Antiproliferative Activity of the Chinese Medicinal Compound, Delisheng, Compared With Rg3 and Gemcitabine in HepG2 Cells

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1 Reserch Pper Antiprolifertive Activity of the Chinese Medicinl Compound, Delisheng, Compred With Rg3 nd Gemcitine in HepG2 Cells S. H. WANG*, Y. C. WANG 1, Y. L. NIE, Y. N. HAI, H. F. SUN, Z. L. YUAN AND K. J. NAN Deprtment of Medicl Oncology, The First Affilited Hospitl of The School of Medicine of Xin Jiotong University, Xin, , Shnxi Province, 1 Center for Cell Biologicl Therpy nd Reserch, Generl Hospitl of Gungzhou Millitry Commnd of PLA, Gungzhou, , Gungdong Province, P.R. Chin Wng, et l.: The Effect of DLS on HepG2 Delisheng consists of rdix ginseng, rdix strgli, venenum ufonis nd mylris. It hs een reported tht delisheng inhiits the prolifertion of denocrcinom cells nd stimultes their poptosis. Delisheng cn lso enhnce the ody s immunity nd induce the redifferentition of crcinom cells. Delisheng inhiited the prolifertion of HepG2 cells in MTT ssy nd promoted poptosis more effectively in contrst to the ctive components of ginseng extrct, Rg3 nd gemcitine. It is possile tht Rg3 hs n importnt role in delisheng ecuse they ll could regulte the cell cycle, poptosis nd expression of endosttin nd VEGFR 2. Delisheng cused the cell cycle to rrest t the S phse, while gemcitine locked the cells t the G0/G1 phse in cell cycle nlysis. Consequently, the poptosis rte of the HepG2 cell line cn e incresed significntly y delisheng in comintion with gemcitine, compred with the single drug. The expression of the procspse proteins, cspse protein, nd dr5 detected y Western lot were incresed while cl 2 nd survivin decresed in the delisheng group, compred with controls. The oservtions suggest tht the delisheng induced poptotic effect might e closely relted to the mitochondril poptosis pthwy, nd the deth receptor signling pthwy. Key words: Apoptosis, Dlisheng, HepG2 cell, prolifertion, trditionl Chinese medicine Heptocellulr crcinom (HCC) is highly mlignnt tumour of the digestive system. At present, its incidence is incresing in the world, nd it is mjor helth concern worldwide ecuse of its high moridity nd mortlity, nd poor prognosis [1]. Potentil curtive tretments for HCC include heptic resection, locl ltive therpies, systemic tretment nd liver trnsplnttion [2]. Surgery is the most effective tretment, ut unfortuntely it is fesile in only 10-20% of ptients, ecuse mjority of HCC ptients present t dvnced or unresectle stges of the disese. Even for those eligile for surgicl resection, the postopertive recurrence rte cn e s high s 50% in 2 yers. Chemotherpy could e n option s n lterntive to improve the prognosis of HCC. In generl, however ptients with HCC do not respond well to chemotherpy, nd get no survivl enefits [3 6]. In ddition, for some intermedite nd terminl ptients, the tolerle doses *Address for correspondence E mil: wsh2003@126.com of chemotherpy re quite low, ecuse of impired liver function nd other complictions. As result, systemic chemotherpy for HCC hs een quite ineffective. Moreover, there re very few therpeutic drugs ginst HCC. Therefore, finding new method or drug which hs etter efficiency nd lower toxicity will mke significnt contriution to the tretment of HCC ptients. Fortuntely, trditionl Chinese medicines hve een shown to hve mrked therpeutic effect ginst mny types of cncers such s esophgel cncer [7], lung cncer [8], heptocellulr cncer [9] nd colonic crcinom [10]. They hve gined wide cceptnce s sfe, pllitive nd effective tretment in Chin ecuse of their unique dvntges [11,12]. Delisheng (DSL), s multicomponent ntitumour drug, is the first trditionl Chinese drug which gins the second kind of new drug certificte in Chin. DSL is composed of rdix ginseng, rdix strgli, venenum ufonis nd mylris. It hs een reported tht DSL hs strong inhiitory effect on the growth 578 Indin Journl of Phrmceuticl Sciences Septemer - Octoer 2013

