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Journal of Ethnopharmacology 137 (2011) 320– 326

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

jo ur nal homep age : www.elsev ier .com/ locate / je thpharm

ro-apoptotic and anticancer properties of Thapring – A Tibetan herbalormulation

enzin Choedona, Dawa Dolmab, Vijay Kumara,∗

Virology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, IndiaTibetan Medical Astro Institute, Dharamsala, Kangra 176215, India

r t i c l e i n f o

rticle history:eceived 8 March 2011eceived in revised form 3 May 2011ccepted 25 May 2011vailable online 31 May 2011

eywords:raditional Tibetan Medicineerbal formulationCCEGFpoptosisytochrome c�m

a b s t r a c t

Aim of the study: To evaluate the pro-apoptotic and anti-tumorigenic properties of Thapring – a TraditionalTibetan Medicine – in hepatoma cells and in a transgenic mouse model of hepatocellular carcinoma.Material and methods: The pro-apoptotic action and growth inhibition property of Thapring were assessedin Huh7, HepG2 and A549 cell lines using flow cytometry and MTT assay, respectively. Confocalmicroscopy for colocalization of cytochrome c and mitochondria was done using dsRed mitotrackerin Huh7 cells. The activation of p38 MAP kinase and p53 pathway was evaluated by Western blotting.Serological studies for liver function, vascular endothelial growth factor and superoxide dismutase wereassessed in the serum of X15-myc transgenic mice. Immuno-histochemical studies for Bcl2 and p21Waf1

expression were also carried out in the liver section of the above mice.Results: Treatment with Thapring inhibited proliferation and accumulation of hepatoma cells in G1 phase.There was increased cytochrome c release from mitochondria and decreased Bcl2 levels – the key markersof apoptotic cell death. Besides activation of p38 MAP kinase and increased p53 expression were alsoobserved. Oral administration of Thapring in transgenic mice lowered serum VEGF levels and conferred

hepatoprotection as evident from normal serum ALT levels. Further, immunohistochemical analysis ofthe liver samples revealed reduced expression of anti-apoptotic protein Bcl2 and over-expression of cellcycle regulator p21Waf1.Conclusions: The ability of Thapring to impose growth arrest and trigger pro-apoptotic death in cell cultureas well as ameliorative effects in vivo provides scientific basis for its usefulness as traditional medicineand its clinical application in adjunct/combination therapy along with other known anticancer drugs.

© 2011 Elsevier Ireland Ltd. All rights reserved.

. Introduction

The Traditional Tibetan Medicine (TTM) is an age old legacy of

erbal and spiritual healing evolved around 7th century and is anssimilation of healing methods from India, China, Greece and Per-ia (Dunkenberger, 2000). Here each treatment is codified in the

Abbreviations: ALT, alanine aminotransferase; ATF, activating transcrip-ion factor 2; ��m, mitochondrial membrane potential; CAM, complemen-ary alternative medicine; DAPI, 4′-6-diamidino-2-phenylindole; DMEM, Dul-ecco’s modified Eagles medium; FBS, fetal bovine serum; HCC, hepatocellulararcinoma; JC1, 5,5′ ,6,6′-tetrachloro-1,1′ ,3,3′-tetraethylbenzimidazolcarbocyanineodide; LFT, liver function tests; MAPK, mitogen-activated protein kinase; MTT,3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliun bromide; PB, pre-bleed; PBS,hosphate-buffered saline; SOD, superoxide dismutase; TTM, Traditional Tibetanedicine; VEGF, vascular endothelial growth factor.∗ Corresponding author. Tel.: +91 11 26741680; fax: +91 11 26742316.

E-mail address: vijay@icgeb.res.in (V. Kumar).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.05.031

form of sacred texts or pharmacopeia that is interpreted with Bud-dhist understanding of herbal cures (Lobsang and Dakpa, 2001).TTM is known to offer cure for chronic diseases like cancer andatherosclerosis (Melzer et al., 2006). Thapring is one herbal for-mulation that has been in use since the beginning of twentiethcentury for the treatment of a variety of cancers (Dawa, 2003).Thapring is a multicomponent herbal formulation composed of Ter-minalia chebula (Rety) (family: Combretaceae), Saussurea lappa (C.B.Clarke) (family: Asteraceae), Acorus calamus (L.) (family: Araceae),Aconitum ferox (Wall. ex Ser) (family: Ranunculaceae), Oxytropismicrophylla (Pall) (family: Fabaceae), Commiphora mukul (Hook)(family: Burseraceae), Acacia catechu (L.F. Willd) (family: Fabaceae),Delphinium brunonianum (Royale) (family: Ranunculaceae) anda mineral ingredient (Dawa, 2003). Keeping in view the tumor

regression property of Thapring and its clinical application as analternative chemopreventive/chemotherapeutic medicine for can-cer, here we have scientifically evaluated the growth inhibitory andpro-apoptotic action of Thapring in cell culture model as well as its

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nti-tumorigenic action in a genetic mouse model of hepatocellulararcinoma.

