Ginsenoside from Panax ginseng Meyer Enhances the Cytotoxic and Apoptotic Effect of Cisplatin in A549 Human Lung Cancer Cells

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1 Ginsenoside from Pnx ginseng Meyer Enhnces the ytotoxic nd Apoptotic Effect of ispltin in A549 Humn Lung ncer ells J. K. PARK, V. ASTRO-AEITUNO, S. AHN, S. Y. SIMU 1, M. H. SIDDIQI 1, D. H. KIM, Y. J. KIM AND D.. YANG 1 * Deprtment of Orientl Medicinl Biotechnology, 1 Grdute School of Biotechnology nd Ginseng Bnk, ollege of Life Sciences, Kyung Hee University, Yongin, , Repulic of Kore Prk, et l.: Ginsenoside Enhnces PP Effect in ncer ells Ginsenosides from Pnx ginseng Meyer hve een used in comintion with cispltin to enhnce nticncer potentil of cispltin. However, the comined effects of ginsenoside nd cispltin hs not een studied so fr. Thus, we evluted the nticncer ctivity of ginsenoside lone nd comined with cispltin y using A549 cell line. Our results showed tht cytotoxicity, rective oxygen species genertion nd poptotic effect of cispltin t 1 µg/ml ws enhnced y ginsenoside long with the increse of p53 expression t protein nd gene level, s well s reduction of the mrna expression levels of Bcl-2 nd Bx ws higher for the comined tretment. Further, phosphoryltion of epithelil growth fctor receptors induced y cispltin lone ws decresed fter exposing the cells to the comined tretment. Similrly, the motility of the cells ws higher decresed fter comining cispltin nd ginsenoside thn single drug tretment. In this study, ginsenoside incresed the nticncer effect of cispltin on A549 cells. Key words: Pnx ginseng; ispltin; Apoptosis; R; Lung cncer ispltin is well-known chemotherpeutic drug used for the tretment of different humn cncers, such s the ldder, lung, ovrin, nd testiculr cncer. The root of cispltin to induce cytotoxicity in cncer cells hs een linked to its ility to interfere with DNA *Address for correspondence E-mil: dcyng@khu.c.kr 468 Indin Journl of Phrmceuticl Sciences This is n open ccess rticle distriuted under the terms of the retive ommons Attriution-Nonommercil-ShreAlike 3. License, which llows others to remix, twek, nd uild upon the work non-commercilly, s long s the uthor is credited nd the new cretions re licensed under the identicl terms Accepted 19 April 217 Revised 4 Jnury 217 Received 14 July 216 Indin J Phrm Sci 217;79(3): My-June 217

