Choedon et al. BMC Complementary and Alternative Medicine 2014, 14:380http://www.biomedcentral.com/1472-6882/14/380

RESEARCH ARTICLE Open Access

Molecular insights into the anti-cancer propertiesof Traditional Tibetan medicine Yukyung KarneTenzin Choedon1,2, Dawa Dolma3, Ganeshan Mathan2 and Vijay Kumar1*

Abstract

Background: Yukyung karne (YK) is a traditional Tibetan formulation used for many centuries for the treatment ofovarian cancer. However, the pharmacological basis of its anticancer property is not well understood. In the presentstudy, the anticancer property of YK was investigated in cell culture.

Methods: The growth inhibitory property of YK was evaluated in SKOV6, IHH, HepG2 and HEK293 cell lines usingMTT assay. The pro-apoptotic activity of drug was analyzed by terminal deoxynuleotidyl transferase dUTP nick endlabeling (TUNEL) and DNA fragmentation assays. Confocal microscopy was used to show the release of cytochromec and its co-localization with mitochondria with the help of dsRed mitotracker in SKOV6 cells. The inhibition in cellproliferation was also visualized by confocal microscopy after BrDU incorporation. The activation of tumor suppressorp53 was evaluated by Western blotting while VEGF levels in culture supernatant were measured by a colorimetricmethod.

Results: YK specifically and efficiently induced apoptotic killing of the human ovarian cancer SKOV6 cells as indicatedby increased DNA fragmentation and nick end DNA labeling. Confocal microscopy suggested inhibition of cellproliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (Δψm).Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, andinhibited VEGF secretion by cells. Interestingly, YK also exhibited a synergy with paclitaxel which is a well-knownanti-cancer therapeutic drug.

Conclusions: The pharmacological properties of YK to impose growth arrest and trigger pro-apoptotic death incells amply justify its usage in primary as well as adjunct therapy for ovarian cancer.

Keywords: Yukyung Karne, Traditional Tibetan medicine, Ovarian cancer, Apoptosis, Mitochondria membrane potential

BackgroundOvarian cancer is one of the most lethal gynecologicalmalignancies and a leading cause of cancer related deathin women [1]. The conventional treatment regimen forovarian cancer includes combination of platinum basedchemotherapy, surgery and radiation [2]. However, noneof these therapies have impacted the overall survival ratesignificantly [3]. Despite several advancements madeduring past few decades, there is enough scope for de-veloping new methods and therapeutic agents for effect-ive treatment of ovarian cancer.Natural products are excellent sources of complex che-

micals with useful properties including great therapeutic

* Correspondence: vijay@icgeb.res.in1Virology Group, International Centre for Genetic Engineering andBiotechnology, Aruna Asaf Ali Marg, New Delhi 110067, IndiaFull list of author information is available at the end of the article

© 2014 Choedon et al.; licensee BioMed CentrCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.

value [4]. Dietary phytochemicals such as curcumin [5]and Silibinin [6] have been identified as two major naturalanticancer agents. Curcumin and Silibinin have been ex-tensively used in the traditional medical system in Asia.Traditional Tibetan medicine (TTM) with over 2000 yearsold legacy of holistic and naturopathic approach integratesdiet, behaviour, lifestyle, herbs and accessory therapies areintegrated to treat the root cause of disease [7]. The mainconstituents of the Tibetan medicine are Terminaliachebula (Aru ra), Terminalia belerica (Baru ra) andEmblica officanalis (Kyuru ra) popularly known in Tibetanas Aru-Baru-Kyuru in a ratio of 2:1:1 just as Triphala inAyurveda [8]. TTM are mostly multi-ingredient formula-tion comprising 3 to 150 herbs and minerals to combatmultifactorial diseases like cancer [9]. Many Tibetan med-icines are considered safe, effective, non-toxic and most of

al Ltd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,

Choedon et al. BMC Complementary and Alternative Medicine 2014, 14:380 Page 2 of 8http://www.biomedcentral.com/1472-6882/14/380

all good therapeutic value for a number of chronic dis-eases [10-12]. Earlier we reported that Thapring, a TTMused for the treatment of chronic liver diseases, can inhibitcell proliferation and induce apoptosis in a transgenicmouse model of hepatocellular carcinoma [13]. Sinceovarian cancer is difficult to treat and relapse rate isquite high, many patients opt for complementary ther-apy to ease stress, symptoms and better survival. Inter-estingly, the therapeutic response of another TTMformulation -Yukyung Karne (YK) [14] has shownpromising results by providing relief to the ovariancancer patients. In the present study, we provide evi-dence based mechanistic insights into the anticancerproperties of YK.

