Hempseed oil induces reactive oxygen species- and C/EBP homologousprotein-mediated apoptosis in MH7A human rheumatoid arthritisfibroblast-like synovial cells

Mini Jeong a, Jaewook Cho a, Jong-Il Shin a, Yong-Joon Jeon a, Jin-Hyun Kim a,Sung-Joon Lee c, Eun-Soo Kim a,b, Kyungho Lee a,b,n

a Department of Biological Sciences, Konkuk University, 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Republic of Koreab Korea Hemp Institute, Konkuk University, 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Republic of Koreac Department of Biotechnology, Graduate School of Life Sciences & Biotechnology, Korea University, Seoul 136-713, Republic of Korea

a r t i c l e i n f o

Article history:Received 25 October 2013Received in revised form27 April 2014Accepted 29 April 2014Available online 9 May 2014

Keywords:Hempseed oilRheumatoid arthritisPolyunsaturated fatty acidsSynovial cellER stressApoptosis

Chemical compounds studied in this article:Palmitic acid (PubChem CID: 985)Oleic acid (PubChem CID: 445639)Linoleic acid (PubChem CID: 5280450)Alpha-linolenic acid (PubChem CID:5280934)

a b s t r a c t

Ethnopharmacological relevance: The medicinal efficacy of hempseed (Cannabis sativa L.), which is rich inpolyunsaturated fatty acids, in atopic dermatitis, inflammation, and rheumatoid arthritis (RA) has beensuggested for centuries. Hempseed has been used as a treatment for these diseases in Korean andChinese folk medicine. The aim of the study is to investigate the effects of hempseed oil (HO) on MH7Ahuman RA fibroblast-like synovial cells.Materials and methods: MH7A cells were used to study the anti-rheumatoid effects of hempseed(Cannabis sativa L., cv. Cheungsam/Cannabaceae) oil by investigating cell viability, apoptosis, lipidaccumulation, oxidative stress, and endoplasmic reticulum (ER) stress-induced apoptosis.Results: HO treatment reduced the survival rate of MH7A cells and promoted apoptotic cell death in atime- and dose-dependent manner. Both lipid accumulation and the level of intracellular reactive oxygenspecies (ROS) increased in HO-treated MH7A cells. Co-treatment with the antioxidant Tiron effectivelyabrogated the cytotoxic effects of HO; the ROS level was reduced, cell viability was recovered, andapoptotic cell death was significantly diminished. Moreover, HO-treated cells exhibited increasedexpression of the major ER stress markers, glucose-regulated protein 78 and C/EBP homologous protein(CHOP). The siRNA-mediated knockdown of CHOP prevented HO-induced apoptosis.Conclusions: Our results suggest that HO treatment induced lipid accumulation, ROS production, CHOPexpression, and apoptosis in MH7A cells, and that CHOP functions as an anti-rheumatoid factordownstream of HO in MH7A cells.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Hempseed (Cannabis sativa L.) has been used as a health food andmedicinal plant resource for centuries (Al-Khalifa et al., 2007;Callaway, 2004; Prociuk et al., 2008; Zias et al., 1993; Zuardi, 2006).For example, hempseed has been used as a treatment for glaucoma,menstrual cramps, asthma, atopic dermatitis, inflammatory diseases,and arthritis (Benhaim, 2000; Callaway et al., 2005; Zuardi, 2006). InKorean and Chinese folk medicine, brewed hemp has traditionallybeen used to reduce chronic knee pain in patients with arthritis.According to the Donguibogam, one of the major resources for

Oriental medicine, the hempseed known as “Mazain” can improveblood circulation, relieve constipation, and build strength in combina-tion with other oriental medicinal herbs (Cho, 2005). These medicinaleffects are closely related to the abundant essential fatty acids andnutrients in hempseed.