2 repir mechnism nd with this induce DNA dmge in cncer cells [1,2]. However, severl studies reported drug resistnce nd severl side effects oserved in ptients during the tretment [1]. Among this, the serch for new therpeutic gents with the cpility to decrese the side effects nd overcome the drug-resistnce of cispltin hs een incresing over the yers. Over the yers, numerous nturl compounds hd een used s nticncer therpeutic gents [3]. Pnx ginseng Meyer, trditionl herl medicine used for thousnds of yers in Est Asin countries, showed vriety of nticncer properties in severl studies [4]. Ginsenosides isolted from P. ginseng, hve een reported to enhnce the nticncer ctivity of cispltin [5]. However, the ctivity of the unique ginsenoside (), isolted only from P. ginseng root, nd its interction with cispltin hs not een reported so fr. Previously, hs demonstrted to induce G2/M phse cell cycle rrest nd poptosis in humn osteosrcom, MG-63 cell line, through the mitochondril pthwy [6]. Thus, we hypothesize tht lone might hve n effect on cell motility, induces cytotoxicity nd poptosis, s well s enhnced the nticncer ctivity of cispltin in A549 lung cncer cells. The ginsenoside, unique compound from P. ginseng; ws received from ginseng nk, Kyung Hee University (South Kore) in powdered with purity of 95%. ispltin (Pltosin) ws otined t 1 mg/ml from Phrmchemie B. V. (GA, Netherlnds). RPMI-164 culture medi ws purchsed from GenDEPOT Inc. (TX, USA). Fetl ovine serum (FBS) nd the ntiiotics, 1 UI/ml penicillin nd 1 µg/ml streptomycin, from Gico-Brl (MD, USA). Non-smll lung crcinom cells (A549), were otined from Koren ell Line Bnk (Seoul, South Kore). The cells were grown in RPMI-164, supplemented with 1% FBS nd 1% of ntiiotics. The cells were mintined in n incutor t 37 with humidified tmosphere of 5% O 2. The evlution of cell toxicity ws done y MTT (3,4,5-dimethylthizol-2-yl)-2-5- diphenyletrzolium romide) ssy. The cells were exposed to cispltin (1 µg/ml) in the presence or sence of ginsenoside for 72 h. Ten microlitres of MTT ssy solution (5 mg/ml) ws dded to ech well nd incuted for 3 h fter the tretment hd finished. Then, old medi ws replced y 1 µl of DMSO nd incuted for 3 min. The mount of formzn formed y vile cells ws mesured y multi-model plte reder (Bio-Tek Instrument, Winooski, VT) t test wvelength of 57 nm with reference wvelength of 63 nm [7]. All experiments were repeted in triplets. A549 cells were cultured in 12-well plte ( cells per well) nd incuted for 24 h t 5% O 2 nd 37 humidified tmosphere. After complete confluence ws reched, old medium ws replced with serum-free growth medi for 24 h. Then, 1 μl sterile pipette tip ws used to mke scrtch in A549 cell cultures lyer. In order to remove ded or floting cells, ech well ws wshed twice with PBS. The cells were stimulted with epiderml growth fctor (, 2 ng/ml) nd treted ccording to our schedule: 2% FBS, 2% FBS+, 2% FBS++cispltin 1 µg/ml, 2% FBS++ 1 µm nd 2% FBS++cispltin 1 µg/ml+ 1 µm. The scrtch gp width t 24 h in ech tretment group ws mesured t two different positions nd compred to the gp width t h. The nlysis of the imges, tken t x1 under n opticl microscope Eclipse ME6L (Nikon Instruments, Melville, NJ), ws done y T-scrtch progrm [8]. The motility of the cells in response to the treted cells ws determined reltive to the vehicle control. A549 cells were exposed to cispltin (1 µg/ml) in the presence or sence of ginsenoside (1 µm) for 48 h. ells were wshed twice with 1x PBS nd fixed with 3.7% (v/v) formldehyde for 5 min t room temperture nd wshed twice with PBS. In order to dye the nucleus, the cells were stined with Hoechst solution (2 µg/ml) for 3 min in drk condition t room temperture. Nucler morphologies of the Hoechst-positive cells were oserved nd photogrphed under fluorescence microscope (x4, Optinity, Koren Ltech) for further nlysis. Totl RNA ws isolted from cultured cells using TriZol LS regents (Invitrogen, rlsd, A, USA) ccording to the mnufcturer s protocol. The firststrnd cdnas ws synthesized y using Thermo Scientific cdna synthesis kit (Oneio, Lithuni EU) [9,1]. Initil denturtion t 95 for 3 min followed y PR cycle of denturtion t 95 for 45 s, nneling t 58 for 1 min nd strnd extension t 72 for 1 min. The numer of cycles ws 3. The finl step included incution t 72 for 1 min. The resultnt PR products were electrophoresed on.8% grose gel nd nlyzed with Imge J softwre [11]. SYBR Green qpr Super Mix UDG kit (Invitrogen, rlsd, A) ws used for quntittive rel-time polymerse chin rection (qrt-pr) mplifiction My-June 217 Indin Journl of Phrmceuticl Sciences 469

in R-orett Rotor-Gene Model 6 (Mortlke, NSW 2137, Austrli). Amplifictions were performed t n initil temperture of 95 for 1 min, followed for 4 cycles t 95 for 1 s, 6 15 s, nd 72 for 2 s.

