2007 antiproliferative, cytotoxic and antitumour activity

JPP 2007 59 719ndash725copy 2007 The AuthorsReceived September 29 2006Accepted January 22 2007DOI 101211jpp5950013ISSN 0022-3573

719

Antiproliferative cytotoxic and antitumour activity of coumarins isolated from Calophyllum brasiliense

Ceacutesar Ruiz-Marcial Ricardo Reyes Chilpa Elizabeth Estrada

Jorge Reyes-Esparza Germaacuten Garrido Farintildea and

Lourdes Rodriacuteguez-Fragoso

Abstract

Among the eight Calophyllum species found on the American continent Calophyllum brasiliense isthe most widely distributed Chemical analysis of this species has shown the presence of xanthoneswith cancer chemopreventive properties and antifungal activity Recently three new coumarinswith antineoplastic properties have been found In this study we have evaluated the biologicaleffects of the antiproliferative activity of coumarins isolated from C brasiliense on the survival cellcycle and apoptosis of cells in-vitro and their antitumour effects in mice The cytological studyshowed that coumarins from C brasiliense reduce the survival of BMK cells (baby mouse kidneycells) by inducing apoptosis and to a lesser degree necrosis The cell cycle was arrested in S-phaseand the division of BMK cells was inhibited Coumarins had caused a reduction of experimentaltumours in 83 of animals by the end of the treatment Therefore coumarins have the potential tobe used alone or in combination with other antineoplastic drugs and they might increase the effec-tiveness of other treatments for cancer

Coumarins comprise a group of natural compounds found in a variety of plant sourcesThey have extensive biological properties that promote health and help reduce the risk ofdisease (Kostova amp Momekov 2006) The coumarins are extremely variable in structuredue to various substitutions in their basic structure which can influence their biological activ-ity Coumarin the parent molecule of dicumarol and a variety of coumarin compounds havenumerous antitumour and antiproliferative effects Coumarin compounds inhibit the prolifera-tion of particular human malignant cell lines in-vitro (Chaya et al 2004 Kostova amp Momekov2006 Riviere et al 2006) as well as affecting the activity of several tumour types in-vivo(Ishihara et al 2006 Kawase et al 2005 Yang et al 2005) In clinical trials these compoundshave shown some activity against prostate cancer malignant melanoma and metastatic renalcell carcinoma (Burgos et al 1999 Agata et al 2004 Lacy amp OrsquoKennedy 2004)

Calophyllum species contain coumarins and have recently received considerable atten-tion pharmacologically Coumarins are a common family of plant secondary metabolitesMany studies on natural products have documented their biological effects and there havebeen many attempts to isolate and purify active compounds It has been reported that cou-marins have a broad spectrum of biological activity including antitumour effects (Kashmanet al 1992 Patil et al 1993 McKee et al 1998)

Among the eight Calophyllum species found on the American continent Calophyllumbrasiliense has the widest distribution growing in the tropical Mexican and Brazilian rain-forests (Stevens 1980) Numerous ethnomedical applications have been recorded for thisspecies throughout Latin America (Sovak et al 2002 Reddy et al 2004 Tsuda et al 2004)Nevertheless none appears related to its antiviral and antitumour effects Previous chemicalanalysis of C brasiliense collected in Brazil has reported the presence of xanthones withcancer chemopreventive properties and antifungal activity In addition three new cou-marins with antineoplastic properties have been found (Ito et al 2002 2003) The organicextract from the leaves of C brasiliense yields coumarins of the mammea type mammea

Introduction

Facultad de Farmacia Universidad Autoacutenoma del Estado de Morelos Cuernavaca Morelos Meacutexico

Ceacutesar Ruiz-Marcial Jorge Reyes-Esparza Lourdes Rodriacuteguez-Fragoso

Instituto de Quiacutemica Universidad Nacional Autoacutenoma Meacutexico Meacutexico City

Ricardo Reyes Chilpa Elizabeth Estrada

FES-Cuautitlaacuten Universidad Nacional Autoacutenoma de Meacutexico Meacutexico

Germaacuten Garrido Farintildea

Correspondence L Rodriacuteguez Fragoso Flavio Garciacutea No 32 Presidentes Ejidales CP 04470 Meacutexico D F E-mail mlrodrig1yahoocommx

