A propionyloxy derivative of 11-keto-β-boswellic acid induces apoptosis in HL60 cells mediated through topoisomerase I & II inhibition

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Chemico-Biological Interactions 189 (2011) 60–71

Contents lists available at ScienceDirect

Chemico-Biological Interactions

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propionyloxy derivative of 11-keto-�-boswellic acid induces apoptosis inL-60 cells mediated through topoisomerase I & II inhibition

ousia Chashooa, Shashank K. Singha,∗, Paraduman R. Sharmaa, Dilip M. Mondhea,bid Hamida, Arpita Saxenaa, Samar S. Andotrab, Bhahwal A. Shahb,aveed A. Qazi c, Subhash C. Tanejab, Ajit K. Saxenaa

Pharmacology Division, Indian Institute of Integrative Medicine, Canal road, Jammu tawi, Jammu and Kashmir180001, IndiaBio-organic Chemistry Division, Indian Institute of Integrative medicine, Canal road, Jammu, IndiaMedicinal chemistry Division, Indian Institute of Integrative medicine, Canal road, Jammu, India

r t i c l e i n f o

rticle history:eceived 31 August 2010eceived in revised form 27 October 2010ccepted 29 October 2010vailable online 4 November 2010

eywords:ropionyloxy derivative of KBAL-60oswellia serrataopoisomerase

a b s t r a c t

Boswellic acids have invariably been reported for their antiproliferative potential in various cell systems.In the present study the growth inhibitory effect of propionyloxy derivative of 11-keto-�-boswellic acid(PKBA; a semisynthetic analogue of 11-keto-�-boswellic acid) on HL-60 promyelocytic leukemia cells isbeing reported for the first time. In the preliminary studies, in vitro cytotoxicity of PKBA was investigatedagainst eight human cancer cell lines viz., IMR-32, SF-295 (both neuroblastoma), PC-3 (prostate), Colo-205 (colon), MCF-7 (breast), OVCAR-5 (ovary), HL-60, Molt-4 (both leukemia) and their respective IC50

values were found to be 5.95, 7.11, 15.2, 14.5, 15, 15.9, 8.7 & 9.5 �g/ml, respectively. For determiningthe mechanism of cell death in HL-60 cells, PKBA was subjected to different mechanistic studies. DNArelaxation assay of PKBA revealed inhibition of both topoisomerases I & II. The fragmentation analysis ofDNA revealed typical ladders indicating the cytotoxic effect to be mediated by induction of apoptosis. The

aspase morphologic studies of PKBA showed the presence of true apoptotic bodies. Apoptosis was confirmedfurther by flow-cytometric detection of sub-G1 peaks and enhanced annexin-V-FITC binding of the cells.The activation of apoptotic cascade by PKBA in HL-60 cells was found to be associated with the lossof mitochondrial membrane potential, release of cytochrome c, activation of initiator and executionercaspases and cleavage of poly ADP ribose polymerase (PARP). In vivo studies of PKBA revealed anti-tumoral activity against both ascitic and solid murine tumor models. These studies thus demonstrate

in H

PKBA to induce apoptosis

. Introduction

Cancer is a complex collection of diseases that can arise in almostny tissue in the body. Vast catalog of cancer cell genotypes is aanifestation of six essential alterations in cell physiology that

ollectively dictate malignant growth, self-sufficiency in growthignals, insensitivity to growth-inhibitory signals, evasion of pro-rammed cell death (apoptosis), limitless replicative potential,ustained angiogenesis, tissue invasion and metastasis. These sixapabilities are shared in common by most and perhaps all typesf human tumors [1]. Since apoptosis is a key defense strategy

gainst the emergence of cancer, the anticancer agents that activatepoptosis in cancer cells could be valuable anticancer therapeu-ics [2]. Anticancer agents may kill cells either by activation ofxtrinsic (death receptor mediated) or intrinsic (mitochondrion

∗ Corresponding author. Tel.: +91 09469709444.E-mail address: [email protected] (S.K. Singh).

009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.10.017

L-60 cells due to the inhibition of topoisomerases I and II.© 2010 Elsevier Ireland Ltd. All rights reserved.

dependent) apoptotic pathways. In extrinsic pathway the engage-ment of death receptors leads to the formation of death inducingsignaling complex (DISC) containing the death receptors, adaptorproteins, caspase-8 and caspase-10. In mitochondrion dependentapoptosis the release of cytochrome c into the cytosol results inthe formation of apoptosome containing cytochrome c, Apaf-1 &caspase-9. Caspase-8, -9 & -10 are believed to be the initiator cas-pases at the top of the caspase signaling cascade. Recruitmentof caspases to DISC and apoptosome leads to their activation bydimer formation. These activated caspases cleave downstream cas-pases and other target proteins as well as activation of DNase thatleads to the fragmentation of DNA [3]. Since last three decadesmuch research is focused in tackling cancer. Many approacheshave been made in this direction. One of the current approaches

is based on the use of natural products or their semisyntheticanalogues to tackle the disease. The role of natural products asfolklore remedies has been recognized since ancient times [4]and still continues to provide essential source of novel drug dis-covery leads [5]. The gum resin of Boswellia serrata, a kind of

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with proteinase K (20 mg/ml) for 15 min. Products were resolved by1% agarose gel electrophoresis in TAE buffer (40 mM Tris-acetate,pH 8.0, 1 mM EDTA) and stained with 0.5 �g/ml ethidium bromide(EtBr) for 15 min and destained with distilled water for 15 min atroom temperature [11].

