Research Article. Mohammad Lalmoddin Mollah, Dong Ki Park, and Hye-Jin Park. 1. Introduction

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1 Evidence-Bsed Complementry nd Alterntive Medicine Volume 212, Article ID , 7 pges doi:1.1155/212/ Reserch Article Cordyceps militris GrownonGermintedSoybenInduces G2/M Cell Cycle Arrest through Downregultion of Cyclin B1 nd Cdc25c in Humn Colon Cncer HT-29 Cells Mohmmd Llmoddin Mollh, Dong Ki Prk, nd Hye-Jin Prk Deprtment of Bioscience nd Biotechnology, Konkuk University, 1 Hwyng-dong, Kwngjin-gu, Seoul , Republic of Kore Correspondence should be ddressed to Hye-Jin Prk, hyejinpk@gmil.com Received 8 November 211; Revised 2 Jnury 212; Accepted 2 Jnury 212 Acdemic Editor: Rffele Cpsso Copyright 212 Mohmmd Llmoddin Mollh et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Cordyceps militris (CM) is n insect-borne fungus tht hs been used in trditionl Chinese medicine becuse of its wide rnge of phrmcologicl ctivities. In this pper, we studied CM grown on germinted soyben (GSC) nd investigted the possible mechnisms underlying ntiprolifertive effect of GSC on HT-29 humn colon cncer cells. In comprison with CM extrcts nd germinted soyben (GS) BuOH extrcts, BuOH extrcts of GSC showed remrkble inhibitory nd ntiprolifertive effects on HT-29 colon cncer cells. After GSC tretment, HT-29 cells becme smller nd irregulr in shpe. High G2/M phse cell popultions were observed in the GSC-treted group. The levels of cyclin B1 nd Cdc25 in the GSC-treted group were lower thn those in the control group. These findings suggest tht GSC BuOH extrcts might ct s n effective nti-prolifertive gent by inducing G2/M cell cycle rrest in colon cncer cells. 1. Introduction Colon cncer is serious public helth problem in the Western world [1]. According to the Americn Cncer Society, colon cncer is the second leding cuse of cncerrelted deths in the United Sttes (US). Ptient survivl is relted to the tumor stge. For exmple, stge I colorectl cncer, in which the crcinom remins loclized in the submucos of the colon epithelium, hs n overll 5-yer survivl rte of over 9% while the 5-yer survivl rte for stge IV cncer is less thn 1%. The dysregultion of cell cycle found in some cncers. A lrge number of cells show modulted expression of the molecules responsible for poptosis nd cell cycle rrest [2], including cyclins nd cyclin-dependent protein kinses (CDKs). Current therpies for cncer re lrgely bsed on cncerspecific surgery, hormone therpy, rdiotherpy, chemotherpy, nd tretment with nticncer drugs. While dvnces continue to be mde in the development of effective strtegies for treting colorectl cncer, chemotherpy is often limited by the severe dverse effects nd dose-limiting toxicity of the drugs. Therefore, it is extremely essentil to identify nd screen compounds from nturl products tht cn effectively tret cncer with no dverse effects. A lrge number of medicinl mushrooms hve been proven to possess nticncer ctivities [3 6]. One such mushroom, Cordyceps militris (CM) hs been used in trditionl Chinese medicine. CM extrcts hve been reported to exert wide rnge of phrmcologicl ctivities, including immunomodultory, nti-inflmmtory, nd ntitumor ctivities [7 1]. Kim et l. [11] ndprketl.[7] reported tht CM hs potent cytotoxic effect ginst cncer cells nd exerts immunostimultory effects. Jin et l. reported tht queous CM extrcts induced poptosis in humn brest cncer MDA-MB231 cells by ctivting cspses nd inctivting Akt [12]. Despite the clinicl nd phrmcologicl importnce of CM, nturlly occurring CM is not esily vilble in lrge quntities becuse of its high cost of production. Therefore, we developed novel methods for cultivting CM. We successfully cultivted CM on germinted soybens (GS), which contin lrge number of nutrients nd ctive