MethodsChemicals, reagents and kitsPaclitaxel was purchased from Calbiochem, Propidium iod-ide, methanol, DMSO and 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) reagents were pro-cured from Sigma Aldrich. DeadEnd Fluorometric TUNELsystem was from Promega. JC1dye-5,5′,6,′-tetrachloro-1,1′, 3, 3′ tetraethylbenzimidazolylcarbocyanine iodide wasfrom Molecular Probes Inc., Eugene, USA. Enhancedchemiluminescence (ECL) reagent and antibodies werefrom Santa Cruz, USA while the VEGF quantikine ELISAkit was from R& D system (Minneapolis, USA). Dulbecco-modified Eagle’s medium (DMEM), fetal bovine serum(FBS), streptomycin and penicillin were from Gibco-BRL,USA. Lipofectamine 2000 was from Invitrogen, USAwhile the BrdU labeling kit was from Roche Diagnos-tics, Indianapolis, USA.

Plant materialYukyung karne was purchased from the Tibetan Medicaland Astrological Institute (TMAI), Dharamshala, India.YK was dissolved in glass-distilled water and diluted ap-propriately for use in our experiments. No extractionstep was involved. According to Tibetan Pharmacopeia[14], YK comprises of a mixture of root of Saussurealappa (C.B. Clarke) (family: Asteraceae), fruit of EmblicaOfficinalis (L) (family: Euphorbiaceae), leaves of Adhatodavasica (NEES) (family: Acanthaceae), seeds of Elletracardomomum (L) (family: Zingiberaceae), fruit of Piperlongum (L) (family: Piperaceae), whole plant part ofDracocephyllum tanguticum (Maxim) (family: Lamiaceae),root of Zingiber officinalis (Roscoe) (family: Zingiberaceae),seed of Coriandrum sativum (L) (family: Apiaceae), wholeplant of Meconopsis horridula (Hook) (Papaveraceae), rootof Corydalis hendersoni (Fedde) (family: Fumariaceae),seeds of Embelia ribes (Burm. F) (Family:Myrsinaceae),Delphinium brunonianum (Royale) (family: Ranunculaceae),fruit of Terminalia chebula (Rety) (family: Combretaceae),root of Acorus calamus (L) (family: Araceae), root of

Aconitum ferox (Wall.ex Ser) (family: Ranunculaceae),resin of Commiphora mukul (Hook) (family: Burseraceae)and a mineral ingredient. All the herbs were identified byDr. Tsering Norbu (Menrampa) and the voucher numbersof plant specimens are available at the herbarium depart-ment of TMAI for reference.

Cell culture and transfectionOvarian cancer cell line SKOV6 was a kind gift of Dr. AnilSuri (National Institute of Immunology, New Delhi). Thehuman hepatoma cells Huh7 was a kind gift from Dr. A.Siddiqui (University of Colorado, Denver). The immortal-ized human hepatocytes (IHH) were kindly provided byDr. F. Danniel, Institut National de la Santé et de laRecherche Médicale Unite 481, Universite Paris 7, Paris,France. HepG2, HEK 293 (CRL-1573) and A549 (CCL-185) cells were purchased from ATCC. All cultures weregrown in DMEM supplemented with 10% FBS, penicillin100 μg/ml and streptomycin (100 μg/ml) incubated at37°C in a humidified chamber and 5% CO2 atmos-phere. Cells were seeded at a density of 0.6 million per60 mm dish and transfected using Lipofectamine 2000(Invitrogen) as per manufacturer’s protocol. For track-ing cytochrome c localization, pEGFP-cytochrome c(1 μg) was co-transfected with pDsRed Mitotracker (1 μg)and analyzed by confocal microscopy (Nikon A1R) Japan.

MTT assayCell viability was analyzed by MTT colorimetric assay asdescribed by van de Loosdrecht et al. [15]. Briefly, cellswere seeded at density of 0.4 × 106 cells per 60 mm dish,allowed to settle overnight and treated with differentconcentrations of YK (1, 10, 100 μg/ml) for 24 h. Cellswere washed with DMEM without phenol red and incu-bated with MTT reagent for 45 min at 37°C in dark.The formazon crystals were solubilized in dimethyl sulf-oxide and the absorbance was recorded at 560 nm. Un-treated cells were used as control of viability (100%).The mean absorbance values of three experiments wereexpressed as percentage of viability in relative to control.

