Extracts from Sageretia Thea Reduce Cell Viability Through Inducing Cyclin

Kim et al. BMC Complementary and Alternative Medicine (2019) 19:43 https://doi.org/10.1186/s12906-019-2453-4 RESEARCH ARTICLE Open Access Extracts from Sageretia thea reduce cell viability through inducing cyclin D1 proteasomal degradation and HO-1 expression in human colorectal cancer cells Ha Na Kim1†, Gwang Hun Park2†, Su Bin Park1, Jeong Dong Kim1, Hyun Ji Eo2, Ho-Jun Son2, Jeong Ho Song2 and Jin Boo Jeong1,3* Abstract Background: Sageretia thea (S. thea) has been used as the medicinal plant for treating hepatitis and fevers in Korea and China. Recently, anticancer activity of S. thea has been reported, but the potential mechanism for the anticancer property of S. thea is still insufficient. Thus, we evaluated whether extracts from the leaves (STL) and branches (STB) of S. thea exert anticancer activity and elucidated its potential mechanism in SW480 cells. Methods: MTT assay was performed for measuring cell viability. Western blot and RT-PCR were used for analyzing the level of protein and mRNA, respectively. Results: Treatment of STL or STB decreased the cell viability and induced apoptosis in SW480 cells. Decreased level of cyclin D1 protein was observed in SW480 cells treated with STL or STB, but no change in cyclin D1 mRNA level was observed with the treatment of STL or STB. MG132 blocked downregulation of cyclin D1 protein by STL or STB. Thr286 phosphorylation of cyclin D1 by STL or STB occurred faster than downregulation of cyclin D1 protein in SW480 cells. When SW480 cells were transfected with T286A-cyclin D1, cyclin D1 degradation by STL or STB did not occur. Inhibition of GSK3β and cyclin D1 nuclear export attenuated STL or STB-mediated cyclin D1 degradation. In addition, STL or STB increased HO-1 expression, and the inhibition of HO-1 attenuated the induction of apoptosis by STL or STB. HO-1 expression by STL or STB resulted from Nrf2 activation through ROS-dependent p38 activation. Conclusions: These results indicate that STL or STB may induce GSK3β-dependent cyclin D1 degradation, and increase HO-1 expression through activating Nrf2 via ROS-dependent p38 activation, which resulted in the decrease of the viability in SW480 cells. These findings suggest that STL or STB may have great potential for the development of anti-cancer drug. Keywords: Anticancer, Cell viability, Cyclin D1, Heme oxygenase-1, Human colorectal cancer, Sageretia thea Background drugs have been developed for clinical use for the treat- Globally, cancer is considered as one of major health ment of colorectal cancer, the long-term use of these anti- problems [1]. Among cancers, colorectal cancer is fourth cancer drugs causes many side effects [3]. For example, most common malignant tumor, the third leading cause of fluorouracil causes neutropenia, stomatitis and diarrhea, cancer deaths worldwide [2]. Although many anticancer and irinotecan induces bone marrow suppression, nausea and alopecia [3]. In addition, oxaliplatin has been reported * Correspondence: [email protected] to cause dysesthesias and renal dysfunction [3], Thus, † Ha Na Kim and Gwang Hun Park contributed equally to this work. medicinal plant resources are being utilized for comple- 1Department of Medicinal Plant Resources, Andong National University, 1375 Gyeongdong-ro, Andong, Gyeongsangbuk-do 36729, Republic of Korea mentary alternative treatment of cancer [4], and have been 3Agricultural Science and Technology Research Institute, Andong National regarded as the promising sources for developing antican- University, Andong 36729, Republic of Korea cer drugs [5]. Indeed, clinically used anticancer drugs such Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. BMC Complementary and Alternative Medicine (2019) 19:43 Page 2 of 11 as taxol, vinblastine, vincristine, irinotecan and camptothe- Methods cin have been developed using plant-derived products [6]. Chemical reagents In cell cycle progression, cyclin D1 complexed with LiCl (GSK3β inhibitor), MG132 (Proteasome inhibitor), CDK4 and 6 induces phosphorylation of retinoblastoma PD98059 (ERK1/2 inhibitor), SB230580 (p38 inhibitor), protein which promotes the progression of cell cycle [7]. leptomycin B (LMB, Nuclear export inhibitor), zinc proto- In addition, cyclin D1 is involved in the transcriptional porphyrin IX (ZnPP, HO-1 inhibitor), 3-(4,5-dimethylthi- activation of genes that induce cell growth, genetic zaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), instability, invasion, and metastasis [7–10]. In addition 5-Fluorouracil (5-FU) and oxaliplatin were purchased in to the cell cycle control function, cyclin D1 has also Sigma Aldrich (St. Louis, MO, USA). Antibodies