2 2 Evidence-Bsed Complementry nd Alterntive Medicine biologicl compounds such s isoflvones [13, 14]. In our previous study, we demonstrted tht CM cultivted on GS (GSC) shows better phrmcologicl ctivity thn nturlly occurring CM [1, 15]. However, the ctivity of GSC ginst colon cncer crcinogenesis hs not been clerly stted. In this study, we investigted the effect of GSC on HT-29 colon cncer cells nd determined the moleculr mechnism underlying this effect. 2. Mterils nd Methods 2.1. Regents nd Chemicls. GSC, CM, nd GS (Kucri 93) were obtined from The Cell Activtion Reserch Institute, Seoul, Republic of Kore. RPMI 164 medium, fetl bovine serum (FBS), penicillin-streptomycin, nd trypsin- EDTA were obtined from Gibco BRL (Grnd Islnd, NY, USA). Propidium iodide (PI), NP-4, nd RNse A were obtined from Sigm Chemicl Co. (St. Louis, MO, USA). Anti-Cdc25c (Cell Signling Technology Inc., Dnvers, MA), nticyclin B1 (Cell Signling Technology Inc., Dnvers, MA), nti-bcl2 (Cell Signling Technology Inc., Dnvers, MA), nticspse-9 (Cell Signling Technology Inc., Dnvers, MA), nti-β-ctin, horserdish peroxidse- (HRP-) conjugted nti-rbbit, nd nti-mouse IgG ntibodies (Snt Cruz Biotechnology, Snt Cruz, CA) were obtined from the respective suppliers. The chemiluminescence detection kits were purchsed from Biosesng (Seoul, Republic of Kore) nd EZ-CyTox ssy kit from Delillb service Co. (Republic of Kore) Preprtion of GSC. GSC ws grown s previously described [1]. An uthenticted voucher specimen of CM (Kucri 93) s deposited in the Herbrium t the College of Bioscience nd Biotechnology, Konkuk University (Seoul, Republic of Kore). Briefly, the CM mycelium (Kucri 96) ws inoculted on germinted soybens (Glycine mx (L.) Merr), nd cultured t 2 25 C for 4 weeks. The powdered mteril (1 kg) ws extrcted under reflux with 8% MeOH (methnol extrct of GSC (GSCM)) for 48 h. The totl extrct (178 g, yield (w/w), 17.8%) ws dissolved with wter. After removing the insoluble solid prticles by filtrtion, the liquid phse ws extrcted sequentilly by solvents with incresing polrity (hexne, EtOAc, BuOH, nd wter; 1 : 1 (w/v) for ll solvents) to yield 4 frctions. The liquid-liquid phse extrction ws performed in Erlenmeyer flsks by shking, nd the extrcts were concentrted to dryness by rotry evportor. Thus, we obtined the following frctions: hexne frction (16 g, yield (w/w) 1.6%), EtOAc frction (4.5 g, yield (w/w).45%), BuOH frction (8.25 g, yield (w/w).825%), nd wter frction (1.86 g, yield (w/w) 1.86%) Cell Culture. The HT-29 cells (humn colon cncer cells) were purchsed from the Americn Type Culture Collection (ATCC, Mnsss, VA, USA). Cells were cultured in RPMI 164 medium (Gibco, Grnd Islnd, NY, USA) supplemented with 1% het-inctivted FBS (Gibco, Grnd Islnd, NY, USA), 1 U/mL of penicillin, nd 1 μg/ml streptomycin t 37 C in humidified incubtor with 5% CO Cell Prolifertion Assy. The effect of GSC, GS, nd CM BuOH extrct on HT-29 cell prolifertion ws mesured using the EZ-CyTox kit (Delillb service Co., Republic of Kore). The ssy ws performed s per the mnufcturer s protocol. HT-29 colon cncer cells (1 1 4 cells/well) were plced in 96-well plte nd incubted with vrious concentrtions (, 25, 5, 75, 1, nd 25 μg/ml) of GSC BuOH, GS BuOH, nd CM BuOH extrcts for 48 h nd 72 h, respectively. A fixed mount (1μL) of EZ-CyTox regent ws dded to ech well nd incubted for n dditionl 1-2 h t 37 C. Cell prolifertion levels were detected t n opticl density (OD) of 45 nm by using n ELISA Multidetection Reder (Tecn, Mnnedorf, Switzerlnd) Morphologicl Anlysis. HT-29 cells were plced in 6- well pltes t density of cells/ml. After 24 h incubtion, the cells were treted with different concentrtions of GSC BuOH extrct. After 