2 of some crcinom cell lines [13,14]. Furthermore, it cn lso stimulte immunity, ugment the effects of surgery, chemotherpy or rdiotherpy, nd improve the clinicl symptoms nd qulity of life in ptients with lte stge HCC [15,16]. As DSL is ttrctive s nturl product for medicinl use, incresing ttention is eing pid to its scientific evlution nd its possile moleculr mechnisms. In this study, we confirmed tht DSL inhiits the prolifertion of the HepG2 cell line. We then compred the role of DSL, nd the ctive components of ginseng extrct, Rg3 nd gemcitine (GEM), in cell cycle, poptosis nd expression of VEGFR 2 nd endosttin (ES). We demonstrted tht oth Rg3 nd DSL hve effects on these processes ut the ltter is more powerful, nd cnnot e sustituted y Rg3. DSL locked the cells in the S phse, while GEM rrested the cells in the G0/G1 phse. Consequently, DSL comined with GEM could strengthen its cpility ginst cncer. Moreover, moleculr nlysis of the poptosis pthwy reveled tht the expression of dr5, pro cspse nd cspse proteins incresed, while the level of cl 2 nd survivin decresed. These oservtions indicted tht DSL induces poptosis most likely vi oth mitochondril nd deth receptor pthwys. MATERIALS AND METHODS The humn heptom cell line HepG2 ws cultured in RPMI 1640 with 10% fetl ovine serum nd 2 mm L glutmine (Gico/BRL). The effects of DSL (Zhengng Phrmceuticl Compny, Beijing, P.R. Chin), Rg3 (Fusheng Nture Phrmceuticl Compny, Dlin, P.R. Chin) nd GEM (Hosen Phrmceuticl Compny, Jingsu, P.R. Chin) were evluted ccording to previously descried method [10]. The IC 50 ws clculted using the SPSS 13.0 softwre to stndrdise drug dministrtion in the following experiments. 3 (4,5 dimethylthizol 2 yl) 2,5 diphenyltetrzolium romide (MTT) ws purchsed from Sigm, St Louise, MO, USA. Mouse monoclonl ntiodies ginst humn ES, VEGFR 2, cspse 3, cspse 8, cspse 9, dr5, cl 2, nd surviving were purchsed from Snt Cruz Biotechnology Inc., Texs, USA. MTT cell viility ssy: Cells were seeded in 96 well pltes t 1000 cells per well in 200 μl of medium the dy efore the experiment. DSL, Rg3 nd GEM were dded to the cells t the indicted concentrtions (DSL: 3.125, 6.25, 12.5, 25, 50 nd 100 μg/ml; Rg3: 5, 10, 20, 40, 80 nd 160 μg/ml; GEM: 0.158, 0.316, 0.625, 1.25, 2.5 nd 5 μg/ml). Cell growth ws ssyed t 24, 48 nd 72 h fter drug tretment, y spiring hlf of the medium (100 μl) nd dding n equl volume of fresh medium contining MTT (1 mg/ml). Cells were incuted further t 37, 5% CO 2 for 4 h, nd 150 μl of dimethyl sulphoxide (DMSO, Sigm, USA) ws dded to ech well, followed y mixing t room temperture for 10 min. The sornce of the superntnt ws mesured t 570 nm using Multifunction microplte reder (POLARstrOPTIMA, BMG, Germny). The inhiition of cell viility ws clculted y the formul, %inhiition=[1-(a570 nm/a'570 nm)] 100, where A570 nm is from the treted cells nd A'570 nm is from the untreted cells. Ech experiment ws repeted t lest three times. Cell cycle nlysis: Cells were treted y drugs (DSL 25 μg/ml, Rg3 80 μg/ml nd GEM 2.5 μg/ml) for 24 h. After 24 h intervention y drugs, treted nd untreted cells ( ) were collected nd wshed with PBS, then fixed in 75% lcohol for 30 min t room temperture. The cells were wshed three times with cold PBS, nd resuspended in 1 ml PBS contining 40 μg propidium iodide (PI, Sigm) nd 100 μg RNse A (Sigm), nd incuted t 37 for 30 min. Smples were nlysed for DNA content using FACScliur TM instrument (BD Immunocytometry Systems, Sn Jose, CA). Ech experiment ws repeted t lest three times. Apoptosis: Following tretment y drugs (DSL 25 μg/ml, Rg3 80 μg/ml nd GEM 2.5 μg/ml) for 24 h, poptotic cells in the popultion were detected using the AnnexinV FITC poptosis Detection KIT I (Phrmingen, Sn Diego, CA), ccording to the mnufcturer s instructions. Briefly, cells were wshed twice with cold cell stining uffer, nd then cells were resuspended in Annexin V inding uffer t concentrtion of cells/ml. Annexin V FITC (5 µl) nd 7 AAD viility stining solution (5 µl) were dded in 100 µl cell suspension in 5 ml test tue. Cells were gently vortexed nd incuted for 15 min t room temperture (25 ) in the drk. 400 µl of Annexin V inding uffer ws dded to ech tue, nd cells were nlysed y flow cytometry with proper mchine settings. Septemer - Octoer 2013 Indin Journl of Phrmceuticl Sciences 579

