Research ArticleAntiproliferative Evaluation of Isofuranodiene onBreast and Prostate Cancer Cell Lines

Michela Buccioni Diego Dal Ben Catia Lambertucci Filippo MaggiFabrizio Papa Ajiroghene Thomas Claudia Santinelli and Gabriella Marucci

School of Pharmacy Medicinal Chemistry Unit University of Camerino Via S Agostino 1 62032 Camerino Italy

Correspondence should be addressed to Gabriella Marucci gabriellamarucciunicamit

Received 21 January 2014 Accepted 1 May 2014 Published 22 May 2014

Academic Editor Ronald M Bukowski

Copyright copy 2014 Michela Buccioni et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The anticancer activity of isofuranodiene extracted from Smyrnium olusatrum was evaluated in human breast adenocarcinomasMDA-MB 231 and BT 474 and Caucasian prostate adenocarcinoma PC 3 cell lines by MTS assay MTS assay showed a dose-dependent growth inhibition in the tumor cell lines after isofuranodiene treatment The best antiproliferative activity of theisofuranodienewas found on PC 3 cells with an IC

50value of 29 120583Mwhichwas slightly less than the inhibition against the two breast

adenocarcinoma cell lines with IC50values of 59 and 55 120583M on MDA-MB 231 and BT 474 respectively Hoechst 33258 assay was

performed in order to study the growth inhibition mechanism in prostate cancer cell line the results indicate that isofuranodieneinduces apoptosis Overall the understudy compound has a good anticancer activity especially towards the PC 3 On the contraryit is less active on Chinese hamster ovary cells (CHO) and human embryonic kidney (HEK 293) appearing as a good candidate asa potential natural anticancer drug with low side effects

1 Introduction

Cancer is the leading cause of death worldwide and itsrelative importance continues to increase In 2008 it caused76 million deaths and the incidence of cancer continues toincrease with an estimated 131 million deaths in 2030 [1]Moreover an increasing proportion of cancer patients areacquiring resistance to traditional chemotherapeutic agentsThis worrying situation requires the development of treat-ment strategies Commercially available anticancer drugswhich can be classified by origin as either chemical syntheticdrugs or natural drugs are derived from organisms or plants[2 3] Often synthetic drugs are the only option for cancerchemotherapy [3ndash5] and their action is not specific for tumorcells since they kill also normal cells generating severe sideeffects [6] Natural antitumor drugs derived from organismsor plants were also proven to be effective and less toxic forcancer therapy [6 7] In particular more than 70 of theapproved anticancer drugs in the United States of America(from 1981 to 2010)were fromnatural origin [8] Screenings ofmedicinal plants used traditionally as anticancer remedy have

provided pharmaceutical industry with effective cytotoxicdrugs

Nonetheless also plants which were historically used forother purposes may deserve attention of scientistsThis is thecase of Smyrnium olusatrum L (Apiaceae) well known aswild celery or Alexanders representing a pot-herb that wascultivated in gardens for many centuries owing to its culinaryproperties and afterwards it was superseded by the improvedform of celery (Apium graveolens L)

From the whole plant it is possible to obtain essentialoils mainly constituted by furanogermacrane-type sesquiter-penes [9] Their occurrence can be explained by the fact thatthese molecules are considered to be precursors of sesquiter-pene lactones [10] which are in turn regarded as markercompounds of the genus Smyrnium [11] The parent com-pound of this class of molecules is isofuranodiene ((5E9E)-3610-trimethyl-47811-tetrahydrocyclodeca[b]furan) CASRegistry Number 57566-47-9 molecular weight 2161514)a thermosensitive molecule which when subjected to hightemperatures undergoes Cope rearrangement to its corre-sponding elemane derivative curzerene [12] Isofuranodiene

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 264829 6 pageshttpdxdoiorg1011552014264829

2 The Scientific World Journal

has been also isolated from leaves of Chloranthus tianmusha-nensis a traditional Chinese medicine used in the treatmentof dermatological disorders [13] and from the coral Lemindamillecra living in Algoa Bay South Africa [14] In a previousinvestigation this molecule showed inhibitory effects againstthe proliferation of human colon carcinoma glioblastomaand breast adenocarcinoma cells while its antioxidant andantimicrobial activity was negligible [15] Taking into accountthat isofuranodiene can induce cell death the in vitro antipro-liferative effect of the molecule on breast and prostate cancerusing MDA-MB 231 and BT 474 breast adenocarcinomaand PC 3 Caucasian prostate adenocarcinoma cells has beeninvestigated in the present work Moreover in the PC 3 cellsit has been examined the effect of isofuranodiene on cellapoptosis

2 Materials and Methods

21 Plant Material and Preparation of Isofuranodiene Isofu-ranodiene (C

15H20O crystals purity 99 as determined by

HPLC) (Figure 1) was isolated by crystallization at minus20∘Cfrom the essential oil of flowers of Smyrnium olusatrum L(Apiaceae) and purified by recrystallization with n-hexaneThe molecular structure was confirmed by comparison of 1Hand 13C-NMR data obtained on a Varian Mercury plus 400Spectrometer using CDCl

3as solvent and the solvent signals

as internal references with those reported in the literature[9] Isofuranodiene was the major constituent accountingfor 48 of the volatile oil (Figure 2) The plant materialfrom which isofuranodiene was isolated was collected inSan Giusto (near Pievebovigliana central Italy 480m abovesea level N 43∘0510158403610158401015840 E 13∘0810158401910158401015840) in April 2012 Thespecimen was confirmed by Dr Maggi using the availableliterature [16] hence it is deposited in the HerbariumUniversitatis Camerinensis (included in the online editionof Index Herbariorum by the New York Botanical Gardenhttpsweetgumnybgorgih) of School of Biosciences andVeterinary Medicine (University of Camerino Italy) underthe accession codex CAME 25675 it is also archived andpublished in the anArchive system (httpwwwanarchiveit)