JPP 59(5)book Page 719 Thursday April 12 2007 402 PM

720 Ceacutesar Ruiz-Marcial et al

ABA ABB BBB COA COB BBA cyclo F BBB cyclo Fand isomammeigin (Reyes-Chilpa etal 2004) Recent studieshave shown that mammea ABA and ABB have high cytotoxicactivity against some tumour cell lines (Reyes-Chilpa etal 2004)

We have studied the biological effects of the antiprolifera-tive activity of mammea ABA and ABB isolated fromC brasiliense on the survival cell cycle and apoptosis of BMKcells We attempted to define their antitumour effects in mice

Chemicals

Vincristine was obtained from Lemery Laboratories (Mexico)DMEM fetal calf serum MTT ((3-(45-dimethyl-2-thia-zolyl)-25-diphenyl-2H-tetrazolium bromide) trypsin trypsininhibitor ribonuclease A (RNase) and propidium iodidewere obtained from Sigma Chemical Co (USA)

Extraction and isolation of mammea ABA and ABB from C brasiliense

C brasiliense (Clusiaceae) was collected at Ejido BenignoMendoza Sierra de Santa Marta Veracruz Mexico A voucherspecimen was deposited in the Herbarium of the Instituto deInvestigaciones Biologicas Universidad Veracruzana at XalapaMexico labelled number 435 Dried leaves (2075g) wereextracted at room temperature over one week with hexane ace-tone and methanol successively Compounds were isolated afterspontaneous crystallization or by column chromatography onSilica Gel-60 and individualized by their spectroscopy (1HNMR 13C NMR IR UV) EIMS and CIMS The results of theseexperiments were compared with Reyes-Chilpa etal (2004)

Hexane extracts (703 g) While in solution the extract showed spontaneous precipita-tion giving a white powder (198 g) Part of this material(51 g) was treated with CH2Cl2 The insoluble part was amixture (583 mg) of friedelin and canophyllol and the solu-ble part afforded a mixture (4 g) of coumarins of two mam-mea types mammea ABA and mammea ABB The extractwas then concentrated in-vacuo (353 g) and 94 g subjectedto column chromatography (200 g) Elution with hexane firstyielded friedelin (459 mg) then a mixture of coumarins(5 mg mammea ABA and mammea ABB)

Identification and chemical structure of mammea ABA and mammea ABB

The compounds isolated after spontaneous crystallization orcolumn chromatography were individually identified Thepurification of compounds (mp 122ndash124degC) was obtained byHPLC using an ODS-column and elution with 90 MeOHChemical structures are shown in Figure 1 The identificationof the mammeas was as follows

1H NMR (200 MHz CDCl3TMS) 756 m 3H (Ar)741 m 2H (Ar) 60 s 1H (H-3) 595 s 1H (OH-5) Isopre-nyl on C-6 509 tm J = 69 Hz 1H (CH) 329 d J = 69 Hz2H (CH2) 165 and 170 both s and 3H (2 CH3)

1 1461 s 1H (OH-7) R = 3-methylbutyryl 319 d J =67 Hz 2H (CH2) 232 m J = 67 Hz 1H (CH) 106 dJ = 66 Hz 6H (2 CH3)

2 1457 s 1H (OH-7) R = 2-methylbutyryl (ABB)395 m J = 66 Hz 1H (CH-CH3) 129 d J = 67 Hz 3H (CH-CH3) 194 m 2H (CH2) 101 t J = 72 Hz 3H (CH3)

EMIE 70 eV mz () 406 M+ (964) [C25H26O5]+363 (556) 351 (760) 293 (100) IR n max (KBr)3985 (OH) 2964 2932 2872 (C-H) 1729 (C= O) 1724(C=O) 1614 (C = C) 1556 (C = C) 1390 (C-O)

For the pharmacological studies a mixture of coumarinsmammea ABA and mammea ABB at a ratio of 21 wasused The mix of mammeas (5 mg mLminus1) was prepared in01 dimethyl sulfoxide (DMSO) (Aldrich USA) at roomtemperature

Cell culture and pharmacological treatments

For in-vitro studies BMK cells were used derived from babymouse kidney These cells were kindly donated by ProfessorSophie Hallez from Brussels University Belgium Cells werecultured in minimal essential medium (Gibco-BRL IncGrand Island NY) supplemented with nonessential amino acids(Gibco-BRL Inc Grand Island NY) 10 fetal calf serum(Gibson-BRL Inc Grand Island NY) 2 mol Lminus1 L-glutamineand antibiotics Cells were plated in 100-mm culture dishes(106 cellsdish) and maintained at 37degC in an atmosphere of5 CO2 in humidified air Subcultures were obtained bytrypsinization (0025 trypsin solution containing 001NN-diethyldithiocarbamic acid sodium salt and EDTA)

