygen species (ROS) apoptotic machinery is complex, and it can be triggered via two different pathways: The extrinsic death-receptor pathway, or the intrinsic mitochondrial pathway (3,4). Therefore, the restoration of these apoptotic pathways using drugs has been accepted as an important molecular Correspondence to: Professor Ki-Tae Ha, Department of Korean Medical Science, School of Korean Medicine, Pusan National target for cancer treatment (1,4). Thus far, numerous anticancer University, 49 Busandaehak-ro, Yangsan, Gyeongnam 50612, drugs targeting apoptotic pathways have been developed (4,5). Republic of Korea However, demand for novel and improved anticancer drugs is E-mail: hagis@pusan.ac.kr growing (5). Traditional herbal medicines and their natural ingredients, including polyphenols, carotenoids and triter- *Contributed equally penes, may be useful resources for developing novel anticancer drugs (6-9). The proapoptotic effects of herbal medicines have Abbreviations: SCE, Sorbus commixta stembark water extract; been researched extensively in experimental and clinical ROS, reactive oxygen species; PARP, poly ADP-ribose polymerase; studies of cancer treatment (8,9). TMRM, tetramethylrhodamine methyl ester perchlorate; Sorbus commixta Hedl. (belonging to the Rosaceae family) DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell is a deciduous tree that is native to Japan, the Russian Far East, sorting; ECL, enhanced chemiluminescence; RT-PCR, reverse transcription-polymerase chain reaction; MMP, mitochondrial and Korea (10). The cortex and barks of S. commixta have been membrane potential; DCFDA, 2',7'-dichlorodihydrofluorescein used for the treatment of diverse diseases, including asthma, diacetate; NAC, N-acetyl cysteine bronchitis, gastritis and edema, in traditional Korean medi- cine (11,12). Several previous studies have demonstrated that Key words: Sorbus commixta, apoptosis, reactive oxygen species, the methanol extract of S. commixta has anti-atherosclerotic mitochondria effects (13-15) and hepatoprotective activity (16). The water extract of S. commixta (SCE) has anti‑inflammatory activity (12), and aqueous-ethanol extracts of S. commixta 4194 MOON et al: Sorbus commixta WATER EXTRACT INDUCES APOPTOTIC CELL DEATH showed neuroprotective action against amyloid β toxicity (17) American Type Culture Collection (Manassas, VA, USA). The and protective effects on ultraviolet-induced photo-aging (18). cells were cultured with Dulbecco's modified Eagle's medium The cancer growth-inhibitory action of S. commixta has not (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supple- been reported until now. However, the triterpenoid compounds mented with L-glutamine (200 mg/l), 10% (v/v), antibiotics isolated from S. commixta, lupeol and lupenone, have been (100 U/ml penicillin and 100 µg/ml streptomycin; Thermo shown to inhibit the activity of protein phosphatase 1B (19), Fisher Scientific, Inc.) and heat‑inactivated fetal bovine serum which is associated with cancer development (20,21). (Sigma-Aldrich; Merck KGaA). The cells were incubated in a Therefore, the present study examined the anticancer humidified cell incubator at 37˚C in an atmosphere of 5% CO2 effect of S. commixta. Firstly, it was demonstrated that SCE prior to experiments. can induce apoptosis in human colorectal carcinoma HCT116 cells. The underlying mechanisms of SCE-induced apoptosis Cell viability assay. The viabilities of the cells incubated with were associated with ROS-dependent expression of proapop- various concentrations of SCE were evaluated by MTT assay. totic proteins, including p53, p21 and B-cell lymphoma-2 Hep3B, HepG2 and HCT116 cells were incubated in 24-well (Bcl-2)-associated X protein (Bax). These results suggested plates with 0, 100, 200, 300, 400 or 500 mg/ml of SCE for 48 h that S. commixta may be a source from which novel anticancer at 37˚C. MTT solution (2.0 mg/ml) was added to each well agents may be developed. and the cells were incubated for 4 h at 37˚C with 5% CO2. The culture media were removed, and cells were lysed in DMSO. Materials and methods The amount of formazan crystals, formed by viable cells, was estimated by measuring the absorbance at 540 nm with a Materials. Antibodies against caspase-3 (cat. no. 9665P), Spectramax M2 microplate reader (Molecular Devices, LLC, caspase-9 (cat. no. 9058P), poly ADP-ribose polymerase Sunnyvale, CA, USA). (PARP; cat. no. 9542) and p27 (cat. no. 3698) were supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA). Cell cycle analysis for the detection of cells in sub‑G1 phase. The antibody against p21 (cat. no. Ab109199) was provided HCT116 cells (2x106 cells) were centrifuged at 2,500 x g and by Abcam (Cambridge, UK). Antibodies against Bcl-2 (cat. 4˚C for 10 min, and washed twice in PBS buffer. The cell no. NB100-56098) and Bax (cat. no. NB100-56095) were pellets were gently resuspended in 100 µl PBS, and fixed for purchased from Novus Biologicals, LLC (Littleton, CO, 30 min at 4˚C via serial addition of 200 µl PBS containing USA). Antibodies against p53 (cat. no. sc-6243), mouse double 10% ethanol/5% glycerol, followed by 200 µl PBS containing minute 2 homology (Mdm2; cat. no. sc-965) and GAPDH (cat. 50% ethanol/5% glycerol. The samples were incubated on no. sc-32233) were supplied by Santa Cruz Biotechnology, ice for 5 min, and 100% ethanol/5% glycerol solution was Inc. (Dallas, TX, USA). The antibodies for goat anti-mouse added to the cell suspension to make a final volume of 1 ml IgG (cat. no. NCI1430KR) and goat anti-rabbit IgG (cat. PBS containing 70% ethanol/5% glycerol. The samples were no. NCI1460KR) were provided by Thermo Fisher Scientific, stored at 4˚C overnight, washed with PBS, centrifuged and Inc. (Waltham, MA, USA). Primary antibodies were diluted resuspended with 12.5 µg of RNase (Sigma-Aldrich; Merck to 1:1,000 and secondary antibodies were diluted to 1:5,000. KGaA) in 250 µl of 1.12% sodium citrate buffer (pH 8.45). The All chemicals, including MTT, tetramethylrhodamine methyl samples were maintained at 37˚C for 30 min prior to staining ester perchlorate (TMRM), and N-acetyl cysteine (NAC) were the cellular DNA with 250 µl of propidium iodide (50 µg/ml) obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, at room temperature for 30 min. The relative DNA contents Germany), unless otherwise indicated. of the stained cells were analyzed on a flow cytometer (BD FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) Extraction and preparation of plant material. Stem and with excitation/emission wavelengths of 495/605 nm. cortex of S. commixta, which was grown and collected in North Gyeongsang Province, South Korea, in 2012, were Nuclear staining and fluorescent microscopic observation. purchased from Omniherb Co., Ltd. (Daegu, South Korea). For detecting morphological changes associated with apop- The plant was authenticated by a botanical expert working in tosis, HCT116 cells (1x105