Investigating Apoptotic Effects of Methanolic Extract of Dorema

Hindawi Publishing CorporationISRN PharmacologyVolume 2013 Article ID 949871 9 pageshttpdxdoiorg1011552013949871

Research ArticleInvestigating Apoptotic Effects of Methanolic Extract ofDorema glabrum Seed on WEHI-164 Cells

Maryam Bannazadeh Amirkhiz12 Nadereh Rashtchizadeh1 Hossein Nazemiyeh3

Jalal Abdolalizadeh1 Leila Mohammadnejad4 and Behzad Baradaran4

1 Drug Applied Research Center Tabriz University of Medical Sciences Tabriz 5165665811 Iran2 Tabriz University of Medical Sciences International Branch (Aras) Jolfa 5441643177 Iran3 Research Center for Pharmaceutical Nanotechnology Tabriz University of Medical Sciences Tabriz 5165665811 Iran4 Immunology Research Center Tabriz University of Medical Sciences Tabriz 5165665811 Iran

Correspondence should be addressed to Nadereh Rashtchizadeh rashtchizadehyahoocomand Behzad Baradaran behzad imyahoocom

Received 16 April 2013 Accepted 11 June 2013

Academic Editors H Cerecetto G Edwards and G Gervasini

Copyright copy 2013 Maryam Bannazadeh Amirkhiz et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

We aimed to investigate the apoptotic effects of the methanolic extract of Dorema glabrum seed on WEHI-164 cancerous cells incomparison with L929 normal cells and compared them with the cytotoxic effects of Taxol So MTT test and DNA fragmentationassay were performed on cultured and treated cells Also electrophoresis which was followed by immunoblotting was done tosurvey the production of Caspase-3 and Bcl

2proteins and to inquire into their relative genes expression RT-PCR was used

According to our findings the methanolic extract of Dorema glabrum seed can alter cells morphology as they shrink and takea spherical shape and lose their attachment too So the plant extract inhibits cell growth albeit in a time- and dose-dependentmanner and results in degradation of chromosomal DNA Induction of apoptosis by the plant extract was proved by the reductionof pro-Caspase-3 and Bcl

2proteins and increase in Caspase-3 gene expression and decrease in that of bcl

2too Our data well

established the antiproliferative effect of methanolic extract of Dorema glabrum seed and clearly showed that the plant extract caninduce apoptosis and not necrosis in vitro These results demonstrated that Dorema glabrum seed might be a novel and attractivetherapeutic candidate for tumor treatment

1 Introduction

Normal cells grow and divide in an ordered fashion in accor-dance with the cell cycle Defective apoptosis (programmedcell death) which results in enhanced growth describesmost cancer cells [1] Several proteins control the timingof the events in the cell cycle which is tightly regulated toensure that cells divide only when necessary The loss of thisregulation is the hallmark of cancer [1 2] Initially somaticcell fusion and nuclear transplantation studies togetherwith the selective use of growth factors and inhibitors ofmacromolecular biosynthesis established the fundamentalparameters of cell cycle regulation [3 4] Our understandingof the complexities of apoptosis and the mechanisms evolvedby tumor cells to resist engagement of cell death has focused

research effort into the development of strategies designed toselectively induce apoptosis in cancer cells [5ndash7]

Several previous studies demonstrated that certain phyto-chemicals present in medicinal herbs exert antitumorigenicactivity by inducing apoptosis in cancer cells [8ndash11] Themechanism of apoptosis are nowmostly well known involv-ing activation of caspases (cysteinyl aspartate-specific pro-teases) which cleave to inactivate or activate target substrateswithin a cell [5] and Bcl

2family members in response to a

wide variety of physiological and injury-induced signals [12]Caspases are synthesized inmost if not all cells as inactive

zymogens which must be proteolytically cleaved at two (orthree in some cases) aspartate residues to generate the activemature enzyme The generation of active caspases forms acascade in which ldquoinitiatorrdquo caspases interact with specific

2 ISRN Pharmacology

adapter molecules to facilitate their own autoprocessingThese now active initiator caspases notably Caspase-8 orFLICE being ldquoapicalrdquo and more susceptible to modifica-tion by endogenous regulatory proteins in turn cleave andactivate the downstream ldquoexecutionerrdquo caspases such asCaspase-3 also known as apopain SCA-1 Yama and CPP32(Alias) which enact the final irreversible commitment todeath [13] These then cleave their target substrates toorchestrate the proteolytic dismantling of the cell [14 15]This sequence of events culminating in the activation ofcaspases has been broadly categorized into two pathwaysthe ldquoextrinsicrdquo pathway characterized by the engagement ofcell surface ldquodeath receptorsrdquo and the ldquointrinsicrdquo pathwayinvolving key mitochondrial events [5 15]

The Bcl2proteins also represent a promising target for

modulating tumor cell sensitivity to apoptosis [5 16] Itwas first human apoptotic protein an inhibitor of apoptosisidentified in 1984 [17 18] High amounts of Bcl

2block

the apoptotic death of a pro-B-lymphocyte cell line ThusBcl2is unique among protooncogenes being localized to

mitochondria and interfering with programmed cell deathindependent of promoting cell division [19] Overexpressionof antiapoptotic Bcl

2proteins is observed in many tumor

types which may contribute to the drug-resistant state andhelp mediate the expansion of a transformed population bydisrupting normal cell turnover [5 20 21]

Chemotherapy drugs are toxic compounds that targetrapidly growing cells So these drugs can also eliminatecertain adult cells that divide more rapidly such as those thatline the gastrointestinal tract bonemarrow cells and hair fol-liclesThis causes some side effects including gastrointestinaldistress low white blood cell count and hair loss [22ndash24]

For several millennia herbal preparations and naturalremedies have been shown to be effective in treating manytypes of maladies [9 25 26] Although herbal therapies arebecoming increasingly popular worldwide we know littleabout the molecular mechanisms and active ingredients inmany of those therapeutic herbs [9 27] Chemical charac-terization and cytotoxicity screening studies on plant-basedmaterials could lead to a discovery of new natural anticancerdrugs [28]