48 h, the cells were fixed with 3% formldehyde nd then observed under phse-light microscope (Olympus, Tokyo, Jpn) nd photogrphed t mgnifiction of 1x to detect ny morphologicl chnges Cell Cycle Anlysis. HT-29 cells (1 1 6 cells/ml) were incubted in 6-well pltes in the presence or bsence of GSC BuOH extrct for 48 h, fter which they were hrvested by trypsiniztion, wshed twice with phosphte buffered sline (PBS), nd fixed with 7% ice-cold ethnol. After centrifugtion, the fixed cells were incubted with stining solution contining.2% NP-4, RNse A (3 μg/ml), nd PI (5 μg/ml) (Sigm, St. Louis, USA) in phosphte-citrte buffer (ph 7.2). Cellulr DNA content ws nlyzed by flow cytometry using Becton Dickinson lser-bsed flow cytometer (Becton Dickinson, New Jersey, USA). At lest 1, cells were used for ech nlysis, nd the results were displyed s histogrms. The verge percentge of cells in ech phse of the cell cycle ws determined over 3 independent experiments Western Blotting Anlysis. The HT-29 cells were treted withgscbuohextrctstconcentrtionsof,25,5, 75, 1, nd 25 μg/ml for 48 h. Cells were lysed in the lysis buffer nd the protein concentrtion ws determined using protein ssy kit (Thermoscientific, Rockford, USA) with bovine serum lbumin s the stndrd. Smples with equl mounts of protein were nlyzed by 12% SDS-PAGE. The proteins were trnsferred to polyvinylidene fluoride (PVDF) membrne using trnsfer buffer. The membrnes were incubted overnight t 4 C with blocking buffer (1 PBS-T nd 5% skim milk) nd then incubted with ntibodies ginst Cdc25c, cyclin B1, Bcl2, cspse-9 (1 : 2,), nd β-ctin (1 : 3,) for 2-3 h t room temperture with constnt shking. The membrnes were wshed three times in 1 PBS-T buffer nd incubted with HRP-conjugted secondry ntibodies (1 : 5,) for 1-2 h. The membrnes were wshed nd detection of the immunorective bnds ws

3 Evidence-Bsed Complementry nd Alterntive Medicine HT-29 cell prolifertion (% of control) HT-29 cell prolifertion (% of control) 12 bb 6 bb bb b bb bbb 4 2 Smple Dox # 6 ## ## ## (µg/ml) 25 (µg/ml) GSC ## 4 GSC 48 h CM 72 h GS () (b) Figure 1: Anti-prolifertive effects of GSC, GS, nd CM BuOH extrct on HT-29 cells. () HT-29 cells (1 14 /well) were treted with vrious concentrtions (, 25, 5, 75, 1, nd 25 μg/ml) of GSC, GS, CM BuOH, extrct nd 25 μg/ml of doxorubicin for 48 h. Doxorubicin, potent nticncer gent, ws used s the positive control. The HT-29 cell prolifertion ws determined by the EZ-CyTox ssy. One-wy ANOVA followed by Dunnett s t-test ws used for comprisons of multiple group mens ( P <.1 or b P <.5 versus blnk). (b) Effect of GSC BuOH extrct on HT-29 cell prolifertion. Time-dependent inhibition of HT-29 cell prolifertion by the GSC BuOH extrcts. The growth inhibition ws clculted s percentge of inhibition nd compred with the vlues for the control. One-wy ANOVA followed by Dunnett s t-test ws used for comprisons of multiple group mens ( P <.1 versus control (48 h), # P <.1 or ## P <.5 versus control (72 h), P <.1 control (48 h) versus control (72 h)). GSC, Cordyceps militris grown on germinted soyben; GS, germinted soyben; CM, Cordyceps militris. µg/ml 25 µg/ml () 75 µg/ml 5 µg/ml (b) 25 µg/ml 1 µg/ml (d) (c) (e) (f) Figure 2: Effect of GSC BuOH extrct on morphologicl chnges in HT-29 colon cncer cells. HT-29 cells were treted with the following concentrtions of GSC BuOH for 48 h. () μg/ml, (b) 25 μg/ml, (c) 5 μg/ml, (d) 75 μg/ml, (e) 1 μg/ml, nd (f) 25 μg/ml of GSC BuOH extrct. The cell morphology ws photogrphed by EVOS inverted microscope t 1x (Advnced Microscopy Group, Mill Creek, USA). The rrow indictes cells with irregulr size nd shpe. The rrowhed indictes single cells nd loss of colony formtion (br = 1 μm). GSC, Cordyceps militris grown on germinted soyben.