For cytological investigation 105 cells were plated andallowed to adhere overnight The medium was then aspiratedand replaced with medium containing vehicle alone ormedium containing the mixture of mammeas (3 5 8 10 1620 and 24 mg mLminus1) and the cells were then incubated for24 h After incubation the cells were collected for furthercytological investigation

Assessment of cell viability

BMK cells were incubated in 96-well plates After 24 h themedium was removed the cells were washed twice withphosphate-buffered saline (PBS) and then incubated with themixture of coumarins (3 5 8 10 16 20 and 24 mg mLminus1)

Materials and Methods

Figure 1 Coumarins isolated from Calophyllum brasiliense leaves

Mammea ABA

HOHO

OHOH

6

78 1

OO

OO

O O

5 43

2 6

7 8 1

5 43

2

Mammea ABB


Antitumour effect of Calophyllum brasiliense 721

After 24 h both cells and conditioned media were collectedand processed Cell viability was measured by the MTT assay(Wang et al 1996) Briefly 20 mL MTT (5 g Lminus1) was added toeach well and incubated for 4 h at 37degC 5 CO2 The culturemedia was then discarded and 200 mL DMSO with 25 mLSorensenrsquos buffer (glycine 01 M NaCl 01 M pH 105) wasadded to each well Once the blue crystals had dissolved theoptical density was determined on a microreader (Bio-RadCo) at 450 nm

Mitotic activity

BMK cells were fixed in 3 paraformaldehyde with 025 M

mannitol (4554 g Lminus1) for 2 h rinsed in PBS and stained withDAPI (4prime6-diamidino-2-phenylindol 480 nm) After rinsingin PBS the cells were embedded in Citifluor mountingmedium The mitotic index was counted with a fluorescentmicroscope (Optiphot 2 Nikon) For each experiment theindices were determined per 1000 cells and with four replicates

Survival of cells and detection of percentage of apoptosis and necrosis

Survival of cells was evaluated based on propidium iodide(PI Sigma P4170) staining (Koopman et al 1994) PI (finalconcentration 5 mg mLminus1) was added to each sample for10 min (room temperature in the dark) The samples (105)were analysed using a FACScalibur flow cytometer at 488 nm(Becton Dickinson USA equipped with an argon laser) Tenthousand cells were analysed using four replicates Theresults were analysed using the CELLQuest program Basedon the penetration of PI and the size of the cells the percent-age of apoptotic and necrotic cells was determined

Analysis of cell cycle

Cells (105) were fixed in 75 ethanol for 24 h and thenwashed in PBS and resuspended in 01 Nonidet P40 (Bio-chemica Fluka) and DNase-free RNase (10 mg mLminus1) for20 min at room temperature (Darzynkiewicz et al 2001) PIwas then added (final concentration 5 mg mLminus1) and incubatedfor 12 h at 4degC in the dark Samples were analysed using aFACScalibur flow cytometer (Becton Dickinson) For eachsample 10 000 cells were analysed using four replicates Theresults were analysed using the CELLQuest program

Tumour formation in mice

Female BALBc mice (6ndash8-weeks-old purchased fromHarlan Mexico Mexico) were fed using a standard basal diet(24 protein) and exposed to a daily cycle of alternating 12-hlight periods The animals were housed in a temperature-and-humidity-controlled environment and food and water werefreely available The experiments were conducted accordingto the principles set forth in ldquoGuide for the Care and Use ofLaboratory Animalsrdquo (National Institutes of Health Publica-tion Number 86-23 Committee on Care and Use of Labora-tory Animals of the Institute for Laboratory AnimalResources 1986) and the Animal Welfare Act of 1966 asamended

Tumour formation in mice was performed as described byDehenhardt et al (2002) Briefly 25 times 107 BMK cells wereinjected into each mouse and tumours were measured regularly

Pharmacological evaluation of coumarins

Mice were randomly divided into four groups each groupcontaining 10 animals Group I control animals that receivedno treatment Group II mice with BMK-cell tumours GroupIII mice with BMK-cell tumours and treated with coumarins(20 mg kgminus1 ip three times per week for three weeks)Group IV mice with BMK-cell tumours and treated with vin-cristine (1 mg mminus2 ip twice per week for two weeks) Cou-marins and vincristine treatments started two weeks after theBMK cells were injected when the tumour size was 5 mm