22 Isofuranodiene Antiproliferative Activity In Vitro Studies

221 Cell Culture Two human breast adenocarcinomasMDA-MB 231 and BT 474 and Caucasian prostate ade-nocarcinoma PC 3 cell lines in comparison with Chinesehamster ovary (CHO) and human embryonic kidney (HEK293) cells were used to study isofuranodiene antiproliferativeactivity Human breast adenocarcinoma cell lines MDA-MB231 and BT 474 were grown adherently and maintainedin Dulbeccorsquos Modified Eaglersquos Medium supplemented with100UmL penicillin 100 120583gmL streptomycin and 10 fetalbovine serum (FBS) Caucasian prostate adenocarcinoma cellline PC 3 was grown adherently andmaintained inminimumessential medium supplemented with 100UmL penicillin100 120583gmL streptomycin and 10 fetal bovine serum (FBS)CHO cells were grown adherently and maintained in Dul-beccorsquos Modified Eaglersquos Medium high glucose supplemented

with 10 FBS 100UmL penicillin 100120583gmL streptomycin25 120583gmL amphotericin and 2mM L-glutamine [17] HEK293 cells were grown adherently and maintained in the samegrowmedia of CHOwith 1mM sodium pyruvate [18] All celllineswere cultured at 37∘Cand aeratedwith 5CO

2 95O

2

222 Evaluation of Antiproliferative Activity Testedcompounds were dissolved in methanol (MeOH) at aconcentration of 10000120583M and diluted with specific cellsunderstudy medium prior to use Ten thousand cells ofeach cell line were suspended in 98 120583L of specific mediumand incubated in a 96-well plate for overnight After theincubation 2120583L of the compound was added to the wellwith the final concentrations of 10ndash150 120583M After 72 hincubation at 37∘C viability of the cells was determined by3-(45-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-tetrazolium (MTS) assay using Cell Titer96 Aqueous One Solution Cell Proliferation Assay (PromegaItalia Srl) [19]

After the addiction ofMTS in combination with the elec-tron coupling agent phenazine methosulfate the cells wereallowed to incubate for 1 h and absorbance was measuredat 492 nm in a microplate reader GeniosPro Cell viabilitywas calculated as a percentage using the formula (mean ODof treated cellsmean OD of control cells)times 100 Results areexpressed as percent of control cells which are not treatedThe growth control (GC) and growth control with MeOH(GCM) were run for each set of cell line For cell countingMDA-MB 231 BT 474 and PC 3 were seeded on to 24-well plates at a density of 7times 104 cells per well The cellswere treated with different concentrations of isofuranodiene(10ndash150 120583M) for 72 hr After the treatment the cells wereharvested counted and compared with GCMThe living cellpopulation was estimated by Trypan blue dye exclusion testIn order to evaluate the kinetics of isofuranodiene the cellswere exposed at different incubation times (6 12 24 48 and72 h) Recovery experiments were performed by treating cellsfor 6 12 24 48 and 72 h with the compound and assessingcell proliferation after the washout of the drug up to reach72 h All experiments were done in triplicate The results areexpressed as IC

50 the concentration that produce the 50

inhibition of cell viability

223 Morphologic Analysis To observe PC 3 cells under-going apoptosis Hoechst 33258 staining was performed asdescribed by Ghavami et al [20] Briefly 35times 105 cells weregrown in each well of a 6-well plate and allowed to adhereAfter treatment with 29 120583M isofuranodiene for 24 h the cellswere fixed with 4 paraformaldehyde for 30min at roomtemperature and then washed twice with PBS Hoechst 33258(1 120583gmL) was added to the fixed cells incubated for 1 hat 37∘C in dark and then washed twice with PBS Cellswere counted and examined by fluorescence microscopyApoptotic cells were identified by their characteristic nucleicondensation and fragmentation whereas nuclei from nor-mal cells demonstrated a normal uniform chromatin patternThepercentage of apoptotic cells was calculated from the ratioof apoptotic cells to total cells counted

The Scientific World Journal 3

O

Figure 1 Crystals and chemical structure of isofuranodiene

(min)10 20 30 40 50

(mAU

)

0

100

200

300

400

500

600

(curzerene120912000002D)

83

57 11

212

16

505

17

026

23

974

245

48 2

847

3 2

953

7

Isofuranodiene

DAD1A Sig = 2304 Ref = 550100

Figure 2 HPLC chromatogram of the essential oil from flowersof Smyrnium olusatrum Isofuranodiene with a retention time of23974min is themain peak (48)The chromatographic conditionsare as follows column Kinetex PFP 100A (100 times 46mm id26 120583m) from Phenomenex (Torrance CA) mobile phases water(A)-acetonitrile (B) (0ndash15min 40B 15ndash30min 60B 30ndash40min60B)with a constant flow rate of 1mLmin injection volume 1 120583Ldetection wavelength 230 nm

23 Statistical Analysis The data are expressed as meanplusmn SDfrom at least three independent experiments Studentrsquos t-test was used for statistical analysis The IC

50values were

determined by regression analysis after plotting a graphof cell viability versus drug concentration Results areconsidered statistically significant at 119875 lt 005

3 Results and Discussion

Human breast adenocarcinomas MDA-MB 231 and BT 474Caucasian prostate adenocarcinoma PC 3 and two nontu-morigenic CHO and HEK 293 cell lines were treated withvarious concentrations (10ndash150 120583M) of isofuranodiene for upto three days and the effect on cell viability was examined incomparison with growth control in MeOH (GCM) Cisplatin[cis-diamminedichloroplatinum (II)] an age old anticancerdrug was used as positive control [21] The of cell viabilityof GCM in comparison with the growth control (GC) was100plusmn 065 indicating that the drug solvent (MeOH) doesnot interferewith the cell viability In contrast isofuranodieneprovided a good in vitro antiproliferative activity against the

Table 1 Antiproliferative activity of isofuranodiene and cisplatinafter 72 h

Cisplatin IC50 [120583M]a Isofuranodiene IC50 [120583M]a

Cell lineMDA-MB 231 39 plusmn 105 59 plusmn 236

BT 474 37 plusmn 165 55 plusmn 254

PC 3 12 plusmn 098 29 plusmn 185

CHO mdash 125 plusmn 394

HEK 293 mdash 130 plusmn 298

aConcentration of compound required for 50 inhibition of cell viabilitydetermined usingMTS assay Cells were treated with concentrations rangingfrom 10 to 150120583M for 72 h The results are reported as the average of threeindependent experiments

tested cancer cell lines and a lower cytotoxicity against thetwo nontumorigenic cell lines (CHO and HEK 293) MTSassay showed a dose-dependent growth inhibition in thetumor cells after isofuranodiene treatment As it is observedin Figure 3(a) growth inhibition effect of isofuranodiene inMDA-MB 231 cells after 72 h of incubation started at 15120583Mand increased up to 120 120583M (cell viability from 91plusmn 410at 15120583M to 20plusmn 245 at 120120583M versus control 100 resp119875 lt 0 05) The effective isofuranodiene concentration for50 inhibition (IC