Cell proliferation assayThe BrdU incorporation assay was performed usingBrdU labelling kit (Roche Diagnostics, Indianapolis, IN,USA) as per manufacturer’s protocol. Nuclei were stainedwith DAPI (blue) while the BrdU incorporation wasdetected using goat anti-mouse-conjugated to FITC(green). The distribution of BrdU positive cells wasshown as bar diagrams.

Detection of DNA fragmentationCells were treated with different concentrations of YKfor 24 h. Assay was carried out as per Peng et al. [16].Briefly cells were harvested and treated with 100 μl lysis

Table 1 Effect of YK on VEGF secretion from SKOV6 cells

Sample VEGF ± SEM (pg/ml)

Control 2.6 ± 0.057

Paclitaxel 2.10 ± 0.01*

Paclitaxel + YK 1.84 ± 0.02*

YK 1.53 ± 0.05*

*, Level of significance p Value <0.001.

Choedon et al. BMC Complementary and Alternative Medicine 2014, 14:380 Page 3 of 8http://www.biomedcentral.com/1472-6882/14/380

buffer and the supernatant was incubated for 2 h withRNase A at 56°C and followed by Proteinase K digestionat 37°C for 2 h. DNA was precipitated with 2.5 volumeof cold absolute ethanol. The DNA pellet was dissolvedin TE buffer and resolved by electrophoresis in a 2%agarose gel.

Cell cycle analysis by FACSEqual number of cells (0.6-0.8 × 106) were seeded ineach 60 mm dish and after 24 h were treated with YK(100 μg/ml). Cell cycle analysis was performed as men-tioned by Mukherji et al. [17]. Briefly cells were fixedand stained with propidium iodide solution (50 μg/mlPI, 10 μg/ml RNase) and the data was acquired using aFACScan flow cytometer equipped with CellQuest soft-ware (Becton Dickinson, San Jose, CA, USA).

TUNEL assaySKOV6 cells were treated with YK and the level of apop-tosis was detected by TUNEL assay using DeadEndTM

Flourometric TUNEL kit (Promega). The assay was doneas per manufacturer’s protocol. Cells were mountedusing Antifade with DAPI and detected localized greenfluorescence of apoptotic cells by confocal microscopy(Nikon A1R) Japan.

Determination of mitochondrial membrane potential(ΔΨm)For analysis of mitochondrial membrane potential, cellswere seeded in 12 well plate overnight followed by treat-ment with YK for another 24 h and then stained withJC1 dye for 15 min at 37°C in 5% CO2 incubator. Cellswere mounted with antifade mounting medium (Invitro-gen, USA) and visualized at 488 nm and 590 nm usingconfocal microscopy (Nikon A1R) Japan.

Estimation of vascular endothelial growth factor (VEGF)The VEGF levels were measured as per manufacturer’sprotocol (R&D system, Minneapolis). Briefly, supernatantof the experimental set of cultured cells were collected forVEGF assay. Each well was washed with wash buffer and100 μl of the VEGF conjugate was added to each well.After repeated washing step, 100 μl of substrate solutionwas added to each well followed by addition of 100 μl ofstop solution and mixed it gently. The OD was measuredat 450 nm.

High performance liquid chromatography (HPLC)The fingerprints of YK samples were monitored on aShimadzu reverse-phase HPLC system (C18 column -250 mm, 4.6 mm) with SCl-10AVp system controllerand SPD-10AVvp UV–vis detector. The mobile phase(acetonitrile and water) was degassed and filtered through0.2 μm membrane filter before pumping into the HPLC

system. A linear gradient of acetonitrile from 5% to 95%over 55 min at a flow rate of 1 ml/min was maintainedand the samples were monitored at 220 and 280 nm usingPhoto Diode Array (PDA) detector. The YK samples(20 μg in 200 μl of glass distilled water) were used forinjection.

Western blottingCell lysates were prepared in cell lysis buffer. (Promega,USA) Protein concentration was determined by Bradfordmethod. Samples with equal amounts of protein wereprepared in 2x sample loading buffer (100 mM Tris–HClpH6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenolblue and 20% glycerol) and resolved on 10-15% SDS-PAGE. The protein bands were visualized using theEnhanced chemiluminescent reagent (Santa Cruz, USA)according to supplier’s protocol and the image capturedby Fluorchem M (Protein Simple, USA).

Statistical analysisStatistical significance of results in Tables 1 and 2were calculated by Duncan’s test using SPSS software(Version 17). All other data were analyzed by Student’st test. p value <0.05 was considered as significant.