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G. Chashoo et al. / Chemico-Biol

eciduous tree grown in the dry parts of China and India, hasraditionally been used for the treatment of inflammatory andrthritic diseases [6]. Following pentacyclic triterpenic acids haveeen isolated from the gum resin of Boswellia serrata: �-boswelliccid, 11-keto-�-boswellic acid, acetyl-�-boswellic acid and acetyl-1-keto-�-boswellic acid. These chemical constituents have beeneported for their anti-inflammatory, antiproliferative and anti-ancer activities [7,8]. Structure activity relationship indicated thathe pentacyclic ring skeleton of boswellic acid is responsible forts anti-topoisomerase activity [8]. The anticancer potential ofoswellic acids (BAs) has been found to be of the order of acetyl11-keto-�-boswellic acid (AKBA) as the most active, followed by1-keto-�-boswellic acid (KBA), acetyl-�-boswellic acid (ABA) andoswellic acid (BA), respectively [9]. Acetyl-11-keto-�-boswelliccid has been shown to induce apoptosis in HL-60 cells by inhibit-ng topoisomerase I [8]. Since AKBA is showing more cytotoxicotential than KBA, attempt was made to synthesize preliminaryropionyloxy derivative of KBA (PKBA) to further increase theotency of the parent molecule and to decipher the role of alkoxyhain length. The propionyloxy derivative of 11-keto-�-boswelliccid (PKBA) was found to have lower IC50 value as compared toKBA [8] and was further evaluated for its anticancer and apop-

otic potential. Its in vitro cytotoxic activity was assessed againstifferent human cancer cell lines and its pharmacodynamic inter-ction against specific cancer targets (topoisomerases I and II) wasvaluated. To know the mechanism of cancer cell killing, PKBA wasubjected to DNA fragmentation and cell cycle analysis in HL-60ells. The observed apoptotic activity of PKBA was found to be asso-iated with the loss of mitochondrial membrane potential, releasef cytochrome c from mitochondrion, activation of intracellular cas-ases and the cleavage of PARP. All these parameters account forhe apoptotic cell death. Further, the in vivo anticancer potential ofKBA was evaluated in different murine tumor models.

. Materials and methods

.1. Chemicals

RPMI-1640, phosphate buffer saline, dimethyl sulphoxideDMSO), ethidium bromide, propidium iodide (PI), DNase-freeNase, proteinase K, camptothecin, 5-fluorouracil, Rh-123, peni-illin, streptomycin, adriamycin, mitomycin-c, sulforhodamine-BSRB), DAPI and staurosporine were purchased from Sigma Chem-cal Co., USA. Fetal bovine serum was obtained from HyCloneCH30160.03, Lot No. CTE0190). Annexin-V-FITC apoptosis detec-ion. Caspase assay kits were from BD Biosciences, USA. Mousenti-human antibodies to PARP-1 (#SC8007), �-actin (#SC-47778)nd goat anti-mouse IgG-HRP (#SC2031) were from Santa Cruziotechnology, USA. Topoisomerase I & II detection kits wereurchased from TopoGEN Inc., Port Orange, USA. Electrophoresiseagents, protein estimation kit and protein markers were fromigma Chemical Co., USA.

.2. Cell lines, cell culture, growth conditions and treatment

Human breast cancer cell line MCF-7, human promyelocyticeukemia cell line HL-60, human lymphoblastic leukemia cell line

OLT-4, human prostate cancer cell line PC-3 and DU-145 and nor-al monkey kidney cell line CV-1 were procured from National

enter for Cell Sciences, Pune, whereas human colon cancer cell

ine Colo-205 and human ovarian cancer cell line OVCAR-5 wererocured from National Cancer Institute, Frederick, USA. Cell linesere grown and maintained in RPMI-1640 medium, pH7.4 with

0% FCS, 100 units/ml penicillin, 100 �g/ml streptomycin and 2 mMlutamine. Cells were grown in CO2 incubator (Heraeus, GmbH,

Interactions 189 (2011) 60–71 61

Germany) at 37 ◦C in the presence of 90% humidity and 5% CO2.PKBA was dissolved in DMSO.

2.3. Synthesis of 3-˛-propionyloxy-11-keto-ˇ-boswellic acid(PKBA)

The synthesis of 3-�-propionyloxy-11-keto-�-boswellic acid(PKBA) was accomplished from 11-keto-�-boswellic acid (KBA), atriterpenic constituent of boswellic acids. KBA was isolated from amixture of boswellic acids by the method reported in the literature[9]. In order to prepare PKBA, KBA (1 g, 2 mmol) was treated withpropionic anhydride (1.2 eq) in the presence of dimethyl aminopyridine (DMAP) as a catalyst in 10 ml dry dichloromethane (DCM)(Scheme 1). After completion, the reaction was treated with 10%sodium bicarbonate (NaHCO3) solution, extracted with DCM (3×100 ml) and the crude product thus obtained after usual workupwas purified by flash chromatography to give PKBA as 95% yield inthe form of white powder.

2.4. Spectral analysis of PKBA

White solid, [˛]25D +66.6 (c 1.0 CHCl3), mp 264–266 ◦C. 1H NMR

(200 MHz, CDCl3): ı 0.83, 0.93, 1.0, 1.12, 1.15, 1.24 (24H, 23, 25,26, 27, 28, 29 & 30 –CH3 & –CH2CH3), 1.40–2.39 (m, nH), 2.33(t, J = 7.3 Hz, 2H, –CH2CO), 5.31 (bs, 1H, H-3), 5.56 (bs, 1H, H-12).13C NMR (50 MHz, CDCl3): ı 9.4, 13.2, 17.5, 18.4, 18.9, 20.5, 21.2,23.5, 23.9, 27.3, 27.5, 28.1, 28.9, 30.9, 32.9, 34.0, 36.9, 37.4, 39.3,39.4, 40.9, 43.8, 45.1, 46.6, 50.6, 59.1, 60.4, 72.8, 130.5, 165.2, 173.9,181.9, 199.2; ESI-MS (m/z): 549 [M+Na]+. Anal. Calc. for C33H50O5:C, 75.25; H, 9.57. Found: C, 75.11; H, 9.72.

2.5. In vitro cytotoxicity screening in human tumor cell lines

In vitro cytotoxicity of PKBA was determined by semi-automatedassay using SRB [10]. IC50 values were determined by non-linearregression analysis using Graph Pad Software (2236 Avenida de laPlaya La Jolla, CA 92037, USA).