against been reported to modulate apoptosis [11]. Inhibition of cyclin D1, phospho-cyclin D1 (Thr286), HA-tag, cyclin D1 expression induced apoptosis of various p-GSK3β, total-GSK3β, p-p38, total-p38, HO-1, Nrf2, cancer cells [12, 13]. On the other hand, upregulation of cleaved PARP, TBP and β-actin were purchased in Cell cyclin D1 inhibited apoptosis in human choriocarcinoma Signaling (Bervely, MA, USA). cells [14]. Cyclin D1 overexpression has been observed in 63.6% of colorectal cancer [15] and cyclin D1 has Preparation of the extracts of branches and leaves from been known to be participated in the growth and differ- S. thea entiation of colorectal cancer [16]. Thus, cyclin D1 has S. thea (voucher number: Jeong 201,804 (ANH)) was been regarded as a hallmark of colorectal cancer and a generously provided and formally identified by Forest potential target of colorectal cancer treatment. Medicinal Resources Research Center, National Institute Recently, HO-1 has been proposed as a potential of Forest Science, Yongju, Korea. Twenty grams of the molecular anticancer target [17]. HO-1, known to as branches or leaves from S. thea were immersed in 500 heat shock protein 32 catalyzed heme degradation into ml of 70% ethanol and then extracted by stirring at the bilirubin, iron ion and carbon monoxide. HO-1 activa- room temperature for 3 days. Then, the ethanol-soluble tion inhibits oxidative cell damage induced by hydrogen fraction was filtered, concentrated to 100 ml volume peroxide and UV, production of inflammatory mediators, using a vacuum evaporator, and freeze-dried. The etha- – and induction of high-glucose [18 21]. However, HO-1 nol extracts from the branches (STB) or leaves (STL) of has been reported to have a multifaceted role in cancer S. thea were stored at − 80 °C until use. development [17]. HO-1 increases the growth of several cancer cells such as pancreatic cancer, melanoma and rhabdomyosarcoma [22–24]. In the opposite effect of Cell culture HO-1, the overexpression of HO-1 exerts anti-proliferative SW480 cells as one of the human colorectal cancer cell activity in breast cancer, prostate cancer and colorectal lines have been widely used to investigate the potency of cancer cells [25–27]. In addition, HO-1 suppresses cell drugs in cancer prevention and treatment [34]. Thus, we migration and xenograft tumor growth in hepatocellular used SW480 cells to investigate anticancer activity of carcinoma [28]. STB or STL. SW480 cells obtained from Korean Cell As one of the Rhamnaceae family, Sageretia thea (S. Line Bank (Seoul, Korea) were maintained in DMEM/ thea) has been commonly known as Chinese sweet plum F-12 (Lonza, Walkersville, MD, USA) with 10% fatal μ or Chinese bird plum [29]. S. thea as traditional herbal bovine serum (FBS), 100 U/ml penicillin and 100 g/ml medicine has been treated for hepatitis and fevers in streptomycin at 37 °C under a humidified atmosphere of Korea and China [29, 30]. In pharmacological study, the 5% CO2. STB or STL was dissolved in dimethyl sulfoxide fruits from S. thea have been reported to exert anti-oxi- (DMSO). DMSO as a vehicle was used in a range not dant, anti-diabetes and anti-melanogenesis activity [30, exceeding 0.1% (v/v). 31]. The leaves of S. thea inhibited the oxidation of low-density lipoprotein through its anti-oxidant activity Cell viability and HIV type 1 protease [30, 32]. Recently, the leaves MTT assay was performed to evaluate the cell viability. and branches from S. thea induced apoptosis in human SW480 cells (3 × 104 cells/well) were cultured in 96-well breast cancer cells, MDA-MB-231 [33]. However, there plate for 24 h and then treated with STB or STL for the have been no studies on the mechanisms of S. thea for additional 24 h. After STB or STL treatment, 50 μlof anticancer activity. Because the elucidation of the mech- MTT solution (1 mg/ml) was added to the cells and anism for anticancer activity of S. thea is essential for allowed to react for 2 h. After 2 h, the media was the development of anticancer agent using S. thea,we removed, and then 100 μl of DMSO was added to the first report the potential mechanisms of branches and cells for resulting crystals. The absorbance was measured leaves of S. thea for the anticancer activity using SW480 using UV/Visible spectrophotometer (Human Cop., colorectal cancer cells. Xma-3000PC, Seoul, Korea) at 570 nm. Kim et al. BMC Complementary and Alternative Medicine (2019) 19:43 Page 3 of 11 Cell cycle analysis USA). The primer sequences of cyclin D1 and GAPDH SW480 cells (5 × 105 cells/well) were cultured in 6-well were as followed: cyclin D1: forward 5’-AACTACCTG plate 24 h.

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