3 Immunohistochemicl stining: The immunohistochemicl stining (IHC) ws performed s per the reported procedure [17]. Briefly, lyer of pproprite size glss coverslips were plced in 24 well pltes, nd cells were seeded in the wells. After tretment y drugs (DSL 25 μg/ml, Rg 3 80 μg/ml nd GEM 2.5 μg/ml) for 24 h, the cells were fixed with 4% polyoxymethylene. Following quenching endogenous peroxidse with 3% H 2 O 2 in methnol for 15 min, the cells were incuted for 30 min using lock uffer (3% ovine serum lumin (BSA) nd 0.1% Triton 100). The cells were then incuted overnight t 4 with mouse monoclonl ntiodies ginst humn ES, VEGFR 2, cspse 3, cspse 8, cspse 9, dr5, cl 2 nd survivin in 1:200 dilution. The next dy, the cells were incuted with secondry ntiody for 2 h t room temperture. The ound secondry ntiody ws visulised y the ctivity of the horserdish peroxidse conjugte using 3,3 diminoenzidine tetrhydrochloride (Sigm, UK) s sustrte. The cells were counter stined with hemtoxylin. For semiquntittive evlution of the IHC y Imge Pro Puls (IPP) 5.1 imge nlysis softwre, 20 rndom visul fields were exmined using the LeicQ550cw imge nlysis system (Germny). Western lotting: After tretment y drugs (DSL 25 μg/ml, Rg3 80 μg/ ml, GEM 2.5 μg/ml) for 24 h, the cells were lysed in the RIPA uffer (50 mm Tris Cl, ph 7.4, 150 mm NCl, 1 mm MgCl 2, 0.5% NP 40, 1 mg/ml BSA, 0.1 mm PMSF). The untreted cells were used s controls. Protein concentrtions in the cell extrcts were determined using the BCA Protein Assy regents (Pierce Biotechnology, Rockford, USA), ccording to the mnufcturer s instructions. Smples were seprted y SDS 10% polycrylmide gel electrophoresis, nd were electrolotted onto polyvinylidene difluoride memrnes. Memrnes were proed with monoclonl mouse ntiodies ginst humn pro cspse 3, pro cspse 8, pro cspse 9, cl 2, survivin nd β ctin protein t pproprite dilutions, followed y incution with horserdish peroxidse conjugted secondry ntirit or ntimouse IgG ntiody (Sigm, USA). Blots were developed using n enhnced chemiluminescence system (Roche, Bsel, Switzerlnd). Sttisticl nlysis: Dt ws reported s mens±sd. The pired t test ws used for sttisticl nlysis nd P<0.05 ws considered sttisticlly significnt. RESULTS DSL locked HepG2 cells in S phse: The sensitivity of HepG2 cell lines to DSL, Rg3 nd GEM ws investigted using the MTT ssy (fig. 1) fter tretment for 24 h (fig. 1), 48 h (fig. 1) nd 72 h (fig. 1c). When drug concentrtions nd the time of incution were incresed, cell inhiition rtios in ll groups incresed. The IC 50 vlues for DSL, Rg3 nd GEM were pproximtely 25, 80 nd 2.5 μg/ml, respectively. DSL nd Rg3 oth cuse the cell cycle to rrest t the S phse. The dt in Tle 1 show tht the percentge of cells rrested in the S phse is higher when they re treted with DSL, compred to tht when treted with Rg3 (P<0.05). By contrst, there were more cells in the G0/G1 phse fter GEM tretment. Consequently, GEM locked HepG2 cell prolifertion t the G0/G1 phse. DSL induced the poptosis of HepG2 cells: DSL, Rg3 nd GEM induced cell poptosis compred with controls (fig. 2). The verge TABLE 1: THE EFFECTS OF DRUG ON CELL CYCLE PROGRESSION Cell cycle G0/G1 S G2/M Control 53.4± ± ±1.86 DSL 26.9± ±1.68* 15.31±1.28 Rg3 34.1± ±1.57* 17.2±2.54 GEM 74.31±2.08* 13.91± ±1.52 Percentge of cells in the different phses of the cell cycle. Ech experiment ws repeted three time (*P<0.05) c Fig. 1: Drug sensitivities of HepG2 cells mesured y the MTT ssy. Cell viility following tretment with DSL(- -), Rg3 (- -) nd GEM (- -) for 24 (), 48 (), or 72h (c). The dose- nd time-dependent effects of the indicted drugs on cell prolifertion re presented s the percentge inhiition of cell viility. Dt represent s mens±sd of three experiments (*P<0.05). 580 Indin Journl of Phrmceuticl Sciences Septemer - Octoer 2013