. Material and methods

.1. Chemicals

Propidium iodide, Silibinin, DMSO, 3-[4,5-dimethylthiazol-2-l]-2,5 diphenyltetrazolium bromide (MTT) and methanol wereurchased from Sigma Chemical Co. (St. Louis, MO) and JC1 dyerom Molecular probe. Dulbecco modified eagle medium (DMEM),etal bovine serum (FBS), streptomycin and penicillin were pur-hased from Gibco BRL, USA. VEGF quantikine ELISA kit wasrocured from R&D system (Minneapolis, USA) and Fugene 6 wasrom Roche diagnostics. Immunohistochemistry kit was procuredrom DAKO and DAB, hematoxylin from Sigma and DPX from BDH.

.2. Plant material

Thapring was procured from the Tibetan Medical and Astro-ogical Institute (TMAI), Dharamsala, India. All the herbs usedn this formulation were identified by Dr. Tsering Norbu (Men-ampa). The voucher numbers of plant specimens used in Thapringre: Terminalia chebula (TMAI/A-1), Sassurea lappa (TMAI/R-7),corus calamus (TMAI/Sha-3), Aconitum ferox (TMAI/G-5), Oxytropisicrophylla (TMAI/Nga-5), Commiphora mukul (TMAI/G-1), Acacia

atechu (TMAI/S-10), and Delphinium brunonianum (TMAI/B-10).

.3. Cell culture and transfection

Human hepatoma cells Huh7 was a kind gift from Dr. A. SiddiquiUniversity of Colarado, Denver), HepG2 cells (HB-8065), AML12ells (immortalized mouse hepatocytes, CRL 2254) HEK293 (CRL-573) cells and A549 (CCL-185) cells were purchased from ATCC.ll cultures were grown in DMEM supplemented with 10% FBS,enicillin 100 �g/ml and streptomycin (100 �g/ml) incubated at7 ◦C in a humidified chamber and 5% CO2 atmosphere. Cells wereeeded at a density of 0.6 million per 60 mm dish and transfectedsing Fugene 6 (Roche) as per manufacturer’s protocol. For trackingytochrome c localization, pEGFP-cytochrome c (1 �g) was cotrans-ected with pDsRed Mitotracker (1 �g) and analyzed by confocal

icroscopy (Nikon A1R).

.4. Cell viability assay

MTT assays were performed as described earlier (Choedon et al.,006). Cells were seeded as above, and after 24 h treated with

ncreasing concentrations (10, 50, 100, and 200 �g/ml) of Thapringor 48 h. Then cells were incubated with MTT (200 �g/ml) for 45 mint 37 ◦C in dark. Formazan crystals were solubilised in DMSO andbsorbance measured at 560 nm. Untreated cells were used as con-rol of cell viability (100%). The mean absorbance value of threexperiments was expressed as percentage of cell viability as com-ared to control.

.5. Animal tumor model

Development of the transgenic mice model (X15-myc) of hepa-ocellular carcinoma (HCC) has been described earlier (Lakhtakiat al., 2003). The transgene positive of mice were identified byCR using tail DNA biopsies and used for evaluating the anti-ancer property of Thapring. Necessary ethical clearance was taken

rom the Institutional Animal Ethics Committee for doing animalxperiments. The animals were maintained under standard con-itions, treated humanely and provided pellet diet and water ab

ibitum. The experimental mice were divided into three groups

rmacology 137 (2011) 320– 326 321

(n = 5 per group), viz., treated, untreated and positive control andadministered daily with Thapring (400 mg/kg) through oral routefor 12 months as described earlier (Choedon et al., 2006). Silib-inin (100 mg/kg) was used as positive control. Mice were housed inhumidity (30–70%) and temperature (74 ± 2 ◦F). The tissue samples(liver) were removed surgically and fixed in 10% buffered forma-lin. Treatment of mice for the histology and immunohistochemistrywork lasted for 3 months.