ResultsInduction of apoptosis in ovarian cancer cellsAn important challenge faced in chemotherapy is the ad-verse effect of drugs on the normal healthy cells. Mostof the anticancer treatment regimen affects both cancer-ous as well as healthy cells. In order to establish the spe-cificity of YK for ovarian cancer, we examined its effecton various cell lines by MTTassay. As shown in Figure 1A,we observed a selective induction of cell death by YK inovarian and cervical cancer cell lines (SKOV6 and HeLacells respectively). Interestingly YK did not induce apop-tosis in IHH cells indicative of its specificity to target can-cerous cells. Since YK exhibited a dramatic cytotoxiceffect on SKOV6 cell line, it was selected for further stud-ies. Paclitaxel a commonly used anticancer drug was usedas a reference drug. As shown in Figure 1B, cells treatedwith paclitaxel (10 nM) showed a significant increase incell death (p <0.005). Further MTT assay showed thattreatment of YK in combination with paclitaxel aug-mented the apoptotic response induced by paclitaxel alone

Table 2 Effect of anticancer drugs on cell cycleprogression of SKOV6 cells

Sample G1 S G2

Control 59.63 ± 2.0 17.58 ± 2.8 17.83 ± 1.09

Paclitaxel 35.0 ± 9.07* 16.65 ± 2.34 19.93 ± 3.3

Paclitaxel + YK 38.6 ± 6.38* 17.0 ± 1.5 18.18 ± 3.8

YK 73.4 ± 1.5 15.46 ± 3.09 14.18 ± 0.64

*, Level of significance: p Value < 0.004 v. control.

Choedon et al. BMC Complementary and Alternative Medicine 2014, 14:380 Page 4 of 8http://www.biomedcentral.com/1472-6882/14/380

(Figure 1B). Since DNA fragmentation is a hallmark char-acteristic feature of apoptosis, we performed DNA frag-mentation assay on the YK treated SKOV6 cells. As anoticeable fragmentation of DNA was seen at 100 μg/mlof YK (Figure 2A), all subsequent experiments were car-ried out at this concentration. We also performed TUNELassay to confirm the observation on YK induced apoptosisof SKOV6 cells. As shown in Figure 2B, a significant num-ber of TUNEL positive cells were detected in the YKtreated cells (p <0.001) whereas no TUNEL positive cellswere observed in untreated control SKOV6 cells. Thesedata confirmed the apoptotic action of YK on ovarian can-cer cell line.

Induction of tumor suppressors and cell cycle regulatorsGiven the key role of p53 as tumor suppressor proteinin cancer prevention [18], we also studied the ability ofYK to enhance p53 expression in the treated cells. Pacli-taxel, a well-known therapeutic anticancer drug, was in-cluded as positive control as well as to evaluate itssynergy with YK. We observed a marked increase in thecellular p53 levels after YK treatment (Figure 2C). How-ever, the level of Mdm2 was down-regulated leading tothe stabilization of p53. Just as p53, we also observed up

A

YK (µg/ml)

0

20

40

60

80

100

120

ControlDoxorubicin 1 10 100

Cel

l via

bilit

y (%

)

Figure 1 Growth inhibitory effect of YK on different cell lines. A. HEK2different doses of YK (1, 10, 100 μg/ml) and analyzed for cell viability by MTand the cell viability was measured as above. Results are represented as mp <0.005.

regulation of pTEN, a potent tumor suppressor and p21,a potent inhibitor of cell proliferation and replication(Figure 2C). These results were further complementedby decrease in proliferation marker PCNA and cell cycleregulator cyclin B suggesting reduced proliferation ofSKOV6 cells upon YK treatment (Figure 2C). Thus, p53the most appealing target for mechanism-driven antican-cer drug discovery, also appeared to play an importantrole in YK induced killing of cancer cell. Further, restor-ation of p53 functions in cancer cells by YK treatmentcould be an important strategy to combat cancer.

Role of mitochondria-dependent intrinsic pathway inapoptosisSince, mitochondria plays a key role in energy metabol-ism, and proving to be the novel target in killing cancer-ous cells [19], we sought to determine the effect of YKon mitochondrial function with the help of cationic JC1dye. Exposure of cells to YK led to the disappearance ofred fluorescence and increase in the green fluorescencein most cells combined with a significant reduction(p <0.001) in mitochondrial membrane potential. (Figure 3A)Further, YK along with Paclitaxel perturbed mitochondrialmembrane potential to a significant level (p <0.05) as com-pared to only paclitaxel treatment (Figure 3B).Since release of cytochrome c from depolarized mito-

chondria and binding to Apaf1 are crucial steps in theformation of apoptosome, next we monitored the releaseof GFP-tagged cytochrome c from YK treated SKOV6cells by confocal microscopy. Interestingly a significantincrease was observed in cytochrome c released fromdsRed Mitotracker-tagged mitochondria as indicated bya significant decrease (p <0.001) in co-localization of cyto-chrome c (GFP) with Mitochondria (dsRed) (Figure 3B).