4 Fig. 2: Cell poptosis ws nlysed y Annexin V stining. () Apoptosis in cells treted with different drug ws detected y nnexin V stining. Numers in the imges indicte the percentge of cells. Control mens untreted HepG2 cells. () Quntifiction of the poptotic cell popultion in A. Dt represent the verge of three experiments. Brs, mens±sd (*P<0.05). percent of poptotic cells treted with DSL, GEM, nd Rg3 were 33.26, 28.76, nd 23.48%, respectively (P<0.05) (fig. 2). Thus, the rte of poptosis is higher in the DSL group thn in the Rg3 nd GEM groups. It is importnt to note tht the percent poptotic poptotic cells incresed to 58.04% in the presence of oth Rg3 nd GEM. The percentge of poptotic cells incresed to 72.25% when the cells were treted with comintion of DSL nd GEM (fig. 2). The nlysis of the percentge of poptotic cells induced y different condition (fig. 2) confirmed tht DSL comined with GEM enhnced the ntitumour ctivity of DSL. Expression of ES nd VEGFR 2 in HepG2 y immunohistochemistry: IHC nlysis demonstrted tht vsculr endothelil growth fctor receptor (VEGFR-2) ws down regulted in DSL group nd Rg3 group compred to control, while ES levels were higher in DSL nd Rg3 treted cells (fig. 3). The expression of VEGFR 2 decresed in the GEM group, lthough the expression of ES ws not significntly different etween the GEM group nd control. Positive stining for VEGFR 2 ws oserved in oth, cell memrne nd cytoplsm. ES ws detected predominntly in the cytoplsm (fig. 3). Further nlysis showed tht the level of VEGFR 2 ws lower (fig. 3c) while the level of ES ws higher (fig. 3) in the DSL group thn tht in the Rg3 nd GEM groups. When the drugs were comined, however, the expression of VEGFR 2 nd c Fig. 3: The expression of ES nd VEGFR 2 in HepG2 exmined y immunohistochemistry. () HepG2 cells treted with different drugs were stined for ES nd VEGFR-2. Cells were counter-stined with hemtoxylin, nd were photogrphed under microscope. The rown point in the picture is positive signl nd the control is HepG2 cells without drug tretment. () (ES) nd (c) (VEGFR-2) re the nlysis of the dt in A. Positive signls were quntified using Imge-Pro Puls 5.1, nd were compred for sttisticl significnce. Dt represent the verge of three experiments.brs, mens ± SD (*P<0.05; n>3). ES did not chnge significntly in comprison with monotherpy. Immunohistochemistry profiling of poptosis relted protein expression in HepG2 cell treted or untreted y DSL: Intense stining for cspse 3, cspse 8 nd cspse 9 ws oserved in cells treted with DSL, compred to the untreted control cells. In prllel, the expression of cl 2 ws lower thn in control cells. Importntly, dr5 ws expressed t high level in the DSL treted cells group, while survivin ws down regulted (fig. 4). Cspse 3, cspse 8, cspse 9, nd cl 2 proteins were expressed oth in the cell memrne nd in the cytoplsm, ut the levels in the cytoplsm were higher. Survivin protein ws detected in cytoplsm, while dr5 ws locted minly in the cell memrne (fig. 4). The expression of pro cspse 3, pro cspse 8, pro cspse 9, dr5, cl 2 nd survivin in HepG2 cells, with or without DSL tretment: We lso exmined y western lot the zymogens of cspse, dr5, cl 2 nd survivin. DSL tretment Septemer - Octoer 2013 Indin Journl of Phrmceuticl Sciences 581

5 Fig. 4: Immunohistochemistry profiling of poptosis relted protein expression in HepG2 cell treted or untreted y DSL. () Representtive stining results re shown for cspse-3, cspse-8, cspse-9, cl-2 nd survivin. Cells were not counter-stined. () Quntittive nlysis of the dt in A, using Imge-Pro Puls 5.1. Dt represent the verge of three experiments. Brs indicte mens±sd (*P<0.05; n>3). Control; DSL induced the expression of procspse 3, procspse 8, procspse 9 nd dr5 nd cused decrese in the expression of survivin nd cl 2 (fig. 5). DISCUSSION Heptocellulr crcinom (HCC) is the sixth most common cncer in the world in terms of incidence, ccounting for pproximtely 630 thousnd