To assess the pharmacological effect of coumarins ontumour growth the size of the tumour was measured everythird day during the treatment After completion of the treat-ment period animals were anaesthetized with diethyl etherand killed by exsanguination A complete post-mortem exam-ination was performed on each animal Samples of liver kid-ney thymus spleen and lungs were weighed and sliced andseveral sections were fixed by immersion in alcohol andembedded in paraffin for histological analysis to identify thepresence of metastasis

Statistical analysis

Data were reported as means plusmn sd of three independentexperiments conducted in quadruplicate Statistical analysiswas performed using a nonparametric analysis of varianceand by Chi-squared testing Individual differences betweentreatments were analysed using Tukeyrsquos test Significantdifferences were established at P lt 0001

Cell survival and induction of apoptosis and necrosis

The coumarin extract caused a significant decrease in thesurvival of BMK cells (Figure 2) The coumarins (mammeaABA and mammea ABB) showed a concentration-dependentinhibitory effect on the growth of BMK cells (Figure 2)(P lt 0001) The lowest concentration produced a 20 reduc-tion in cell survival (P lt 0001) Concentrations of 5 8 and10mgmLminus1 reduced the survival by approximately 30(Plt0001) A reduction in cell survival greater than 50 wasobserved with concentrations higher than 20mgmLminus1 (Plt0001)

During incubation with all concentrations of coumarinsthe decrease in the percentage of live cells was accompaniedby a proportional increase in the percentage of apoptotic andnecrotic cells (Table 1) Treatment with the lowest concentra-tion did not produce any important change in the relationshipbetween live and dead cells Treatment of cells with concen-trations higher than 5 mg mLminus1 gave a higher increase in thepercentage of apoptotic cells than the increase in necroticcells (Table 1) All concentrations gave a higher percentageof apoptotic cells than necrotic cells (P lt 0001)

Results



Cell cycle analysis

Cell cycle analysis following treatment with coumarins(mammea ABA and mammea ABB) showed that duringincubation with the extract the proportion of the BMK cellpopulation in each of G1 and S phases varied with the con-centration of the extract (Figure 3) The proportion of G1phase cells reduced while the percentage of cells at S phaseof the cell cycle increased (Figure 3) This change wasdependent on the concentration of the coumarins and wasaccompanied by the appearance of apoptotic cells at the sub-phase G1 (data not shown)

Treatment with the lower concentrations of coumarins(3 and 5 mg mLminus1) caused a reduction in G1 and G2M phasesafter 24 h incubation (Figure 3 P lt 005) Concentrations ofcoumarins higher than 5 mg mLminus1 caused an almost 50reduction of G1 phase cells (P lt 005) In contrast a signific-ant increase of the percentage in S phase cells was observedwith all concentrations P lt 0001

Mitotic index after treatment with coumarins

The coumarins (mammea ABA and mammea ABB) at allchosen concentrations reduced the percentage of cell divisions

or led to their almost total inhibition in a concentration-dependent manner (Figure 4) The lowest concentrations ofcoumarins (3 and 5 mg mLminus1) lowered the mitotic activity ofthe BMK cells by 18 and 30 respectively after incuba-tion for 24 h However concentrations higher than 8 mg mLminus1

produced a reduction of cell division in a dose-dependentmanner The two highest concentrations used (20 and24 mg mLminus1) caused almost total inhibition of cell division(P lt 0001)


The subcutaneous injection of viable BMK cells led to thedevelopment of tumours in mice Tumour growth did not pro-duce any significant change in body weight compared withthe control group Similarly it did not lead to the death of anyanimals

Treatment with coumarins caused a reduction in the devel-opment of tumours this was evident seven days after thetreatment started (Figure 5) Tumours reached an average size

Figure 2 Changes in cell survival after incubation for 24 h in differ-ent concentrations of coumarins from Calophyllum brasiliense(control = 100 survival) The results are presented as means plusmn sd ofthree independent experiments P lt 0001 as compared with all groupsP lt 0001 as compared with control 3 16 20 and 24 mg mLminus1

120

100

80

60

40

20

0

Surv

ival

(

of

con

tro

l)

Concn (microg mLndash1)

lowastlowast

lowast

lowastlowast

Control 3 5 8 10 16 20 24

Table 1 Changes in the percentage of normal apoptotic and necroticBMK cells after incubation for 24 h in different concentrations ofcoumarins from Calophyllum brasiliense extract