2.6. DNA topoisomerase-I & -II assay

Supercoiled PryG DNA was incubated with 20 �g/ml of PKBA andfour units of human topoisomerase I & II separately (TOPO GEN),in relaxation buffer containing 10 mM Tris–HCl, pH 7.9, 10 mMEDTA, 1.5 mM NaCl, 0.1 mg/ml bovine serum albumin, 1 mM sper-midine and 50% glycerol. Camptothecin (100 �M) and etoposide(100 �M) were used as a positive controls for topoisomerase I &II, respectively. Each reaction volume was made up to 20 �l withH2O, incubated at 37 ◦C for 30 min and stopped by the additionof SDS to a final concentration of 1% which was treated further

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Scheme 1. PKBA synthesis.

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.7. DNA gel electrophoresis

DNA fragmentation was determined by electrophoresis ofxtracted genomic DNA from HL-60 cells. Exponentially growingells (2 × 106 cells/ml) were treated with test compound at con-entration of 10, 15 and 20 �g/ml, respectively, in 6 well platesor 24 h. Cells were harvested, washed with PBS, pellets were dis-olved in lysis buffer [10 mM EDTA, 50 mM Tris pH 8.0, 0.5% (w/v)DS and proteinase K (0.5 mg/ml)] and incubated at 50 ◦C for 1 h.he lysate was incubated with RNase A (0.5 mg/ml) at 37 ◦C for 2 h.he DNA, thus, obtained was heated rapidly at 70 ◦C, supplementedith loading dye and resolved immediately on 1.5% agarose gel at

0 V for 2–3 h [12].

.8. 4′-6-Diamidino-2-phenylindole (DAPI) staining of cells foruclear morphology

HL-60 cells (2 × 105/2 ml/well) were seeded in 6-well platesnd treated with 5 &10 �g/ml PKBA for 18 h. Cells were collected,ashed with PBS and smears were prepared. For fluorescent 4′-

-diamidino-2-phenylindole (DAPI) staining, air dried slides werexed in methanol at −20 ◦C for 20 min, air dried and stained with�g/ml DAPI (Sigma) for 20 min in the dark, mounted in glycerolBS (9:1) and observed under inverted fluorescence microscopeOlympus 1× 70) [13].

.9. Scanning electron microscopy

HL-60 cells were incubated with 10 �g/ml PKBA for 24 h androcessed for scanning electron microscopy [14,15]. Briefly, theells on a coverslip were fixed with 2.5% glutaraldehyde in 0.1 Mhosphate buffer (pH 7.2) at 4 ◦C for 1 h, post-fixed with 1% osmiumetraoxide in the same buffer for 1 h, dehydrated with gradedthanol solutions, dried in a critical point drier using carbon diox-de (Balzer’s Union) and coated with gold using a sputter coaterPolaron). The specimens were examined with an electron micro-cope (JEOL-100CXII) with ASID at 40 kV.

.10. Flow-cytometric analysis of apoptosis and necrosis

PKBA induced apoptosis in HL-60 cell line was determined usingITC-labeled Annexin V antibody by flowcytometry [16]. Expo-entially growing HL-60 cells (2 × 106/ml/well) were treated withifferent concentrations of PKBA for 24 h, washed with PBS andtained with annexin V-FITC antibody and PI as per the instructionsiven by the manufacturer. The cells were scanned for fluorescencentensity in FL-1 (FITC) and FL-2 (PI) channels. The fractions of cellopulation in different quadrants were analyzed using quadranttatistics. Cells in the lower right quadrant represented apoptosishile cells in the upper right quadrant represented necrosis or post

poptotic necrosis [17].

.11. Quantification of DNA damage using comet assay

Comet assay was performed to quantify DNA damage [18]. Expo-entially growing HL-60 cells (2 × 106cells/ml/well) were treatedith different concentrations of PKBA and incubated for 24 h. The

ells were harvested and washed twice with PBS and its 50 �lliquot was embedded in 450 �l of warm (45 ◦C) low melting pointgarose (0.75%). The resulting mixture was spread over precoatedicroscopic slides (0.1% agarose routine). The gel was covered

ith glass cover slip and left to set at 4 ◦C for 5–10 min. Gel

mbedded cells were lysed in lysing buffer (2.5 M NaCl, 100 mMisodium EDTA, 10 mM Trizma base, 8 g/l NaOH, pH 10) for 20 mint 4 ◦C to allow DNA unwinding. Electrophoresis was performed at00 mA and 50 V for 20 min. The slides were stained with 20 �g/ml

Interactions 189 (2011) 60–71

ethidium bromide and examined under an Olympus fluorescencemicroscope (I× 41) equipped with an excitation filter (BP 510 nm)and a barrier filter (590 nm). Slides were analyzed using computer-ized imaging analysis system (KOMET 5.5). To evaluate the amountof DNA damage, computer generated tail moment values were used.Approximately hundred cells were used to access the DNA dam-age by (a) olive tail moment, (b) tail length and (c) tail coefficientvariance.

2.12. Flow-cytometric analysis of nuclear DNA

HL-60 cells (2 × 106/ml) were treated with test compound for24 h at 1, 5, 10, 30 & 50 �g/ml, respectively, and washed twicewith ice-cold PBS, harvested, fixed in cold 70% ethanol in PBS andstored at −20 ◦C for 30 min. After fixation, the cells were incubatedwith RNase A (0.1 mg/ml) at 37 ◦C for 30 min, stained with pro-pidium iodide (50 �g/ml) for 30 min on ice in dark [19] and thenmeasured for nuclear DNA content using BD-LSR Flowcytometer(Becton Dickinson, USA) equipped with electronic doublet discrim-ination capability using blue (488 nm) excitation from argon laser.The fluorescence intensity of sub-G1 cell fraction represented theapoptotic cell population.