Iran has unique plant varieties yet to be studied foranticancer components Just to mention Valiyari et alstudied apoptosis-inducing properties of dichloromethanoicand methanolic extracts of Scrophularia oxysepala in MCF-7human breast cancer cells [8] and Samavati et al investigatedantitumoral effects of Ornithogalum cuspidatum on WEHI-164 cells [29]

Dorema glabrum a medicinal plant of the family ofApiaceae which grows in Transcaucasia (Nakhichevan andArmenia zones) and north west of Iran (Azerbaijan) inloamy or rocky slopes of Aras river was chosen to study itsapoptotic effects on mouse fibrosarcoma cell line WEHI-164cells in comparison with its effects on L929 mouse normalcells The plant has extensive uses for example as an herbalremedy or food additive in the mentioned regions [30] Thisstudy was conducted according to the common folk beliefsof Armenian and Azeri people that Dorema glabrum cancure many anomalies especially different kinds of cancer

Of course it should be mentioned that in a preliminarywork the crude extract of the plant demonstrated antioxidantactivity and antilipidemic effects [31]

2 Materials and Methods

21 Plant Material Seeds of Dorema glabrum Fisch CAMey were collected during the fruiting stage from rockyslopes of Aras River bank Jolfa Eastern Azerbaijan (383010158409210158401015840 45 27101584036210158401015840 1590m 15 km from Jolfa to St StephanusChurch) Iran Air-dried and finely powdered seeds weresubjected to extraction by refluxing methanol in a soxhlet inorder to obtain its ooze Then the extract was dried using aRotary Evaporator 20mg of dried extract was dissolved in100 120583L DMSO and diluted with 390mL RPMI-1640 to give aconcentration of 5000 120583gmLThis was used to treat the cells

22 Cell Culture WEHI-164 cells mouse fibrosarcoma cellline (NCBI codeC200) and L929 cells mouse normal adiposetissue cell line (NCBI code C161) were obtained from IranPasture institute Both cell lines were cultured in RPMI-1640(Sigma pH = 72) containing 10 FCS (fetal calf serum) andantibiotic (100UmL penicillin 100 120583gmL streptomycin)placed in 37∘C and 5 CO

2in an incubator overnight

23 MTT Test MTT assay is one of the most useful testsfor investigating cells viability and cytotoxic effects of drugscosmetics and food additives MTT (3-[4 5-dimethyl-2-thiazolyl]-2 5 diphenyl tetrazoliumbromide) which is yellowand soluble in water can be reduced by mitochondrialdehydrogenases of live cells to give a bluish purple andinsoluble salt called Formosan that can easily and rapidlybe quantitated by an ELISA plate reader at 570 nm toevaluate growth and viability of cells and cytotoxic effects ofinterventor agents [13 32]

WEHI-164 and L929 cells were separately seeded in atriplicate manner in 96-well microplates (5000 cellswell) asabove After 6 hours both cells were treated with differentconcentrations (10 30 50 100 200 300 and 400120583gmL)of methanolic extract of Dorema glabrum seeds with dif-ferent time periods (6 24 and 36 hours) No plant extractwas added to negative controls but the same amount ofDMSO was added to eliminate its intervening effects if anyPositive control cells were treated with Taxol as the sameconcentrations of plant extract in test cells Taxol whichcontains paclitaxel as the main active compound is used inchemotherapy of cancer Of course prior to treatment thecells viability was determined by counting on a Neubauerslide (Hemocytometer) with the aid of Trypan blue Trypanblue can penetrate into dead cellsrsquo membrane and colourthem purple

After desired time the supernatants of all wells werediscarded and washed with PBS then 100120583L of RPMI and50120583L of MTT solution (2mgmL) were added to each wellFollowing incubation at 37∘C for 4 hours the liquid phase ofwells was discarded again After adding 200 120583L DMSO and25 120583L Sorensenrsquos glycine buffer (01M glycine 01M NaClpH = 105) the plates were incubated at 37∘C in the dark for

ISRN Pharmacology 3

another half an hour At the end absorbencies of wells weredetermined at 570 nm wavelength using a microplate reader(Awareness Technology USA)

24 Electrophoresis and Immunoblotting To survey pro-duction of proteins involved in apoptosis SDS-PAGE andimmunoblotting were performed Electrophoresis and re-lated applications have contributed greatly to the under-standing of themolecular bases of cell structure and functionTo enhance resolution and discrimination of proteins onthe basis of molecular size rather than charge or shapethe proteins were denatured by SDS prior to electropho-resis [33]

The western blot is a widely accepted analytical techniqueused to detect specific proteins in the given sample Proteinsseparated by electrophoretic technique are electrophoreti-cally transferred (ldquoelectroblottedrdquo) onto a membrane Themembrane which is now a replica of the polyacrylamide gelis subsequently probed with antibodies to specific proteinsThe primary antibodies can be revealed by an additionalincubation with HRP conjugated followed by enhancedelectrochemiluminescent (ECL) detection [33]

241 Protein Extraction Prior to electrophoresis we need toextract cellsrsquo proteins henceWEHI-164 cells were cultured in6-well plates (106 cells per well) with RPMI 1640 containing10 FCS and antibiotic (total volume of 5mL) Six hourslater treatments with different concentrations of plant extract(0 30 50 and 100 120583gmL) with different time periods (24and 36 hours) were done According to MTT results andIC50

value of the plant extract there was no need to treatcells with concentrations more than 100 120583gmL The wellswith no extract considered as negative controls and toeliminate effects of DMSO the same amount of DMSOconcentrations in test cells were added to them And as MTTtest positive control cells were treated with Taxol as the sameconcentrations of extract in test cells