4 4 Evidence-Bsed Complementry nd Alterntive Medicine Cell number () GSC µg/ml (b) GSC 25 µg/ml (c) GSC 5 µg/ml Sub-Gl: 1.87% Sub-Gl:.95% Sub-Gl: 1.51% G1/G: 73.7% G1/G: 81.1% G1/G: 61.38% G1/G G1/G G1/G 12 S: 1.77% 12 S: 5.33% 12 S: 15.65% 8 S G2/M: 1.34% 8 S G2/M: 12.28% 8 S G2/M: 21.69% (d) GSC 75 µg/ml (e) GSC 1 µg/ml (f) GSC 25 µg/ml Sub-Gl: 1.46% Sub-Gl: 1.97% Sub-Gl: 3.41% G1/G G1/G: 64.74% G1/G: 59.61% G1/G G1/G: 49.25% G1/G S: 12.98% S: 11.79% S: 17.8% 8 S G2/M: 21.8% 8 S G2/M: 26.69% 8 S G2/M: 3.21% DNA contents µg/ml Sub-G1 G1/G S G2/M 48 h Control GSC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.65 The results were represented s percentge of totl treted cells. Dt vlues re expressed s men ± SD (n = 3). Significntly different from control t P<.5. Figure 3: GSC BuOH extrct induced G2/M phse cell cycle rrest in HT-29 cells. Cell cycle progression ws exmined by flow cytometry. () μg/ml, (b) 25 μg/ml, (c) 5 μg/ml, (d) 75 μg/ml, (e) 1 μg/ml, nd (f) 25 μg/ml. One representtive of 3 independent experiments is shown (48 h tretments). Histogrms show the findings during sub-g1, G1/G, S, nd G2/M phses of HT-29 cells. One-wy ANOVA followed by Dunnett s t-test ws used for comprisons of multiple group mens ( P<.5 or P<.1 versus control). performed using the enhnced chemiluminescence western blotting detection system (Biosesng, Seoul, Republic of Kore) Sttisticl Anlysis. Vlues re presented s percentge ± SD of control. Student s t-test or one-wy ANOVA/Dunnett s t-test ws used to nlyze the sttisticl significnce between the CM-treted nd control groups. Sttisticl nlysis ws performed using SPSS, version 12 (SPSS Inc., Chicgo, IL, USA). 3. Results 3.1. GSC Tretment Inhibited HT-29 Colon Cncer Cell Prolifertion nd Induced Chnges in HT-29 Cell Morphology. In comprison with the GS nd CM BuOH extrcts, GSC BuOH extrct showed significnt inhibitory effect on the growth of HT-29 cells (Figure 1()). After 48 h tretment with 1 μg/ml of GSC BuOH, GS BuOH, nd CM BuOH extrcts, the prolifertion of HT-29 cells reduced by 46.56%± 3.55%, 42.56% ± 2.99%, nd 36.23% ±.46%, respectively. Therefore, further experiments were conducted using the GSC BuOH extrct. As shown in Figure 1(b), GSCBuOH extrcts significntly inhibited HT-29 cell prolifertion in concentrtion- nd time-dependent mnner. However, GSC BuOH extrcts did not ffect cell vibility of mouse mcrophge RAW264.7 (dt not shown). We lso found morphologicl chnges in HT-29 cells fter GSC BuOH extrct tretment (Figure 2). After tretment with GSC BuOH (, 25, 5, 75, 1, nd 25 μg/ml) for 48 h, the cells showed morphologicl fetures such s loss of colony formtion nd irregulr size nd shpe, which were not found in the cells of the control group GSC Induced G2/M Phse Cell Cycle Arrest in HT-29 Cells. Uncontrolled cell prolifertion is chrcteristic of cncer cells [16]. To prove the inhibitory mechnism of GSC BuOH extrct ginst colon cncer cells, we exmined the cell cycle ltertion of HT-29 cells by flow cytometry. After GSC BuOH extrct tretment for 48 h, the DNA contents of the G2/M phse of HT-29 cells incresed in concentrtion-dependent mnner thn those of the control group. The percentge of cells in G2/M phse were 12.28% ± 1.5%, 21.69% ± 5.37%, 21.8% ± 4.8%, 26.69% ±.92%, nd 3.21% ±.65% compred to tht in control (1.34% ± 1.6%), when treted with 25, 5, 75, 1, nd 25 μg/ml of GSC BuOH extrct for