3 in R-orett Rotor-Gene Model 6 (Mortlke, NSW 2137, Austrli). Amplifictions were performed t n initil temperture of 95 for 1 min, followed for 4 cycles t 95 for 1 s, 6 15 s, nd 72 for 2 s. The nlysis ws done y follow the delt cycle threshold (t) method. ells were plted t density of cells per wells in 96 well plte, llowed to ttch overnight nd exposed to tretment for 72 h. The cells were stined with 1 µm H 2 DFDA for 3 min t 37, nd the fluorescence intensity of the cells ws determined using multi-model plte reder. A549 cells were seeded in 1 mm dish culture plte t cells per dish. After 24 h incution, the cells were suject to serum strvtion for 2 h following y 48 h of tretment. Externl stimultion ws done t 2 ng/ml for 3 min prior protein isoltion. After stimultion nd the tretment time ws finished, the cells were rinsed twice with ice-cold PBS. The totl proteins were soluilized with 2X sodium dodecyl sulphte (SDS) loding uffer (1 mm Tris-l (ph 6.8), 4% (w/v) SDS,.2% (w/v) romophenol lue, 2% glycerol nd 2 mm β-mercptoethnol) for 5 min t room temperture (RT). Then, the protein ws dentured t 95 for 1 min nd storge t 2 [12]. For immunolotting, proteins of totl cell lystes were loded nd resolved in 1% SDS-polycrylmide gel electrophoresis nd run t 12 V. The proteins were then trnsferred to nitrocellulose memrnes (Millipore) t 1 V for 2 h. The memrnes were locked t room temperture (RT) for 1 h with 5% skim milk. After locking, the lots were incuted with specific ntiodies (Phospho- Receptor (Tyr168), receptor, p53, nd β-ctin) overnight t 4. The lots were then wshed seven times with TBS-T, followed y got ntimouse or ntirit IgG secondry ntiody for 2 h t RT. Immunolelling ws visulized y enhnced chemiluminescence detection (EMD Millipore). Bnd densities were mesured using ImgeJ softwre [11]. The sttisticl nlyses were performed using GrphPd 6.4 softwre (L Joll, A 9237, USA). Results re expressed s men±sem. The sttisticl significnce of differences etween vlues ws evluted y one-wy ANOVA. The differences were considered significnt t P.5. In the present study, we investigte the ility of ginsenoside () to increse the nticncer ctivity of cispltin. The effect ws ssocited with the 47 Indin Journl of Phrmceuticl Sciences inhiition of cell growth, cell motility; nd induction of poptosis t in vitro level. Previously, ws reported the ility of compounds derived from medicinl plnts to enhnce the cytotoxicity of cispltin in A549 lung cncer cells [13]. Furthermore, it hd een reported tht ginsenosides from P. ginseng enhnced the nticncer ctivity of cispltin [13,14]. In this study, s result of the tretment with t 1 µm in the presence of cispltin t 1 µg/ml, significnt reduction in the cell viility compred to individul nd cispltin tretments ws oserved (fig. 1). In ddition, the comined tretment significntly enhnced the ROS genertion compred to the individul tretments (fig. 2). This result indicted tht ginsenoside my enhnce the cytotoxicity of cispltin through incresing the genertion of rective oxygen species. ell viility (% of control) G 5 1 Fig. 1: ytotoxicity nd phrmcologicl interction of ginsenoside nd cispltin Results re representtive of three independent experiments. Dt re shown s men±sem. ***P<.5 vs. control. *P<.5 vs. cispltin lone. G : Ginsenoside (μm) nd : cispltin (1 μg/ml) ROS Genertion (%) Fig. 2: Intrcellulr ROS genertion induced y ginsenoside nd cispltin The production of intrcellulr ROS in treted A549 cells ws detected y using H2DFDA. Dt re shown s men±sem. ***P<.5 vs. control. #P<.5 vs. cispltin lone. ROS: rective oxygen species; : ginsenoside (1 μm) nd : cispltin (μg/ml) My-June 217

The induction of poptosis y cispltin through induction of DNA dmge hd een well documented [15,16]. Besides, the induction of poptosis y in osteosrcom cells hd een lredy reported [1,15-17].

expression of poptotic mrkers s well s evlute its effect on the nucleus morphology. A higher reduction in mrna expression levels of Bx (fig. 3) nd Bcl-2 (fig.

