A novel parthenin analog exhibits anti-cancer activity: Activation of apoptotic signaling events through robust NO formation in human leukemia HL-60 cells

A novel parthenin analog exhibits anti-cancer activity: Activation of apoptotic signaling events through robust NO formation in human leukemia HL-60 cells
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  A novel parthenin analog exhibits anti-cancer activity: Activation of apoptoticsignaling events through robust NO formation in human leukemia HL-60 cells Ajay Kumar a , Fayaz Malik a , Shashi Bhushan a , Bhahwal A. Shah b , Subhash C. Taneja b , Harish C. Pal a ,Zahoor A. Wani a , Dilip M. Mondhe a , Jagdeep Kaur c , Jaswant Singh a, ⇑ a Division of Pharmacology, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu Tawi 180001, India b Division of Bio-organic Chemistry, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu Tawi 180001, India c Department of Biotechnology, Panjab University, Chandigarh 160014, India a r t i c l e i n f o  Article history: Received 29 January 2011Received in revised form 8 June 2011Accepted 15 June 2011Available online 29 June 2011 Keywords: Parthenin analog (P19)ApoptosisNitric oxide (NO)NACNF- j B a b s t r a c t This study describes the anti-cancer activity of P19, ananalog of parthenin. P19induced apoptosis in HL-60cellsandinhibitedcellproliferationwith48hIC50of3.5 l M.At10mg/kgdose,itdoubledthemediansurvival time of L1210 leukemic mice and at 25mg/kg it inhibited Ehrlich ascites tumor growth by 60%.Investigation of themechanismof P19inducedapoptosis inHL-60cells revealed that N-acetyl- L  -cysteine(NAC) and s-methylisothiourea (sMIT) could reverse several molecular events that lead to cell death byinhibiting nitric oxide (NO) formation. It selectively produced massive NO in cells while quenching thebasal ROSlevelswithconcurrent elevationof GSH. P19disruptedmitochondrial integrityleadingtocyto-chrome  c   release and caspase-9 activation. P19 also caused caspase-8 activation by selectively elevatingthe expression of DR4 and DR5. All these events lead to the activation of caspase-3 leading to PARP-1cleavageandDNAfragmentation.However,knockingdownofAIFbysiRNAalsosuppressedtheapoptosissubstantially thus indicating caspase independent apoptosis, too. Further, contrary to enhanced iNOSexpression, its transcription factor, NF- j B (p65) was cleaved with a simultaneous increase in cytosolicI j B-alpha. In addition, P19 potently inhibited pro-survival proteins pSTAT3 and survivin. The multi-modal pro-apoptoticactivityof P19raises itspotential usefulnessasapromisinganti-cancer therapeutic.   2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Cancer cells accumulate several genetic and epigenetic muta-tions to cleverly evade self demise by deregulating apoptosis andsetting a stage for initiation, promotion and progression of themalignant growth [1]. Agents that can trigger the process of apop-tosisincancercellsaretherefore,consideredpotentiallyimportantfor the development of anti-cancer chemotherapeutics [2]. In thisconcern, we focusedour attentiononparthenin, amajor sesquiter-pene lactone from the obnoxious weed  Parthenium hysterophorus Linn. (Asteraecae) that grows wild in different regions of India.The plant causes contact dermatitis and allergic rhinitis in animals[3]. Despite the fact that  Parthenium  is considered a toxic plant itstraditional uses are amply reported in literature [4]. One of itschemical constituent parthenin is reported to be responsible forcytotoxic effects of this plant [5,6]. Parthenin has also been re-ported to exhibit anti-tumor activity in mice [7]. In view of the re-ported toxicity of