B

HEK293T

A549

HepG2

Hela

IHH

SKOV6 Cel

l via

bilit

y (%

)

0

20

40

60

80

100

120

Control Paclitaxel Pac+YK

*

93T, A549, HepG2, Hela, IHH and SKOV6 cells were treated withT assay. B. SKOV6 cells was treated with YK and/ or paclitaxel (10 nM)ean of three independent experiments ± S.E.M. Level of significance; *,

µg

µg

Figure 2 Induction of apoptosis by YK in SKOV6 ovarian cells. A, Cells were treated with indicated doses of YK and/or paclitaxel [Lanes: 1-Control,2-Paclitaxel 10nM, 3-Pac + YK (10nM+ 100 μg/ml), 4–7 YK (1, 10, 100, and 200 μg/ml respectively) and 8-50 bp ladder] for 24 h. Genomic DNA was isolatedand resolved by agarose gel electrophoresis. B, Cells were treated with different doses of YK (1, 10, 100 μg/ml) was visualized by TUNEL assay. TheTUNEL positive cells (stained by FITC) were counted from random fields and are presented in the graph. Nuclei were visualized by DAPIstaining (blue). Results are represented as mean of three independent experiments ± SD. Level of significance; *, p <0.001. C, Induction ofcell cycle regulators. SKOV6 cells were treated for 24 h with YK (100 μg/ml), Paclitaxel (Pac) (10 μM) or both and the expression of p21, p53,Mdm2, cyclin B1, and PCNA was verified by western blot. GAPDH was used as internal control.

Choedon et al. BMC Complementary and Alternative Medicine 2014, 14:380 Page 5 of 8http://www.biomedcentral.com/1472-6882/14/380

Taken together, these observations suggested that themitochondria-dependent intrinsic apoptotic pathway isinvolved in YK-induced cell death.

Anti angiogenic effect of YKSustained angiogenesis is another hallmark of cancer.Neo-angiogenesis is essential for supplying nutrients tothe fast growing cancerous cells. Therefore starving thecancer cells is one of the most promising approach tofight cancer [20]. Of various growth factors that regulateangiogenesis, VEGF is believed to be the most importantfactor. To explore if YK could block angiogenesis, weused ELISA to measure the levels of VEGF secreted bythe YK treated cells. Duncan analysis of these results(Table 1) revealed that all the four groups were signifi-cantly different from each other and that the VEGF levelsin treated groups were lower than control (p <0.05).Another most prominent change seen in cancer is the

deregulation of cell cycle [21]. Anticancer agents on theother hand, may exert their effect by halting the cellcycle progression [22]. Therefore, next we studied thecell cycle distribution of SKOV6 cells treated with YK byFACS. Duncan test revealed that two groups of significantdifference exist. The paclitaxel- and paclitaxel + YK-treated

groups are not different from each other but are differentfrom control- and YK-treated groups in the cells of G1phase (p <0.05). Further, the control and YK groups arenot different from each other. No significant difference wasobserved in four groups of cells of S and G2 phases.Since, YK could abrogate cell cycle progression we also

checked cell proliferation status using BrdU incorpor-ation. YK treatment led to a significant reduction in thenumber of the BrdU positive cells. Further combinedtreatment with YK and paclitaxel showed the maximumreduction in proliferating cells. (Figure 4B) Together,these observations suggested that YK can inhibit prolif-eration by inducing cell cycle arrest at G1 phase andthus could be useful as an effective anticancer agent.

DiscussionDespite significant advances made in the area of cancerchemotherapy, chemo resistance continues to be a majorproblem associated with the outcome of treatment [23].Further, efficient targeting of cancer cells still remains amajor challenge for the scientists. Therapies which arecapable of inducing selective apoptosis in cancer cellsare gaining attention as new potential alternative. Theconventional treatment for ovarian cancer includes the

Control Paclitaxel Pac+Yk YK

TRITC

FITC

DAPI

Merged

B Control Paclitaxel Pac+ YK YK

Mitotracker -dsRED

Cytochrome C -GFP

Merged

A

0

0.2

0.4

0.6

0.8

1

1.2

1.4

* *

Leve

l of M

itoch

ondr

ial

mem

bran

e po

tent

ial (Δψm

)