4 The induction of poptosis y cispltin through induction of DNA dmge hd een well documented [15,16]. Besides, the induction of poptosis y in osteosrcom cells hd een lredy reported [1,15-17]. Thus, in order to determinte whether the cytotoxicity of the comined tretment of nd cispltin cn e relted to the induction of poptosis, we evluted the effect of the single nd comined tretment on the expression of poptotic mrkers s well s evlute its effect on the nucleus morphology. A higher reduction in mrna expression levels of Bx (fig. 3) nd Bcl-2 (fig. 3) ws oserved in the comined tretment group thn the single ones. In ddition, highly numer of poptotic cells ws oserved in the present of cispltin nd RF thn these drugs lone (fig. 4). Further, protein expression nlysis of p53 protein showed Averge Reltive Density Bx. ** Averge Reltive Density Bel-2. Bx Bel-2 GADPII GADPII Fig. 3: Expression of pro- nd ntipoptosis genes in A549 cells The density of PR nds of reltive gene expression of poptotic genes ws done y ImgeJ softwre. Dt re shown s men±sem. ***P<.5 vs. control. : ginsenoside (1 μm); : cispltin (1 μg/ml) A B p53 (DO-1) β-ctin D Reltive density normlized to β-ctin c d e p53 mrna p21 mrna spse-3 mrna 2. Fig. 4: Anlysis of poptotic ctivity of the single nd comined drug tretment in A549 cells () Morphologicl chnges in the nucleus were oserved y Hoechst ssy fter the following tretment: A. untreted cells, B. t 1 µm,. cispltin t 1 µg/ml nd D. cispltin t 1 µg/ml + t 1 µm. Apoptotic cells re indicted with rrows. Scle r: 1 μm. () Western lot nlysis of expression of the pro-poptotic p53 protein. (c-e) mrna expression nlysis of poptotic relted genes normlized to GAPDH. Results re representtive of three independent experiments. Dt re shown s men±sem. **P<.5 vs. control. ## P<.5 vs. cispltin lone. : ginsenoside (1 μm); : cispltin (1 μg/ml) My-June 217 Indin Journl of Phrmceuticl Sciences 471

5 R mrna Reltive density normlized to R significnt increse in the presence of the comined tretment (fig. 4). Also, mrna expression of p53, p21 nd cspse 3 genes ws higher in the comined group (fig. 4c-e). In previous studies, ws reported tht the genes, Bcl-2 nd Bx, cn e cple of independent regultion of common poptotic pthwy [18]. Besides, some studies indicted tht decrese in the expression levels of Bx is ssocited with cispltin resistnce nd Muttion of p53 gene [19]. This muttion of the p53 gene ws previously reported in A549 cells [2]. For this reson, we suggest tht the independent regultion of Bx nd Bcl-2 genes nd increse in the pro-poptotic mrkers in A549 cells oserved during the comined tretment with nd cispltin might e involved in the induction of morphologicl chnges of the nucleus visulized through Hoechst stining. The ctivtion of epithelil growth fctor receptors (R) y cispltin hd een reported previously [21]. This phosphoryltion of R leds to the ctivtion of different pthwys nd with this the initition of other processes, such s migrtion nd invsion [22]. Since metstsis represent the mjor prolem in the tretment of cncer nd involves multiple processes such cell migrtion [23], evlution of the ility of the comined tretment with nd cispltin to reduce the phosphoryltion of R nd cell migrtion ws evluted. It ws found tht (lone) did not modify the expression of phospho-r. On the other hnd, the comined tretment with nd cispltin reduced the expression of phospho-r previously enhnced y cispltin (fig. 5). Also, ws oserved tht the comined drug tretment induces higher decresed on cell migrtion thn single drug tretment (fig. 6 nd ). Next, in order to define if the scrtch ssy result were relted to cell migrtion nd not to the, evlution of mrna levels of cdherin, snil nd slug genes, which re relted to epithelilmesenchyml trnsition (EMT) process ecuse of its reltion to wound heling nd cncer progression [24]. Our results hve shown the increse in the expression of e-cdherin gene (fig. 6c) nd the decrese of snil nd slug genes (fig. 6d-e) for the comined drug tretment, this effect ws significntly different in comprison with the cispltin tretment lone. This finding suggested n ntimigrtory ctivity of in the presence of cispltin though possile shift of motile mesenchyml cells, -stimulted A549 cells; into epithelil cells. This study exposed for the first time the effect of 472 Indin Journl of Phrmceuticl Sciences ginsenoside () in the comintion of cispltin ginst A549 lung-crcinom cell line. It ws oserved tht t 1 µm enhnced the cytotoxicity, poptotic nd the effect on cell motility of cispltin t 1 µg/ml. It is envisged tht further studies re needed y the use of other cncer cell lines to determinte whether the effect is oserved in other cncer cells or if is specific to A549 lung cncer cells. onflict of interest: The uthors report no declrtions of interest. Acknowledgements: This work ws supported y the Kore Institute of Plnning nd Evlution for Technology in Food, Agriculture, Forestry nd Fisheries, Repulic of Kore (grnt numer SB1). Finncil support: Nil. c phospho R R Fig. 5: Anlysis of epithelil growth fctor receptor (R) pthwy () Immunolotting of phospho-r nd R proteins in presence or sence of tretment. () Density nlysis of PR nds ws done y Imge J softwre. (c) mrna expression of R gene normlized to GAPDH. Dt re shown s men±sem. **P<.5 vs. control. : ginsenoside (1 μm); : cispltin (1 μg/ml) My-June 217

(2 ng/ml) h 1 8 6 4 2 Migrtion re (%) 24 h c 2. d 2. e c-cdherin mrna Snil mrna Slug mrna.. Fig.

6 (2 ng/ml) h Migrtion re (%) 24 h c 2. d 2. e c-cdherin mrna Snil mrna Slug mrna.. Fig. 6: Ginsenoside nd cispltin reduce cell migrtion in A549 cells () Photogrphs of A549 cells were tken t the eginning of the scrtch ssy nd fter 24 h. Scle r: 5 μm. () Percentge of the scrtch gp width fter 24 h. (c-e) Evlution of the mrna levels for epithelil nd mesenchyml genes. Ech column represents the men±sem. ++ P<.5 ontrol versus -ontrol. **P<.5 versus -control. ## P<.5 versus cispltin lone. : epiderml growth fctor (2 ng/ml); : ginsenoside (1 μm); : cispltin (1 μg/ml) REFERENES 1. Dsri S, Tchounwou PB. ispltin in cncer therpy: moleculr mechnisms of ction. Eur J Phrmcol 214;74: Kellnd L. The resurgence of pltinum-sed cncer chemotherpy. Nt Rev ncer 27;7: Murthy KN, Sultnpur M, Ro RM. Nturl molecules s tumour inhiitors: Promises nd prospects. J Her Med 214;4: Leung KW, Wong AS. Phrmcology of ginsenosides: literture review. hin Med 21;5:2. 5. hen S, Wng Z, Hung Y, O'Brr SA, Wong RA, Yeung S, et l. Ginseng nd nticncer drug comintion to improve cncer chemotherpy: criticl review. Evid Bsed omplement Alternt Med 214;214: Xu X, Zhng Y, Qu D, Jing T, Li S. Osthole induces G2/M rrest nd poptosis in lung cncer A549 cells y modulting PI3K/Akt pthwy. J Exp lin ncer Res 211;3: Ahn S, Siddiqi MH, Aceituno V, Simu SY, Zhng J, Perez ZE, et l. Ginsenoside Rg5: Rk1 ttenutes TNF-α/IFNγ-induced production of thymus-nd ctivtion-regulted chemokine (TAR/L17) nd LPS-induced NO production vi downregultion of NF-κB/p38 MAPK/STAT1 signling in humn kertinocytes nd mcrophges. In Vitro ell Dev Biol Anim 216;52: Geäck T, Schulz MM, Koumoutskos P, Detmr M. TScrtch: novel nd simple softwre tool for utomted nlysis of monolyer wound heling ssys. Biotechniques 29;46: Ahn S, Siddiqi MH, Noh HY, Kim YJ, Kim YJ, Jin G, et l. Antiinflmmtory ctivity of ginsenosides in LPS-stimulted RAW cells. Science Bulletin 215;6: Sirj FM, Ntrjn S, Huq MA, Kim YJ, Yng D. Structurl investigtion of ginsenoside with PPARγ mjor trnscriptionl fctor of dipogenesis nd its impct on dipocyte. J Ginseng Res 215;39: ollins TJ. ImgeJ for microscopy. Biotechniques 27;43: Duon MJ, Prk KS. Sustnce P enhnces the prolifertion nd migrtion potentil of murine one mrrow derived mesenchyml stem cell like cell lines. Exp Ther Med 215;9: Ozturk O, Bozcuk H, Burgucu D, Ekinci D, Ozdogn M, Akc S, et l. ispltin cytotoxicity is enhnced with zoledronic cid in A549 lung cncer cell line: preliminry results of n in vitro study. ell Biol Int 27;31: Li Y, Zhou T, M, Song W, Zhng J, Yu Z. Ginsenoside metolite compound K enhnces the efficcy of cispltin in lung cncer cells. J Thorc Dis 215;7: Arny I, Megyesi JK, Kneto H, Price PM, Sfirstein RL. ispltin-induced cell deth is R/src/ERK signling dependent in mouse proximl tuule cells. Am J Physiol Renl Physiol 24;287:F543-F Sorenson M, Estmn A. Mechnism of cisdimminedichloropltinum (II)-induced cytotoxicity: role of G2 rrest nd DNA doule-strnd reks. ncer Res 1988;48: Shnggun WJ, Li H, Zhng YH. Induction of G2/M phse cell cycle rrest nd poptosis y ginsenoside in humn osteosrcom MG 63 cells through the mitochondril pthwy. Oncol Rep 214;31: ho DT, Korsmeyer SJ. BL-2 fmily: regultors of cell deth. Annu Rev Immunol 1998;16: Perego P, Girol M, Righetti S, Supino R, serini, My-June 217 Indin Journl of Phrmceuticl Sciences 473

7 Deli D, et l. Assocition etween cispltin resistnce nd muttion of p53 gene nd reduced x expression in ovrin crcinom cell systems. ncer Res 1996;56: Supino R, Perego P, Gtti L, serini, Leonetti, olntuono M, et l. A role for c-myc in DNA dmge-induced poptosis in humn TP53-mutnt smll-cell lung cncer cell line. Eur J ncer 21;37: Benhr M, Engelerg D, Levitzki A. ispltin-induced ctivtion of the receptor. Oncogene 22;21: irdiello F, De Vit F, Orditur M, Tortor G. The role of R inhiitors in nonsmll cell lung cncer. urr Opin Oncol 24;16: Krmer N, Wlzl A, Unger, Rosner M, Krupitz G, Hengstschläger M, et l. In vitro cell migrtion nd invsion ssys. Mutt Res 213;752: Lmouille S, Xu J, Derynck R. Moleculr mechnisms of epithelil-mesenchyml trnsition. Nt Rev Mol ell Biol 214;15:178. My-June 217 Indin Journl of Phrmceuticl Sciences 474

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Prentl doi:.8/nture57 Figure S HPMECs LM Cells Cell lines VEGF (ng/ml) Prentl 7. +/-. LM 7. +/-.99 LM 7. +/-.99 Fold COX induction 5 VEGF: - + + + Bevcizum: - - 5 (µg/ml) Reltive MMP LM mock COX MMP LM+