2.13. Flow-cytometric determination of mitochondrial membranepotential

Detection of mitochondrial permeability transition event pro-vides an early indication of the initiation of cellular apoptosis. Thisprocess is typically confined to the collapse of the electrochemi-cal gradient across the mitochondrial membrane as measured bythe change in the membrane potential (�� m). The loss of themitochondrial membrane potential is indicative of apoptosis andcan be measured after staining with Rhodamine-123 [20,21] Expo-nentially growing HL-60 cells (1 × 106/ml/well) were treated withPKBA for 24 h. Rhodamine-123 (200 nM) was added 1 h before thetermination of the experiment. Cells were collected, washed in PBSand incubated with propidium iodide (5 �g/ml) for 15 min. Thedecrease in fluorescence intensity due to the loss of mitochondrialmembrane potential was analyzed in FL-1 channel.

2.14. Caspase assays

The activation of intracellular caspases was measured bycaspase colorimetric kits. HL-60 cells (2 × 106/ml/well) were incu-bated with PKBA at 10 and 20 �g/ml, respectively, for 3, 6, 12 and24 h. Cells were collected, washed in PBS and lysed in lysis buffer.Activities of caspases-1, -2, -3, -6, -8, -9 & -10 in cell lysate weredetermined as per the instructions of manufacturer. Camptothecin(5 �M) was used as reference compound.

2.15. Preparation of total cell lysates

After incubation with PKBA for 24 h, HL-60 cells (2 × 106cells)were collected, washed with cold PBS and incubated with cold lysisbuffer (50 mM Tris; pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Non-idet P-40, 1 mM PMSF and 1% (v/v) eukaryotic protease inhibitorcocktail) for 30 min on ice [22]. Cells were centrifuged at 12,000 × gfor 10 min at 4 ◦C and the resulting supernatant was collected aswhole cell lysate for western blot analysis of various proteins.

2.16. Preparation of cytosolic lysates

For the analysis of cytochrome c, immunoblotting cytosolicfractions were obtained by selective plasma membrane perme-abilization with digitonin [22]. Briefly, 2 × 106 cells were lysedfor 1–2 min in lysis buffer (75 mM NaCl, 8 mM Na2HPO4, 1 mM

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Table 1In vitro cytotoxicity studies to determine the influence of PKBA on the proliferation of various human cancer cell lines.

Tissue Cell line IC50 value (�g/ml)

PKBA Doxorubicin Etoposide Camptothecin 5-FU Mitomycin C

Neuroblastoma IMR-32 5.95 ± 0.04 0.015 ± 0.012 nd nd nd ndSF-295 7.11 ± 0.05 0.019 ± 0.05 nd nd nd nd

Prostate PC-3 15.2 ± 0.02 nd nd nd nd 0.5 ± 0.11Colon Colo-205 14.5 ± 0.05 nd nd nd 0.74 ± 0.01 0.6 ± 0.3Breast MCF-7 15 ± 0.05 nd nd 0.031 ± 0.22 0.19 ± 0.06 ndOvary OVCAR-5 15.9 ± 0.07 nd nd nd 0.72 ± 0.31 ndLeukemia HL-60 8.7 ± 0.021 0.21 ± 0.32 1.76 ± 0.02 0.29 ± 0.21 nd 0.059 ± 0.64

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former acts by stabilizing DNA cleavable enzyme complexes lead-ing to DNA break and later acts by stabilizing enzyme where bothDNA strands remain intact and no DNA breaks occur, resulting inapoptosis. PKBA at 20 �g/ml inhibits the enzymatic activity of bothtopoisomerases I & II (Fig. 1) in a way similar to that of known topo-

Fig. 1. PKBA inhibits topoisomerase I & II activity. Inhibitory assay of topoiso-merase I & II was performed using topoisomerase I & II kits from Topogen. The

Molt-4 9.5 ± 0.032 6.31 ± 0.011

KBA showed an IC50 > 100 �g/ml in normal monkey kidney CV-1 cell line.d; Not determined. The values reported here in are the mean values of three expe

aH2PO4, 1 mM EDTA, and 350 �g/ml digitonin and 1% (v/v)ukaryotic protease inhibitor cocktail). The lysates were cen-rifuged at 12,000 × g for 1 min, and the supernatant was collecteds cytosolic fraction and stored at −70 ◦C.

.17. Western blotting

For the immunoblotting analysis, 60 �g of protein was run on2% SDS-polyacrylamide gel and then transferred on PVDF mem-rane. Non-specific binding was blocked by 5% non-fat milk in Trisuffered saline containing 0.1% Tween-20 (TBST). The blots wererobed with primary antibodies of cytochrome c and PARP (1:1000)nd then incubated with goat anti mouse horseradish peroxidase-onjugated secondary antibodies. Signals were detected using ECLlus chemiluminescence’s kit on radiography film in dark.

.18. In vivo anticancer activity

In vivo anti-cancer activity of PKBA was evaluated in murineodels. Non-inbred Swiss albino mice from an in-house colonyere used in the present study. The animals were housed under

tandard laboratory conditions and provided pelleted mice feedM/s Ashirwad Industries, Chandigarh, India) and autoclaved waterd libitum. The number of animals for this study was approvedy the Institutional Animal Ethics Committee, Indian Institute of

ntegrative Medicine, Jammu. Ehrlich ascites carcinoma (EAC) andarcoma-180 (S-180) cells were collected from the peritoneal cav-ty of the Swiss albino mice harbouring 8–10 days old ascitic tumor.

ale animals weighing 18–23 g were selected for the present study.n case of solid tumor experiments, the animals were injected (i.m.)

ith 1 × 107 EAC cells in the right thigh on day 0, the animals wereandomized and divided into four groups including one controlroup. Out of four, two groups were treated intraperitoneally with.2 ml of 100 and 150 mg/kg of PKBA, emulsified with 0.5% gumcacia, respectively, from days 1 to 9. The third test group received-fluorouracil (22 mg/kg, i.p.) and it served as positive control. Theontrol group was sham treated with normal saline. On day 13,umor weight was determined as per the standard procedure [23].scitic tumors were produced by injecting EAC and S-180 asciticells (107 cells/animal, i.p.), respectively, in Swiss albino mice. Theroup of animals and treatment with test compound was the sames described for solid tumor experiments, except for 5-fluorouracil,hich was administered at a dose of 20 mg/kg (i.p.). All the animalsere sacrificed on 12th day and the ascitic fluid was collected from

he peritoneal cavity of each mouse for the evaluation of tumorrowth [23].