2.6. SOD estimation

The SOD activity was determined spectrophotometrically inserum samples by measuring the autooxidation of epinephrine for3 min at 480 nm (Misra, 1987). The assay mixture contained 50 mMglycine buffer pH 10.4 with epinephrine (20 �g/ml).

2.7. Assessment of liver functions

For liver function test, blood samples from the experimentalmice were collected by retro-orbital bleeding at 0, 2, 4, 6, 8, 10months post treatment. The serum alanine aminotransferase (ALT)levels were measured by autoanalyzer while the VEGF levels weremeasured as per manufacturer’s protocol (R&D system, Minneapo-lis).

2.8. Determination of mitochondrial membrane integrity

For analysis of changes in mitochondrial transmembrane poten-tial, Huh7 cells were incubated with JC1 (100 nM) for 15 min at 37 ◦Cin dark and then washed with phosphate buffered saline and ana-lyzed in fluorescence activated cell sorter (FACS)-FL1 green and redFL2 channel.

2.9. Cell cycle analysis

Cell cycle analysis was performed by the method previouslydescribed (Mukherji et al., 2007). Briefly, Huh7 cells were fixedin 70% ethanol and stained with Propidium iodide (50 �g/ml) andsubjected to apoptosis analysis in a FACScan (Becton Dickinson, SanJose, CA). Based on propidium iodide staining, the percentage of cellcycle distribution was determined using FlowJo software.

2.10. Western blot analysis

Western blotting was done according to Khatter and Kumar(2010). Briefly, Huh7 cells were directly lysed in 2× sample load-ing buffer (100 mM Tris–Cl pH 6.8, 200 mM dithiothreitol, 4% SDS,0.2% bromophenol blue and 20% glycerol) and protein concentra-tions were determined by Bradford method. The samples wereelectrophoresed and protein bands were visualized by enhancedchemiluminiscence western blotting detection system (Santa Cruz,USA).

2.11. Immunohistochemistry studies

Paraffin sections of embedded liver tissues of 4–5 �m thicknesswere cut, dewaxed and then treated with proteinase K (20 mg/ml)for 20 min at 37 ◦C and kept at room temperature for 10 min. Afterthree quick washing with PBST, the slides were blocked in 0.3%hydrogen peroxide followed by 2 washes in PBST for 5 min each. The

slides were then incubated with primary antibodies (1:150 dilu-tions) in PBS for 2 h at RT. After a quick rinse with PBS, slides wereoverlaid with secondary antibody (DAKO) and developed usingDAB. Slides were then counterstained with hematoxylin, dehy-

322 T. Choedon et al. / Journal of Ethnopha

Fig. 1. Induction of apoptosis and growth arrest of cells in the presence of Thapring.(A) Huh7, Hek 293 and A549 cells were treated with increasing concentrations (10,50, 100, 200 �g/ml of Thapring and the level of cytotoxicity was measured by MTTassay. Silibinin (50 �g/ml) was used as positive control. (B) Huh7 cells were treatedwws

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ith indicated concentration of Thapring. After 48 h, cells were fixed and stainedith propidium iodide and their DNA content analyzed by flow cytometry. Data are

hown as mean ± SD (n = 3). Level of significance: *, p < 0.03.

rated and then mounted with DPX and viewed under Nikon 80iicroscope.

.12. Statistical analysis

Results were expressed as mean ± SEM and all statistical com-arisons were made by student ‘t’ test. p-Values less than or equalo 0.05 were considered as significant.

. Results

.1. Pro-apoptotic and growth inhibition properties of Thapring

Selective modulation of the apoptotic machinery is consideredrucial for selecting an anticancer agent whose development usu-lly involves empirical screening for cytotoxic activity in cancerells (Suggitt and Bibby, 2005). The cytotoxic property of Thapringas evaluated in hepatoma (HepG2 and Huh7) and non-hepatoma

ells (A549, HEK293, and AML12) over a range of concentration10–200 �g/ml) and the cell viability was measured after 48 h.s shown in Fig. 1A, treatment with Thapring led to a significant

ncrease in cell death (p < 0.001) and 50 per cent inhibition wasbserved at 0.1 mg/ml in both Huh7 and HepG2 cells. Interestingly,ittle or no change was observed in the viability of A549 and HEK293ells under these conditions. Likewise, untransformed AML12 cellsere also spared (data not shown). Thus, the present study suggests

hat Thapring can induce selective killing of hepatoma cells.Deregulation of cell cycle progression in cancer cells by anti-

ancer agents is an effective strategy to halt tumor growthMalumbres and Carnero, 2003). Therefore, we next examined theell cycle distribution of Huh7 cells in the presence of Thapring.

low cytometry analysis showed a marked growth inhibition andajority of cells (p < 0.01) showed arrested in G1 phase (Fig. 1B).

urther, their DNA content also increased to 14% in G0/G1 phases compared to control. The number of cells in S phase went down

rmacology 137 (2011) 320– 326

significantly due to G1 arrest. This clearly suggested that Thapringalso carries a growth inhibition/anti proliferative property.

