Antiproliferation of Cryptocarya Concinna-Derived Cryptocaryone Against Oral Cancer Cells Involving Apoptosis, Oxidative Stress
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Chang et al. BMC Complementary and Alternative Medicine (2016) 16:94 DOI 10.1186/s12906-016-1073-5 RESEARCH ARTICLE Open Access Antiproliferation of Cryptocarya concinna- derived cryptocaryone against oral cancer cells involving apoptosis, oxidative stress, and DNA damage Hsun-Shuo Chang1,2†, Jen-Yang Tang3,4,5†, Ching-Yu Yen6,7, Hurng-Wern Huang8, Chang-Yi Wu9, Yi-An Chung10, Hui-Ru Wang10, Ih-Sheng Chen1,2, Ming-Yii Huang3,4* and Hsueh-Wei Chang10,11,12,13* Abstract Background: Cryptocarya-derived crude extracts and their compounds have been reported to have an antiproliferation effect on several types of cancers but their impact on oral cancer is less well understood. Methods: We examined the cell proliferation effect and mechanism of C. concinna-derived cryptocaryone (CPC) on oral cancer cells in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial depolarization, and DNA damage. Results: We found that CPC dose-responsively reduced cell viability of two types of oral cancer cells (Ca9-22 and CAL 27) in MTS assay. The CPC-induced dose-responsive apoptosis effects on Ca9-22 cells were confirmed by flow cytometry- based sub-G1 accumulation, annexin V staining, and pancaspase analyses. For oral cancer Ca9-22 cells, CPC also induced oxidative stress responses in terms of ROS generation and mitochondrial depolarization. Moreover, γH2AX flow cytometry showed DNA damage in CPC-treated Ca9-22 cells. CPC-induced cell responses in terms of cell viability, apoptosis, oxidative stress, and DNA damage were rescued by N-acetylcysteine pretreatment, suggesting that oxidative stress plays an important role in CPC-induced death of oral cancer cells. Conclusions: CPC is a potential ROS-mediated natural product for anti-oral cancer therapy. Keywords: Cryptocarya concinna, Cryptocaryone, Oral cancer, Apoptosis, Oxidative stress, γH2AX Background demonstrated to have an antiproliferative ability against Oral cancer is the sixth most common cancer in the cancer. For example, methanolic extracts of the leaves of world [1, 2]. Oral cancer is easy to detect clinically but is C. griffithiana and the roots of C. concinna can inhibit frequently ignored by patients resulting in high mortality cell proliferation of human HL60 promyelocytic rates [3]. Although several oral tumor markers have leukemia cells [7] and oral cancer cells [8], respectively. been reported [4, 5], these efforts focused on detection The ethanolic extracts of the fruit and trunk bark of C. rather than therapy and effective anti-oral cancer therap- obovata have been reported to have an antiproliferative ies are still needed. effect against human KB cells [9]. Cryptocarya (family Lauraceae) is widely found in the Several Cryptocarya-derived compounds have been tropics and subtropics [6]. Its crude extracts have been found to have diverse biological functions, i.e., anti- dengue virus by alkylated flavanones [10], anti-HIV by * Correspondence: miyihu@gmail.com; changhw@kmu.edu.tw phenanthroindolizidine alkaloids [11], anti-tuberculosis by † Equal contributors pinocembrin [12], anti-plasmodial by (+)-N-methylisoco- 3Department of Radiation Oncology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan claurine, atherosperminine, and 2-hydroxy-atherosperminine 10Department of Biomedical Science and Environmental Biology, Kaohsiung [13], anti-trypanosomal by 7′,8′-dihydroobolactone [14], and Medical University, Kaohsiung, Taiwan Full list of author information is available at the end of the article © 2016 Chang et al. 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. Chang et al. BMC Complementary and Alternative Medicine (2016) 16:94 Page 2 of 10 anti-inflammatory by (2S)-5,7-dihydroxyflavanone and cryp- Cell cultures and chemicals tocaryanone B [15]. Two human oral cancer cell lines (Ca9-22 [21] and CAL Recently, Cryptocarya-derived compounds have been 27 [22]), purchased from the Cell Bank, RIKEN BioRe- reported to have an antiproliferation effect on cancer. source Center (Tsukuba, Japan) and the American Type For example, the proliferation of leukemia cells was Culture Collection (ATCC; Virginia, USA), respectively, inhbitied by C. costata-derived 2′,4′-dihydroxy-5′,6′- were incubated in DMEM/F12 (3:2) medium (Gibco, dimethoxychalcone and isodidymocarpin [16] and C. Grand Island, NY, USA) supplemented with 10 % fetal konishii-derived by desmethylinfectocaryone, infecto- bovine serum (Gibco), 100 U/ml penicillin, 100 μg/ml caryone, and cryptocaryone (CPC) [17]. Among these streptomycin, and 0.03 % glutamine. Normal gingival Cryptocarya-derived compounds, we are interested in fibroblast (HGF-1) was purchased from ATCC and the anticancer