Hempseed contains about 30% oils and 25% proteins in additionto dietary fiber, vitamins, and minerals. Hempseed consists ofmany fatty acids including palmitic acid, stearic acid, oleic acid,linoleic acid (LA, 18:2 omega-6), α-linolenic acid (AL, 18:3omega-3), arachidic acid, gamma-linolenic acid (GLA), and stear-idonic acid; the fatty acid patterns vary slightly depending on thehempseed genotype (Callaway, 2004; Carvalho et al., 2006;Kriese et al., 2004). The protein content and amino acid values ofhempseed were previously reported (Callaway, 2004) and a pro-teomic profiling of hempseed proteins from Cheungsam, a nondrug-type hemp, was also recently described (Park et al., 2012).

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Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.04.0520378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

n Corresponding author at: Department of Biological Sciences, Konkuk University,1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Republic of Korea. Tel.: þ82 2 450 3423;fax: þ82 2 3436 5432.

E-mail address: kyungho@konkuk.ac.kr (K. Lee).

Journal of Ethnopharmacology 154 (2014) 745–752

Hempseed oil (HO) is composed of 480% polyunsaturated fattyacids (PUFAs) and is a good source of the two essential fatty acids,LA and AL (Callaway, 2004). Omega-3 fatty acids, which areessential for development and growth, have curative effects oninflammation, cardiovascular disease, autoimmune diseases, andcancer (Berquin et al., 2008; Leaf and Weber, 1988; Simopoulos,1991, 2002b). A lower ratio of omega-6:omega-3 fatty acids hasimportant beneficial effects on disease (Simopoulos, 2002a). SinceHO contains 480% PUFAs and its ratio of omega-6 to omega-3 isabout 2.5:1, which is quite low, there have been many attempts touse HO as a drug for rheumatoid arthritis (RA), cancer, and otherinflammatory diseases. Additionally, hempseed meal affects bodyweight, cell growth, and neurodegenerative disease in Drosophilamelanogaster (Lee et al., 2010, 2011).

RA is an autoimmune inflammatory disease characterized bychronic synovial tissue inflammation, synovial tissue hyperplasia,and progressive articular cartilage destruction (Choy, 2012; Liu andPope, 2003; Malaviya and Ostor, 2012; Scott and Steer, 2007). Inthe joints of patients with RA, the aggressive front of synovialtissue, called pannus, invades and destroys the articular tissue.Pannus is composed mainly of fibroblast-like synoviocytes (FLSs)combined with an infiltrate of lymphocytes and macrophages.Both increased proliferation and insufficient apoptosis of FLSscontributes to synovial tissue hyperplasia (Baier et al., 2003;Choy, 2012; Kawakami and Eguchi, 2002). Since synovial hyper-plasia is primarily caused by inflammation, immunosuppressiveagents have been suggested as antiarthritis treatments to inhibitthe production of inflammatory cytokines and abnormal FLSproliferation (Hui et al., 1997; Jeong et al., 2009; Libby, 2008;Lorenz and Kalden, 1999; Ma et al., 2002). Recent studies havedemonstrated the efficacy of PUFAs in decreasing the diseaseseverity of RA in animal studies and beneficial effects on someclinical outcomes of RA patients (Ruggiero et al., 2009).

Recently, we showed that immunoglobulin binding protein(BiP)/glucose-regulating protein 78 (GRP78), a representativeendoplasmic reticulum (ER) chaperone, plays a critical role insynoviocyte proliferation and angiogenesis (Yoo et al., 2012). BiPworks as a regulator of three ER stress sensor proteins and hasbeen recovered as an autoantigen in RA (Panayi and Corrigall,2006). The unfolded protein response (UPR) is induced by theactivation of three mammalian UPR sensor proteins, inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6(ATF6), and PKR-like ER kinase (PERK) (Kaufman, 1999; Kaufmanet al., 2002). The UPR is caused by the accumulation of unfoldedproteins in the ER lumen under conditions of insufficient proteinfolding. IRE1α induces the transcription of ER chaperones andfolding enzymes, including BiP/GRP78, through the bZIP transcrip-tion factor XBP1 (Lee et al., 2002). C/EBP homologous protein(CHOP)/growth arrest and DNA damage 153 (GADD153), a UPR-regulated protein, is a transcription factor that forms heterodimerswith C/EBP family members through its bZIP domain and controlsthe expression of genes that function in cell survival or cell death(Oyadomari and Mori, 2004; Yamaguchi and Wang, 2004). CHOPfunctions as a proapoptotic factor by downregulating Bcl-2 expres-sion and by disrupting the cellular redox state via the depletion ofcellular glutathione (Kim et al., 2006; McCullough et al., 2001).CHOP also acts as a regulator of inflammation, sometimes withoutinducing cell death, in several cell types (Endo et al., 2006; Jeonget al., 2009). Despite several pieces of evidence indicating thatCHOP works as a proapoptotic factor, CHOP-mediated apoptosis iscontroversial.