parthenin we attempted to prepare variousanalogs of parthenin modifying its functional groups to obtainsome potent anti-cancer analog of lesser toxicity. In this endeavorwe screened several semi-synthetic analogs of parthenin primarilyfor their apoptotic index and selected P19 among themas the pro-spectivepro-apoptotic candidate for its detailedstudies todevelopit into a promising anticancer therapeutic lead. About 50% of thecancers are known to have p53 mutated. Therefore, we employedp53 null, human acute myeloid leukemia HL-60 cells [8]. Thus,the agents that can induce apoptosis via a  p53 -independent path-way should have a broader therapeutic potential not only in AML patients but also in other cancer diseases.Severalearlierstudieshavedemonstratedthatnitricoxide(NO)isanimportantsignalingmessengerthatplaysanimportantroleinmanyphysiological andpathologicalconditions. EndogenousNOisgenerated from  L  -arginine by three major types of NO synthases(NOS), i.e. endothelial NOS, neural NOS, and inducible NOS (iNOS)while another form of NO has recently been reported from 0009-2797/$ - see front matter    2011 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2011.06.006  Abbreviations:  AIF, apoptosis inducing factor; AML, acute myeloid leukemia;DR4, death receptor 4; DR5, death receptor 5; DRP-1, dynamin related protein-1;GSH, glutathione reduced; iNOS, inducible nitric oxide synthase; NAC, N-acetyl- L  -cysteine; NF- j B, nuclear factor-kappa B; PARP-1, poly (ADP ribose) polymerase-1;pSTAT3, phosphorylated signal transducer and activator of transcription protein-3;ROS, reactive oxygen species; sMIT, s-methylisothiourea; TNFR1, tumor necrosisfactor receptor 1. ⇑ Corresponding author. Tel.: +91 191 2569000x291; fax: +91 191 2569333. E-mail address: (J. Singh).Chemico-Biological Interactions 193 (2011) 204–215 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage:  mitochondria [9,10]. Several studies have amply demonstratedthat NO can induce apoptosis in a variety of tumor cells [11,12],by mechanisms involving induction of stress proteins, mitochon-drial disruption, release of cytochrome  c  , and caspase activation[13]. In the recent past, we reported a lignan composition from Cedrus deodara  that caused NO-mediated apoptotic cell death of Molt-4 cells by activating both intrinsic and extrinsic pathways[14]. Nitric oxide in vivo may contribute to paracrine tumor-suppressive activities by triggering various signaling pathways. Aknown example is tamoxifen, which potently induced iNOS andNO formation in myoepithelial cells in culture in an estrogenreceptor- b -dependent manner [15]. This chemotherapeutic is usedfor high risk population of women with familial history of breastcancer. One potent transcription factor accounting for iNOS induc-tion is the nuclear factor-kappa-B (NF- j B), which is constitutivelyactivated in most of the cancer cell lines and in several cancertissues while its level is very low in resting cells [16]. Cells thatexpressconstitutivelyactivatedNF- j Bareresistanttovariousche-motherapeuticagentsandradiationtreatment[16].ManypreviousstudieshaveimplicatedNF- j Binthedevelopmentandprogressionofcancer.NF- j Bisalsoknowntoregulatetheexpressionofseveralanti-apoptotic genes such as IAPs, Bcl-2, Bcl-xL and survivin [17].In addition NF- j B regulates the expression of several genes in-volved in cancer cell proliferation, interestingly, some of thesegenes are also regulated by STAT3 (signal transducer and activatorof transcription protein-3). In fact STAT3 and NF- j B are closelylinked and both these transcription factors require each other fortheir persistent activation in the cancer cells [18]. STAT3 is foundto be crucial for growth of several cancer types but its expressionis dispensable for normal cells in postembryonic stage [19]. Thismakes NF- j B and STAT3 an ideal target for development of anti-cancer therapeutics. The present studies describe the multi-modalaction of P19 in killing cancer cells by targeting several such apop-totic signaling pathways where the selective generation of NO ap-pears indispensable initiating event in activating pro-apoptoticcascades while simultaneously inhibiting the activity of NF- j Band STAT3. 2. Materials and methods  2.1. Chemicals and antibodies Dihydrorhoethidium (DHE), 2,7-dichlorodihydrofluoresceindiacetate (DCFH-DA), 4,5-diaminofluorescene-diacetate (DAF-2-DA),  L  -buthionine-S,R-sulfoximine (BSO), ethidium bromide,propidium iodide (PI), DNase-free RNase, proteinase K, 3-(4,5,-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT),N-acetyl- L  -cysteine (NAC), s-methylisothiourea (sMIT), stauro-sporine, camptothecin, Fetal bovine serum were purchased fromM/s Sigma–Aldrich, USA; other reagents used were of analyticalgrade and available locally. Annexin V-FITC apoptosis detectionkit, Mitochondrial Membrane Sensor Kit and Apo Alert glutathionedetectionkitwereobtainedfromM/sBDBioscienceswhileApoalertcaspasesassaykitswerefromM/s B.D. Clontech;Anti-humananti-bodies to Bax (#SC-20067), Bid (#SC-6538), DRP-1 (#SC-21804),PARP-1(#SC-8007), Bcl-2 (SC-7382), TNFR1(#SC-1070), FAS (#SC-8009), DR4 (#SC-6824), DR5 (#SC-7192), NF- j B p65 (#SC-8008),NF- j Bp50(#SC-8414), pSTAT-3(Ser727)(#SC-21876), CASPASE-3(#SC-7272), CASPASE-8 (#SC-56070), CASPASE-9 (#SC-56077), AIF(SC-5586), AIF siRNA (h) (SC-29193) survivin (#SC-8806), actin(#SC-8432), goat anti-rabbit IgG-HRP (#SC2030) and goat anti-mouse IgG-HRP (#SC2031), were from M/s Santa Cruz, USA; iNOS(#610432, clone 54) and cytochrome  c   (#556433, clone 7H8.2C12)were from M/s BD, Pharmingen, USA; actin (#CP01), iNOS(#482755) and rabbit anti-goat IgG-HRP (#401504) were fromCalbiochem, Germany; electrophoresis reagents and protein mark-erswerefromM/sBIO-RAD,USAwhileHyperfilmandECLreagentsfromM/s AmershamBiosciences, UK. The sources for other chemi-calsandreagentsaresameasusedearlier[20,21].  2.2. Synthesis of parthenin analog P19 Parthenin, a sesquiterpene lactone was isolated from a plantweed P. hysterophorus .P19[2 0 -(4-chlorophenyl)-3-(4-chloropheny-lidene)-5,10-dimethyl-12-methylene-decahydro-1,2-(1 0 ,3 0 -dioxo-cyclopenta[c])azuleno(4,5-b)furan-4,11-dione] (Scheme 1) wassynthesized from parthenin as reported earlier [22].  2.3. Cell culture, growth conditions and treatment  Human promyelocytic leukemia cell line HL-60 was obtainedfrom NCI, USA; MCF-7 and HeLa cells were obtained from NCCSPune, India; Human Gingival Fibroblast (hGF) cell line developedfrom healthy gingival [23] was provided kindly by Dr. Anil Bala-pure, Scientist, CDRI, Lucknow, India. The cells were grown inRPMI-1640 medium containing 10% FCS, 100units pencillin/100 l g streptomycin per ml medium in CO 2  incubator at 37  Cwith95%humidityand5%CO 2 gasenvironment.Cellsweretreatedwith P19 dissolved in DMSO while the untreated cultures receivedonly the vehicle (DMSO, <0.2%, v/v).  2.4. Housing and care of animals Swissalbinomice(22–25g), DBA/2femalemiceandCDF1malemice (18–23g) were housed in standard size polycarbonate cagesfed with standard pellet diet (Gold Muhor, Lipton India Ltd.) andautoclaved water was given ad libitum. They were housed in con-trolled conditions of temperature (25±2  C), humidity (50–60%)and12:12hof light:darkcycle. The studies andnumberof animalsused were approved by the institutional ethics committee.  2.5. Cell proliferation assay Cell proliferation was determined using 3-(4,5-dimethylthia-zole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as describedearlier [20]. HL-60 cells (2.0  10 4 /200 l l) and adherent cultures(10 4 /200 l l) of MCF-7, HeLa and hGF cells in 96 well culture platesweretreatedwithvariousconcentrationsofP19for48horvarioustime periods. The MTT formazancrystals formed were dissolved in200 l l of DMSO; OD measured at 570nm. The cytotoxicity of P19was expressed as the relative viability (% of untreated controlcells). OOOOOClCl Scheme 1.  Structure of parthenin analog P19.  A. Kumar et al./Chemico-Biological Interactions 193 (2011) 204–215  205   2.6. In vivo anti cancer studies The studies for in vivo anti cancer activity in mouse modelswere conducted as per the protocols of National cancer Institute(NCI), USA [24].  2.7. Anti-cancer activity of P19 in L1210 lymphoid leukemia model inmice L1210 lymphoid leukemia cells grown in the peritoneal cavityof DBA/2 female mice were collected from the animal harboring6–7days old ascites. For testing, CDF1 males were used.5  10 5 cells were injected intraperitoneally in 18 CDF1 malesweighing 18–23g on day 0. The next day, animals were random-ized and divided into three groups, containing seven animals each.Group I were treated with P19 at the dose of 10mg/kg (i.p.) fornineconsecutivedays,GroupIIreceived20mg/kg(i.p.)of5-fluoro-uracilaspositivecontrol.GroupIIIservedascontrolanditreceived0.2ml normal saline (i.p.) for nine consecutive days. The mediansurvival time of animals in each group was calculated as describedearlier [24].  2.8. The effect of P19 on Ehrlich ascites tumor (solid) models in mice Ehrlich Ascites Carcinoma (EAC) cells maintained in the perito-nealcavityofSwissalbinomicewerecollectedfromananimalhav-ing 8–10days old ascitic tumor by aspirating ascetic fluid. Mice of single sex weighing 18–23g were selectedfor the experiment, andwere injected with 1  10 7 EAC cells intramuscularly in the rightthigh of each animal on day 0 and the treatment of P19 (15 and25mg/kg,i.p.)startedfromday1.Thetumorweightwascalculatedonday13intheanimalstreatedfor9daysintraperitoneally(i.p.)ineach animal on day 0. On day 1, the animals were randomized anddivided into different groups. Treatment groups contained sevenanimals each and a control group contained 15 animals. P19 waspreparedin1%Gumacaciainnormalsalineandwasgivenintraper-itoneallyfromday1to9.Similarly,Controlgroupreceived1%Gumacacia in normal saline, whilst 5-fluorouracil (5FU), 22mg/kg i.p.,was used as a positive control. The average tumor weight for eachgroup was calculated, and the percent tumor growth inhibition intreatedgroupswascalculatedasexplainedearlier[24].  2.9. The effect of P19 on Sarcoma-180 (solid) model in mice Sarcoma-180 cells maintained in the peritoneal cavity of balb/cmice were collected from an animal having 8–10days old ascitictumor by aspirating ascetic fluid and 1  10 7 Sarcoma-180 cellswere injected intramuscularly into the right thigh of each animalon day 0 and the treatment of P19 (15mg/kg, i.p.) started fromday 1. Treatment with 5-fluorouracil (5FU), 22mg/kg i.p., wasgiven to a group of mice serving as positive control. Rest of theconditions used were same as that in EAT model.  2.10. Flow cytometric analysis of apoptosis and necrosis HL-60 cells (1  10 6 /ml) were treated with or without 10 l MP19and5mMNACforindicatedtimeperiods.Cellswerecollected,washed twice with PBS and suspended in 0.1ml binding bufferprovided with apoptosis detection kit. Cells were stained with an-nexin V-FITC antibody and propidium iodide as per instructions of the manufacturer and scanned in FL-1 (FITC) vs. FL-2 (PI) channelson BD-LSR flow cytometer using quadrant analysis for evaluatingapoptotic and necrotic cell populations [14].  2.11. DNA cell cycle analysis Cells were treated for various time periods with indicatedconcentrations of P19, processed, stained with PI and analyzedfor PI-DNA fluorescence by flow cytometry as described earlier[20]. The hypodiploid sub-G0/G1 DNA fraction predicts an apopto-tic cell fraction.  2.12. DNA agarose gel electrophoresis DNA fragmentation was assessed by electrophoresis of extracted genomic DNA and visualized in BIO-RAD Gel Documen-tation System as described earlier [20].  2.13. Measurement of intracellular peroxides/ROS, nitric oxide andmitochondrial membrane potential ( D w m ) in HL 60 cells by flowcytometry The levels of intracellular peroxides/ROS were analyzed byusing dihydroethidium (DHE) and 2 0 ,7 0 -dichlorodihydrofluoresceindiacetate (DCFH-DA). Intracellular generation of nitric oxide wasmeasured by using cell permeable dye 4,5-diaminofluorescene-diacetate (DAF-2-DA). Mitochondrial membrane potential wasmeasured by using a Mitochondrial Membrane Sensor Kit contain-ing JC-1 dye as described earlier [20,21].  2.14. Measurement of GSH contents in cells Intracellular levels of GSH were estimated using the BD Apo-Alert™ glutathione detection kit employing monochlorobimane(MCB) reagent. Briefly, cells after various treatments were lysedand incubated with 2mM MCB for 3h at 37  C. Reduced glutathi-one levels were assayed fluorometrically at 395/480nm accordingto manufacturer’s protocol.  2.15. Caspase assays For fluoremetric enzyme assay cells (2  10 6 ) were incubatedwith10 l MP19forindicatedtimeperiods.Attheendoftreatmentcells were washed in PBS and pellets lysed in the cell lysis buffer.Activities of caspase-3, -9/6 and -8 in the cell lysates were deter-mined fluorometrically, using BD ApoAlert caspase fluorescentassay kits. Caspase-3 and -8 employed fluorochrome conjugatedpeptidesDEVD-AFCandIETD-AFCas substrates, respectivelywhilecaspase-9 employed LEHD-AMC. Release of AFC (7-amino-4-trifluoromethyl coumarin) and AMC (7-aminomethylcoumarin)was assayed according to the instructions provided in the manualbythesupplier. Specificinhibitorswereusedasnegativecontroltodetermine whether fluorescence intensity changes were specificfortheactivityof caspases. ThepeptidebasedinhibitorsusedwereDEVD-CHO for caspase-3, IETD-fmk for caspase-8 and LEHD-CHOfor caspase-9/6. Activation of caspases-3, -9 and -8 was also con-firmed by western blotting from the whole cell lysates.  2.16. Use of AIF-siRNA and caspase inhibitors to validate thecontribution of AIF and caspases to apoptosis Expression of AIF was knocked-down in HL-60 cells by usingsiRNAaccordingtomanufacturer’sprotocol(SantacruzBiotechnol-ogy Inc., USA). For inhibition of caspases cell permeable pan cas-pase inhibitor Z-VAD-fmk at 50 l V was added to cell cultures 1hbefore 6h treatment with 10 l V  of P19 or 50 l V  of   b -boswellicacid butyrate (BA-145), used as positive control. 206  A. Kumar et al./Chemico-Biological Interactions 193 (2011) 204–215   2.17. Preparation of cell lysates for western blots analysis Treated and untreated HL-60 cells were centrifuged at 400  g   at4  C, washed in PBS and cell pellets processed for preparation of cytosolic, mitochondrial, nuclear and whole cell lysate fractionsas described earlier [21]. The conditions for the analysis of theexpression of various proteins by western blot analysis using spe-cific antibodies were same as reported earlier [21].  2.18. Statistical analysis Dataarepresentedasmean±S.D.ofthenumberofexperimentsindicated. The comparisons were made between control and trea-tedgroupsandstatisticalsignificancewascalculatedbyusingBon-ferroni test. The difference was considered to be statisticallysignificant if the  p  value was  ⁄  p  <0.05,  ⁄⁄  p  <0.01,  ⁄⁄⁄  p  <0.001. 3. Results  3.1. P19 inhibits cell proliferation P19inhibitedcellproliferationwithlowestIC50valueforHL-60cells incomparisontofewother cancer cell lines of different srcin(Fig. 1A). The 48h IC50 value of P19 was found to be lower in HL-60 cells than in HeLa and MCF-7 cells. HL-60 cells demonstrated atime dependent increase in toxicity of with IC50 values of 9.5, 6.0,5.0and3.5 l V at6,12, 24and48h,respectively(Fig.1B).TheIC50value for normal human cell line hGF however, was higher to theorder of 10-fold when compared to HL-60 cells (Fig. 1A). DMSO,used as a delivery vehicle (<0.2% v/v), did not affect cell growth.Therefore, for all practical purposes HL-60 cells were used forunderstanding the mechanism of action.  3.2. P19 inhibits cancer growth in vivo P19exhibitedanti-canceractivityindifferent mousemodels. InL1210 lymphoid leukemia model in mice it significantly extendedthe median life span of animals to 15.5days (198%) when admin-istered for nine consecutive days at the i.p. dose of 10mg/kg of body weight. Whereas 5-fluorouracil (5-FU) at 20mg/kg of b. wt.increased the median survival time to 18days (230%) (Table 1).Anti-cancer activity of P19 was also observed in other mice tumormodels where P19at 15mg/kg b. wt. (i.p.) produced about 64% tu-mor growth inhibition in Sarcoma-180 (solid) bearing mice; 42%and 63% in the Ehrlich ascites tumor (solid at the doses of 15 and25mg/kg b. wt. (i.p.) (Table 1). Animals treated with P19 appearedhealthyandactive,andnomortalityoccurredduringthetreatmentperiod.  3.3. Induction of apoptosis To elucidate the mechanism of P19 action, we postulated thatcancer cell toxicity might be due to the induction of apoptosis.For this purpose, we analyzed various stereotype biological end-points of apoptosis during an early 6h period of exposure of HL-60 cells to P19 as described in the following.  3.3.1. Induction of DNA fragmentation typical of apoptosis P19 induced concentration dependent DNA fragmentation typ-ical of apoptotic cells. The minimal concentration of P19 inducingDNA fragmentation within 6h apparently was   3 l M (Fig. 1C). The lactone caused DNA damage producing high molecular weightlarger fragments, which appeared as a smear followed byfragments of 180bp or multiples. Fig. 1.  P19 induces cytotoxicity and apoptosis in HL-60 cells. (A) Inhibition of cell proliferation. Influence of P19 on cell proliferation employing MTT assay was evaluated inHL-60, MCF-7and HeLa cancer cell lines and in Primary Human Gingival Fibroblast (hGF) cell line. Cells were treated for 48hand IC50 value for eachcell line was calculatedintermsof50%ofcell viabilityascomparetountreatedcontrol. (B)TimedependentIC5OvalueofP19inHL-60cells; dataaremean±S.D. ofthreeindependentexperiments.(C) P19inducesDNAladdertypical of apoptosis. HL-60cells(2  10 6 )wereincubatedwithindicatedconcentrationsof P19andwith4 l Mof camptothecin(positivecontrol)for 6h followed by DNA extraction and electrophoresis on 1.8% agarose gel as described in Section 2.  A. Kumar et al./Chemico-Biological Interactions 193 (2011) 204–215  207   3.3.2. P19 induced apoptosis assayed by annexin V binding  Annexin-V binding of phosphatidyl serine of exposed cells is anearly indicator of cells undergoing apoptosis. Therefore, cells wereexposed to P19 for different time periods, and the percentage of cells undergoing apoptosis/necrosis was determined by dual stain-ing with annexin V-FITC and PI by flow cytometry. Cells observedtime-dependent increase in apoptotic cell population, which was60% at the end of 6h (Fig. 2A). Interestingly, pretreatment of cellswith 5mM NAC reduced the apoptotic population from 60% toabout 15%.  3.3.3. P19 induced generation of sub-G0/G1 population is inhibited byNAC despite augmentation of GSH contents Treatment of HL-60 cells with P19 observed an increase of about 60% in hypodiploid sub-G0/G1 DNA fraction, indicatingapoptotic population. Pretreatment with NAC, however, reducedthis fraction to about 19%. Treatment with other antioxidants suchastironandtroloxdidnotproduceanyinhibitoryeffectonsub-G0/G1 population (Table 2). NAC, a potent antioxidant, is also thesubstrate utilized by cells in the synthesis of cellular glutathione;it was therefore, of interest to examine the role of GSH in the  Table 1 Comparative influence of P19 on tumor growth inhibition in three different mouse models. For L1210 lymphoid leukemia model in mice the animals were divided into threegroups. Group I was treated with P19 at the dose of 10 mg/kg (i.p) for nine consecutive days. Group II served as control and it received 0.2 ml normal saline (i.p.) for nineconsecutive days Group III treated with 5 FU, 20 mg/kg (i.p.) served as positive control. The median survival time of animals in each group was calculated as described in Section 2.Different doses of P19 were given i.p. to the tumor bearing animals (Swiss albino mice for Ehrlich ascites tumor (Solid) and balb/c for Sarcoma-180) for 9 days and tumor and bodyweight were observed on day 13 and tumor growth inhibition (%) was calculated. Animals treated with 5 FU, 22 mg/kg (i.p.) served as positive control. For Sarcoma-180 andEhrlich ascites tumor each value is the mean ± S.D. of seven observations for animals treated with drug and 15 observations for control groups and represented as tumor weight(mg). TreatmentgroupsDose, mg/kg,i.p.Average tumor weight(mg)Median survival time(days)Tumor growth inhibition(%)Increaseinmediansurvivaltime(%) L1210 lymphoid leukemia Control – – 7.8 – –P19 10 – 15.5 – 1985-Fluorouracil 20 – 18 – 231 Sarcoma-180, solid Control – 1346 – – –P19 15 481 *** – 64 –5-Fluorouracil 22 520 *** – 61 – Ehrlich ascites tumor, solid Control – 1412 – – –P19 15 808 *** – 43 –25 512 *** – 64 –5-Fluorouracil 22 676 *** – 52 – ***  p  <0.001 for treated vs. untreated controls. Other conditions were same as given in Section 2. Fig. 2.  P19 induced apoptosis is protected by NAC despite P19 augmentation of GSH content. (A) NAC protects P19 induced apoptosis assayed by annexin-V binding. HL-60cells(1  10 6 /ml) wereincubatedwith10 l MofP19for indicatedtimeperiods. NAC, 5mMwas addedtocell cultures1hpriortothetreatment withP19. Cells werestainedwith annexin-V-FITC/PI and analyzed for apoptotic/necrotic cell population by flow cytometry. Cells in the lower right quadrant represented apoptosis while in the upperright quadrant indicated post-apoptotic necrosis. FACSCan is representative of one of three similar experiments. (B and C) P19 induces robust increase in GSH contents of cells.HL-60cells(3  10 6 /well,6-wellplate)weretreatedwith10 l MP19forindicatedtimeperiods(B)orwithvariousconcentrationsofP19for6h(C).Cellswereextractedfor determination of reduced glutathione contents using monocholorobimane (MCB). HL-60 cells treated with 5mMNAC alone were considered as positive control. Data aremean±S.D. of three similar experiments.  ⁄⁄⁄  p  <0.001 for treated vs. control; and  @@@  p  <0.001 when cells treated with P19 and NAC were compared tocells treated with P19only.208  A. Kumar et al./Chemico-Biological Interactions 193 (2011) 204–215
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