.19. Protein measurement

Protein was measured employing BCA protein assay kit fromigma.

0.64 ± 0.6 0.0028 ± 0.6 3.2 ± 0.031 nd

ts each carried in triplicate.

2.20. Statistical analysis

Data expressed as mean ± S.D., unless otherwise indicated. Com-parisons were made between control and treated groups unlessotherwise indicated using ANOVA and p-values <0.01 were consid-ered significant.

3. Results

3.1. In vitro cytotoxicity assay

Inhibitory effect of PKBA was evaluated against different humancancer cell line using a conventional SRB based colorimetric cyto-toxicity assay [10]. PKBA treated cells produced concentrationdependent inhibition of cell proliferation of IMR-32, SF-295, PC-3, Colo-205, MCF-7, OVCAR-5, HL-60 & MOLT-4 cell lines and theirIC50 values were determined as 5.95, 7.11, 15.2, 14.5, 15, 15.9, 8.7& 9.5 �g/ml, respectively (Table 1).

3.2. Topoisomerase I & II inhibitory assay

Topoisomerases are the essential enzymes that control andmodify the topological state of DNA. Topoisomerases I and II actby sequential breakage and reunion of single and double strands ofDNA, respectively [24,25]. The topoisomerase targeting drugs caneither be classified as topo poisons or topo catalytic inhibitors. The

assay was performed according to the instructions provided by the supplier. Lanes1 & 2 (Supercoiled DNA + Topo-I & Supercoiled DNA + Topo-II, respectively). Lanes 3and 4 (Supercoiled DNA + Topo-I + camptothecin 100 �M & Supercoiled DNA + Topo-II + Etoposide 100 �M, respectively). Lanes 5 & 6 (Supercoiled DNA + Topo-I + PKBA20 �M & Suprecoiled DNA + Topo-II + PKBA 20 �M, respectively). Lane 7 (RelaxedDNA as Marker). Lane 8 (Supercoiled DNA).

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64 G. Chashoo et al. / Chemico-Biological Interactions 189 (2011) 60–71

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ig. 2. (a) DNA fragmentation assay in HL-60 cells. Cells (2 × 106/ml/well) were incuells + 5 �M camptothecin), lanes 3–5 (HL-60 cells + 10 15 & 20 �g/ml PKBA). (b) DNarious concentrations for 24 h. Lane 1 (untreated cells), lanes 2–6 (PKBA 5, 10, 20,

& -II inhibitors, camptothecin (100 �M) and etoposide (100 �M),espectively. AKBA on the other hand has been found to inhibit thenzymatic activity of topoisomerase I at a higher concentration of25 �g/ml [8].

.3. PKBA induced DNA fragmentation in HL-60 cells

Apoptosis or programmed cell death is a regulated physiologicalrocess whereby a cell can be removed from a population markedy DNA fragmentation. During apoptosis endonucleases cleave the

ntra nucleosomal chromatin in multiples of 180 bp leading to DNAragmentation resulting in a typical DNA ladder formation. HL-60ells when incubated with PKBA for 24 h exhibited a typical DNAadder formation at 10, 15 and 20 �g/ml (Fig. 2a). AKBA has beenound to induce the same at higher concentration of >25 �g/ml8]. Camptothecin (5 �M), as reference exhibited a discrete ladderattern after 6 h of treatment, however, no ladder formation wasbserved in untreated cells (Fig. 3). DNA isolated from untreatednd PKBA treated normal monkey kidney CV-1 cells did not showny DNA ladder formation (Fig. 2b).

.4. PKBA induces altered nuclear morphology

In contrast to normal cells, the nuclei of apoptotic cells containighly condensed chromatin material which when stained withAPI, a DNA binding dye represents the entire nucleus as a singler a group of featureless, bright spherical beads and these mor-hological changes can be visualized by fluorescence microscopy.uclei of HL-60 cells appeared round in shape (Fig. 3a), while those

reated with PKBA showed the formation of apoptotic bodies, chro-atin condensation, shrinkage of cells and bleb formation, (Fig. 3c,

) thus, depicting internuclesomal DNA fragmentation and sub G1ccumulation.

.5. Scanning electron microscopic (SEM) analysis

HL-60 cells after SEM examination were found to be sphericaln shape with a few surface projections (Fig. 4a, b, c) while PKBA

with PKBA at various concentrations for 24 h. Lane 1 (untreated cells), lane 2 (HL-60mentation assay in CV-1 cells. Cells (2 × 106/ml/well) were incubated with PKBA at

50 �g/ml, respectively).

treated cells showed smoothening of cell surface, decrease in cellsize and a clear apoptosis (Fig. 4d, e, f).

3.6. Flow-cytometric estimation of apoptosis and necrosis

Loss of plasma membrane integrity is an early event in apop-tosis, independent of the cell type, resulting in the exposure ofphosphatidylserine (PS) residues at the outer plasma membrane[26]. Annexin V in the presence of calcium interacts strongly andspecifically with PS and can be used to detect apoptosis by target-ing for the loss of plasma membrane asymmetry. Annexin V-FITCanalysis revealed a basal apoptotic population in the untreated cul-ture as 3.5%, however, after treatment with PKBA a dose dependantincrease in apoptotic population was observed which was found tobe 70.74% and 96.74%, respectively, at 10 and 20 �g/ml after 24 hof incubation (Fig. 5). Apoptosis thus appeared to be the primarymode of cell death induced by PKBA.