Twenty-four and 36 hours after treatment the super-natants of all wells were gathered and kept in separate tubesThis time we did not discard the supernatants because weneeded the dead cells that were affected by plant ooze toextract their proteins All wells were washed with PBS andcells were detached from bottom of platesrsquo wells using Trp-EDTA and all liquids and cells from each well was addedto its own supernatant The tubes were centrifuged at 2000RPM for 10min and then washed two times with PBS at 2500RPM for 5minThe cells pellets were homogenized by gentlevortexing and removed tomicrotubes with 200120583L of PBS andcentrifuged at 1500 RPM for 5min The supernatants werediscarded and pellets were completely homogenized by gentlevortexing 200120583L of lysis buffer was added to each tube andfollowing mixing gently left on the rotator in 4∘C for halfan hour The tubes were centrifuged again at 15000 RPM for15minThen supernatants were removed to freshmicrotubesand pellets were discarded To obtain protein concentrationof each sample absorbance of each tube at 280 nm wave-length was read using nanodrop spectrophotometer againstlysis buffer blank

242 SDS-PAGE and Electroblotting As mentioned aboveto investigate production of proteins involved in apoptosisSDS-PAGE and western blotting should be performed SDS-PAGEwas performed under reducing conditions using 125polyacrylamide slab gels Then proteins on the gel weretransferred to the PVDF membrane using a semidry elec-troblotting tankThen themembranewaswashed three times(every time 5 minutes) with wash buffer (005 Tween 20 inPBS) and was soaked in the primary antibody (Abcam USA)with a dilution of 11000 on the shaker for two hours followedby again three times washing with wash buffer 003 of HRPconjugated secondary antibody (Abcam USA 3 120583L in 10mLwash buffer at 15000 dilution) was used to incubate themembrane on the shaker away from direct light for 2 hoursAfter discarding the secondary antibody also the membranewas washed three times with wash buffer The protein bandsin the membrane were developed using ECL substrate onradiography film

25 Investigating bcl2and Caspase-3 Genes Expression In

order to investigate expression of bcl2and Caspase-3 genes

to confirmWestern blotting results RT-PCR was performedHence PCR was performed on synthesized cDNAs usingWEHI-164 cellsrsquo mRNA

251 RNA Extraction The cells were cultured and treatedas mentioned above Since mRNA is the first thing to besynthesized in genes expression and also mRNAs are veryunstable briefly (3 and 6hours) treated cellswere used aswellAfter detaching cells from the plates and doing the washingprocess with PBS their precipitants were transferred toRNase-freemicrotubes Total RNAwas extracted using RNX-Plus solutions according to the manufacturerrsquos protocol andbecause RNA is very unstable work always should be doneon ice Purified RNAs were dried and then dissolved in 50 120583LDEPC-water Then cDNAs were transcribed from RNAs asdescribed below

252 cDNA Synthesis Prior to cDNA synthesis RNA con-tents of samples were determined using a nanodrop UVspectrophotometer Then keeping in mind the samples con-centrations the desired volume (for 20120583L reaction a volumeequivalent to 3120583g RNA) of each sample was added to reactionmixture containing 100 pmole oligo dt in a sterile RNase-free tube Then the mixtures were incubated at 70∘C for 5minutes After transferring the mixtures to AccuPower RTPreMix tubes they filled up to 20 120583L with DEPC distilledwater The mixtures were vortexed to dissolve the pelletsand briefly spun down To avoid samples evaporation onedrop of mineral oil was added to each sample before puttingin thermal cycler apparatus The strips were put in thethermal cycler apparatus under the following conditionscDNA synthesis at 42∘C for 60 minutes and 94∘C for 5minutes to inactivate RTase and terminate the reaction

253 cDNA PCR The volume of each reaction was consid-ered to be 20120583L The Ampliqon master mix that is 2X was

4 ISRN Pharmacology

used to make reaction mixture as follows 2 120583L of each cDNAsample was added to 10 120583L of master mix then 1120583L of eachforward and reverse primers (10mM) and 6 120583L of distilleddeionised sterile water were added to make the final volumeof 20120583L andMgCl

2concentrations of 15mM In the end one

drop of mineral oil was added to the top of each sample toavoid evaporation of samples

Thenmicrotubes were put in the thermal cycler apparatusunder the following conditions initial denaturation at 94∘Cfor 4 minutes followed by 35 amplification cycles eachconsisting of denaturation at 94∘C for 30 seconds annealingat 58∘C for 30 seconds and extension at 72∘C for 30 secondswith an additional extension step at the end of the procedureat 72∘C for 5 minutes

All RT-PCR products were visualized by electrophoresisthrough 2 agarose gel followed by ethidium bromide stain-ing

26 DNA Fragmentation Assay The biochemical hallmarkof apoptosis is the fragmentation of the genomic DNA anirreversible event that commits the cell to die and occursbefore changes in plasma membrane permeability (prelyticDNA fragmentation) The DNA laddering technique is usedto visualize the endonuclease cleavage products of apoptosisThis assay involves extraction of DNA from a lysed cellhomogenate followed by agarose gel electrophoresis

261 DNA Extraction After culturing and treating cellswith different concentrations of plant extract (0 30 50 and100 120583gmL) for different time periods (24 and 36 hours) theirgenomic DNA contents were extracted by firstly lysing thecells using 500120583L of lysis buffer Then 10 120583L of proteinaseK (Fermentas Life Sciences 20mgmL) were added and thesamples were incubated at 56∘C overnight Next day 40 120583Lof saturated NaCl (5M) was added and mixed completelyfollowed by incubation at 4∘C for 10 minutes After 20-minute centrifugation in 12000 RPM their upper liquidswere transferred to a fresh microtube 1mL of cold ethanol100 (stored in minus20∘C) was added and then incubated atminus20∘C for 10 minutes After centrifugation for 15 minutesin 12000 RPM the ethanol in upper phase was removedcompletely Following adding 1mL of ethanol 70 (kept in 4∘C)and centrifugation for 10 minutes in 12000 RPM the ethanolwas removed completely againThe samples were left at roomtemperature or 37∘C for 10ndash20 minutes to dry The pelletswere dissolved in 100 120583L distilled deionized and sterile wateror TE (TrisEDTA)

262 Agarose Electrophoresis After determining the samplesconcentrations using a nanodrop UV spectrophotometer theequivalent amount of DNA samples diluted with the 6XDNAloading dye (supplied with the ladder) were subjected to 15agarose submarine electrophoresis in company with DNAladder marker (Fermentas Life Sciences 1 kb DNA Ladder)In the end following ethidium bromide staining the frag-mentedDNAs bands were visualized by UV transilluminator

3 Results

31 Cell Culture Fusiform or spindle-like natural and liveWEHI-164 cells (Figure 1(a)) undergo morphological chan-ges after treatment with Taxol or methanolic extract ofDorema glabrum seed and because of chromatin condensa-tion and other changes they shrink and take a spherical shape(Figure 1(b)) characteristics of apoptotic cells

32 MTT Test MTT assay showed a time- and dose-depend-ent inhibition of the cell growth by plant extract (Figure 2)As the figure shows IC

50value the concentration that causes

50 loss of cell viability in WEHI-164 cell line is about50120583gmL in 36 hours By contrast the plant extract had higherIC50

value (about 100 120583gmL in 36 hours) for normal L929cells meaning that it is less toxic to the normal cells thanWEHI-164 cells

Statistical analysis using independent 119905-test whichresulted in 119875 lt 00001 showed that cytotoxicity effects of50120583gmL plant extract in 36 hours on WEHI-164 and L929cells are significantly different

33 Electrophoresis and Immunoblotting In order to comparethe effects of different concentrations of plant extract on theproduction of Bcl

2and Caspase-3 all samples were unified

using 120573-actin amount as an intrinsic factor As it can beseen fromFigure 3 because antizymogenCaspase-3 was usedas primary antibody in the samples related to treated cellsCaspase-3 zymogene band is weakened due to its cleavageAs mentioned previously most caspases are synthesized asinactive zymogens and must be cleaved at two or threeaspartate residues to generate the active enzyme

The figure also shows the reduction of Bcl2antiapoptotic

protein in treated cells So both western blotting resultsconfirmed that methanolic extract of Dorema glabrum seedcan induce apoptosis in WEHI-164 cells

34 Investigating bcl2and Caspase-3 Genes Expression All

PCR products of synthesised cDNAs were subjected toelectrophoresis through 2 agarose gel followed by ethidiumbromide staining in order to be visualized Figure 4 indicatesthe increasement of Caspase-3 and reduction of bcl

2genes

expression Again all samples were unified using 120573-actin asan intrinsic control

35 DNA Fragmentation DNA fragmentation can be anal-ysed by the typical ldquoDNA ladderrdquo formation for which DNAis extracted from the apoptotic cells and separated in anagarose gel As shown in Figure 5 treatment with Doremaglabrum seed extract resulted in degradation of chromosomalDNA into small internucleosomal fragments a biochemicalhallmark of cells undergoing apoptosis

4 Discussion

Despite a period in which pharmaceutical companies cutback their use of natural products in drug discovery there are

ISRN Pharmacology 5

(a) (b)Figure 1 (a) Spindle-like natural and live WEHI-164 40x (b) spherical apoptotic WEHI-164 cells 40x

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()

Viability of treated cells after 6 hours

WEHI-164 viability treated with plant extractWEHI-164 viability treated with TaxolL929 viability treated with plant extractL929 viability treated with Taxol

Concentrations (120583gmL)

(a)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(b)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(c)Figure 2 The viability of WEHI-164 and L929 cells treated with the different concentrations of plant extract in the different time periods incontrast with that of the cells treated with Taxol

6 ISRN Pharmacology

12085

50

35

25

20

(kDa)

120573-Actin 42 kDa

(a)

12085

50

35

25

20

(kDa)

Pro-Caspase-3 32 kDa

(b)

12085

50

35

25

20

(kDa)

Bcl2 25 kDa

(c)

Figure 3Western blot analysis of (a)120573-actin (b) pro-Caspase-3 and (c) Bcl2production inWEHI-164 cells From the left first and fifth Lanes

are negative controls Lanes 2ndash4 and 6ndash8 are treated cells with different concentrations (30 50 and 100120583gmL) of plant extract respectivelyafter 24 and 36 hours

many promising drug candidates in the current developmentpipeline that are of natural origin Technical drawbacksassociated with natural product research have been lessenedand there are better opportunities to explore the biologicalactivity of previously inaccessible sources of natural productsWith the increasing acceptance that the chemical diversity ofnatural products is well suited to provide the core scaffoldsfor future drugs there will be further developments in theuse of novel natural products and chemical libraries based onnatural products in drug discovery campaigns [34] After alltraditional cytotoxic chemotherapy although kills cancer cellsby indirectly inducing apoptosis unfortunately side effectsare brutal and most tumors become resistant [17 23 24]Therefore drugs that restore the apoptotic pathways have thepotential for effectively treating tumors Herbal plants havebeen the basis for nearly all medicinal therapies since ancient

times and constitute a common alternative for cancer pre-vention and treatment in many countries around the world[35] A number of chemotherapeutic agents with propertiesincluding apoptosis induction and antiangiogenesis havebeen isolated from natural products and characterized toinhibit the development of malignancies such as curcuminfrom Curcuma longa epicatechin gallate from tea paclitaxelfrom Pacific yew [36] Emodin a natural anthraquinonederivative from Rheum palmatum L [37] and Honokiola biphenyl extract fromMagnolia obovata bark [36]

Dorema glabrum is a perennial medicinal plant grow-ing in loamy or rocky slopes commonly in ArmeniaNakhichevan and north west of Iran that is currently used asa remedy for treating cancerous diseases in folkmedicine andalso as a green vegetable in domestic use [30] To evaluate theeffects of Dorema glabrum seed extract on cell proliferation

ISRN Pharmacology 7

120573-Actin

(a)

Caspase-3

(b)

bcl2

(c)

Figure 4 120573-Actin (a) Caspase-3 (b) and bcl2(c) gene expression Caspase-3 gene upregulated expression and bcl

2downregulated expression

show time- and concentration-related manner From left lane 1 negative control lanes 2ndash5 6ndash9 10ndash13 and 14ndash17 are related to cells treatedwith different concentrations (10 30 50 and 100120583gmL of extract) after different time periods (3 6 12 and 24 hours) End lanes in (a) and(b) and middle lane in (c) are Ladder

1000080006000500040003500300025002000

1500

1000750

500

250

Figure 5 DNA ladder formation From left lane 1 negative controllanes 2ndash4 and treated cells with different concentrations (30 50 and100120583gmL) of plant extract in 24 hours and lanes 5ndash7 and treatedcells in 36 hours and lane 8 Ladder (1 kb)

and identify its therapeutic potential we demonstrated forthe first time the potent cytotoxicity activity of differentconcentrations of methanolic extract of Dorema glabrum

seed against WEHI-164 mouse fibrosarcoma cell line andL929 normal cell line A successful anticancer drug shouldkill or incapacitate cancer cells without causing excessivedamages to normal cells meaning minimum side effectsThis ideal situation is achievable by inducing apoptosis incancer cells Understanding the modes of action of thesecompounds should provide useful information for theirpossible applications in cancer prevention and perhaps incancer therapy [38] Cell cycle modulation by various naturaland synthetic agents is gaining widespread attention in recentyears [38] So multiple techniques were used to assess theantiproliferative and apoptotic effects of D glabrum seedmethanolic extract on cancer cells Usually cells undergoingapoptosis display a very similar pattern of morphologicalchangesThese include blebbing loss of cell membarane sym-metry and attachment cell shrinkage nuclear fragmentationand chromatin condensation [35]

In the present study MTT assay was performed whichshowed that the methanolic extract of Dorema glabrum seedcaused growth inhibition in the WEHI-164 cells in a dose-and time-dependent manner But it appeared less toxic inlow concentrations to normal or nonmalignant cells in vitrobecause IC

50value of the plant extract for WEHI-164 cells is

50120583gmL and for L929 cells is 100120583gmL in 36 hours Thisclaimwas confirmed by statistical analysis using independent119905-test that resulted in 119875 lt 00001 meaning that the meandifferences of cytotoxicity effects of 50120583gmL plant extractin 36 hours on WEHI-164 and L929 cells are significantThirty-sixh hours treatment was selected because in shortertimes higher concentrations of plant extract were needed tocause 50 loss of cell viability Since concentrations more

8 ISRN Pharmacology

than 50 120583gmL affect L929 cells viability too it is preferedto choose 36-hour treatment with 50 120583gmL plant extract inorder to avoid massive damages to normal cells Also wecompared the effects of plant extract with the effects of Taxolan anticancer and apoptosis inducer drug and it should bementioned here that the effects of plant extract on both celllines followed the same pattern as Taxol effects on the cells(Figure 2)

Microscopic studies showed morphological changes ofthe cells too Chromatin condensation cell shrinkage andother alterations characteristics of apoptotic cells cause themorphology of treated cells with the plant extract changefrom spindle like to spherical shape and also make them losetheir attachment (Figure 1) In conclusion the plant extractinduced apoptosis in treated cells and not necrosis

Also apoptosis induction was confirmed by DNA laddertechnique Treatment with the plant extract resulted indegradation of chromosomal DNA into smaller fragments(Figure 5) a biochemical hallmark of cells undergoing apop-tosis [6]Oncemore induction of apoptosis andnot necrosisby plant extract was confirmed because electrophoresis ofnecrotic cellsrsquo DNA results in smear not ladder

As it was referred apoptosis is a consequence of a highlycomplex and sequential cascade of cellular events andCaspase-3 has been implicated in the execution phase ofapoptosis cleaving over 100 substrates [35] Due to ourinvestigation results the immunoblotting data since 32KDCaspase-3 precursor was decreased in time- and concen-tration-dependent manner methanolic extract of Doremaglabrum seed can induce Caspase-3 activation via its prote-olytic cleavage into active subunits which enact the final irre-versible commitment to death Also from the immunoblot-ting results the decrease in the amount of antiapoptotic Bcl

2

protein is clear in the cells exposed to plant extract (Figure 3)It was mentioned that overexpression of Bcl

2proteins is

seen in different types of tumors which can contribute todrug-resistant state It is believed that prevailing a blockadeinduced by Bcl

2or Bcl-xl could restore normal cellular home-

ostasis reverse the drug-resistant phenotype and restoretumor cell sensitivity to conventional chemotherapeutics [520 21]

We have also performed RT-PCR technique and demon-strated that the plant extract-dependent apoptosis wasaccompanied with significant increase of Caspase-3 mRNAand hence its expression and decrease of that of Bcl

2protein

(Figure 4)

5 Conclusion

In conclusion our data well established the antiproliferativeeffect of methanolic extract of Dorema glabrum seed andclearly showed that the plant extract can induce apoptosis andnot necrosis in vitro but its activities remained unknown invivo These results demonstrated that Dorema glabrum seedwith antiproliferative properties especially with IC

50value

for cancerous cells lower than that of normal cells mightbe a novel and attractive therapeutic candidate for tumortreatment in clinical practice

Authorsrsquo Contribution

Nadereh Rashtchizadeh and Behzad Baradaran equally con-tributed to this work

Acknowledgments

The authors appreciate the financial support provided byDrug Applied Research Center of Tabriz University of Medi-cal Sciences TabrizIran Also special thanks go to Dr HamedHamishekar for his pleasant statistical analysis

References

[1] L H Hartwell andM B Kastan ldquoCell cycle control and cancerrdquoScience vol 266 no 5192 pp 1821ndash1828 1994

[2] A Kamb N A Gruis J Weaver-Feldhaus et al ldquoA cell cycleregulator potentially involved in genesis of many tumor typesrdquoScience vol 264 no 5157 p 436 1994

[3] G Stein andA PardeeCell Cycle andGrowthControl Biomolec-ular Regulation and Cancer John Wiley amp Sons Hoboken NJUSA 2nd edition 2004

[4] G I Evan and K H Vousden ldquoProliferation cell cycle andapoptosis in cancerrdquo Nature vol 411 no 6835 pp 342ndash3482001

[5] S Kasibhatla and B Tseng ldquoWhy target apoptosis in cancertreatmentrdquo Molecular Cancer Therapeutics vol 2 no 6 pp573ndash580 2003

[6] S Elmore ldquoApoptosis a review of programmed cell deathrdquoToxicologic Pathology vol 35 no 4 pp 495ndash516 2007

[7] S Cory and J M Adams ldquoThe BCL2family regulators of the

cellular life-or-death switchrdquo Nature Reviews Cancer vol 2 no9 pp 647ndash656 2002

[8] S Valiyari B Baradaran A Delazar A Pasdaran and FZare ldquoDichloromethane and methanol extracts of scrophulariaoxysepala induces apoptosis in MCF-7 human breast cancercellsrdquo Drugs vol 15 article 20 2012

[9] H Gao J Lamusta W F Zhang et al ldquoTumor cell selectivecytotoxicity and apoptosis induction by an herbal preparationfrom brucea javanicardquo North American Journal of Medicine ampScience vol 4 no 2 pp 62ndash66 2011

[10] M Yamamoto N Miura N Ohtake et al ldquoGenipin ametabolite derived from the herbal medicine Inchin-ko-toand suppression of Fas-induced lethal liver apoptosis in micerdquoGastroenterology vol 118 no 2 pp 380ndash389 2000

[11] D Fau M Lekehal G Farrell et al ldquoDiterpenoids fromgermander an herbal medicine induce apoptosis in isolatedrat hepatocytesrdquoGastroenterology vol 113 no 4 pp 1334ndash13461997

[12] A H Wyllie ldquolsquowhere o death is thy stingrsquo A brief review ofapoptosis biologyrdquoMolecular Neurobiology vol 42 no 1 pp 4ndash9 2010

[13] A H Wyllie ldquoCell Death In Apoptosis Cell Death and CellProliferationrdquo Roche Applied Science 3rd edition

[14] A-O Hueber M Zornig D Lyon T Suda S Nagata and GI Evan ldquoRequirement for the CD95 receptor-ligand pathway inc-myc-induced apoptosisrdquo Science vol 278 no 5341 pp 1305ndash1309 1997

ISRN Pharmacology 9

[15] N A Thornberry ldquoThe caspase family of cysteine proteasesrdquoBritish Medical Bulletin vol 53 no 3 pp 478ndash490 1997

[16] X Luo I Budihardjo H Zou C Slaughter and X Wang ldquoBida Bcl2interacting protein mediates cytochrome c release from

mitochondria in response to activation of cell surface deathreceptorsrdquo Cell vol 94 no 4 pp 481ndash490 1998

[17] K Garber ldquoNew apoptosis drugs face critical testrdquo NatureBiotechnology vol 23 no 4 pp 409ndash411 2005

[18] J C Reed ldquoBcl-2 and the regulation of programmed cell deathrdquoJournal of Cell Biology vol 124 no 1-2 pp 1ndash6 1994

[19] DHockenbery G Nunez CMilliman R D Schreiber and S JKorsmeyer ldquoBcl-2 is an innermitochondrial membrane proteinthat blocks programmed cell deathrdquo Nature vol 348 no 6299pp 334ndash336 1990

[20] Z N Oltvai C L Milliman and S J Korsmeyer ldquoBcl-2heterodimerizes in vivo with a conserved homolog Bax thataccelerates programed cell deathrdquo Cell vol 74 no 4 pp 609ndash619 1993

[21] A Gross J M McDonnell and S J Korsmeyer ldquoBCL-2family members and the mitochondria in apoptosisrdquoGenes andDevelopment vol 13 no 15 pp 1899ndash1911 1999

[22] Cell Biology and Cancer REDISCOVERING BIOLOGY-Molecular to Global Perspective

[23] M A Jordan and L Wilson ldquoMicrotubules as a target foranticancer drugsrdquoNature Reviews Cancer vol 4 no 4 pp 253ndash265 2004

[24] T Kawabe ldquoG2 checkpoint abrogators as anticancer drugsrdquoMolecular Cancer Therapeutics vol 3 no 4 pp 513ndash519 2004

[25] A Y Oubre T J Carlson S R King and G M Reaven ldquoFromplant to patient an ethnomedical approach to the identificationof new drugs for the treatment of NIDDMrdquo Diabetologia vol40 no 5 pp 614ndash617 1997

[26] H Yano A Mizoguchi K Fukuda et al ldquoThe herbal medicinesho-saiko-to inhibits proliferation of cancer cell lines by induc-ing apoptosis and arrest at the G0G1 phaserdquo Cancer Researchvol 54 no 2 pp 448ndash454 1994

[27] S Zahri S M Razavi F H Niri and S Mohammadi ldquoInduc-tion of programmed cell death by Prangos uloptera amedicinalplantrdquo Biological Research vol 42 no 4 pp 517ndash522 2009

[28] M Yousefzadi M Heidari M Akbarpour M H Mirjalili AZeinali andM Parsa ldquoIn vitro cytotoxic activity of the essentialoil of Dorema ammoniacum D Don Middle-Eastrdquo Journal ofScientific Research vol 7 no 4 pp 511ndash514 2011

[29] M Samavati Z Babaloo A Delazar et al ldquoCytotoxic and apop-totic effects of Ornithogalum cuspidatum methanolic extracton WEHI-164 fibrosarcoma cancer cell linerdquo PharmaceuticalSciences vol 16 no 3 pp 149ndash156 2010

[30] S Asnaashari E Dadizadeh A H Talebpour M Eskandaniand H Nazemiyeh ldquoFree radical scavenging potential andessential oil composition of the Dorema glabrum Fisch CAmey roots from Iranrdquo BioImpacts vol 1 no 4 pp 241ndash244 2011

[31] G Dehghan G Fatholahi N Sheikhzadeh and N AhmadiaslldquoHypocholesteremic and antioxidant effects ofDorema glabrumextract in rats fed high cholesterol dietrdquo Journal of the IranianChemical Society vol 6 pp 115ndash143 2009

[32] H Wan R Williams P Doherty and D F Williams ldquoA studyof the reproducibility of the MTT testrdquo Journal of MaterialsScience vol 5 no 3 pp 154ndash159 1994

[33] ldquoCurrent Protocols in Cell Biologyrdquo John Wiley amp Sons Inc2002 httponlinelibrarywileycom

[34] A L Harvey ldquoNatural products in drug discoveryrdquo DrugDiscovery Today vol 13 no 19-20 pp 894ndash901 2008

[35] A Nawab M Yunus A A Mahdi and S Gupta ldquoEvaluation ofanticancer properties of medicinal plants from the Indian sub-continentrdquo Molecular and Cellular Pharmacology vol 3 no 1pp 21ndash29 2011

[36] Z Li Y Liu X Zhao et al ldquoHonokiol a natural therapeuticcandidate induces apoptosis and inhibits angiogenesis of ovar-ian tumor cellsrdquo European Journal of Obstetrics Gynecology andReproductive Biology vol 140 no 1 pp 95ndash102 2008

[37] Y-T Su H-L Chang S-K Shyue and S-L Hsu ldquoEmodininduces apoptosis in human lung adenocarcinoma cells througha reactive oxygen species-dependent mitochondrial signalingpathwayrdquo Biochemical Pharmacology vol 70 no 2 pp 229ndash2412005

[38] M H Abdolmohammadi S Fouladdel A Shafiee G AminS M Ghaffari and E Azizi ldquoAnticancer effects and cell cycleanalysis on human breast cancer T47D cells treated withextracts of Astrodaucus persicus (Boiss) Drude in comparisonto doxorubicinrdquo Daru vol 16 no 2 pp 112ndash118 2008

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of

2 ISRN Pharmacology

adapter molecules to facilitate their own autoprocessingThese now active initiator caspases notably Caspase-8 orFLICE being ldquoapicalrdquo and more susceptible to modifica-tion by endogenous regulatory proteins in turn cleave andactivate the downstream ldquoexecutionerrdquo caspases such asCaspase-3 also known as apopain SCA-1 Yama and CPP32(Alias) which enact the final irreversible commitment todeath [13] These then cleave their target substrates toorchestrate the proteolytic dismantling of the cell [14 15]This sequence of events culminating in the activation ofcaspases has been broadly categorized into two pathwaysthe ldquoextrinsicrdquo pathway characterized by the engagement ofcell surface ldquodeath receptorsrdquo and the ldquointrinsicrdquo pathwayinvolving key mitochondrial events [5 15]

The Bcl2proteins also represent a promising target for

modulating tumor cell sensitivity to apoptosis [5 16] Itwas first human apoptotic protein an inhibitor of apoptosisidentified in 1984 [17 18] High amounts of Bcl

2block

the apoptotic death of a pro-B-lymphocyte cell line ThusBcl2is unique among protooncogenes being localized to

mitochondria and interfering with programmed cell deathindependent of promoting cell division [19] Overexpressionof antiapoptotic Bcl

2proteins is observed in many tumor

types which may contribute to the drug-resistant state andhelp mediate the expansion of a transformed population bydisrupting normal cell turnover [5 20 21]

Chemotherapy drugs are toxic compounds that targetrapidly growing cells So these drugs can also eliminatecertain adult cells that divide more rapidly such as those thatline the gastrointestinal tract bonemarrow cells and hair fol-liclesThis causes some side effects including gastrointestinaldistress low white blood cell count and hair loss [22ndash24]

For several millennia herbal preparations and naturalremedies have been shown to be effective in treating manytypes of maladies [9 25 26] Although herbal therapies arebecoming increasingly popular worldwide we know littleabout the molecular mechanisms and active ingredients inmany of those therapeutic herbs [9 27] Chemical charac-terization and cytotoxicity screening studies on plant-basedmaterials could lead to a discovery of new natural anticancerdrugs [28]

Iran has unique plant varieties yet to be studied foranticancer components Just to mention Valiyari et alstudied apoptosis-inducing properties of dichloromethanoicand methanolic extracts of Scrophularia oxysepala in MCF-7human breast cancer cells [8] and Samavati et al investigatedantitumoral effects of Ornithogalum cuspidatum on WEHI-164 cells [29]

Dorema glabrum a medicinal plant of the family ofApiaceae which grows in Transcaucasia (Nakhichevan andArmenia zones) and north west of Iran (Azerbaijan) inloamy or rocky slopes of Aras river was chosen to study itsapoptotic effects on mouse fibrosarcoma cell line WEHI-164cells in comparison with its effects on L929 mouse normalcells The plant has extensive uses for example as an herbalremedy or food additive in the mentioned regions [30] Thisstudy was conducted according to the common folk beliefsof Armenian and Azeri people that Dorema glabrum cancure many anomalies especially different kinds of cancer

Of course it should be mentioned that in a preliminarywork the crude extract of the plant demonstrated antioxidantactivity and antilipidemic effects [31]

2 Materials and Methods

21 Plant Material Seeds of Dorema glabrum Fisch CAMey were collected during the fruiting stage from rockyslopes of Aras River bank Jolfa Eastern Azerbaijan (383010158409210158401015840 45 27101584036210158401015840 1590m 15 km from Jolfa to St StephanusChurch) Iran Air-dried and finely powdered seeds weresubjected to extraction by refluxing methanol in a soxhlet inorder to obtain its ooze Then the extract was dried using aRotary Evaporator 20mg of dried extract was dissolved in100 120583L DMSO and diluted with 390mL RPMI-1640 to give aconcentration of 5000 120583gmLThis was used to treat the cells

22 Cell Culture WEHI-164 cells mouse fibrosarcoma cellline (NCBI codeC200) and L929 cells mouse normal adiposetissue cell line (NCBI code C161) were obtained from IranPasture institute Both cell lines were cultured in RPMI-1640(Sigma pH = 72) containing 10 FCS (fetal calf serum) andantibiotic (100UmL penicillin 100 120583gmL streptomycin)placed in 37∘C and 5 CO

2in an incubator overnight

23 MTT Test MTT assay is one of the most useful testsfor investigating cells viability and cytotoxic effects of drugscosmetics and food additives MTT (3-[4 5-dimethyl-2-thiazolyl]-2 5 diphenyl tetrazoliumbromide) which is yellowand soluble in water can be reduced by mitochondrialdehydrogenases of live cells to give a bluish purple andinsoluble salt called Formosan that can easily and rapidlybe quantitated by an ELISA plate reader at 570 nm toevaluate growth and viability of cells and cytotoxic effects ofinterventor agents [13 32]

WEHI-164 and L929 cells were separately seeded in atriplicate manner in 96-well microplates (5000 cellswell) asabove After 6 hours both cells were treated with differentconcentrations (10 30 50 100 200 300 and 400120583gmL)of methanolic extract of Dorema glabrum seeds with dif-ferent time periods (6 24 and 36 hours) No plant extractwas added to negative controls but the same amount ofDMSO was added to eliminate its intervening effects if anyPositive control cells were treated with Taxol as the sameconcentrations of plant extract in test cells Taxol whichcontains paclitaxel as the main active compound is used inchemotherapy of cancer Of course prior to treatment thecells viability was determined by counting on a Neubauerslide (Hemocytometer) with the aid of Trypan blue Trypanblue can penetrate into dead cellsrsquo membrane and colourthem purple

After desired time the supernatants of all wells werediscarded and washed with PBS then 100120583L of RPMI and50120583L of MTT solution (2mgmL) were added to each wellFollowing incubation at 37∘C for 4 hours the liquid phase ofwells was discarded again After adding 200 120583L DMSO and25 120583L Sorensenrsquos glycine buffer (01M glycine 01M NaClpH = 105) the plates were incubated at 37∘C in the dark for

ISRN Pharmacology 3














4 ISRN Pharmacology









3 Results














2genes



4 Discussion


ISRN Pharmacology 5


0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(a)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(b)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()





6 ISRN Pharmacology

12085

50

35

25

20

(kDa)

120573-Actin 42 kDa

(a)

12085

50

35

25

20

(kDa)


(b)

12085

50

35

25

20

(kDa)

Bcl2 25 kDa

(c)






ISRN Pharmacology 7

120573-Actin

(a)

Caspase-3

(b)

bcl2

(c)




1000080006000500040003500300025002000

1500

1000750

500

250







8 ISRN Pharmacology





2


2proteins is





2protein

(Figure 4)

5 Conclusion


50value




Acknowledgments


References
















ISRN Pharmacology 9






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Pharmacology 3














4 ISRN Pharmacology









3 Results














2genes



4 Discussion


ISRN Pharmacology 5


0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(a)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(b)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()





6 ISRN Pharmacology

12085

50

35

25

20

(kDa)

120573-Actin 42 kDa

(a)

12085

50

35

25

20

(kDa)


(b)

12085

50

35

25

20

(kDa)

Bcl2 25 kDa

(c)






ISRN Pharmacology 7

120573-Actin

(a)

Caspase-3

(b)

bcl2

(c)




1000080006000500040003500300025002000

1500

1000750

500

250







8 ISRN Pharmacology





2


2proteins is





2protein

(Figure 4)

5 Conclusion


50value




Acknowledgments


References
















ISRN Pharmacology 9






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

4 ISRN Pharmacology









3 Results














2genes



4 Discussion


ISRN Pharmacology 5


0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(a)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(b)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()





6 ISRN Pharmacology

12085

50

35

25

20

(kDa)

120573-Actin 42 kDa

(a)

12085

50

35

25

20

(kDa)


(b)

12085

50

35

25

20

(kDa)

Bcl2 25 kDa

(c)






ISRN Pharmacology 7

120573-Actin

(a)

Caspase-3

(b)

bcl2

(c)




1000080006000500040003500300025002000

1500

1000750

500

250







8 ISRN Pharmacology





2


2proteins is





2protein

(Figure 4)

5 Conclusion


50value




Acknowledgments


References
















ISRN Pharmacology 9






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Pharmacology 5


0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(a)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()




(b)

0

20

40

60

80

100

0 100 200 300 400 500

Viab

ility

()





6 ISRN Pharmacology

12085

50

35

25

20

(kDa)

120573-Actin 42 kDa

(a)

12085

50

35

25

20

(kDa)


(b)

12085

50

35

25

20

(kDa)

Bcl2 25 kDa

(c)






ISRN Pharmacology 7

120573-Actin

(a)

Caspase-3

(b)

bcl2

(c)




1000080006000500040003500300025002000

1500

1000750

500

250







8 ISRN Pharmacology





2


2proteins is





2protein

(Figure 4)

5 Conclusion


50value




Acknowledgments


References
















ISRN Pharmacology 9






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

6 ISRN Pharmacology

12085

50

35

25

20

(kDa)

120573-Actin 42 kDa

(a)

12085

50

35

25

20

(kDa)


(b)

12085

50

35

25

20

(kDa)

Bcl2 25 kDa

(c)






ISRN Pharmacology 7

120573-Actin

(a)

Caspase-3

(b)

bcl2

(c)




1000080006000500040003500300025002000

1500

1000750

500

250







8 ISRN Pharmacology





2


2proteins is





2protein

(Figure 4)

5 Conclusion


50value




Acknowledgments


References
















ISRN Pharmacology 9






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Pharmacology 7

120573-Actin

(a)

Caspase-3

(b)

bcl2

(c)




1000080006000500040003500300025002000

1500

1000750

500

250







8 ISRN Pharmacology





2


2proteins is





2protein

(Figure 4)

5 Conclusion


50value




Acknowledgments


References
















ISRN Pharmacology 9






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

8 ISRN Pharmacology





2


2proteins is





2protein

(Figure 4)

5 Conclusion


50value




Acknowledgments


References
















ISRN Pharmacology 9






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Pharmacology 9






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Investigating Apoptotic Effects of Methanolic Extract of Dorema