In this study, we investigated the anti-rheumatoid effect ofHO and the UPR using MH7A human FLSs from patients with RA(RA-FLS). Since hempseed has been used to prevent RA or toimprove the symptoms of RA for years via an unknown mechan-ism, understanding the molecular mechanism of HO in RA-FLS

would improve the application of hempseed to RA treatment. Inour attempt to identify the factors involved in HO-mediated celldeath, we found that the induction of reactive oxygen species(ROS) and CHOP play critical roles in the apoptotic death of HO-treated RA-FLS.

2. Materials and methods

2.1. Chemicals, reagents, cells, and cell culture

20,70-Dichlorodihydrofluorescein diacetate (DCFH-DA) was pur-chased from Molecular Probes (Eugene, OR). DAPI, 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron), and Oil Red O were purchasedfrom Sigma (St. Louis, MO). Anti-poly(ADP-ribose) polymerase(PARP) and –GADD153 (CHOP) antibodies were obtained fromSanta Cruz Biotechnology (Santa Cruz, CA). Anti-KDEL antibodies,which recognize both GRP78 and GRP94, were obtained fromStressGen Biotechnologies Co. (Victoria, BC, Canada). HO wasextracted from Cheungsam (Cannabis sativa L., cv. Cheungsam/Cannabaceae), a nondrug-type hemp variant. Cheungsam wasdeveloped by crossing the IH3 Netherland variety with a localKorean variety (Moon et al., 2002). Hempseeds were provided byDangjin Agricultural Technology Center (DATC), a branch office ofthe local government in Korea. We obtained government permis-sion to use hempseeds from the Ministry of Food and Drug Safety(permission # 2007-230). Hexane-extracted HO was prepared aspreviously described by Jeun et al. (2011). Briefly, hempseed waswashed, dried, and finely ground with a high-speed mixer. Hemp-seed powder was mixed with three volumes of hexane for 24 hwith mild stirring. The extract was serially filtered using a No.2 filter (Whatman International Ltd., Madistone, Kent, UK) and anylon filter (0.45 μm, Whatman International Ltd.). The extractionprocedure was repeated twice with the residue. The solvent wasevaporated using a rotary vacuum evaporator (Eyla, Tokyo, Japan)at 35 1C. Residual solvent was re-evaporated twice at 70 1C for10 min. To increase the solubility of HO extract in culture media,two volumes of ethanol were added to the hexane-extracted HOand mixed by vortexing for 5 min at room temperature. The upperethanol phase was used in this work. The final concentration ofHO-containing ethanol in most cases was 2% in culture. All of thechemicals used for the Griess assay were purchased from Sigma.The MH7A cells, immortalized human RA fibroblast-like synovio-cytes (human RA-FLS) with SV40 T antigen (Miyazawa et al., 1998),were grown in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY) and 1%penicillin/streptomycin (Gibco) under a humidified atmosphere in5% CO2 at 37 1C.