effect of CPC, which is one of the major maintained in DMEM medium (Gibco, Grand Island, constituents in the commonly distributed evergreen NY, USA) with a similar supplement with 1 mM pyru- plant C. concinna in Taiwan [18]. vate as described above. Cells were incubated in humidi- Although the anticancer effect of CPC, a kind of dihy- fied air at 37 °C with 5 % CO2. N-acetylcystein (NAC) drochalcone, had been reported in some cancer types was purchased from Sigma (St. Louis, MO, USA) for such as murine leukemia [17] and prostate cancer [19], pretreatment before CPC application. Passage numbers few studies have addressed its antiproliferative effect on of the oral cancer (Ca9-22 and CAL 27) cells used in this oral cancer. Moreover, the cell killing mechanism of study were 15–22 and 8–15, respectively. cryptocaryone in cancer remains unclear. Recently, re- active oxygen species (ROS) generation was reported to Cell viability be involved in Corema album-derived dihydrochalcone Cell viability was determined using the CellTiter 96® induced cytotoxicity for colon cancer cells [20]. Accord- AQueous One Solution Cell Proliferation Assay (MTS) ingly, the relationship between ROS generation and CPC (Promega Corporation, Madison,WI, USA) as previously effect for oral cancer cells is worth examining. described [21]. Two oral cancer cell lines (Ca9-22 and This study evaluates possible anticancer functions of CAL 27) were seeded at 1 × 105 cells per well and HGF- CPC and explores its drug mechanisms in terms of cell 1 cells were seeded at 4 × 104 cells per well in a 6-well viability, cell cycle analysis, apoptosis, ROS generation, plate, respectively. After seeding for 24 h, cells were mitochondrial depolarization, and DNA damage detec- treated with CPC at indicated concentrations for 24 h tion. The role of oxidative stress in CPC’s effect on oral and cell viability was determined by an ELISA reader at cancer cells is also addressed. 490 nm. Methods Determination of cell cycle distribution Plant material and isolation Propidium iodide (PI) (Sigma, St Louis, MO, USA) was C. concinna was identified by one of the authors (Ih-Sheng added to stain the cellular DNA content [23]. In brief, Chen) and its roots were collected at Mudan, Pingtung 3×105 cells per well in 6 well plates were seeded over- County, Taiwan, in May 2004. A voucher specimen night and then treated with the vehicle (0.05 % DMSO) (Chen6153) has been deposited in the Herbarium of the or 3, 6, 9, 12 μM of CPC for 24 h. After cells were har- School of Pharmacy, College of Pharmacy, Kaohsiung vested and washed twice with PBS, they were fixed Medical University. The dried roots (7.7 Kg) of C. con- overnight with 70 % ethanol. Subsequently, the cell cinna were processed by slicing and cold methanol- pellets were resuspended in 50 μg/ml PI for 30 min at extraction three times at room temperature. Finally, the 37 °C in darkness. The cell cycle distribution was eval- solution was evaporated under reduced pressure to yield uated by a flow cytometer (BD Accuri™ C6; Becton- the methanolic extract (800 g; yield: methanolic extract/ Dickinson, Mansfield, MA, USA) and BD Accuri™ C6 dried roots = 10.4 %) [8]. CPC (5.7 g; yield: CPC/methano- software (version 1.0.264). lic extract = 0.7 %) was isolated from the root of C. con- cinna as described previously [19]. In brief, the methanolic Determination of apoptosis by annexin V/PI extract was partitioned between chloroform/water (1:1) to Apoptosis was detected by annexin V (Strong Biotect yield a chloroform fraction and a water fraction. The Corporation, Taipei, Taiwan)/PI (Sigma, St Louis, MO, chloroform fraction was subjected to silica gel column USA) as described in [24]. Briefly, 3 × 105 cells per well chromatography and eluted with a gradient of chloro- in 6 well plates were seeded for 24 h and then treated form–methanol to produce 13 fractions (A-1–A-13). CPC with the vehicle or indicated concentrations of CPC for was then obtained from fraction A-3 (chloroform-metha- 24 h. Cells were then incubated with 100 μl binding nol 100:1) and the structure of CPC was determined by buffer containing 2 μl of annexin-V-fluorescein isothio- spectral analyses (Additional file 1). cyanate (FITC) stock (0.25 μg/μl) and 2 μl of PI stock Chang et al. BMC Complementary and Alternative Medicine (2016) 16:94 Page 3 of 10 (1 mg/ml) for 30 min. Finally, it was suspended with cells were resuspended in 20 μg/ml of PI for flow cytome- 400 μl PBS for flow cytometry analysis (BD Accuri™ C6; try analysis (BD Accuri™ C6; Becton-Dickinson). Becton-Dickinson). Statistical analysis Determination of apoptosis by pancaspase activity Group differences of the same drug treatment with differ- The apoptosis was also detected by the measurement of ent concentrations were analyzed by one-way ANOVA caspase activation [25].