50) in these cells was 59 120583M As shown

in Figure 3(a) isofuranodiene induced a reduction also inthe BT 474 cell viability (89plusmn 520 at 15120583M to 19plusmn 805at 120 120583M versus control 100 resp 119875 lt 005) showingan IC

50of 55 120583M In addition isofuranodiene induced a

significant inhibition of PC 3 cell proliferation (90plusmn 510at 15120583M to 13plusmn 124 at 100 120583M versus control 100 resp119875 lt 005) with an IC

50of 29 120583M(Figure 3(a))The IC

50values

of isofuranodiene are reported in Table 1 Data show thatthe IC

50values of isofuranodiene are comparable to those of

the positive control cisplatin (MDA-MB 231 59 versus 39 120583MBT 474 55 versus 37 120583M and PC 3 29 versus 12 120583M) Resultsindicate that the molecule shows antiproliferative activityagainst all the three cell lines suggesting that isofuranodienecould be considered an anticancer agent such as cisplatinNotably the major effect is exerted against the PC3 prostateadenocarcinoma cells

Moreover data reported in Figures 3(b) 3(c) and 3(d)show the rapid onset of the compound and in general theisofuranodiene induced 50 of cell death in a range of 60ndash90 120583M after 12ndash24 h This means that the isofuranodiene is

4 The Scientific World Journal

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

MDA-MB 231BT 474PC 3

Effect of isofuranodiene after 72h

Conc (120583M)

(a)

Effect of isofuranodiene in MDA-MB 231

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(b)

Effect of isofuranodiene in BT 474 cells

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(c)

Effect of isofuranodiene in PC 3 cells

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(d)

Figure 3 Cell viability in MDA-MB 231 BT 474 and PC 3 cell lines (a) Percentage of cell viability after treatment with isofuranodieneat doses of 10ndash150 120583M after 72 h of incubation (b) (c) and (d) Percentage of cell viability after treatment with isofuranodiene at doses of10ndash150120583M at different incubation times (2 6 12 24 48 and 72 h) The results are reported as (viability of treated cells)(viability of controlcells)times 100 and represent the average of three independent experiments with a maximum SD lower than plusmn80

able to induce cell death quite quickly In addition whenisofuranodiene was added at CHO and HEK 293 cells thecytotoxicity observed after 72 h was fairly low IC

50of 125 120583M

and 130 120583M respectively (Table 1) The concentration of60 120583M of isofuranodiene which in turn provided generallythe 50 of antiproliferative activity in breast cancer cell linesproduced only 20 of inhibition in CHO and HEK 293cell viability even if the maximum concentration 150120583Mproduced an inhibition of 70plusmn 45 and 75plusmn 36 cell via-bility respectivelyThe isofuranodiene antiproliferative effectdifference observed at IC

50concentrations (30 and 60 120583M in

PC 3 andMDA-MB 231 BT 474 cells resp) among the tumorcell lines and the two noncancer cell lines is significant Thissuggests that since the toxicity of the isofuranodiene is beingless in noncancer cells it could be a potential anticancer drugwith low side effects

In order to understand the kinetic of the compoundand the reversibility of the antiproliferative effect the iso-furanodiene was evaluated in drug washout experimentsFor this purpose the human cancer cell lines were treatedwith various concentrations (10ndash150 120583M) of isofuranodieneat different incubation times (6 12 24 48 and 72) Cellswere then subsequentlywashedwith buffer (PBS) fresh drug-free medium was added and residual inhibitory activitywas evaluated at 72 h from incubation As shown in Figures4(a) 4(b) and 4(c) the data obtained led to the followingconclusions (1) the antiproliferative effect of isofuranodieneincreases with the increase of its concentration (10ndash150 120583M)and with time of treatment up to 72 h (2) after washing theinhibitory activity is partially reversed after low incubationtimes but not after 48 and 72 h (3) in general the dose thatproduced the 50 of antiproliferative activity after washing

The Scientific World Journal 5

Effect of isofuranodiene in MDA-MB 231 after washing at different times

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(a)

Effect of isofuranodiene in BT 474 cells after washing at different times

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(b)

Effect of isofuranodiene in PC 3 cells after washing at different times

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(c)

Figure 4 Cell viability assay in MDA-MB 231 (a) BT 474 (b) and PC 3 (c) cell lines expressed in percentage of cell survival after treatmentwith isofuranodiene at doses of 10ndash150 120583M and at different incubation times (2 6 12 24 48 and 72 h) After incubation isofuranodiene waswashed out with PBS and fresh drug-free medium was added Analysis was performed after 72 h in all the cell lines results are reported as(viability of treated cells)(viability of control cells)times 100 and represent the average of three independent experiments with a maximum SDlower than plusmn80

(a) (b)

Figure 5 Morphological changes in the nuclei of PC 3 cells (a) PC 3 control the majority of cells had uniformly stained nuclei after stainingwith Hoechst (b) PC 3 cells after 24 h exposure to 29120583M isofuranodiene induced morphological changes typical of apoptosis

6 The Scientific World Journal

is shifted approximately to 80ndash100120583Mexcept for 2 h inwhichthe antiproliferative effect is very low and 48ndash72 h in whichthe IC

50is very similar before and after washing

Since antiproliferative effect of isofuranodiene in PC 3cell line was noteworthy an additional study was carriedout to explore the cell growth inhibition mechanism Forthis reason the Hoechst 33258 assay was performed in orderto investigate whether the compound induced cell growthinhibition by cell apoptosis In this experiment PC 3 cellswere treated with 29 120583M (IC

50) of isofuranodiene for 24 h

and apoptotic cell death was analyzed by Hoechst 33258staining and quantified by using fluorescence microscopy

In the control most cells contained intact genomic DNA(Figure 5(a)) however in isofuranodiene-treated cells manycells had condensed chromatin (Figure 5(b)) Approximately355 of isofuranodiene-treated cells showed DNA changesbut in the controls only 18 of cells were apoptoticThis sig-nificant difference between the control and isofuranodiene-treated cells (Figures 5(a) and 5(b) 119875 lt 005) suggests thatisofuranodiene is able to induce apoptosis in prostate cancercell lines

4 Conclusions

The antiproliferative effect of isofuranodiene at MDA-MB231 BT 474 and PC 3 cells is comparable to that of cisplatinThis effect is dose dependent increasing with the isofurano-diene concentrations The best antiproliferative activity wasexhibited on PC 3 prostate cancer cells where the death isinducedwith an apoptoticmechanismThe lower cytotoxicityagainst the two nontumorigenic CHO and HEK 293 cellswith respect to the cancer ones allows to hypothesize thatisofuranodiene could be an anticancer agent endowed withlow side effects

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by Fondo di Ricerca di Ateneo (Uni-versity of Camerino) and by aGrant of the ItalianMinistry forUniversity and Research (PRIN2010-11 n∘ 20103W4779 003)

References

[1] I Soerjomataram J Lortet-Tieulent DM Parkin et al ldquoGlobalburden of cancer in 2008 a systematic analysis of disability-adjusted life-years in 12 world regionsrdquoThe Lancet vol 380 no9856 pp 1840ndash1850 2012

[2] C-C Chang W-C Chen T-F Ho H-S Wu and Y-H WeildquoDevelopment of natural anti-tumor drugs bymicroorganismsrdquoJournal of Bioscience and Bioengineering vol 111 no 5 pp 501ndash511 2011

[3] X Ma and Z Wang ldquoAnticancer drug discovery in the futurean evolutionary perspectiverdquo Drug Discovery Today vol 14 no23-24 pp 1136ndash1142 2009

[4] V T DeVita Jr and E Chu ldquoA history of cancer chemotherapyrdquoCancer Research vol 68 no 21 pp 8643ndash8653 2008

[5] B A Chabner and T G Roberts Jr ldquoChemotherapy and thewaron cancerrdquoNature Reviews Cancer vol 5 no 1 pp 65ndash72 2005

[6] G M Cragg P G Grothaus and D J Newman ldquoImpactof natural products on developing new anti-cancer agentsrdquoChemical Reviews vol 109 no 7 pp 3012ndash3043 2009

[7] A G Ravelo A Estevez-Braun H Chavez-Orellana E Perez-Sacau andDMesa-Siverio ldquoRecent studies on natural productsas anticancer agentsrdquo Current Topics in Medicinal Chemistryvol 4 no 2 pp 241ndash265 2004

[8] D J Newman and G M Cragg ldquoNatural products as sourcesof new drugs over the 30 years from 1981 to 2010rdquo Journal ofNatural Products vol 75 no 3 pp 311ndash335 2012

[9] F Maggi L Barboni F Papa et al ldquoA forgotten vegetable(Smyrnium olusatrum L Apiaceae) as a rich source of isofura-nodienerdquo Food Chemistry vol 135 no 4 pp 2852ndash2862 2012

[10] J Kawabata Y Fukushi S Tahara and J Mizutani ldquoIsolationand structural elucidation of four sesquiterpenes fromChloran-thus japonicus (Chloranthaceae)rdquo Agricultural and BiologicalChemistry vol 49 no 5 pp 1479ndash1486 1985

[11] A A El-Gamal ldquoSesquiterpene lactones from Smyrniumolusatrumrdquo Phytochemistry vol 57 no 8 pp 1197ndash1200 2001

[12] W N Setzer ldquoAb initio analysis of the Cope rearrangement ofgermacrane sesquiterpenoidsrdquo Journal of Molecular Modelingvol 14 no 5 pp 335ndash342 2008

[13] B Wu J Chen H Qu and Y Cheng ldquoComplex sesquiter-penoids with tyrosinase inhibitory activity from the leaves ofChloranthus tianmushanensisrdquo Journal of Natural Products vol71 no 5 pp 877ndash880 2008

[14] K L McPhail M T Davies-Coleman and J StarmerldquoSequestered chemistry of the Arminacean nudibranchLeminda millecra in Algoa Bay South Africardquo Journal ofNatural Products vol 64 no 9 pp 1183ndash1190 2001

[15] L Quassinti M Bramucci G Lupidi et al ldquoIn vitro biologicalactivity of essential oils and isolated furanosesquiterpenes fromthe neglected vegetable Smyrnium olusatrum L (Apiaceae)rdquoFood Chemistry vol 138 no 2-3 pp 808ndash813 2013

[16] S Pignatti Flora drsquoItalia vol 2 Edagricole Bologna Italy 1982[17] R Volpini M Buccioni D Dal Ben et al ldquoSynthesis

and biological evaluation of 2-alkynyl-N6-methyl-51015840-N-methylcarboxamidoadenosine derivatives as potent and highlyselective agonists for the human adenosine A3 receptorrdquoJournal of Medicinal Chemistry vol 52 no 23 pp 7897ndash79002009

[18] M Buccioni G Marucci D D Ben et al ldquoInnovative func-tional cAMP assay for studying G protein-coupled receptorsapplication to the pharmacological characterization of GPR17rdquoPurinergic Signalling vol 7 no 4 pp 463ndash468 2011

[19] T L Riss and R A Moravec ldquoComparison of MTT XTT anda novel tetrazolium compound MTS for in vitro proliferationand chemosensitivity assaysrdquoMolecular Biology of the Cell vol3 A184 1992

[20] S Ghavami C Kerkhoff M Los M Hashemi C Sorgand F Karami-Tehrani ldquoMechanism of apoptosis induced byS100A8A9 in colon cancer cell lines the role of ROS and theeffect of metal ionsrdquo Journal of Leukocyte Biology vol 76 no 1pp 169ndash175 2004

[21] A Maccio and C Madeddu ldquoCisplatin an old drug with anewfound efficacymdashfrom mechanisms of action to cytotoxic-ityrdquo Expert Opin Pharmacother vol 14 no 13 pp 1839ndash18572013

4 The Scientific World Journal

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

MDA-MB 231BT 474PC 3

Effect of isofuranodiene after 72h

Conc (120583M)

(a)

Effect of isofuranodiene in MDA-MB 231

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(b)

Effect of isofuranodiene in BT 474 cells

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(c)

Effect of isofuranodiene in PC 3 cells

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(d)

Figure 3 Cell viability in MDA-MB 231 BT 474 and PC 3 cell lines (a) Percentage of cell viability after treatment with isofuranodieneat doses of 10ndash150 120583M after 72 h of incubation (b) (c) and (d) Percentage of cell viability after treatment with isofuranodiene at doses of10ndash150120583M at different incubation times (2 6 12 24 48 and 72 h) The results are reported as (viability of treated cells)(viability of controlcells)times 100 and represent the average of three independent experiments with a maximum SD lower than plusmn80

able to induce cell death quite quickly In addition whenisofuranodiene was added at CHO and HEK 293 cells thecytotoxicity observed after 72 h was fairly low IC

50of 125 120583M

and 130 120583M respectively (Table 1) The concentration of60 120583M of isofuranodiene which in turn provided generallythe 50 of antiproliferative activity in breast cancer cell linesproduced only 20 of inhibition in CHO and HEK 293cell viability even if the maximum concentration 150120583Mproduced an inhibition of 70plusmn 45 and 75plusmn 36 cell via-bility respectivelyThe isofuranodiene antiproliferative effectdifference observed at IC

50concentrations (30 and 60 120583M in

PC 3 andMDA-MB 231 BT 474 cells resp) among the tumorcell lines and the two noncancer cell lines is significant Thissuggests that since the toxicity of the isofuranodiene is beingless in noncancer cells it could be a potential anticancer drugwith low side effects

In order to understand the kinetic of the compoundand the reversibility of the antiproliferative effect the iso-furanodiene was evaluated in drug washout experimentsFor this purpose the human cancer cell lines were treatedwith various concentrations (10ndash150 120583M) of isofuranodieneat different incubation times (6 12 24 48 and 72) Cellswere then subsequentlywashedwith buffer (PBS) fresh drug-free medium was added and residual inhibitory activitywas evaluated at 72 h from incubation As shown in Figures4(a) 4(b) and 4(c) the data obtained led to the followingconclusions (1) the antiproliferative effect of isofuranodieneincreases with the increase of its concentration (10ndash150 120583M)and with time of treatment up to 72 h (2) after washing theinhibitory activity is partially reversed after low incubationtimes but not after 48 and 72 h (3) in general the dose thatproduced the 50 of antiproliferative activity after washing

The Scientific World Journal 5

Effect of isofuranodiene in MDA-MB 231 after washing at different times

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(a)

Effect of isofuranodiene in BT 474 cells after washing at different times

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(b)

Effect of isofuranodiene in PC 3 cells after washing at different times

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(c)

Figure 4 Cell viability assay in MDA-MB 231 (a) BT 474 (b) and PC 3 (c) cell lines expressed in percentage of cell survival after treatmentwith isofuranodiene at doses of 10ndash150 120583M and at different incubation times (2 6 12 24 48 and 72 h) After incubation isofuranodiene waswashed out with PBS and fresh drug-free medium was added Analysis was performed after 72 h in all the cell lines results are reported as(viability of treated cells)(viability of control cells)times 100 and represent the average of three independent experiments with a maximum SDlower than plusmn80

(a) (b)

Figure 5 Morphological changes in the nuclei of PC 3 cells (a) PC 3 control the majority of cells had uniformly stained nuclei after stainingwith Hoechst (b) PC 3 cells after 24 h exposure to 29120583M isofuranodiene induced morphological changes typical of apoptosis

6 The Scientific World Journal

is shifted approximately to 80ndash100120583Mexcept for 2 h inwhichthe antiproliferative effect is very low and 48ndash72 h in whichthe IC

50is very similar before and after washing

Since antiproliferative effect of isofuranodiene in PC 3cell line was noteworthy an additional study was carriedout to explore the cell growth inhibition mechanism Forthis reason the Hoechst 33258 assay was performed in orderto investigate whether the compound induced cell growthinhibition by cell apoptosis In this experiment PC 3 cellswere treated with 29 120583M (IC

50) of isofuranodiene for 24 h

and apoptotic cell death was analyzed by Hoechst 33258staining and quantified by using fluorescence microscopy

In the control most cells contained intact genomic DNA(Figure 5(a)) however in isofuranodiene-treated cells manycells had condensed chromatin (Figure 5(b)) Approximately355 of isofuranodiene-treated cells showed DNA changesbut in the controls only 18 of cells were apoptoticThis sig-nificant difference between the control and isofuranodiene-treated cells (Figures 5(a) and 5(b) 119875 lt 005) suggests thatisofuranodiene is able to induce apoptosis in prostate cancercell lines

4 Conclusions

The antiproliferative effect of isofuranodiene at MDA-MB231 BT 474 and PC 3 cells is comparable to that of cisplatinThis effect is dose dependent increasing with the isofurano-diene concentrations The best antiproliferative activity wasexhibited on PC 3 prostate cancer cells where the death isinducedwith an apoptoticmechanismThe lower cytotoxicityagainst the two nontumorigenic CHO and HEK 293 cellswith respect to the cancer ones allows to hypothesize thatisofuranodiene could be an anticancer agent endowed withlow side effects

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by Fondo di Ricerca di Ateneo (Uni-versity of Camerino) and by aGrant of the ItalianMinistry forUniversity and Research (PRIN2010-11 n∘ 20103W4779 003)

References

[1] I Soerjomataram J Lortet-Tieulent DM Parkin et al ldquoGlobalburden of cancer in 2008 a systematic analysis of disability-adjusted life-years in 12 world regionsrdquoThe Lancet vol 380 no9856 pp 1840ndash1850 2012

[2] C-C Chang W-C Chen T-F Ho H-S Wu and Y-H WeildquoDevelopment of natural anti-tumor drugs bymicroorganismsrdquoJournal of Bioscience and Bioengineering vol 111 no 5 pp 501ndash511 2011

[3] X Ma and Z Wang ldquoAnticancer drug discovery in the futurean evolutionary perspectiverdquo Drug Discovery Today vol 14 no23-24 pp 1136ndash1142 2009

[4] V T DeVita Jr and E Chu ldquoA history of cancer chemotherapyrdquoCancer Research vol 68 no 21 pp 8643ndash8653 2008

[5] B A Chabner and T G Roberts Jr ldquoChemotherapy and thewaron cancerrdquoNature Reviews Cancer vol 5 no 1 pp 65ndash72 2005

[6] G M Cragg P G Grothaus and D J Newman ldquoImpactof natural products on developing new anti-cancer agentsrdquoChemical Reviews vol 109 no 7 pp 3012ndash3043 2009

[7] A G Ravelo A Estevez-Braun H Chavez-Orellana E Perez-Sacau andDMesa-Siverio ldquoRecent studies on natural productsas anticancer agentsrdquo Current Topics in Medicinal Chemistryvol 4 no 2 pp 241ndash265 2004

[8] D J Newman and G M Cragg ldquoNatural products as sourcesof new drugs over the 30 years from 1981 to 2010rdquo Journal ofNatural Products vol 75 no 3 pp 311ndash335 2012

[9] F Maggi L Barboni F Papa et al ldquoA forgotten vegetable(Smyrnium olusatrum L Apiaceae) as a rich source of isofura-nodienerdquo Food Chemistry vol 135 no 4 pp 2852ndash2862 2012

[10] J Kawabata Y Fukushi S Tahara and J Mizutani ldquoIsolationand structural elucidation of four sesquiterpenes fromChloran-thus japonicus (Chloranthaceae)rdquo Agricultural and BiologicalChemistry vol 49 no 5 pp 1479ndash1486 1985

[11] A A El-Gamal ldquoSesquiterpene lactones from Smyrniumolusatrumrdquo Phytochemistry vol 57 no 8 pp 1197ndash1200 2001

[12] W N Setzer ldquoAb initio analysis of the Cope rearrangement ofgermacrane sesquiterpenoidsrdquo Journal of Molecular Modelingvol 14 no 5 pp 335ndash342 2008

[13] B Wu J Chen H Qu and Y Cheng ldquoComplex sesquiter-penoids with tyrosinase inhibitory activity from the leaves ofChloranthus tianmushanensisrdquo Journal of Natural Products vol71 no 5 pp 877ndash880 2008

[14] K L McPhail M T Davies-Coleman and J StarmerldquoSequestered chemistry of the Arminacean nudibranchLeminda millecra in Algoa Bay South Africardquo Journal ofNatural Products vol 64 no 9 pp 1183ndash1190 2001

[15] L Quassinti M Bramucci G Lupidi et al ldquoIn vitro biologicalactivity of essential oils and isolated furanosesquiterpenes fromthe neglected vegetable Smyrnium olusatrum L (Apiaceae)rdquoFood Chemistry vol 138 no 2-3 pp 808ndash813 2013

[16] S Pignatti Flora drsquoItalia vol 2 Edagricole Bologna Italy 1982[17] R Volpini M Buccioni D Dal Ben et al ldquoSynthesis

and biological evaluation of 2-alkynyl-N6-methyl-51015840-N-methylcarboxamidoadenosine derivatives as potent and highlyselective agonists for the human adenosine A3 receptorrdquoJournal of Medicinal Chemistry vol 52 no 23 pp 7897ndash79002009

[18] M Buccioni G Marucci D D Ben et al ldquoInnovative func-tional cAMP assay for studying G protein-coupled receptorsapplication to the pharmacological characterization of GPR17rdquoPurinergic Signalling vol 7 no 4 pp 463ndash468 2011

[19] T L Riss and R A Moravec ldquoComparison of MTT XTT anda novel tetrazolium compound MTS for in vitro proliferationand chemosensitivity assaysrdquoMolecular Biology of the Cell vol3 A184 1992

[20] S Ghavami C Kerkhoff M Los M Hashemi C Sorgand F Karami-Tehrani ldquoMechanism of apoptosis induced byS100A8A9 in colon cancer cell lines the role of ROS and theeffect of metal ionsrdquo Journal of Leukocyte Biology vol 76 no 1pp 169ndash175 2004

[21] A Maccio and C Madeddu ldquoCisplatin an old drug with anewfound efficacymdashfrom mechanisms of action to cytotoxic-ityrdquo Expert Opin Pharmacother vol 14 no 13 pp 1839ndash18572013

The Scientific World Journal 5

Effect of isofuranodiene in MDA-MB 231 after washing at different times

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(a)

Effect of isofuranodiene in BT 474 cells after washing at different times

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(b)

Effect of isofuranodiene in PC 3 cells after washing at different times

0

20

40

60

80

100

120

10 15 20 30 40 60 80 100 120 130 150

Cel

l via

bilit

y (

)

2h6h12h

24h48h72h

Conc (120583M)

(c)

Figure 4 Cell viability assay in MDA-MB 231 (a) BT 474 (b) and PC 3 (c) cell lines expressed in percentage of cell survival after treatmentwith isofuranodiene at doses of 10ndash150 120583M and at different incubation times (2 6 12 24 48 and 72 h) After incubation isofuranodiene waswashed out with PBS and fresh drug-free medium was added Analysis was performed after 72 h in all the cell lines results are reported as(viability of treated cells)(viability of control cells)times 100 and represent the average of three independent experiments with a maximum SDlower than plusmn80

(a) (b)

Figure 5 Morphological changes in the nuclei of PC 3 cells (a) PC 3 control the majority of cells had uniformly stained nuclei after stainingwith Hoechst (b) PC 3 cells after 24 h exposure to 29120583M isofuranodiene induced morphological changes typical of apoptosis

6 The Scientific World Journal

is shifted approximately to 80ndash100120583Mexcept for 2 h inwhichthe antiproliferative effect is very low and 48ndash72 h in whichthe IC

50is very similar before and after washing

Since antiproliferative effect of isofuranodiene in PC 3cell line was noteworthy an additional study was carriedout to explore the cell growth inhibition mechanism Forthis reason the Hoechst 33258 assay was performed in orderto investigate whether the compound induced cell growthinhibition by cell apoptosis In this experiment PC 3 cellswere treated with 29 120583M (IC

50) of isofuranodiene for 24 h

and apoptotic cell death was analyzed by Hoechst 33258staining and quantified by using fluorescence microscopy

In the control most cells contained intact genomic DNA(Figure 5(a)) however in isofuranodiene-treated cells manycells had condensed chromatin (Figure 5(b)) Approximately355 of isofuranodiene-treated cells showed DNA changesbut in the controls only 18 of cells were apoptoticThis sig-nificant difference between the control and isofuranodiene-treated cells (Figures 5(a) and 5(b) 119875 lt 005) suggests thatisofuranodiene is able to induce apoptosis in prostate cancercell lines

4 Conclusions

The antiproliferative effect of isofuranodiene at MDA-MB231 BT 474 and PC 3 cells is comparable to that of cisplatinThis effect is dose dependent increasing with the isofurano-diene concentrations The best antiproliferative activity wasexhibited on PC 3 prostate cancer cells where the death isinducedwith an apoptoticmechanismThe lower cytotoxicityagainst the two nontumorigenic CHO and HEK 293 cellswith respect to the cancer ones allows to hypothesize thatisofuranodiene could be an anticancer agent endowed withlow side effects

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by Fondo di Ricerca di Ateneo (Uni-versity of Camerino) and by aGrant of the ItalianMinistry forUniversity and Research (PRIN2010-11 n∘ 20103W4779 003)

References

[1] I Soerjomataram J Lortet-Tieulent DM Parkin et al ldquoGlobalburden of cancer in 2008 a systematic analysis of disability-adjusted life-years in 12 world regionsrdquoThe Lancet vol 380 no9856 pp 1840ndash1850 2012

[2] C-C Chang W-C Chen T-F Ho H-S Wu and Y-H WeildquoDevelopment of natural anti-tumor drugs bymicroorganismsrdquoJournal of Bioscience and Bioengineering vol 111 no 5 pp 501ndash511 2011

[3] X Ma and Z Wang ldquoAnticancer drug discovery in the futurean evolutionary perspectiverdquo Drug Discovery Today vol 14 no23-24 pp 1136ndash1142 2009

[4] V T DeVita Jr and E Chu ldquoA history of cancer chemotherapyrdquoCancer Research vol 68 no 21 pp 8643ndash8653 2008

[5] B A Chabner and T G Roberts Jr ldquoChemotherapy and thewaron cancerrdquoNature Reviews Cancer vol 5 no 1 pp 65ndash72 2005

[6] G M Cragg P G Grothaus and D J Newman ldquoImpactof natural products on developing new anti-cancer agentsrdquoChemical Reviews vol 109 no 7 pp 3012ndash3043 2009

[7] A G Ravelo A Estevez-Braun H Chavez-Orellana E Perez-Sacau andDMesa-Siverio ldquoRecent studies on natural productsas anticancer agentsrdquo Current Topics in Medicinal Chemistryvol 4 no 2 pp 241ndash265 2004

[8] D J Newman and G M Cragg ldquoNatural products as sourcesof new drugs over the 30 years from 1981 to 2010rdquo Journal ofNatural Products vol 75 no 3 pp 311ndash335 2012

[9] F Maggi L Barboni F Papa et al ldquoA forgotten vegetable(Smyrnium olusatrum L Apiaceae) as a rich source of isofura-nodienerdquo Food Chemistry vol 135 no 4 pp 2852ndash2862 2012

[10] J Kawabata Y Fukushi S Tahara and J Mizutani ldquoIsolationand structural elucidation of four sesquiterpenes fromChloran-thus japonicus (Chloranthaceae)rdquo Agricultural and BiologicalChemistry vol 49 no 5 pp 1479ndash1486 1985

[11] A A El-Gamal ldquoSesquiterpene lactones from Smyrniumolusatrumrdquo Phytochemistry vol 57 no 8 pp 1197ndash1200 2001

[12] W N Setzer ldquoAb initio analysis of the Cope rearrangement ofgermacrane sesquiterpenoidsrdquo Journal of Molecular Modelingvol 14 no 5 pp 335ndash342 2008

[13] B Wu J Chen H Qu and Y Cheng ldquoComplex sesquiter-penoids with tyrosinase inhibitory activity from the leaves ofChloranthus tianmushanensisrdquo Journal of Natural Products vol71 no 5 pp 877ndash880 2008

[14] K L McPhail M T Davies-Coleman and J StarmerldquoSequestered chemistry of the Arminacean nudibranchLeminda millecra in Algoa Bay South Africardquo Journal ofNatural Products vol 64 no 9 pp 1183ndash1190 2001

[15] L Quassinti M Bramucci G Lupidi et al ldquoIn vitro biologicalactivity of essential oils and isolated furanosesquiterpenes fromthe neglected vegetable Smyrnium olusatrum L (Apiaceae)rdquoFood Chemistry vol 138 no 2-3 pp 808ndash813 2013

[16] S Pignatti Flora drsquoItalia vol 2 Edagricole Bologna Italy 1982[17] R Volpini M Buccioni D Dal Ben et al ldquoSynthesis

and biological evaluation of 2-alkynyl-N6-methyl-51015840-N-methylcarboxamidoadenosine derivatives as potent and highlyselective agonists for the human adenosine A3 receptorrdquoJournal of Medicinal Chemistry vol 52 no 23 pp 7897ndash79002009

[18] M Buccioni G Marucci D D Ben et al ldquoInnovative func-tional cAMP assay for studying G protein-coupled receptorsapplication to the pharmacological characterization of GPR17rdquoPurinergic Signalling vol 7 no 4 pp 463ndash468 2011

[19] T L Riss and R A Moravec ldquoComparison of MTT XTT anda novel tetrazolium compound MTS for in vitro proliferationand chemosensitivity assaysrdquoMolecular Biology of the Cell vol3 A184 1992

[20] S Ghavami C Kerkhoff M Los M Hashemi C Sorgand F Karami-Tehrani ldquoMechanism of apoptosis induced byS100A8A9 in colon cancer cell lines the role of ROS and theeffect of metal ionsrdquo Journal of Leukocyte Biology vol 76 no 1pp 169ndash175 2004

[21] A Maccio and C Madeddu ldquoCisplatin an old drug with anewfound efficacymdashfrom mechanisms of action to cytotoxic-ityrdquo Expert Opin Pharmacother vol 14 no 13 pp 1839ndash18572013

6 The Scientific World Journal

is shifted approximately to 80ndash100120583Mexcept for 2 h inwhichthe antiproliferative effect is very low and 48ndash72 h in whichthe IC

50is very similar before and after washing

Since antiproliferative effect of isofuranodiene in PC 3cell line was noteworthy an additional study was carriedout to explore the cell growth inhibition mechanism Forthis reason the Hoechst 33258 assay was performed in orderto investigate whether the compound induced cell growthinhibition by cell apoptosis In this experiment PC 3 cellswere treated with 29 120583M (IC

50) of isofuranodiene for 24 h

and apoptotic cell death was analyzed by Hoechst 33258staining and quantified by using fluorescence microscopy

In the control most cells contained intact genomic DNA(Figure 5(a)) however in isofuranodiene-treated cells manycells had condensed chromatin (Figure 5(b)) Approximately355 of isofuranodiene-treated cells showed DNA changesbut in the controls only 18 of cells were apoptoticThis sig-nificant difference between the control and isofuranodiene-treated cells (Figures 5(a) and 5(b) 119875 lt 005) suggests thatisofuranodiene is able to induce apoptosis in prostate cancercell lines

4 Conclusions

The antiproliferative effect of isofuranodiene at MDA-MB231 BT 474 and PC 3 cells is comparable to that of cisplatinThis effect is dose dependent increasing with the isofurano-diene concentrations The best antiproliferative activity wasexhibited on PC 3 prostate cancer cells where the death isinducedwith an apoptoticmechanismThe lower cytotoxicityagainst the two nontumorigenic CHO and HEK 293 cellswith respect to the cancer ones allows to hypothesize thatisofuranodiene could be an anticancer agent endowed withlow side effects

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by Fondo di Ricerca di Ateneo (Uni-versity of Camerino) and by aGrant of the ItalianMinistry forUniversity and Research (PRIN2010-11 n∘ 20103W4779 003)

References

[1] I Soerjomataram J Lortet-Tieulent DM Parkin et al ldquoGlobalburden of cancer in 2008 a systematic analysis of disability-adjusted life-years in 12 world regionsrdquoThe Lancet vol 380 no9856 pp 1840ndash1850 2012

[2] C-C Chang W-C Chen T-F Ho H-S Wu and Y-H WeildquoDevelopment of natural anti-tumor drugs bymicroorganismsrdquoJournal of Bioscience and Bioengineering vol 111 no 5 pp 501ndash511 2011

[3] X Ma and Z Wang ldquoAnticancer drug discovery in the futurean evolutionary perspectiverdquo Drug Discovery Today vol 14 no23-24 pp 1136ndash1142 2009

[4] V T DeVita Jr and E Chu ldquoA history of cancer chemotherapyrdquoCancer Research vol 68 no 21 pp 8643ndash8653 2008

[5] B A Chabner and T G Roberts Jr ldquoChemotherapy and thewaron cancerrdquoNature Reviews Cancer vol 5 no 1 pp 65ndash72 2005

[6] G M Cragg P G Grothaus and D J Newman ldquoImpactof natural products on developing new anti-cancer agentsrdquoChemical Reviews vol 109 no 7 pp 3012ndash3043 2009

[7] A G Ravelo A Estevez-Braun H Chavez-Orellana E Perez-Sacau andDMesa-Siverio ldquoRecent studies on natural productsas anticancer agentsrdquo Current Topics in Medicinal Chemistryvol 4 no 2 pp 241ndash265 2004

[8] D J Newman and G M Cragg ldquoNatural products as sourcesof new drugs over the 30 years from 1981 to 2010rdquo Journal ofNatural Products vol 75 no 3 pp 311ndash335 2012

[9] F Maggi L Barboni F Papa et al ldquoA forgotten vegetable(Smyrnium olusatrum L Apiaceae) as a rich source of isofura-nodienerdquo Food Chemistry vol 135 no 4 pp 2852ndash2862 2012

[10] J Kawabata Y Fukushi S Tahara and J Mizutani ldquoIsolationand structural elucidation of four sesquiterpenes fromChloran-thus japonicus (Chloranthaceae)rdquo Agricultural and BiologicalChemistry vol 49 no 5 pp 1479ndash1486 1985

[11] A A El-Gamal ldquoSesquiterpene lactones from Smyrniumolusatrumrdquo Phytochemistry vol 57 no 8 pp 1197ndash1200 2001

[12] W N Setzer ldquoAb initio analysis of the Cope rearrangement ofgermacrane sesquiterpenoidsrdquo Journal of Molecular Modelingvol 14 no 5 pp 335ndash342 2008

[13] B Wu J Chen H Qu and Y Cheng ldquoComplex sesquiter-penoids with tyrosinase inhibitory activity from the leaves ofChloranthus tianmushanensisrdquo Journal of Natural Products vol71 no 5 pp 877ndash880 2008

[14] K L McPhail M T Davies-Coleman and J StarmerldquoSequestered chemistry of the Arminacean nudibranchLeminda millecra in Algoa Bay South Africardquo Journal ofNatural Products vol 64 no 9 pp 1183ndash1190 2001

[15] L Quassinti M Bramucci G Lupidi et al ldquoIn vitro biologicalactivity of essential oils and isolated furanosesquiterpenes fromthe neglected vegetable Smyrnium olusatrum L (Apiaceae)rdquoFood Chemistry vol 138 no 2-3 pp 808ndash813 2013

[16] S Pignatti Flora drsquoItalia vol 2 Edagricole Bologna Italy 1982[17] R Volpini M Buccioni D Dal Ben et al ldquoSynthesis

and biological evaluation of 2-alkynyl-N6-methyl-51015840-N-methylcarboxamidoadenosine derivatives as potent and highlyselective agonists for the human adenosine A3 receptorrdquoJournal of Medicinal Chemistry vol 52 no 23 pp 7897ndash79002009

[18] M Buccioni G Marucci D D Ben et al ldquoInnovative func-tional cAMP assay for studying G protein-coupled receptorsapplication to the pharmacological characterization of GPR17rdquoPurinergic Signalling vol 7 no 4 pp 463ndash468 2011

[19] T L Riss and R A Moravec ldquoComparison of MTT XTT anda novel tetrazolium compound MTS for in vitro proliferationand chemosensitivity assaysrdquoMolecular Biology of the Cell vol3 A184 1992

[20] S Ghavami C Kerkhoff M Los M Hashemi C Sorgand F Karami-Tehrani ldquoMechanism of apoptosis induced byS100A8A9 in colon cancer cell lines the role of ROS and theeffect of metal ionsrdquo Journal of Leukocyte Biology vol 76 no 1pp 169ndash175 2004

[21] A Maccio and C Madeddu ldquoCisplatin an old drug with anewfound efficacymdashfrom mechanisms of action to cytotoxic-ityrdquo Expert Opin Pharmacother vol 14 no 13 pp 1839ndash18572013

Submit your manuscripts athttpwwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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