3.7. Comet assay analysis

Comet assay measures, double as well as single strandbreaks, alkali labile sites, oxidative DNA base damage,DNA–DNA/DNA–protein/DNA–Drug cross linkage and DNArepair. The principle of the assay rests on strand breakage of thesupercoiled double helical DNA which leads to the reduction inthe size of large molecules where in strands of the DNA can bestretched out by electrophoresis. Comets form when the brokenends of the negatively charged DNA molecule become free tomigrate in the electric field towards the anode [27]. PKBA treatedHL-60 cells revealed the formation of comets (Fig. 6c) with headDNA of 70.15% and 43.21% at concentration of 10 and 20 �g/ml,respectively. Tail DNA that revealed the actual DNA damagewas found to be 29.85% and 56.79% at the above-mentioned

concentrations. The olive tail movement was found to be 2.83%and 4.46% while the tail length was found to be 10.06% & 27.07%,respectively (Table 3). Camptothecin (5 �M) taken as a posi-tive control also induced comet formation (Fig. 6b) in HL-60with a head DNA of 66.66% and tail DNA of 33.34% (Table 3),

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G. Chashoo et al. / Chemico-Biological Interactions 189 (2011) 60–71 65

Fig. 3. (A) Influence of PKBA on nuclear morphology and apoptotic bodies formation in HL-60 cells. Cells (2 × 105/2 ml/6 well plate) were treated with indicated dose of PKBAfor 18 h stained with DAPI and visualized for nuclear morphology and apoptotic bodies. (a) Untreated control cells showing large sized nuclei. (b) Cells treated with 5 �Mcamptothecin indicate condensation both in cytoplasm and in nuclei and thus apoptosis (↑). (c) Cells treated with 5 �g/ml PKBA indicate apoptosis (↑). (d) Cells treated with1 Bar (—a ** < 0.

h(

3

cd3mc

3

m

0 �g/ml PKBA showing increase in apoptotic population (↑). Magnification 400×.poptosis. Data are Mean ± S.D. from three similar experiments. p-Value: ** < 0.01, *

owever, no comet formation was observed in untreated cellsFig. 6a).

.8. Cell cycle analysis by flowcytometry

Using human leukemia HL-60 cells the effect of PKBA on DNAontent was assessed by cell cycle phase distribution. The hypoiploid sub-G1 DNA fraction (<2n DNA) was found to increase from.32% to 95.81% in a concentration dependent manner after treat-ent with of PKBA (Fig. 7), the blocking of G2/M phase of the cell

ycle was also observed (Table 2).

.9. PKBA induces loss of mitochondrial membrane potential

HL-60 cells exposed to PKBA for 24 h when analyzed foritochondrial membrane potential loss (�� m) employing Rh-

) 20 �M. (B) Relationship between alteration in nuclear morphology and extent of001 compared to untreated control.

123 uptake by flowcytometry revealed that almost all the cellswere functionally active with high Rh-123 uptake fluorescencein untreated cells while the cells treated with PKBA causedmitochondrial damage resulting in the loss of mitochondrialmembrane potential. As is evident from the results the mito-chondrial membrane potential loss was found to be 64.73%and 91.38%, respectively at a concentration of 10 and 20 �g/ml(Fig. 8).

3.10. PKBA stimulates caspase activities in HL-60 cells

Caspases are the central regulators of both intrinsic and extrin-sic apoptotic pathways. PKBA produced optimal caspases activationthrough 12 h incubation and the prolonged incubation up to 24 henhanced the activity further (Fig. 9). PKBA produced a dose andtime dependant increase in caspase-1, -2, -3, -6, -8, -9 & -10. Results

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66 G. Chashoo et al. / Chemico-Biological Interactions 189 (2011) 60–71

F tivelym lli andb

rea-awa

TT

ig. 4. SEM of control HL-60 cells (a, b, c) at 4000×, 8000× and 10,000×, respecagnifications. The untreated control cells (a, b, c) show rough surface and microvi

lebbing (↑) of the plasma membrane and apoptosis (d, e, f).

evealed that HL-60 cells treated with 20 �g/ml PKBA for 24 hnhanced the activation of caspase-1, -9 and -6 by 2-fold (Fig. 9)nd that of caspase-2 and -3 by 10-fold (Fig. 9) while caspase-8 and

10 were activated to a lesser extent (Fig. 9). Activation of caspase-3nd -9 revealed PKBA to induce apoptosis through intrinsic path-ay but the activation of caspase-6, -8 and -10 revealed that it can

lso undergo apoptosis via. extrinsic pathway. Further it might be

able 2he cell cycle phase distribution of PKBA in HL-60 cells.

Sample Sub G1 (%)/Apoptotic (%)

Untreated cells 3.32Camptothecin 5 �M 56.08PKBA 1 �g/ml 2.91PKBA 5 �g/ml 57.13PKBA 10 �g/ml 78.16PKBA 30 �g/ml 80.10PKBA 50 �g/ml 95.81

and HL-60 cells treated with PKBA (10 �g/ml) for 24 h (d, e, f) at the respectivethe treatment, after 24 h causes reduction in cell size, smoothening of cell surface,

possible that the activation of one pathway may be responsible forthe activation of the other.

3.11. Western blotting

PKBA induced loss of mitochondrial membrane potential has itsrelevance towards the opening of PTP and release of cytochrome cfrom mitochondria (Fig. 10). Initially cytochrome c is known to acti-

G1/G0 (%) S (%) G2/M (%)

49.42 22.54 27.4132.17 6.93 5.7651.42 23.34 29.2126.81 16.13 3.2210.56 11.44 1.0716.28 2.84 1.74

5.24 1.32 0.56

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G. Chashoo et al. / Chemico-Biological Interactions 189 (2011) 60–71 67

F s wereV ptotica

vatma

3

iirimtm

Fc

ig. 5. PKBA induced apoptosis in HL-60 cells using Annexin V-FITC/PI. HL-60 cell-FITC/PI to analyze apoptotic and necrotic cell population as described earlier. Apore representative of one of the two similar experiments.

ate caspase-9 and then executioner caspase-3 which used PARPs a substrate that inhibited the repair of damaged DNA. In PKBAreated HL-60 cells the cleavage of PARP occurs in a dose dependent

anner (Fig. 10) thereby, confirming the involvement of intrinsicpoptotic pathway.

.12. In vivo studies

These studies revealed PKBA to induce 100% growth inhibitionn Ehrlich ascites carcinoma (EAC) model at a dose of 100 mg/kgn Swiss albino mice. In solid tumor models the inhibition was

eported to be dose dependant up to the tune of 33.78% and 66.74%n Ehrlich Tumor (EAT) and 34.25% and 68.02% in S-180 solid tumor

odels at 100 and 150 mg/kg, respectively (Fig. 11), on the basis ofhe tumor weight which was measured on day 13th of the experi-

ent (Table 4).

ig. 6. Comet assay analysis. (a) Untreated cells, (b) camptothecin treated HL-60 cells forells (a) showed no comet formation and the treatment with PKBA after 24 h induced the

treated with indicated concentrations of PKBA for 24 h and stained with annexinpopulation was found to be 70.06% and 96.74% at 10 & 20 �g/ml, respectively. Data

4. Discussion

The present study illustrated the pro-apoptotic potential of apropionyloxy derivative of 11-keto-�-boswellic acid (PKBA) onhuman leukemia HL-60 cells for the first time. From the currentobservation it was found that PKBA was more potent cytotoxic andapoptotic agent than other boswellic acids such as AKBA. As evi-denced from the results PKBA inhibited the cell growth and inducedapoptosis as measured by various biological end points [21] includ-ing DNA fragmentation, apoptotic body’s formation and annexin V-FITC binding in HL-60 cells at much lower concentration than AKBA[8]. Moreover, no such effect in terms of in vitro cytotoxicity and

DNA fragmentation was observed in PKBA treated monkey kidneynormal cell line CV-1. In addition, PKBA induced a dose dependentDNA double strand breaks, presented by “comet tail” in neutralcomet assay in HL-60 cells. Considering the potential that PKBAoffers in its development as anticancer agent, we further sought to

6 h at 5 �M, (c) PKBA treated HL-60 cells for 24 h at 20 �g/ml. The untreated controlformation of actual comets in HL-60 cells (c).

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68 G. Chashoo et al. / Chemico-Biological Interactions 189 (2011) 60–71

Table 3The single cell gel electrophoresis of PKBA.

Head DNA Tail DNA Olive tail movement Tail length

Untreated cells 91.08 ± 1.028 8.92 ± 1.028 0.440 ± 0.031 1.22 ± 0.238Camptothecin 5 �M 66.66 ± 0.021 33.34 ± 0.026 7.85 ± 0.052 33.25 ± 0.042PKBA 10 �g/ml 70.15 ± 0.038 29.85 ± 0.023 2.83 ± 0.042 10.06 ± 0.031PKBA 20 �g/ml 43.21 ± 0.045 56.79 ± 0.031 4.46 ± 0.052 27.07 ± 0.042

The single cell gel electrophoresis of PKBA treated HL-60 cells after 24 h of incubation revealed a head DNA of 70.15% and 43.21%, Tail DNA of 29.85% and 56.79% at 10 and2 s 2.83c 6% ane

utGicoscodmlot

Faf

0 �g/ml, respectively which signifies the DNA damage. The olive tail movement waoncentrations. Camptothecin taken as a positive control showed a head DNA of 66.6xperiments.

nderstand early events associated with cell death. We observedhat PKBA blocked cancer cell proliferation by arresting cells at2/M phase of cell cycle. Mitochondria are particularly affected

n the early apoptotic process and are thought to act as centralo-ordinators of cell death. Mitochondrial dysfunction induces thepening of mitochondrial permeability transition pore (PTP), dis-ipation of �� m and release of apoptogenic proteins (cytochrome). In this study, we demonstrated that PKBA disrupted the functionf mitochondria in the early apoptotic process in a concentrationependent manner. PKBA lead to the release of cytochrome c from

itochondria into the cytosol. The review of literature has shown

oss of mitochondrial membrane potential towards the activationf caspases [28,29] and the present study also showed activa-ion of different upstream and downstream caspases. The reduced

ig. 7. Flow-cytometric analysis of DNA content in PKBA treated HL-60 cells. HL-60 cellsnd stained with PI to determine DNA fluorescence and cell cycle phase distribution as derom FL2-A vs. cell counts is shown (%). Data are representative of one of three similar ex

% and 4.46% while a tail length of 10.06% and 27.07% was observed at the respectived tail DNA of 33.34% at 5 �M concentration. Data are mean ± S.D. from three similar

mitochondrial membrane potential, cytochrome c release from themitochondria to cytosol [30], the activated caspase-9 and -3 whichlead to PARP cleavage [24], DNA damage and fragmentation andeventually the induction of apoptosis. Since the review of litera-ture and the scientific data available reveal that these classes ofagents induce apoptosis by inhibiting topoisomerases, PKBA wastherefore subjected to topoisomerase inhibition studies, whereinit was found that PKBA inhibits both topoisomerases I & II stronglyand at much lower concentration than AKBA [8]. The dual inhibitoryactivity of PKBA may be due to the recognition of structural motifs

present on both topoisomerases I & II, however, there is as yet nodetailed understanding of the factor that result in selective or dualinhibition, but the structure–activity studies in several classes haveshown that the changes in the structure can influence topo I/II selec-

(2 × 106 cells/ml/well) were exposed to indicated concentrations of PKBA for 24 hscribed in materials and methods. Fraction of cells for sub-G1 population analyzedperiments.

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G. Chashoo et al. / Chemico-Biological Interactions 189 (2011) 60–71 69

Fig. 8. PKBA-induced loss of mitochondrial membrane potential (�� m) in HL-60 cells. Cells (1 × 106/ml/6 well plates) were incubated with indicated doses of PKBA for24 h.Thereafter, cells were stained with Rhodamine-123 (10 nM) for 1 h and analyzed in FL-1 vs. FL-2 channels of flowcytometer. Data are representative of one of threesimilar experiments.

Table 4In vivo anticancer activity of PKBA against (A) Ehrlich Ascities carcinoma, (B) Ehrlich tumor, and (C) Sarcoma-180.

Sample Dose (mg/kg i.p.) Animals/Mortality Tumor volume (ml) Cell count (107) % Tumor growth inhibition

(A) Ehrlich Ascities carcinomaControl NS 7/0 3.11 ± 0.35 100.26 ± 3.26 –PKBA 100 7/0 1.32 ± 0.16 1.33 ± 5.60b 100% ± 0.56b

5 FU 20 7/0 0.45 ± 0.15 7.98 ± 1.1b 92.45 ± 1.5b

Sample Dose (mg/ kg i.p.) Animals/Mortality Body weight (g) Tumor weight (mg) % Tumor growth inhibition

(B) Ehrlich tumorPKBA 100 7/0 21.25 ± 0.40 1205.28 ± 51.10a 33.78 ± 0.47b

150 7/0 22.18 ± 0.61 598.44 ± 49.22c 66.74 ± 0.51c

5 FU 22 7/0 19.11 ± 0.50 989.64 ± 45.3b 45.13 ± 1.5c

(C) PKBA against Sarcoma-180Control NS 7/0 22.33 ± 0.35 1972.16 ± 76.24 –PKBA 100 7/0 21.45 ± 0.40 1296.59 ± 52.91a 34.25 ± 0.46b

150 7/0 18.38 ± 0.61 630.69 ± 50.01c 68.02 ± 0.44%c

5 FU 22 7/0 18.65 ± 0.50 1039.33 ± 52.11b 47.3 ± 0.44%c

The values reported here in are the mean values of three experiments each carried in triplicate. Data are mean ± S.D.a (p < 0.05).b (p < 0.01).c (p < 0.001).

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70 G. Chashoo et al. / Chemico-Biological Interactions 189 (2011) 60–71

0

0.05

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0.4

0.45

0.5

3 h 6 h 12 h 24 h

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alue

Time, h

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Camptothecin 5µM

PKBA 10 µg/ml

PKBA 20 µg/ml

0

0.2

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0.6

0.8

1

1.2

1.4

3 h 6 h 12 h 24 h

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0

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1.2

1.4

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PKBA 20µg/mL

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

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PKBA 10µg/mL

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00.10.20.30.40.50.60.70.80.9

1

3 6 12 24

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alue

Time, h

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Camptothecin 5µM

PKBA 10µg/mL

PKBA 20µg/mL

0

0.05

0.1

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0.25

0.3

0.35

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00.050.1

0.150.2

0.250.3

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3 6 12 24

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ase-

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PKBA 10µg/mL

PKBA 20µg/mL

Fig. 9. PKBA induces the activation of initiator caspases-1, -8, -9 and -10, effector caspases -2,-3, and -6. HL-60 cells (2 × 106/2 ml/6-well plates) in culture were exposed to1 termini by the

taImcTt

0 and 20 �g/ml of PKBA for indicated time periods. The caspases activities were den methodology. All assays were performed according to the instructions provided

ivity. These findings therefore demonstrate that the induction ofpoptosis by PKBA is mediated via inhibition of topoisomerases& II. The above studies nevertheless provide important infor-

ation about the pro apoptotic nature of PKBA prospecting this

andidate for developing into a potential anti-cancer therapeutic.he anticancer therapeutic potential of PKBA was further substan-ially supported by in vivo findings that reveal its growth inhibitory

ed colorimetrically using caspase-colorimetric kits from R & D system as describedsupplier. Data are Mean ± S.D. from three similar experiments.

effect on ascitic and solid tumor models. Taken together, thesestudies provide us the basis for the potential use of PKBA as ananti-tumor agent. Since the current study significantly determines

the role of alkoxy chain length on the apoptotic potential of KBA,our future emphasis will be on the synthesis of other semisyntheticderivatives of KBA with further increase in alkoxy chain length andevaluating their anticancer potential.

Page 12

G. Chashoo et al. / Chemico-Biological

Fig. 10. Immunoblot analysis of cytochrome c and PARP of PKBA. HL-60 cells(2 × 106/3 ml/6-well plates) were treated with indicated doses of PKBA for 24 h anddifferent fraction were prepared and immunoblotted as described in Section 2. �-actin used as internal control to represent the same amount of proteins applied forSDS-PAGE. Specific antibodies were used for detection of cytochrome c and PARP.Data are representative of one of three similar experiments.

Fig. 11. The in vivo anti-cancer studies of PKBA indicate a dose dependant inhibitionof different tumor models in swiss albino mice. At 100 mg/kg body wt. PKBA showed100% growth inhibition of ascitic tumor model (Ehrlich ascites carcinoma). Thegrowth inhibition caused by the PKBA treatment was observed to be 33.78% & 66.74%itEC

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[7 (2002) 313–319.

[29] D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science 281 (1998)

n Ehrlich tumor and 34.25% & 68.02% in S-180 tumors at 100 and 150 mg/kg, respec-ively. The growth inhibition caused by 5-flurouracil was 92.45%, 45.13% & 47.3% inAC, ET and S-180 tumor models, respectively, at 20, 22 & 22 mg/kg respectively.ontrol vs. treatment. p-Values: ** < 0.01, *** < 0.001.

. Conclusion

The in vitro studies performed above lead us to conclude thatKBA exhibited significant cytotoxic potential on a wide range ofuman cancer cell lines. The anti-topoisomerase and the apoptotictudies in human leukemia HL-60 cells, further confirmed its pro-poptotic features. Moreover, it requires four to five fold higheroncentrations of PKBA to kill normal cells than cancer cells as haseen observed in this study. The in vivo findings further confirm

ts anticancer potential. To conclude, the present investigationseem to provide a substantial support for the use of PKBA in cancerhemotherapy.

onflict of interest statement

None.

cknowledgements

The authors are indebted to Dr. Ram Avtar Vishwakarma, Direc-or, Indian Institute of Integrative Medicine, Jammu, for his keennterest and encouragement. They also express their sincere thankso Dr. Ravinder Kaur Bhatia, Department of Pharmacology of thisnstitute for her assistance in writing this manuscript.

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