5 Evidence-Bsed Complementry nd Alterntive Medicine 5 Cyclin B1 Cdc25c Cspse-9 Bcl2 Actin GSC (µg/ml) Cyclin B1 control (%) Cspse-9 control (%) GSC (µg/ml) GSC (µg/ml) (C) (A) GSC (µg/ml) Cdc25c control (%) Bcl2 control (%) GSC (µg/ml) (D) () (B) 25 Cyclin B1 Cdc25c 12 Action GSC-1 (µg/ml) (h) Cyclin B1 Percentge of control (%) Time (h) (A) Cdc25c Percentge of control (%) Time (h) (B) (b) Figure 4: Effect of GSC BuOH extrct on the protein expression levels of cyclin B1 nd Cdc25c in HT-29 cells. () HT-29 cells were treted with the indicted concentrtions (, 25, 5, 75, 1, nd 25 μg/ml) of GSC BuOH extrct for 48 h. Cell lystes were processed for western blot nlysis with nti-cyclin B1, nti-cdc25c, nti-bcl2, nticspse-9, nd nti-β-ctin ntibodies. Densitometric ((A) (D)) nlysis of 3 independent western blots (men ± SD) expressed in terms of percentge of the vlues for the control groups ( P<.5; P<.1; P<.5). (b) HT-29 cells were treted with 1 μg/ml of GSC BuOH extrct for 2, 6, 12, 24, nd 48 h. Cell lystes were processed for western blot nlysis with nti-cyclin B1, nti-cdc25c, nd nti-β-ctin ntibodies. β-actin ws used s n internl control. Figures re representtive of 3 independent experiments. Densitometric nlysis of the bnds of three western blots (men ± SD) expressed in terms of percentge of the vlues for the control groups ( P<.5; P<.1; P<.5).

6 6 Evidence-Bsed Complementry nd Alterntive Medicine 48 h, respectively (Figure 3). These results indicte tht GSC BuOH extrct cused G2/M cell cycle rrest in HT-29 cells GSC Reduced the Protein Levels of Cyclin B1 nd Cdc25c in HT-29 Cells. To determine the moleculr mechnisms underlying the G2/M rrest by GSC BuOH extrct, the levels of the molecules involved in the G2/M phse of the cell cyclewerechecked.gscbuohextrct-tretedcellsstrongly blocked the expression of cyclin B1 nd Cdc25c protein in HT-29 cells in concentrtion-dependent (, 25, 5, 75, 1, nd 25 μg/ml) nd time-dependent (, 2, 6, 12, 24, nd 48 h) mnner (Figures 4() nd 4(b)). However, the GSC BuOH extrct hd no effect on the levels of poptosis-relted proteins like Bcl2 nd cspse-9 (Figure 4()). 4. Discussion Due to the incresing incidences nd reltively low remission rtes of colon cncers, there is need to estblish more effective tretment regimens by dopting novel nd innovtive pproches [16]. The use of ctive medicinl compounds or extrcts from trditionl medicines or nturl sources is considered s one such lterntive tretment pproch. Mny nturlly derived compounds/extrcts re considered sfe s they re obtined from commonly consumed foodstuffs. CM is well-known medicinl mushroom tht hs been used in orientl medicine for treting vrious diseses, including cncers. Previous studies hve demonstrted tht CM hs wide rnge of phrmcologicl ctivities, including immunomodultory nd nti-inflmmtory ctivities [7 11]. Cncer cells generlly exhibit few chrcteristics such s high prolifertion, migrtion, nd mtrix-invsion potentils [17, 18]. Inhibition of tumor growth is one of the therpeutic trgets in the development of nticncer gents. The regultion of tumor cell growth nd the induction of cell deth re the 2 mjor wys to inhibit tumor growth [19]. The present study evluted whether GSC BuOH extrcts hd nti-prolifertive ctivities ginst HT- 29 cells. Although GSC BuOH, GS, nd CM BuOH extrcts