2.2. Measurement of cell viability

EZ-cytox (Daeiltech Co., Seoul, Korea) was used to determinethe relative number of viable cells. MH7A cells were seeded in48-well cell culture plates and incubated for 16 h. The cells weretreated with 0–2.5% HO for 24 h or 0–20 μM methotrexate (MTX)for 24 as a positive control. When needed, the cells werepretreated with the antioxidant Tiron for 1 h, and then transfectedwith siRNA oligos or plasmids for 18 h before HO treatment. Afterwashing the cells with phosphate-buffered saline (PBS), 300 μl offresh medium was added to each well together with 15 μl ofEZ-cytox and incubated for 30 min at 37 1C. The absorbance of thesoluble substrate was measured at 450/690 nm using an ELISAReader (UYM 340; ASYS Hitech, Salzburg, Austria).

M. Jeong et al. / Journal of Ethnopharmacology 154 (2014) 745–752746

2.3. Plasmids, siRNA oligos, and transfection

CHOP cDNA was obtained from MH7A cells by reverse tran-scription (RT)–PCR. A 0.5-kb BamHI–HindIII fragment was sub-cloned into the same sites of the pcDNA 3.1 plasmid. siRNAoligonucleotides specific for CHOP (siC1 and siC4) were purchasedfrom Sigma-Proligo (The Woodlands, TX) and scrambled oligonu-cleotides (nonspecific control, NC) were purchased from Bioneer(Daejeon, Korea). Transfection of the CHOP plasmid (pcDNA-CHOP) was carried out using Lipofectamine 2000 (Life Technolo-gies Co., Carlsbad, CA) according to the manufacturer's protocols.

2.4. Identification of apoptotic cells by microscopy and fluorescence-activated cell sorting (FACS) analysis

To monitor apoptotic cell death by fluorescence microscopy,control or HO-treated cells were washed twice with PBS and fixedwith 4% (w/v) paraformaldehyde in PBS for 1 h at room tempera-ture. The cells were then stained with 1 μg/ml DAPI in PBS for10 min at room temperature. After washing three times with PBS,nuclear morphology was observed under a fluorescence micro-scope (ECLIPSE TS-200; Nikon, Tokyo, Japan) equipped with aninverted fluorescence optics lens (400� ). Cells showing fragmen-ted or condensed nuclei were considered apoptotic cells. For theevaluation of apoptotic cell death by FACS, attached and floatingcells grown in 60-mm culture dishes were pooled in conical tubes,pelleted by centrifugation, washed with PBS, and fixed with cold70% ethanol at 4 1C overnight. Cells were washed, re-suspended in1 ml of a propidium iodide (PI) solution containing 20 μg/ml RNaseA and 100 μg/ml PI, incubated for 30 min at 37 1C, and assayedusing a Becton Dickinson Flow Cytometer (Becton–DickinsonBiosciences, San Jose, CA) at 488 nm. The data were analyzed withWinMDI version 2.9 software (Joe Trotter, Scripps Research Insti-tute, La Jolla, CA). Sub-G1 cells were considered to be apoptotic.The percentage of apoptotic cells was calculated as the ratio ofsub-G1 cells to that of the whole cell population.

2.5. Oil Red O staining and Griess assay

Cells cultured in 12-well plates were treated with HO for 24 h,fixed with 4% (w/v) paraformaldehyde for 30 min at room tem-perature, and washed with 60% isopropanol three times. A work-ing solution of Oil Red O was incubated with the completely driedsamples for 10 min. After removal of the Oil Red O solution, waterwas added immediately and the cells were washed four times.Stained lipid droplets in the cells were observed using a fluores-cence microscope (ECLIPSE TS-200; Nikon) equipped with aninverted fluorescence optics lens (200� ). For the Griess assay,Oil Red O was eluted from the cells with 100% isopropanol for10 min. The absorbance of Oil Red O was measured at 500 nm(corrected for the background absorbance at 690 nm) using amicroplate reader (UYM 340; ASYS Hitech).