new cses per yer; in ddition, HCC is the third most common cuse of cncer deth. The stndrd tretment in the erly stges of the disese, such s surgicl resection, locl ltion nd liver trnsplnttion, re le to cure proportion of ptients, ut most cses of HCC present in dvnced stges, precluding the use of such tretments with curtive intent. In these dvnced stges, systemic tretments re commonly used. Unfortuntely, chemotherpy with conventionl cytotoxic gents, such s doxoruicin, cispltin, nd cpecitine, is ineffective nd does not seem to modify the nturl history of disese. Trditionl Chinese medicines hve een used cliniclly for more thn 5000 yers in Chin nd Asi. There is undnt evidence tht trditionl Chinese medicine hs mrked effect on the tretment of severl diseses, including tumours [11,12]. DSL is common Fig. 5: The expression of pro cspse 3, pro cspse 8, pro cspse 9, dr5, cl 2, nd survivin in HepG2 cells, with or without DSL tretment. Cell lystes were prepred from HepG2 cells treted with DSL or untreted. () Western lotting nlysis ws performed to detect the expression of the indicted proteins, with β-ctin s reference control. () Quntifiction nd summry of the poptotic cell popultion in 5. Control; DSL Chinese medicinl compound, whose composition includes rdix ginseng, rdix strgli, venenum ufonis, nd mylris. Anlysis of mny clinicl reports hs shown tht DSL cn e used in ptients who re unwilling to or cnnot ccept surgery, chemotherpy or rdiotherpy [13,14]. Moreover, DSL cn reinforce the immunity of ptients, nd ugment the effects of surgery, chemotherpy or rdiotherpy. The mechnisms of ction of DSL, however, re still uncler. We hve found tht DSL cn inhiit cell prolifertion in dose nd time dependent fshion. Although DSL hs potentil dvntges, its irrittion of veins hs hmpered its ppliction in clinicl prctice. Pnxoside, is one of the ctive components of ginseng extrct nd hs cused widespred concern in the lst two decdes. Recent studies hve shown tht ginsenosides (Rg3, RH2) re the min ntitumour gents in ginseng extrct [18 22]. Some studies hve demonstrted tht Rg3 could not only induce tumour cells differentition, ut lso inhiit tumour growth nd promote poptosis of tumour cells [23,24]. Therefore, we estimted the role of DSL nd Rg3 in inhiiting the growth of crcinom cells in vitro. To our surprise, oth Rg3 nd DSL regulte the cell cycle 582 Indin Journl of Phrmceuticl Sciences Septemer - Octoer 2013

6 nd poptosis. The percentge of cells treted y DSL or Rg3 in the S phse is significntly higher thn tht of control, which suggests tht oth DSL nd Rg3 induce the HepG2 cells to strt their cell cycle, ut lock them in the S phse. The dt, however, showed tht the rte of poptosis in the DSL group is 33.26%, ut tht in the Rg3 group is 23.48%, demonstrting DSL is more powerful in inducing poptosis. Cui et l. [14] reported tht DSL could increse the expression of ES in HepG2. We lso found tht the expression of ES is upregulted nd the expression of VEGFR 2 is downregulted in HepG2 cells treted with DSL or Rg3, compred to controls. These oservtions confirm tht Rg3 hs some ntitumour effect. It is possile tht Rg3 is one of mjor ctive constituents of DSL, ut DSL is more powerful ctivtor ecuse of synergism with other components in the complex mixture. DSL cn increse the ody s immunity, ugment the effects of surgery, chemotherpy or rdiotherpy, nd improve the clinicl symptoms nd the qulity of life of ptients. Thus, DSL is usully comined with vrious chemotherpy drugs. GEM is deoxycytidine nlogue, with cycle specific drug fetures. Experiment with pncretic crcinom nd lung cncer confirmed tht GEM depresses cell growth [25 27]. According to our study, GEM could inhiit the prolifertion nd induce the poptosis of HepG2 cells. Interestingly, DSL cuses cells to e rrested in the S phse, while cell popultions of G0/ G1 phse previl following tretment with GEM. The trget of GEM is known to e the cell popultions in the DNA synthesis stge [28]. Thus, we thought tht if the DSL is comined with