3.2. The proapoptotic action of Thapring is mediated bymitochondria

Mitochondria is an integral part of apoptotic machinery andevents such as disruption of electron transport, loss of mitochon-drial membrane potential, release of cytochrome c resulting inactivation of caspases are classical evidence for apoptosis (Greenand Reed, 2000; Martinou, 2000). To study the mitochondrial mem-brane integrity, cells were treated with Thapring and stained withJC1 followed by FACS analysis. Treated cells showed marginal shiftin ��m (Fig. 2A). However, confocal microscopy of these cellsshowed disruption of ��m and a significant decrease in cell via-bility as evident from increased monomeric form of JC1 dye upontreatment with Thapring (more green fluorescence) as opposedto untreated cells (more red fluorescence due to aggregated dye)(Fig. 2B and C).

To confirm whether cytochrome c was released from mito-chondria following Thapring treatment, we expressed dsREDmitotracker and cytochrome c GFP in hepatoma cells. We observeda significant increase in cytochrome c release with increasing con-centration of Thapring along with a concomitant decrease in levelof colocalization of cytochrome c and mitochondria (Fig. 3A andB). To support this observation, we also looked into the canonicalmarkers of anti-apoptosis such as Bcl2 and pro-apoptotic proteinsuch as Bid (Youle and Strasser, 2008). As expected Bcl2 levelsdecreased with increasing drug dose while Bid levels increased onlymarginally (Fig. 3C). Further, a decline in Bcl2 expression in the livertissue of Thapring-treated transgenic mice (Fig. 3D), reconfirmedthe pro-apoptotic action of the herbal drug that was mitochondria-dependent.

3.3. Anticancer property of Thapring

As Thapring showed proapoptotic and anti-oncogenic proper-ties in cell culture, we further examined the anticancer propertiesof Thapring in a HBV-related mouse model of HCC. The X15-myctransgenic mice were treated with Thapring and assessed for criticalhepatic functions as liver function tests (LFT). The most commonlyelevated blood test of liver damage is ALT level. LFT is usually ele-vated in HCC. The ALT levels in both treatment groups of miceshowed a significant reduction in its levels at 10 months (p < 0.001)indicating the ameliorative effects of Thapring against HCC develop-ment suggesting restoration of normal liver function after Thapringtreatment (Fig. 4A). Cancer cells are also characterized by theiraltered redox state. SOD is an enzyme that catalyses the reductionof superoxide anion to hydrogen peroxide crucial for maintainingthe cell integrity (Hogg, 2004). Analysis of SOD levels in serum ofthe Thapring-treated transgenic mice showed a significant reduc-tion (p < 0.002) at 6 months treatment (Fig. 4B). Thus, Thapringexhibited an antioxidant property essential for any anticancer andhepatoprotective agent.

3.4. Antiangiogenic action of Thapring

Emerging preclinical evidence continues to shed light on impor-tant questions in angiogenesis research, including how to optimallytarget critical angiogenic growth factors. Increased angiogenesis isconsidered as hallmark of cancer. Several factors are responsible

for increased vascularisation. Among these factors, VEGF has beenidentified as the most predominant. VEGF expression in hyper-vascular tumor such as HCC has been considered to be associatedwith size and grade of tumor (Yamaguchi et al., 1998). Interest-

T. Choedon et al. / Journal of Ethnopharmacology 137 (2011) 320– 326 323

Fig. 2. Effect of Thapring on mitochondrial membrane potential (��m). Huh7 cells were treated with indicated concentrations (10, 50 and 100 �g/ml) of Thapring and thenincubated in the presence of JC1 dye for 30 min at 37 ◦C. The ��m was measured by flow cytometry (A). (B) Parallel set of control and Thapring-treated cells (100 �g/ml)w h sho

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ngly, the proangiogenic VEGF levels were markedly reduced in thehapring treated transgenic mice (Fig. 4C).