Concn extracted coumarins (mg mL-1)

Normal cells ()

Apoptotic cells ()

Necrotic cells ()

Control 87 plusmn 65 85 plusmn 12 45 plusmn 12 3 86 plusmn 32 10 plusmn 21 43 plusmn 14 5 79 plusmn 33 16 plusmn 21 55 plusmn 12 8 74 plusmn 37 18 plusmn 28 87 plusmn 30 10 70 plusmn 34 21 plusmn 29 90 plusmn 21 16 64 plusmn 65 24 plusmn 25 105 plusmn 2620 62 plusmn 41 28 plusmn 33 97 plusmn 38 24 52 plusmn 53 30 plusmn 40 201 plusmn 20

Figure 3 Percentage of cells in G1 S G2M phases of the cell cycleafter incubation for 24 h in different concentrations of coumarins fromCalophyllum brasiliense The results are presented as means plusmn sd ofthree independent experiments P lt 0001 vs control values

Cel

l po

pu

lati

on

(

)

80

60

40

20

0


Control 3 5 8 10 16 20 24

G1SG2M

lowast

lowastlowast lowast lowast lowast lowast

lowastlowast lowast lowast lowast

lowastlowast

lowast

Figure 4 Changes in the mitotic index of cells after incubation for 24 hin different concentrations of coumarins from Calophyllum brasilienseThe results are presented as means plusmn sd of three independent experi-ments P lt 0001 as compared with all groups P lt 0001 as comparedwith 3 16 20 and 24 mg mLminus1 ampP lt 0001 as compared with 3 5 8 and10 mg mLminus1

6

5

4

3

2

1

0

Mit

oti

c in

dex

(

)

Control 3 5 8 10 16 20 24

lowastlowast

lowast

lowastlowastamp

ampamp

lowastlowast




of 8 mm in animals treated with coumarins Compared withanimals with no treatment a reduction of 83 was observedin tumour size at the end of treatment with coumarins and thetumour size was approximately 2 mm when the animals werekilled In contrast vincristine stopped the development oftumours which was evident after the first dose (Figure 5)The tumours reached a size of 6 mm (average) during thetreatment with vincristine and they continued to reduce insize after the treatment A reduction of 100 was observed intumour size at the end of four weeks

Histopathological analysis revealed the presence of meta-static cells in hepatic tissue and to a lesser degree in kidneytissue in tumour-bearing mice receiving no treatment (datanot shown) Animals with tumours and treated with cou-marins did not show any evidence of metastasis to any of thetissues analysed including the liver Animals with tumourand treated with vincristine had livers showing large areas ofmetastatic cells (data not shown)

Cancer is one of the main causes of death in the world Tworelated fields are being explored through basic and clinicalresearch the identification of the molecular mechanismsinvolved in the illness and the discovery of drugs for preven-tion or treatment (Kirsner 2003) In the area of drug discov-ery there is increasing interest in cancer-prevention agents ofplant origin (Newman et al 2003)

Coumarins comprise a vast array of biologically activecompounds ubiquitous in plants many of which have beenused in traditional medicine for thousands of years The cou-marins constitute an important class of compounds with sev-eral types of pharmacological actions possessing anticanceranti-HIV anticoagulant spasmolytic and antibacterial activ-ity among others However their most prominent actions aretheir antioxidant and antiproliferative effects (Mattern et al1999 Kostova amp Momekov 2006)

Antineoplastic drugs are designed either to inhibit abnor-mal cell proliferation or to cause the death of abnormal cells

Due to the complex biochemical pathways and the specificphases of cellular life cycles there are numerous opportuni-ties for these drugs to exert a beneficial effect Some types ofcoumarin show antitumour activity in-vivo and in-vitro (Guiletet al 2000 Kawaii et al 2001 Finn et al 2002) however therehave been no investigations regarding the effect of coumarinsfrom C brasiliense on the mitotic index survival apoptosisinduction or cell cycle of cells in-vitro The results of ourinvestigation have shown that the coumarins isolated fromC brasiliensemdasha mixture of mammea ABA and ABBmdashwere capable of producing antiproliferative and cytotoxiceffects in-vitro and reduced tumour development in mice