exhibited nti-prolifertive ctivity ginst HT-29 cells, GSC BuOH extrcts showed nti-prolifertive ctivity with the lowest IC 5 (1 μg/ml) vlue. Severely distorted HT-29 cells nd loss of colony formtion bility were observed fter GSC BuOH tretment. We nlyzed the chnges in HT-29 cell cycle progression fter GSC BuOH tretment by flow cytometry. Cellulr prolifertion is controlled by vrious geneticlly defined checkpoints, which ensure the progression of cells through the vrious stges of the cell cycle [2]. In cncer cells, cell cycle checkpoint control systems re known to be disrupted through the ccumultion of muttions [21]. In the G2/M phse, dmged cells hve the opportunity to repir DNA or permnently rrest cell growth if the degree of dmge is severe [22]. Severl nticncer gents rrest the cell cycle in G2/M phse, nd then induce poptosis nd necrosis, resulting in cell deth [23, 24]. In generl, the G2/M trnsition is regulted by complex of cell-division cyclins, nmely, Cdc2 nd B-type cyclin [22]. The protein tyrosine phosphtse, Cdc25c, plys the role of mitotic ctivtor by dephosphorylting Cdc2/p34, which forms the Cdc2/cyclin B1 complexes tht permit cells to enter into mitosis [25]. Mny studies showed tht Cdc2/p34 kinse ctivity ws enhnced in some humn cncer cells becuse of their genetic nd epigenetic ltertions [22]. Therefore, we investigted whether the expressions of the molecules involved in G2/M trnsition in HT-29 cells were ltered fter GSC BuOH extrct tretment. We found tht GSC tretmentresultedindownregultionofcdc25cndcyclin B1 expression in HT-29 colon cncer cells. The results of the present study indicted tht GSC cused G2/M-phse cell cycle rrest long with decrese in the levels of cyclin B1 nd Cdc25c, which re involved in cell cycle progression from the G2/M phse. In ddition, the identifiction of such compounds will improve our understnding of the ntiprolifertive ctivities of GSC. Further experiments need to be done to clrify the nti-prolifertive mechnisms of these identified compounds. 5. Conclusion In conclusion, GSC BuOH extrcts might ct s n effective nti-prolifertive gent by inducing G2/M cell cycle rrest in colon cncer cells. Conflict of Interests The uthors declre tht there is no conflict of interests. Authors Contributions M. L Mollh nd D. K. Prk contributed eqully to this pper. Acknowledgments This pper ws supported by the SMART Reserch Professor Progrm of Konkuk University, Seoul, Republic of Kore. References [1] E. T. Hwk nd B. Levin, Colorectl cncer prevention, Journl of Clinicl Oncology, vol. 23, no. 2, pp , 25. [2] L. Frhn, M. Dwson, A. K. Rishi et l., Cyclin B nd E2F-1 expression in prostte crcinom cells treted with the novel retinoid CD437 re regulted by the ubiquitin-medited pthwy, Cncer Reserch, vol. 62, no. 13, pp , 22. [3] Y. C. Hseu, S. C. Chen, P. C. Tsi et l., Inhibition of cyclooxygense-2 nd induction of poptosis in estrogennonresponsive brest cncer cells by Antrodi cmphort, Food nd Chemicl Toxicology, vol. 45, no. 7, pp , 27. [4] C. Y. Jin, Y. H. Choi, D. O. Moon et l., Induction of G2/M rrest nd poptosis in humn gstric epithelil AGS cells by queous extrct of Agricus blzei, Oncology Reports, vol. 16, no. 6, pp , 26. [5]K.C.Kim,J.S.Kim,J.K.Son,ndI.G.Kim, Enhnced induction of mitochondril dmge nd poptosis in humn

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