*

Leve

l of c

oloc

aliz

atio

n

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

* *

Figure 3 Alterations in mitochondrial functions following YK treatment of SKOV6 cells. A, Confocal images of JC1 stained cells treated withYK for 24 h. The green fluorescence shows depolarized mitochondria (monomer), whereas red fluorescence shows hyperpolarized (aggregates)mitochondria. B, Cells treated with YK for 24 h showing the release of cytochrome c from mitochondria during apoptosis. Cells were co-transfectedwith cytochrome c-GFP fusion construct and dsRed mitotracker and visualized by confocal microscopy. Green represents cytochrome c, red mitochondriaand orange indicates co-localization of cytochrome c and mitochondria. Results in both panels are represented as mean of three independentexperiments ± SD Level of significance; *, p <0.05; **, p <0.001.

Choedon et al. BMC Complementary and Alternative Medicine 2014, 14:380 Page 6 of 8http://www.biomedcentral.com/1472-6882/14/380

administration of paclitaxel or cisplatin as a standardpostoperative chemotherapy for advanced cancer pa-tients [24], but the adverse effects are often inevitable. Inthis report, we provide a scientific basis for the clinicaloutcome of YK, a TTM that has been used in regularclinical practice for targeted therapy of ovarian cancer.Besides we studied the complementary action of YK witha well-known anticancer drug paclitaxel. We found thatYK can induce cytotoxicity specifically in cancer cellsand spared immortalized cell lines such as IHH. TheYK- treated cells exhibited typical characteristic of apop-tosis such as DNA fragmentation and apoptotic celldeath as evident from the MTT and TUNEL assays(Figures 1 and 2).Given the central role of mitochondria in initiating the

apoptotic process by releasing cytochrome c, we studied theeffect of YK on mitochondria and asked if the YK-induced

apoptosis was mitochondria dependent. We observed thatYK perturbed the mitochondrial membrane potential whichtriggered the release of cytochrome c which in turn acti-vated the apoptotic machinery [25].One of the key mechanisms of action of anticancer

drug on cell is to halt cell cycle progression [26]. Wealso investigated the effect of YK on cell cycle progres-sion in cancer cells by FACS analysis. Interestingly, wefound that treatment with YK led to the arrest of SKOV6 cells in G1 phase suggesting its growth inhibitoryproperty (Figure 4A). This was further supported by thedown regulation of cyclin B in the YK- treated cellswhich otherwise allows the cell cycle progression to Sphase. Nevertheless, these results did not rule out thepossibility of activation of some crucial cell cycle check-point inhibitors in the treated cells in order to preventtheir proliferation. In this context it was interesting to

Control Paclitaxel Paclitaxel+YK YK

-10

0

10

20

30

40

50

60

Ctrl Pac Pac+YK YK 100μg

Prol

ifera

ting

cells

(%)

* * *

*

FITC

DAPI

Merged

Figure 4 Inhibition of cell proliferation in the presence of YK. SKOV 6 cells were treated with paclitaxel (10nM) and/or YK 100 μg/ml for 24 hand analyzed by confocal microscopy for BrdU incorporation for DNA synthesis. Results are represented as mean of three independentexperiments ± SD. Level of significance; *, p <0.02, **, p <0.002.

Choedon et al. BMC Complementary and Alternative Medicine 2014, 14:380 Page 7 of 8http://www.biomedcentral.com/1472-6882/14/380

observe the restoration of p53 levels in the YK-treatedcells which may be responsible for their G1 arrest as alsosupported by the down regulation of PCNA (prolifera-tion marker) in these cells [27]. The most important ob-servation made in the present study was the ability ofYK to synergize with the action of paclitaxel - a widelyused anticancer drug. Since most anticancer therapiesact by induction of apoptosis which often lead to resist-ance, the combinatorial treatment could be advanta-geous as a potent strategy to bypass resistance [28]. Thesynergy between YK and paclitaxel was seen at all levelsincluding the inhibition of cell cycle progression andeven activation of p53 (Figure 2C). Angiogenesis is a keyrequirement for tumorigenesis and blocking the processappears to be an effective strategy to combat cancer[29]. Many tumors show increased expression of VEGFcompared to normal tissues [30]. Therefore, therapiesdirected against VEGF or its receptors hold great prom-ise in cancer treatment. Incidentally, YK treatment alsoled to a reduction in VEGF secretion by cancer cells in-dicating the anti-proliferative signaling by YK (Table 1).Since the analytical profiling of YK reproducibly showedthe presence of some specific components in this formu-lation (Additional file 1: Figure S1), it will be desirable tostudy each molecular component of this formulation tounderstand the pharmacological principles of YK.