.5. Activation of p38 MAPK and induction of p53 by Thapring

Since p38 mitogen-activated protein kinase (MAPK) pathways well known to play a mediatory role in pro-apoptotic actionPadhan et al., 2008; Cuadrado and Nebreda, 2010), we next exam-ned the levels of phosphorylated p38 in cells treated with Thapring.s shown in Fig. 5A, there was a marked increase in the levels ofhosphorylated p38 and its downstream target ATF2 suggestinghat p38 MAPK is necessary for Thapring-mediated apoptosis.

The tumor suppressor p53 protein is a critical mediator of manyellular functions including the response to genotoxic stress, dif-erentiation, senescence and apoptosis (Menendez et al., 2010).nactivation of p53 is commonly seen in tumors (Goh et al., 2011).53 has become the focus on most intensive cancer based research.

nterestingly, we observed an increase in the levels of p53 uponreatment with Thapring along with increased phosphorylation ofts negative regulator mdm2 which is indicative of its inactivationFig. 5B). The increased p53 level also coincided with increase ints downstream target p21Waf1 (Fig. 5B). Further our immunohisto-hemistry studies in the liver of X15-myc transgenic mice showed

levated levels of p21Waf1 upon treatment as compared to controlFig. 5C). This observation further validated our above observationsn cell culture that Thapring activates apoptotic pathway in cancerells.

wing relative viability of cells shown in panel A. *, p < 0.01.

4. Discussion

Herbal medicines and remedies were the most commonly usedcomplementary alternative medicine (CAM) therapies against can-cer. The majority of cancer patients use CAM to increase the body’sability to fight cancer or improve physical and emotional well-being, and many seemed to have benefited from its usage. Tibetanformulations are mostly seen in the context of CAM. The dosagesof individual components in a herbal formulation are very low andare apparently without side-effects. It is believed that when sev-eral herbs or herbal extracts are formulated together, it resultsin highly effective recipe against some disease and such formu-lations are considered integral to the therapy. In terms of modernmedical research, one such formulation best known as Padma 28– a multicomponent Tibetan herbal formulation – with woundhealing property (Aslam et al., 2010), anti-inflammatory activity(Moeslinger et al., 2000) and used in the treatment of some circula-tory disorders (Melzer et al., 2006) and chronic hepatitis B infection(Gładysz et al., 1993). Padma Lax is a related formulation that isreported to have anti-proliferative/anti-cancer property (Hofbaueret al., 2006). In the present study, we have used another Tibetanherbal formulation called Thapring that has been effectively usedagainst a variety of cancer (Dawa, 2003). Here, we provide thepharmacological basis of its beneficial effects using cell culture and

preclinical mouse models.

Induction of apoptosis is considered as one of the key mech-anisms for the targeted therapy of various cancers. Cancer drugsare believed to kill target cells either by inducing death receptor

324 T. Choedon et al. / Journal of Ethnopharmacology 137 (2011) 320– 326

Fig. 3. Intracellular localization of cytochrome c in the presence of Thapring. Huh7 cells were co-transfected with plasmids pEGFP-Cyt c, pDsRed-mito. After 36 h, cells weretreated further for 24 h with indicated concentrations of Thapring and analyzed by confocal microscopy (A) for the distribution of DsRed-Mitotracker (Red) and pEGFP-c eatmes ic Bcl2( 5-my

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ytochrome c (Green). (B) Quantitative data of the confocal images showing post trignificance: *, p < 0.05; **, p < 0.01. (C) Levels of pro-apoptotic Bid and anti-apoptotIHC) for Bcl2 expression in the liver of untreated (control) and Thapring-treated X1

athway or interfering with mitochondrial pathways (Constantinit al., 2000). Our cell cycle analysis of Thapring-treated cells showedelective killing of hepatoma cells and their arrest in G1 phaseFig. 1). The induction of apoptosis was accompanied by stimu-ation of p38 MAPK signaling pathway and its effector moleculeTF2 (Fig. 5A) that is well known to mediate apoptotic cell death

Cuadrado and Nebreda, 2010). Our subsequent studies indicatedhat Thapring can also promote the intrinsic death pathway byhanging the permeability of mitochondrial outer membrane inrder to facilitate the release of cytochrome c (Fig. 2). As expected,e observed the release of key apoptogenic factor cytochrome c

rom mitochondria (Fig. 3A and B) which is essential for the activa-ion of caspases and induction of apoptosis (Green and Reed, 2000;

artinou, 2000). The Thapring-treated cells also showed decreasedxpression of canonical anti-apoptotic markers like Bcl2. It is wellnown that the Bcl-2 family includes both pro-apoptotic (Bad,ak and Bax) and anti-apoptotic (Bcl-2 and Bcl-XL) proteins thatre critical modulators of the intrinsic death pathway (Youle andtrasser, 2008).