GEM, the resulting ntiprolifertive effect would e stronger; this ws indeed oserved in our experiments. The percentge of cell poptosis is 33.26% when the cells re treted with DSL lone, while the rte increses to 72.25% when the cells re treted with DSL comined with GEM. This comintion, however, did not ffect the expression of VEGFR 2 nd ES in HepG2 cells, most likely ecuse GEM does not regulted the expression of VEGFR 2 nd ES in these cells. These oservtions indicte tht the comintion of DSL nd GEM my e of significnt vlue. Nevertheless, the DSL/GEM comintion my not e useful in suppressing ngiogenesis. Consistent with our study, numer of investigtions hve shown tht DSL cn induce poptosis. The moleculr mechnisms of poptosis induced y DSL re not cler. Clssic poptosis pthwys include the intrinsic mitochondril pthwy, nd the extrinsic deth receptor medited pthwy. The extrinsic pthwy is triggered y the ctivtion of deth receptors in response to n extrcellulr signl, which initites the chin ctivtion of the cspses [29]. Firstly, the dimeristion of cspses 8 nd 10 were promoted y incresing the locl concentrtion of procspse monomers [30], followed y the clevges of the zymogens of cspses 3, 6, or 7. The cspse cscde cn lso e ctivted y the so clled mitochondri dependent pthwy [31]. In response to the poptotic stimuli, the permeility of the mitochondril memrne increses, nd the mitochondri relese series of molecules, including cytochrome C. In the cytosol, the ssocition of cytochrome C with the dptor protein Apf 1 nd severl procspse 9 molecules gives rise to the formtion of the poptosome. Cspse 9 is thus le to recruit nd ctivte cspse 3 [32], which is n effector of oth pthwys. We nlysed the criticl molecules in the two poptosis pthwys, nmely cspse 3, cspse 8, cspse 9, pro cspse 3, pro cspse 8, pro cspse 9, nd dr5. It is surprising tht DSL not only regultes the expression of proteins involved in the extrinsic pthwy (deth receptor dr5 nd cspse 8), ut lso protein in the intrinsic pthwy (cspse 9). Bsed on these oservtions, we propose tht the poptosis of HepG2 cells induced y DSL is regulted y oth poptosis pthwys, ut not y either of them lone. The expression of cspse 3 nd zymogens of cspse, including pro cspse 3, pro cspse 8 nd pro cspse 9, were incresed sustntilly over the control, confirming our presumption. Severl studies hve shown tht the intrinsic nd extrinsic pthwys re not mutully exclusive [33]. It is reported the regultion of the mitochondril poptotic pthwy is governed y the cl 2 protein fmily [34,35]. Survivin inhiits poptosis y locking the ctivtion of effector cspses in oth the extrinsic nd intrinsic pthwys of poptosis. The high expression of survivin in HCC is significntly higher thn tht in djcent cirrhosis tissues nd norml tissues [36]. DSL reduced the levels of cl 2 nd survivin in HepG2 cells. Thus, DSL might effect poptosis y the regulting the expression of poptosis proteins, including cl 2 nd survivin. Septemer - Octoer 2013 Indin Journl of Phrmceuticl Sciences 583

7 In summry, DSL inhiited the prolifertion nd promoted the poptosis of HepG2 cells, prtly through the ction of Rg3. Our results indicte tht etter therpeutic effect could e otined y the comintion of DLS nd GEM. They lso illuminte the mechnisms of poptosis induced y DSL; this my help us in the development of new pproches to the therpy of HCC. ACKNOWLEDGEMENTS We thnk Professor Hu Hn for his guidnce on the experimentl design nd performnce of the project. This work ws gretly supported y the Ntionl Nturl Science Foundtion (No nd No ). REFERENCES 1. Chn SL, Mok T, M BB. Mngement of heptocellulr crcinom: Beyond sorfeni. Curr Oncol Rep 2012;14: Ppouls M, Theochris S. Primry liver tumors: Origin nd trget therpy. Expert Opin Ther Trgets 2009;13: Shire AM, Roerts LR. Prevention of heptocellulr crcinom: Progress nd chllenges. Minerv Gstroenterol Dietol 2012;58: Rougier P, Mitry E, Brre JC, Tie J. Heptocellulr crcinom (HCC): An updte. 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