ConclusionsYK is an ovarian cancer-specific and effective traditionalTibetan formulation bearing the following anticancerproperties: (i) imposes G1 arrest of cells by activatingp53, (ii) induces cytochrome c release from mito-chondria, and (iii) inhibits angiogenesis. Further, YKcould be of immense pharmacological significance as

it complements the action of known anticancer drugslike paclitaxel which may allow improving the efficacyand potency of conventional drugs and reduce side ef-fects. Thus, YK appears to be a strong candidate asnovel therapeutic for the ovarian cancer patients.

Additional file

Additional file 1: Figure S1. Analytical profile of Yukyung Karne (YK). YKsample (300 mg in water) was analyzed by reverse phase-HPLC (Shimadzu)on a C18 column using 2% acetonitrile gradient from 5% to 95% over a 55minute run (flow rate of 1.0ml/min). Sample detection was done at 220 and280nm under ambient temperature conditions.

AbbreviationsDMEM: Dulbecco-modified Eagle’s medium; ECL: Enhanced chemiluminescence;FBS: Fetal bovine serum; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazoliumbromide; TTM: Traditional Tibetan medicine; TUNEL: Terminal deoxynuleotidyltransferase dUTP nick end labeling; VEGF: Vascular endothelial growth factor;YK: Yukyung Karne.

Competing interestsThe authors have declared that they have no competing interest.

Authors’ contributionsTC and GM carried out experiments and drafted the manuscript. DDconceived the project and provided the research material. VK designed thestudy, arranged funds and finalized the manuscript. All authors have readand approved the final manuscript.

AcknowledgementsThis work was supported by the J.C. Bose fellowship to VK from theDepartment of Science of Technology, Government of India, New Delhi. Theauthors thank Dr. S. Jameel (Virology Group, ICGEB) for kindly providingpEGFP-cytochrome c and pDsRed-Mitotracker plasmids and Dr. Anil Suri(Cancer Microarray, Genes and Proteins Laboratory, National Institute ofImmunology, New Delhi) for kindly providing SKOV6 cells.

Author details1Virology Group, International Centre for Genetic Engineering andBiotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. 2Department

Choedon et al. BMC Complementary and Alternative Medicine 2014, 14:380 Page 8 of 8http://www.biomedcentral.com/1472-6882/14/380

of Biomedical Science, Bharathidasan University, Tiruchirappalli 620024, India.3Tibetan Medical Astro Institute, Dharamsala, Kangra 176215, India.

Received: 17 March 2014 Accepted: 3 October 2014Published: 7 October 2014

References1. Januchowski R, Wojtowicz K, Sujka-Kordowska P, Andrzejewska M, Zabel M:

MDR gene expression analysis of six drug resistant ovarian cancer celllines. Biomed Res Int 2013, 2013:241763.

2. Kim A, Ueda Y, Naka T: Therapeutic strategies in epithelial ovarian cancer.J Exp Clin Cancer Res 2012, 31:14.

3. Dobbin ZC, Landen CN: The importance of the PI3K/AKT/MTOR pathwayin the progression of ovarian cancer. Int J Mol Sci 2013, 14:8213–8227.

4. Song MK, Roufogalis BD, Huang THW: Modulation of diabetic retinopathypathophysiology by natural medicines through PPAR-γ-relatedpharmacology. Br J Pharmacol 2012, 165:4–19.

5. Lin YG, Kunnumakkara AB, Nair A, Meritt WM, Han LY, Armaiz-Pena GN,Kamat AA, Spannuth WA, Gershenson DM, Lutgendorf SK, Aggarwal BB,Sood AK: Curcumin inhibits tumor growth and angiogenesis in ovariancarcinoma by targeting the Nuclear Factor-kB pathway. Clin Cancer Res2007, 13:3423–3430.

6. Cheung CW, Gibbons N, Johnson DW, Nicol DL: Silibinin—a promisingnew treatment for cancer. Anticancer Agents Med Chem 2010, 10:186–195.

7. Choedon T, Kumar V: Medicinal plants used in the practice of Tibetanmedicine. In Recent progress in Medicinal plants, Phytoconstituents andPhysiological processes, Volume 34. USA: Stadium Press LLC; 2012:385–402.

8. Sandhya T, Lathika KM, Pandey BN, Mishra KP: Potential of traditionalayurvedic formulation, Triphala, as a novel anticancer drug. Cancer Lett2006, 231:206–214.

9. Keith CT, Borisy AA, Stockwell BR: Multicomponent therapeutics fornetworked systems. Nat Rev Drug Discov 2005, 4:71–78.