Another interesting observation in this study included the up-egulation of p53 gene and its target and cell cycle inhibitor p21Waf1

nd inactivation of its negative regulator Mdm2 (Fig. 5B). p53s considered as guardian of cells owing to its tumor suppressorroperty (Menendez et al., 2010). The mutational inactivation or

nt per cent colocalization of the mitochondrial marker and cytochrome c. Level of in cells treated with Thapring. C, control, Sn, silibinin. (D) Immunohistochemistry

c (at 400×).

down-regulation of p53 is a common scene in many cancers (Gohet al., 2011). Therefore, sustained elevated levels of p53 along withincreased phosphorylation of its negative regulator Mdm2 upontreatment with Thapring are good indicators of its growth inhibitionproperties.

Interestingly, our cell culture studies were adequately sup-ported by in vivo studies in a genetic mouse model of HCC. Oraltreatment of X15-myc mice with Thapring resulted in a significantreduction in the serum levels of ALT (Fig. 4A) which is considered asa hallmark of hepatotoxicity (Ozer et al., 2008). Suppression of ALTlevels indicated that Thapring also confers hepatoprotection. Theherbal formulation also exhibited a strong anti-oxidative propertyby preventing oxidative damage to liver as seen in Fig. 4B. Moreimportantly, there was a significant reduction in the serum VEGFlevels of the oncomice treated with Thapring (Fig. 4C) which is con-sidered an important arm in the management of cancer (Greenbergand Cheresh, 2009; Kaseb et al., 2009). Besides, there was a dramaticreduction in the tissue expression of anti-apoptotic protein Bcl2(Fig. 3D) and improvement in the expression of growth inhibitoryp21Waf1 (Fig. 5C) in support of the pro-apoptotic action of Thapringin vivo.

Synergistic interactions are documented for constituents withina total extract of a single herb, as well as between differentherbs in a formulation like Thapring. The concept, that a whole or

T. Choedon et al. / Journal of Ethnopharmacology 137 (2011) 320– 326 325

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heir sera were analyzed for the levels of ALT (A), VEGF (B) and SOD (C). Data are sho

artially purified extract of a herb offers advantages over a sin-

le isolated ingredient, also underpins the philosophy of herbaledicine. Therefore, multi-target drug design combined with a

etwork-dependent approach is a promising concept to combat

ig. 5. Activation of apoptotic markers in the presence of Thapring. Huh7 cells were treateA) phosphorylated p38 and ATF2 and (B) p21Waf1, p53 and phosphorylated and total mdntreated (control) and Thapring-treated X15-myc (at 400×).

ent age groups were treated orally with Thapring as described under Section 2 and mean ± SD (n = 4). PB, pre-bleed serum. Level of significance: *, p < 0.02, **, p < 0.03.

multifactorial diseases like cancer. The control of a complex dis-

ease system should involve simultaneous disruption of multipletargets located in distant cellular networks (Keith et al., 2005). Thus,Thapring as a TTM promises to offer all that for treating cancer.

d with indicated concentrations of Thapring for 24 h and analyzed for the levels of:m2. (C) Immunohistochemical (IHC) analysis of p21Waf1 expression in the liver of

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26 T. Choedon et al. / Journal of Eth

. Conclusions

Thapring is used as an anticancer and hepatoprotective agentn TTM. The present investigation provides scientific basis in sup-ort of its therapeutic applications. The present study suggests thathapring possesses a strong anti-cancer activity (growth inhibi-ion, cell cycle arrest, pro-apoptotic activity) in hepatoma cells andhows minimal cytotoxic effect on non-hepatoma cells and non-ransformed AML12 hepatocytes. These results warranted an in vivotudy in a genetic mouse model of HCC. Oral administration of therug in mice revealed a clear hepatoprotection and conferred anti-arcinogenic and anti-angiogenic effects. Thus, Thapring appears toe a good and reliable candidate as CAM for clinical application asdjunct/combination therapy in cancer.

cknowledgements

We thank Dr. S. Jameel for kindly providing pEGFP-cytochrome and pDsRed-Mitotracker plasmids. This work was supported byhe core fund of ICGEB, New Delhi.

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