Our results showed that coumarins at concentrations of5ndash10 mg mLminus1 had a cytotoxic effect on BMK cells (30)However with higher concentrations (15ndash24 mg mLminus1) thecytotoxic effect was the greatest The cytotoxicity producedby other types of coumarin have been reported using a non-small-cell bronchial carcinoma line (Kofinas et al 1998Loacutepez-Gonzaacutelez et al 2000) carcinoma epidermoid cells(Guilet et al 2001) human renal cell lines (Kawase et al2005) and cells from the uterine cervix (HeLa) larynx(Hep2) prostate gland (PC3) lymphoma (K562) and neuraltissue (U251) (Reyes-Chilpa et al 2004)

One aspect of neoplastic cells is their proclivity to replicateDepending on their mechanism of action antineoplastic agentsmay exert their effects specifically during one of the phases orthey may act during any phase of the cell cycle to cause celldeath (Dictor etal 2004) We analysed the cell cycle in cellstreated with coumarins Interestingly in BMK cells we observedarrest in the S-phase of the cell cycle Damage to DNA may pre-vent replication by altering DNA RNA or their function Agentsthat work by this mechanism inhibit the proliferation of neoplas-tic cells (they cannot divide) Other drugs may cause damage toDNA which can result in cell death After treatment inhibitionof the mitotic division of BMK cells occurred The inhibitiondepended on the concentration of the coumarins This inhibitionwas accompanied by the appearance of large numbers of apop-totic cells Therefore it can be speculated that the cells enterapoptosis at the border of the G1 phase and cells that have alter-ations in their DNA are arrested so to repair the damage

The effects of coumarins on the cell cycle have been stud-ied previously Coumarins and 7-hydroxycoumarin have anti-tumour actions in-vitro and in-vivo (Marshall etal 1999 Lacy ampOrsquoKennedy 2004 Elinos-Baez et al 2005) Coumarins and7-hydroxycoumarin inhibit cell growth by inducing cell cyclearrest in the G1 phase in lung carcinoma cell lines (Loacutepez-Gonzaacutelez et al 2004) Jimenez-Orozco et al (2001) observedthat 7-hydroxycoumarin 1 mM inhibited the G1S transition ofthe cell cycle in the human lung adenocarcinoma cell lineA-427 They also found that 7-hydroxycoumarin significantlyreduced cyclin D1 expression which appeared to indicate anaction of 7-hydroxycoumarin in early events of phase G1Finally studies using nitro-derivatives of coumarins showedthat they caused dose-dependent inhibition of the S-phaseregulatory protein cyclin A in an irreversible manner There-fore these and other nitro-derivatives of 7-hydroxycoumarinpossess selective and irreversible cytotoxicity (Finn et al2004) Recent studies using C brasiliense collected in Brazilhave shown that it inhibited the growth of leukaemic cells andinduced caspase-mediated and p53-independent apoptosis

Discussion

Figure 5 Effect of coumarins on tumour size Viable BMK cells wereinjected subcutaneously into mice The size of the tumours wasmeasured every third day during the treatment Mean tumour diameter(mm) was expressed relative to time after cell injection The results arepresented as means plusmn sd n = 10 animals

20

16

12

8

4

0

TumourTumour + vincristineTumour + coumarins

BMK cellsinjection

Mea

n t

um

ou

r d

iam

eter

(m

m)

Vincristine

Start

Coumarins

Killed

3 6 9 12 15 18 21 24 27 30 33 36 39 Time (day)



(Kimura et al 2005) Our results agreed with those previousstudies we found that coumarins isolated from C brasiliensecollected in Mexico displayed potent cytotoxic and antiprolif-erative activity in-vitro

Tumorigenesis is an extremely complex multistep processthat leads to the pathological expansion of a tissue Tumourcells gain an unlimited replicative potential because of an imbal-ance between finely tuned proliferate growth-inhibitory andapoptotic signals Animal models that accurately represent thecellular and molecular changes associated with the initiationand progression of cancer have significant potential to facilitatethe development of better methods for the early detection andtreatment of this illness Chemotherapeutic drugs can be dividedinto several categories based on the way they act Antineoplas-tic agents act primarily on rapidly dividing and growing cells

To date there are no reports regarding the assay of theeffects of coumarins in animal models In this study we haveevaluated the antitumoral effects of coumarins in tumoursinduced by subcutaneous injection of BMK cells into miceOur in-vitro results demonstrated that coumarins isolatedfrom C brasiliense had cytotoxic and antiproliferativeeffects Additionally we demonstrated that coumarins werebetter than vincristine in inhibiting tumour growth