10. Randal J: Diagnosis, Tibetan style, underlies small herbal study ofadvanced breast cancer. J Natl Cancer Inst 1999, 91:587–588.

11. Sallon S, Namdul T, Dolma S, Dorjee P, Dolma D, Sadutshang T, Ever-Hadani P,Bdolah-Abram T, Apter S, Almog S, Roberts S: Mercury in traditional Tibetanmedicine- panacea or problem? Hum Exp Toxicol 2006, 25:405–412.

12. Ginsburg I, Koren E, Horani A, Mahamid M, Doron S, Muhanna N, Amer J,Safadi R: Amelioration of hepatic fibrosis via Padma Hepaten isassociated with altered natural killer T lymphocytes. Clin Exp Immunol2009, 157:155–164.

13. Choedon T, Dolma D, Kumar V: Proapoptotic and anticancer properties ofThapring- a Tibetan herbal formulation. J Ethnopharmacol 2011, 137:320–326.

14. Dawa: Bod kyi Gso Ba Rigpa Las Sman Rdzas Sbyor lag len Gsan Sgo byed PaiLde Mig. Dharamsala, India: RigDrag publication; 2003.

15. Van de Loosdrecht AA, Beelen RH, Ossenkoppele GJ, Broekhoven MG,Langenhuijsen MM: A tetrazolium-based colorimetric MTT assay to quantitatehuman monocyte mediated cytotoxicity against leukemic cells from celllines and patients with acute myeloid leukemia. J Immunol Methods 1994,174:311–320.

16. Peng B, Chang Q, Wang L, Hu Q, Wang Y, Tang J, Liu X: Suppression ofhuman ovarian SKOV-3 cancer cell growth by Duchesnea phenolicfraction is associated with cell cycle arrest and apoptosis. GynecolOncol 2008, 108:173–181.

17. Mukherji A, Janbandhu VC, Kumar V: HBx –dependent cell cyclederegulation involves interaction with cyclin E/A-cdk2 complex anddestabilization of p27Kip1. Biochem J 2007, 401:247–256.

18. Menendez D, Inga A, Resnick MA: Potentiating the p53 network. DiscovMed 2010, 10:94–100.

19. Giannattasio S, Guaragnella N, Arbini AA, Moro L: Stress-relatedmitochondrial components and mitochondrial genome as targets ofanticancer therapy. Chem Biol Drug Des 2013, 81:102–112.

20. Greenberg JI, Cheresh DA: VEGF as an inhibitor of tumor vesselmaturation: implications for cancer therapy. Expert Opin Biol Ther 2009,9:1347–1356.

21. Nakayama KI, Nakayama K: Ubiquitin ligases: cell cycle control and cancer.Nat Rev Cancer 2006, 6:369–380.

22. Darwiche N, El-Banna S, Gali-Muhtasib H: Cell cycle modulatory and apoptoticeffect of plant–derived anticancer drugs in clinical use or development.Expert Opin Drug Discov 2007, 2:361–379.

23. Longley DB, Johnston PG: Molecular mechanism of drug resistance. J Pathol2005, 205:275–292.

24. Mouratidou D, Gennatas C, Michalaki V, Papadimitriou A, Andreadis CH,Sykiotis C, Tsavaris N: A Phase III Randomized study comparing Paclitaxeland Cisplatin versus Cyclophosphamide and Cisplatin in patients withadvanced ovarian cancer. Anticancer Res 2007, 27:681–686.

25. Martinou JC, Desagher S, Antonsson B: Cytochrome c release frommitochondria: all or nothing. Nat Cell Biol 2000, 2:E41–E43.

26. Malumbres M, Carnero A: Cell cycle deregulation: a common motif incancer. Prog Cell Cycle Res 2003, 5:5–18.

27. Ehrhardt H, Wachter F, Grunert M, Jeremias I: Cell cycle –arrested tumorcells exhibit increased sensitivity towards TRAIL- induced apoptosis.Cell Death Dis 2013, 4:e661.

28. Fulda S, Debatin KM: Sensitization for anticancer drug- induced apoptosisby betulinic acid. Neoplasia 2005, 7:162–170.

29. Ferrara N, Gerber HP, LeCouter J: The biology of VEGF and its receptors.Nat Med 2003, 9:669–676.

30. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell 2011,144:646–674.

doi:10.1186/1472-6882-14-380Cite this article as: Choedon et al.: Molecular insights into the anti-cancerproperties of Traditional Tibetan medicine Yukyung Karne. BMC Complementaryand Alternative Medicine 2014 14:380.

Submit your next manuscript to BioMed Centraland take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit