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Hesperidin Induces Paraptosis Like Cell Death in Hepatoblatoma, Hepg2 Cells: Involvement of ERK1/2 MAPK

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Hesperidin Induces Paraptosis Like Cell Death in Hepatoblatoma, Hepg2 Cells: Involvement of ERK1/2 MAPK Hesperidin Induces Paraptosis Like Cell Death in Hepatoblatoma, HepG2 Cells: Involvement of ERK1/2 MAPK Silvia Yumnam1, Hyeon Soo Park1, Mun Ki Kim1, Arulkumar Nagappan1, Gyeong Eun Hong1, Ho Jeong Lee1, Won Sup Lee2, Eun Hee Kim3, Jae Hyeon Cho1, Sung Chul Shin4, Gon Sup Kim1* 1 Research Institute of Life Science, College of Veterinary Medicine (BK21 plus project), Gyeongsang National University, Gazwa, Jinju, Republic of Korea, 2 Department of Internal Medicine, Institute of Health Sciences, Gyeongsang National University School of Medicine, Gyeongnam Regional Cancer Center, Gyeongsang National University Hospital, Jinju, Republic of Korea, 3 Department of Nursing Science, International University of Korea, Jinju, Republic of Korea, 4 Department of Chemistry, Research Institute of Life Science, Gyeongsang National University, Jinju, Republic of Korea Abstract Hesperidin, a natural flavonoid abundantly present in Citrus is known for its anti-cancer, anti-oxidant and anti-inflammatory properties. In this study we examined the effect of hesperidin on HepG2 cells. HepG2 cells treated with various concentration of hesperidin undergo a distinct type of programed cell death. Cytoplasmic vacuolization, mitochondria and endoplasmic reticulum swelling and uncondensed chromatin were observed in hesperidin treated cells. DNA electrophoresis show lack of DNA fragmentation and western blot analysis demonstrates lack of caspase activation and PARP cleavage. It was observed that hesperidin induced cell death is nonautophagic and also activate mitogen activated protein kinase ERK1/2. Taken together, the data indicate that hesperidin induces paraptosis like cell death in HepG2 cells with the activation of ERK1/2. Thus our finding suggests that hesperidin inducing paraptosis may offer an alternative tool in human liver carcinoma therapy. Citation: Yumnam S, Park HS, Kim MK, Nagappan A, Hong GE, et al. (2014) Hesperidin Induces Paraptosis Like Cell Death in Hepatoblatoma, HepG2 Cells: Involvement of ERK1/2 MAPK. PLoS ONE 9(6): e101321. doi:10.1371/journal.pone.0101321 Editor: Diego Calvisi, Institut fu¨r Pathologie, Greifswald, Germany Received March 11, 2014; Accepted June 5, 2014; Published June 30, 2014 Copyright: ß 2014 Yumnam et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by a grant from the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT & Future Planning (No. 2012M3A9B8019303 and No. 2012R1A2A2A06045015) and National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs, Republic of Korea (No. 0820050). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction absent. It typically does not response to caspase inhibitors z- VAD.fmk, BAF, p53, xiap, Bcl-XL nor does it involve activation of Liver cancer is one of the most prevalent cancers and the third caspases [30], [37]. Paraptosis has also been described to be leading cause of cancer related deaths worldwide [6]. Hepato- mediated by mitogen-activated protein kinases [31] and can also blastoma is the most common primary liver tumor in children and be triggered by the TNF receptor family member TAJ/TROY accounts for 25–45% of the liver tumor [43]. It is of a major [35]. concern due to the poor prognosis and low rate of long term According to the WHO about 65% of the world’s population survival. And it is also highly chemoresistant to currently available relies on plant derived traditional medicines for their primary chemotherapeutic agents. Preventive approaches are therefore health care [5]. The use of natural products as therapeutic agents sorely needed. against cancer has become very popular in the recent years One of the most effective cancer therapy methods is induction considering the toxicity of chemotherapeutics. Natural products of apoptosis using various cytotoxic agents [13]. Apoptosis is a are considered to be safe and also reduce the mutagenicity in programmed cell death (PCD) characterized by cell shrinkage, normal cells [19]. In our previous studies we have also reported membrane blebbing, chromatin condensation, DNA fragmenta- Citrus aurantium extracts, Scutellaria baccalensis extracts and polyphe- tion, and the formation of apoptotic bodies [12], [34]. In recent nolic extracts of Lonicera japonica to possess anticancer activity in years, alternative types of PCD have also been described. Among human gastric cancer, lung cancer and liver cancer cells these paraptosis which is a non-apoptotic PCD has been a new respectively [16], [26], [27]. area of interest in the study of cancer related therapy. Unlike Flavonoids are natural polyphenolic compounds widely occur- apoptosis, paraptosis is characterized by cytoplasmic vacuolation ring in fruits and vegetables. And in the recent years the use of that begins with progressive swelling of mitochondria and flavonoids as anti-cancer compounds has received considerable endoplasmic reticulum. In this type of cell death, the formation attention [1], [22]. The flavonoid is sub-grouped into flavones, of apoptotic bodies, or other characteristics of apoptotic morphol- flavanols, isoflavones, flavanols, flavanones and flavanonols [28]. ogy such as chromatin condensation and DNA fragmentation is Hesperidin (5, 7, 39-trihydroxy-49-methoxy-flavanone7-rhamno PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e101321 Hesperidin Induces Paraptosis in HepG2 Cells glucoside) (Fig. 1A) is a flavanone glycoside widely found in Citrus (St. Louis, MO, USA). Goat anti-mouse and goat anti-rabbit fruits and vegetables [9]. Hesperidin has reported to exhibit secondary antibody were purchased from Enzo Life Sciences. 49, diverse biological and pharmacological properties including 6-Diamidino-2-phenylindole (DAPI) was purchased from Vector antianalgesic, anti-inflammatory [7], antidepressant [29], antiox- Laboratories Inc. (Burlingame, CA). Pro-caspases 3 and active idant and anticarcinogenic activity [10], [38]. Hesperidin inducing caspases 3, ERK1/2 and pERK1/2, AIF, cathepsin D, LC3B and apoptosis has been reported in various cancer cells including PI3K inhibitor LY294002 were purchased from Cell Signaling colon, pancreatic and mammary cancer cells [23], [25] but that of (Beverly, MA, USA). The pan-caspase inhibitor, z-VAD.fmk was inducing paraptosis is still yet to be explored. purchased from Promega (Madison, WI, USA) and U0126 The main aim of the study was to determine the effect of ERK1/2 inhibitor was purchased from Tocris Biosciences (Bristol, hesperidin on HepG2 cells and to evaluate its anticancer potential. UK) In the present study we observed that hesperidin induces cytoplasm vacuolation and mitochondrial swelling in HepG2 cells. Cell Culture and Treatment In addition we also observed the non-involvement of caspase, AIF, Liver cancer cells, HepG2, Hep3B and normal human liver cell, cathepsin D, lack of apoptotic body formation, chromatin Chang liver cell lines were obtained from the Korean Cell Line condensation and DNA fragmentation. It was also observed the Bank (Seoul, Korea). The cells were maintained in DMEM cell death in HepG2 cells were nonautophagic. The results suggest supplemented with 10% heat inactivated FBS and 1% penicillin/ that hesperidin induces paraptosis like cell death in HepG2 cells streptomycin at 37uC in a 5% CO2 incubator. Cells were treated with the activation of ERK1/2 protein kinase. And as far as we with vehicle alone (1% DMSO) or 0.1, 0.5, 1, 1.5 and 2 mM of know this would be the first reported study of hesperidin inducing hesperidin dissolved in 1% DMSO. paraptosis like cell death in HepG2 cells. Cell Viability Assay Materials and Methods Cell viability was determined using MTT assay. HepG2, Hep3B 5 Chemicals and Reagents and Chang liver cells were seeded at a density of 1610 cells/well Dulbecco’s Modified Eagles medium (DMEM) media was in 12 well plates. After overnight incubation at 37uC in a 5% CO2 purchased from Hyclone (Logan, UT, USA). Antibiotics (strepto- incubator cells were treated with 0.1, 0.5, 1, 1.5 and 2 mM of mycin/penicillin) and fetal bovine serum (FBS) were obtained hesperidin or vehicle alone. MTT assay was carried out after 24 h from Gibco (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2- of incubation. 100 ml of 0.5% (w/v) MTT dissolved in 1X PBS yl)-2,5-diphenyltetrazolium bromide (MTT), Propidium iodide was added to each well and incubated for 3 h at 37uC. The medium was aspirated and the formazan contained in the cell was (PI), DiOC6 and hesperidin were purchased from Sigma–Aldrich solubilized by 500 ml of DMSO. After 15 min of shaking, absorbance at 540 nm was measured with a microplate reader. Cell viability was expressed as a percentage of proliferation versus controls (untreated cells), which was set at 100%. Cell cycle analysis Flow cytometer was used to analyze the cell cycle distribution. Cells grown overnight were treated with 0.1, 0.5, 1, 1.5 and 2 mM hesperidin or vehicle alone for 24 h in complete media. Whole HepG2 cells were harvested and fixed with 70% ethanol for 1 h at 4uC. Fixed cells were washed in phosphate-buffered saline (PBS). Cells were then incubated with 1 U/ml of RNase A (DNase free) and 5 mg/ml of propidium iodide (PI; Sigma–Aldrich) for 30 min at 4uC in the dark. FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the cell cycle distribution. Approximately 10000 cells were analyzed for each sample. DAPI staining HepG2 cells were seeded on 12 well plates. After overnight incubation at 37uC the cells were treated with 0, 0.1 mM, 1 mM and 2 mM hesperidin.
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