Conclusion

Coumarins isolated from C brasiliense had cytotoxic effectsand were able to alter the G1S transition of the cell cycle in-vitro In addition they had antitumour effects Thereforecoumarins have the potential to be used alone or in combina-tion with other antineoplastic drugs and they might increasethe effectiveness of other treatments against cancer

Agata N Nogi H Milhollen M Kharbanda S Kufe D (2004)2-(8-Hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl)propionicacid a small molecule isocoumarin potentiates dexamethasone-induced apoptosis of human multiple myeloma cells Cancer Res64 8512ndash8516

Burgos A Alcaide A Alcoba C Azcona J M Garrido JLorente C Moreno E Murillo E Olsina-Pavia J Olsina-Kissler J Samaniego E Serra M (1999) Comparative study ofthe clinical efficacy of two different coumarin dosages in the man-agement of arm lymphedema after treatment for breast cancerLymphology 32 1ndash2

Chaya N Terauchi K Yamagata Y Kinjo J Okabe H (2004)Antiproliferative constituents in plants 141) Coumarins and acri-done alkaloids from Boenninghausenia japonica NAKAI BiolPharm Bull 27 1312ndash1316

Committee on Care and Use of Laboratory Animals of the Institutefor Laboratory Animal Resources (1986) Commission on Life Sci-ences National Research Council Guide for the care and use oflaboratory animals Publication 86-23 18 and the Animal WelfareAct of 1966 as amended

Darzynkiewicz J Robinson P Crissman H A (2001) Analysis ofcell cycle In Flow cytometry Part A Vol 41 Plenum PressLondon pp 223ndash254

Dehenhardt K Cheng G Lindsten T White E (2002) Bax andBak mediate p53-independent suppression of tumorigenesis Can-cer Cell 2 193ndash203

Dictor M Ehribger M Mertens F Akervall J Wenneberg JDorai T Aggarwal B B (2004) Role of chemopreventive agentsin cancer therapy Cancer Lett 215 129ndash140

Elinos-Baez C M Leon F Santos E (2005) Effects of coumarinand 7 OH-coumarin on bcl-2 and Bax expression in two humanlung cancer cell lines in vitro Cell Biol 29 703ndash708

Finn G J Kenealy E Creaven B S Egan D A (2002) In vitrocytotoxic potential and mechanism of action of selected cou-marins using human renal cell lines Cancer Lett 183 61ndash68

Finn G J Creaven B S Egan D A (2004) A study of the role ofcell cycle events mediating the action of coumarin derivatives inhuman malignant melanoma cells Cancer Lett 214 43ndash54

Guilet D Heacutelesbeux J J Seacuteraphin D Seacutevenet T Richomme PBruneton J (2000) Novel cytotoxic 4-phenylfuranocoumarinsfrom Calophyllum dispar J Nat Prod 64 10ndash15

Guilet D Seacuteraphin D Rondeau D Richomme P Bruneton J(2001) Cytotoxic coumarins from Calophyllum dispar Phyto-chemistry 58 571ndash575

Ishihara M Yokote Y Sakagami M (2006) Quantitative struc-ture-cytotoxicity relationship analysis of coumarin and its deriva-tives by semiempirical molecular orbital method Anticancer Res26 2883ndash2886

Ito C Itoigawa M Mishina Y Filho V C Mukainaka TTokuda H Nishino H Furukawa H (2002) Chemical constitu-ents of Calophyllum brasiliense structure elucidation of sevennew xanthones and their cancer chemopreventive activity J NatProd 65 267ndash272

Ito C Itoigawa M Mishina Y Filho V C Enjo F Tokuda HNishino H Furukawa H (2003) Chemical constituents of Calo-phyllum brasiliense 2 Structure of three new coumarins and can-cer chemopreventive activity of 4-substituted coumarins J NatProd 66 368ndash371

Jimenez-Orozco F A Loacutepez-Gonzaacutelez J S Nieto-Rodriguez AVelasco-Velazquez M A Molina-Guarneros J A Mendoza-Patino N Garcia-Mondragon M J Elizalde-Galvan P Leon-Cedeno F Mandoki J J (2001) Decrease of cyclin D1 in thehuman lung adenocarcinoma cell line A-427 by 7-hydroxycou-marin Lung Cancer 34 185ndash194

Kashman Y Gustafson K R Fuller R W Cardellina J HMcMahon J B Currens M J Buckheit R W Hughes S HCragg G M Boyd M R (1992) The calanolides a novelHIV-inhibitory class of coumarin derivatives from the tropicalrainforest tree Calophyllum lanigerum J Med Chem 352735ndash2743