2.6. Immunoblot analysis

Cells were harvested using RIPA lysis buffer (150 mM NaCl, 1%Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris–HCl,and 2 mM EDTA) with 1% phosphatase inhibitors and proteaseinhibitors. The protein concentration was quantified using theBradford method (Bio-Rad, Hercules, CA). Proteins boiled in 1�sample buffer [500 mM Tris–HCl (pH 6.8), 10% SDS, 20% glycerol,0.05% bromophenol blue, and 1% β-mercaptoethanol] for 5 minat 100 1C were separated on SDS-polyacrylamide gels. The proteinswere then electrotransferred to Immobilon-P membranes (Milli-pore Corp., Bedford, MA) and blotted with the indicated anti-bodies at 4 1C overnight in Tris-buffered saline containing 0.08%

Tween 20 (TBST) and 1% nonfat milk. The membranes were thenincubated with horseradish peroxidase-conjugated antibodies atroom temperature for 2 h, and the band signal was detected usingan LAS-3000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan).To ensure equal loading of the samples, the blots were strippedin stripping buffer [100 mM β-mercaptoethanol, 2% SDS, and62.5 mM Tris–HCl (pH 6.8)] at 50 1C for 30 min, washed twicewith TBST buffer for 15 min each, and re-probed with anti-β-actinantibodies.

2.7. Measurement of intracellular ROS levels

Intracellular ROS production was detected using DCFH-DA as anintracellular probe. Briefly, cells were treated with 5 μM DCFH-DAfor 1 h at 37 1C. After washing twice with PBS, the cells weretreated with HO for 3 h at 37 1C. When needed, Tiron was added1 h before HO treatment. The fluorescence intensity of dichloro-fluorescein was observed under a fluorescence microscope(ECLIPSE TS-200; Nikon) using 488 nm for excitation.

2.8. Observation of endocytosis

Endocytosis was observed using dextran staining. Cells werepre-stained with 5 μM dextran for 1 h, washed with 1� PBS twice,and treated with 1% and 2% HO or ethanol (as the control) for 24 h.The cells were then observed under a fluorescence microscope at560 nm.

2.9. Statistical analysis

A one-way analysis of variance was used for all statisticalanalyses with independent experiments. Following this, post hocanalysis by Student's or Duncan's t-test was conducted using SPSS12.0KO for Windows (SAS Institute Inc., Cary, NC). P-values o0.05were considered to be significant, unless otherwise specified. Alldata are expressed as the mean7the standard error of the mean(SEM) of three independent experiments performed in triplicate.

3. Results

HO containing high levels of unsaturated fatty acids has beenused as a source of food and medicine for centuries withoutknowledge of its mechanism of action. In this study, we examinedthe effect of HO on MH7A human RA-FLS cells. We first investi-gated the effect of HO on cell viability and apoptosis using RA-FLScells. To increase the solubility of the HO extract in culture media,HO extracted with hexane was further extracted with ethanol (seeSection 2). MH7A cells treated with increasing concentrations ofHO for 24 h showed decreased viability (Fig. 1A, right). When thecells were treated with MTX, a drug used for treatment of RA, for24 h as a positive control, cell viability was reduced in a dose-dependent manner (Fig. 1A, left).

When the cells treated with 2% HO were observed for apoptoticmarkers, fragmentation of PARP and nuclear condensation wereobserved (Fig. 1B and C), suggesting that HO inhibited theproliferation of MH7A cells by inducing apoptosis.

To verify whether apoptotic cell death was induced by HO, wemeasured lipid accumulation in MH7A cells using Oil Red O, whichstains oxidized lipid droplets in cells (Laughton, 1986). Microscopicobservation of the HO-treated MH7A cells revealed that thenumber of lipid droplets was increased in a dose-dependentmanner (Fig. 2A). The same pattern was observed when theamount of the Oil Red O extracted from the stained cells wasmeasured quantitatively using a microplate reader (Fig. 2B). Itseems that the uptake of HO by the cells occurred through

M. Jeong et al. / Journal of Ethnopharmacology 154 (2014) 745–752 747

endocytosis, as shown by dextran staining of HO-treated cells(Fig. 2C). These results suggest that HO-mediated lipid accumula-tion in the cells caused apoptotic cell death by increasing oxidativestress through lipid peroxidation.

We tested whether oxidative stress was increased in the HO-treated MH7A cells. First, we investigated the induction of intracel-lular ROS in HO-treated MH7A cells using DCFH-DA, a fluorescent

ROS probe. By fluorescence microscopy, the ROS signal was clearlyincreased in the HO-treated cells (Fig. 3A). To determine whetherHO-induced cell death was relevant to the generation of ROS, wemeasured the viability of cells co-treated with HO and the anti-oxidant Tiron. While the HO-treated cells showed reduced viability,consistent with our previous results (Fig. 1), the viability of the cellspretreated with Tiron just before HO treatment was similar to that

Fig. 1. Hempseed oil (HO) inhibited the cell proliferation and induced apoptosis in MH7A cells. (A) Cells were treated with various concentrations of HO for 24 h (right) ormethotrexate (MTX) for 24 h (left, as a positive control). Cell survival was estimated. (B) Cells were treated with 2% HO for various periods and cell lysates were subjected toimmunoblot analysis. The fold increase represents the relative intensity of PARP normalized with β-actin. An arrowhead indicates the cleaved fragment of PARP. (C) Cellstreated with 2% HO or ethanol (as a control) for 24 h were stained with DAPI and observed by microscopy (400� ) to detect chromatin condensation.

0

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0 0.2 0.5 1 HO (%)2

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* * p<0.01

Fig. 2. Hempseed oil (HO) increased lipid accumulation in MH7A cells. (A) Cells were treated with various concentrations of HO for 24 h and the accumulated lipid dropletswere observed under an interference light microscope (200� ) after staining with Oil Red O. (B) The Oil Red O in the cells was dissolved in isopropanol and the absorbancewas measured. (C) Cells were stained with 5 μM dextran for 1 h and then treated with 1% and 2% HO for 24 h. Ethanol was used as a control. Endocytosis was observed usinga fluorescence microscope (400� ).

M. Jeong et al. / Journal of Ethnopharmacology 154 (2014) 745–752748

of the control cells. This demonstrates that treatment with Tironinhibited HO-induced cell death in MH7A cells (Fig. 3B). Lastly, weexamined the sub-G1 population, a hallmark of apoptosis, usingflow cytometry to confirm whether HO treatment induced ROS-mediated apoptosis. The proportion of HO-treated cells in sub-G1was �21%, while that of cells co-treated with Tiron and HO was�14%, indicating recovery by treatment with the antioxidant(Fig. 3C). These results indicate that HO induces ROS-mediatedapoptotic cell death in MH7A cells.

Since HO is rich in PUFAs, and PUFAs were reported to induceER stress in some cancer cells (Jakobsen et al., 2008; Pan et al.,2004), we investigated the relationship among HO, ER stress, andER stress-induced apoptosis. Western blot analyses using antibo-dies specific for KDEL, which recognizes both GRP78 and GRP94,and CHOP, which is involved in the ER stress pathway, revealed theinduction of GRP94 and CHOP by HO treatment in a dose-dependent manner (Fig. 4A). Expression of CHOP was increasedby HO treatment in a time-dependent manner. CHOP is a proa-poptotic factor that downregulates Bcl-2 or upregulates otherproapoptotic factors, although there are some discrepanciesdepending on the cell type. Therefore, we investigated the roleof CHOP in cell viability and apoptosis in HO-treated MH7A cellsby knocking down CHOP expression using siRNAs specific forCHOP. Our cell viability assay results showed that the siRNA-mediated knockdown of CHOP inhibited HO-induced death inMH7A cells (Fig. 4B and C), and FACS analysis showed thatknocking down CHOP clearly reduced the sub-G1 population inHO-treated cells from 25.9% to �11%, suggesting that CHOP acts asa proapoptotic factor (Fig. 4D). In contrast, overexpression of CHOPslightly increased the percentage of sub-G1 cells. Taken together,these results suggest that CHOP functions as a proapoptotic factorin HO-treated MH7A cells.

4. Discussion

Hempseed is rich in PUFAs and essential amino acids and haslong been used as food and a medicinal resource. In this study, weinvestigated the mechanism of action for HO in MH7A synovialcells and found that HO reduced cell viability, increased lipidaccumulation and oxidative stress, and induced CHOP expressionand apoptosis. These results support the idea that hempseed is agood resource for the prevention or treatment of RA, consistentwith traditional beliefs from Korean and Chinese medicine. Webelieve that our results are closely tied to the effects of the omega-3 fatty acids contained in hempseed because HO contains 480%PUFAs, including the omega-3 fatty acids, which have anti-inflammatory properties and beneficial effects on some clinicaloutcomes of RA patients (Ruggiero et al., 2009; Simopoulos,2002b). Indeed, AL is converted to docosahexaenoic acid (DHA,C22:6n3) and eicosapentaenoic acid (EPA, C20:5n3) in someeukaryotic cells. DHA and EPA are also known to have anti-inflammatory effects and to suppress the proliferation of synovio-cytes in patients with RA (Hamaguchi et al., 2008). Kim (2011), andKim and Lee (2011) have shown that Cheungsam hempseedcontains �32.5% oil and that the major constituents of HO, suchas palmitic acid, stearic acid, oleic acid, and LA, are detected inCheungsam HO, similar to previous reports on different hempgenotypes (Borhade, 2013; Callaway, 2004; Carvalho et al., 2006;Kim, 2011; Kim and Lee, 2011; Kriese et al., 2004; Park et al., 2012).In addition, the total percentages of PUFAs and the two essentialfatty acids, LA and AL, are similar to those previously reported(Callaway, 2004; Karimi and Hayatghaibi, 2006; Kriese et al.,2004). For these reasons, along with the reasons discussed above,we believe that our results are tightly correlated with the effectsof the omega-3 fatty acids. At this point, whether any single

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Fig. 3. Hempseed oil (HO) induced ROS-mediated cell death in MH7A cells. (A) MH7A cells pretreated with DCFH-DA were treated with 2% HO for 2 h and the production ofintracellular ROS was observed by fluorescence microscopy (400� ). Cells were treated with or without Tiron for 1 h before exposure to HO. ((B) and (C)) Cells pretreatedwith or without Tiron were treated with 2% HO for 24 h. Cell viability was determined using a WST-1 assay (B). Cells stained with PI were subjected to FACS analysis.Apoptosis is expressed as the percentage of cells counted in sub-G1 phase as indicated by the bar in the histogram (C).

M. Jeong et al. / Journal of Ethnopharmacology 154 (2014) 745–752 749

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Fig. 4. CHOP is involved in hempseed oil (HO)-induced cell death in MH7A cells. (A) Cells were treated with various concentrations of HO for 24 h (top) or with 2% HO forvarious periods (bottom) and cell lysates were subjected to immunoblot analysis. Thapsigargin (Tg, 1 μM) was used as a positive control. (B) Cells were transiently transfectedwith siRNA oligonucleotides specific for CHOP (siC1 or siC4) or scrambled oligonucleotides (nonspecific control, NC). At 18 h post-transfection, the cells were treated with 2%HO for 24 h and cell viability was determined. ((C) and (D)) Cells were transiently transfected with a pcDNA-CHOP plasmid or siRNA oligonucleotides specific for CHOP (siC1and siC4). (C) Cell lysates were subjected to immunoblot analysis using antibodies specific for CHOP (indicated by the arrowhead) or β-actin. (D) Cells stained with PI weresubjected to FACS analysis. Apoptosis is expressed as the percentage of cells in sub-G1 phase as indicated by the bar in the histogram.

M. Jeong et al. / Journal of Ethnopharmacology 154 (2014) 745–752750

compound or combination of compounds derived from PUFAscaused the effects observed in our study is unclear. One of thepossible candidates that exerted anti-proliferative effects is GLAbecause hempseed oil contains about 4% GLA and it can beconverted to dihomo-δ-linolenic acid (DGLA), and eventually toprostaglandin E1 (PGE1), which is known to have anti-proliferativeproperties (Callaway, 2004; Fan and Chapkin, 1998; Wang et al.,2012). Currently, we are assessing the effects of several purecompounds found in HO on synovial cells.

One possible explanation for HO-mediated cell death is lipidperoxidation and ROS accumulation. We observed lipid accumula-tion (Fig. 2A and B), ROS production (Fig. 3A), and apoptosis(Fig. 1B and C) in HO-treated synovial cells. An analogous inter-pretation of these results is that significant portions of theaccumulated lipids were converted to highly reactive and oxida-tive molecules by lipid peroxidation, and that the resultantaldehydes (e.g., 4-hydroxyhexenal) served as ROS donors. Theseproducts, which are relatively stable in the cell, would serve asalkyl donors, causing ROS-mediated cell death (Lee et al., 2006).The roles of ROS are complicated because ROS can be induced bymany different sources and exert diverse effects depending onthe cell type. Nevertheless, the results of this study show thatROS are mediators of apoptosis in HO-treated, lipid-accumulatedsynovial cells. These results are consistent with our previousreport regarding apigenin-mediated synovial cell death in theinduction of ROS and its apoptotic role in synovial cells (Shinet al., 2009).

An interesting feature of HO-mediated apoptotic cell death isthe involvement of CHOP. CHOP is a transcription factor thatfunctions in the cell death pathway under ER stress conditions.CHOP is known to induce apoptosis by downregulating transcrip-tion of the anti-apoptotic factor Bcl-2, by disrupting the cellularredox state, and by upregulating proapoptotic factors such as Bax,as well as the calcium-mediated activation of calcium–calmodulinkinase II (CaMKII) (Kim et al., 2006; McCullough et al., 2001). Theprimary regulators of CHOP expression in HO-treated synovialcells are not known, but if the UPR in synovial cells follows thetypical pattern in eukaryotic cells, CHOP expression is regulated bythe PERK-eIF2α-ATF4 pathway rather than IRE1α-XBP1 or ATF6,two other UPR pathways with relatively weak activation. Due totheir cancer and inflammatory cell-like features, however, it isunlikely that the synovial cell UPR is similar to that of normaleukaryotic cells. Downstream effectors of CHOP-mediated apop-tosis are complicated and the main effectors in synovial cells areunknown. Possible targets are ERO1α-mediated hyperoxidation ofthe ER lumen, inositol trisphosphate receptor 1 (IP3R)-mediatedcalcium release from the ER, and the resultant activation of CaMKIIand Bax–Bak-mediated mitochondrial permeabilization (Hetz,2012; Marciniak et al., 2004; Tabas and Ron, 2011). Since ourresults showed that ROS are involved in HO-mediated cell death,which is suppressed by Tiron, it is obvious that at least CHOP andERO1α contribute to the induction of oxidative stress markers andapoptosis. However, we cannot rule out the possibility that thereare synovial cell-specific roles for CHOP because, as mentionedabove, synovial cells have both cancer cell-like and inflammatorycell-like features where the intercellular and intracellular environ-ments are prone to ER stress due to the uncontrolled cell cycle. Itwill be necessary to elucidate which pathway is involved in oursystem by specifically blocking downstream targets or usingmutants for ERO1α, IP3R, and CaMKII. By defining the roles ofeffectors other than CHOP (e.g., GADD34, DR5, JNK, TRB3, andRIDD) in synovial cells and by identifying the primary componentof HO that exerted the effects shown in this study, our under-standing of synoviocyte survival and death will be significantlyimproved and the opportunity to use HO-derived drugs for RAtreatment will be increased.

Acknowledgment

This paper was supported by Konkuk University in 2010.

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