Kawaii S Tomono Y Ogawa K Sugiura M Yano MYoshizawa Y (2001) The antiproliferative effect of coumarins onseveral cancer cell lines Anticancer Res 21 917ndash924

Kawase M Sakagami H Motohashi N Hauer H Chatterjee S SSpengler G Vigyikanne A V Molnar A Molnar J (2005)Coumarin derivatives with tumor-specific cytotoxicity and multi-drug resistance reversal activity In Vivo 19 705ndash711

Kimura S Ito C Jyoko N Segawa H Kuroda J Okada MAdachi S Nakahata T Yuasa T Filho V C Furukawa HMaekawa T (2005) Inhibition of leukemic cell growth by a novelanti-cancer drug (GUT-70) from Calophyllum brasiliense that actsby induction of apoptosis Int J Cancer 113 158ndash165

Kirsner M K (2003) Cancer new therapies and new approaches torecurring problems AANA J 71 55ndash62

Kofinas C Chinou I Loukis A Harvala C Roussakis CMaillard M Hostettman K (1998) Cytotoxic coumarins fromthe aerial parts of Tordylium apulum and their effects on a non-small-cell bronchial carcinoma line Planta Med 64 174ndash176

Koopman G Reutelingsperger C P M Kuijten G A MKeehnen R M J Pals S T van Oers M H J (1994) Annexin Vfor flow cytometric detection of phosphatidylserine expression onB cells undergoing apoptosis Blood 84 1415ndash1420

References



Kostova I Momekov G (2006) New zirconium (IV) complexes ofcoumarins with cytotoxic activity Eur J Med Chem 41 717ndash726

Lacy A OrsquoKennedy R (2004) Studies on coumarins and cou-marin-related compounds to determine their therapeutic role in thetreatment of cancer Curr Pharm Des 10 3797ndash3811

Liu R H (2004) Potential synergy of phytochemicals in cancer pre-vention mechanism of action J Nutr 134 3479Sndash3485S

Loacutepez-Gonzaacutelez J S Garcia H P Caacutezares D A Guarneros J MMandoki J J Morales F M (2000) Efecto en el ciclo celular de liacuteneasde adenocarcinoma pulmonar por cumarina y 7-hidroxicumarina RevInst Nal Enf Resp Mex 13 92ndash197

Loacutepez-Gonzaacutelez J S Prado-Garciacutea H Aguilar-Caacutezares D Molina-Guarneros J A Morales-Fuentes J Mandoki J J (2004) Apoptosisand cell cycle disturbances induced by coumarin and 7-hydroxycou-marin on human lung carcinoma cell lines Lung Cancer 43 275ndash283

Marshall M E Kervin K Benefield C Umerani A Albainy-Jenei SZhao Q (1994) Growth-inhibitor effects of coumarin (12-benzo-pyrone) and 7-hydroxycoumarin in human malignant cell lines invitro J Cancer Res Clin Oncol 120 3ndash10

Mattern U Luer P Kreusch D (1999) Biosynthesis of coumarinTaken from Comprehensive Natural Products Chemistry 1st ednElsevier pp 623ndash637

McKee T C Covington C D Fuller R W Bokesch H R YoungS Cardellina J H I Kadushin M R Soejarto D D Stevens P FCragg G M Boyd M R (1998) Pyranocoumarins from tropical spe-cies of the genus Calophyllum a chemotaxonomic study of extracts inthe National Cancer Institute Collection J Nat Prod 6 1252ndash1256

Newman D J Cragg G M Snader K M (2003) Natural productsas sources of new drugs over the period 1981ndash2002 J Nat Prod66 1022ndash1037

Patil A D Freyer A J Eggleston D S Haltiwanger R CBean M F Taylor P B Caranfa M J Breen A L Bartus H R

Johnson R K (1993) The inophyllums novel inhibitors of HIV-1reverse transcriptase isolated from Malaysian tree Calophylluminophyllum Linn J Med Chem 36 4131ndash4138

Reddy N S Reddy M M Cosenza S Gumireddy K Bell S CReddy E P Ramana M V (2004) Synthesis of new coumarin3-(N-aryl) sulfonamides and their anticancer activity BioorganMed Chem Lett 14 4093ndash4097

Reyes-Chilpa R Estrada-Muntildeiz E Ramiacuterez-Apan T Amekraz BAumelas A Jankowski Ch Vazquez-Torres M (2004) Cyto-toxic effects of mammea type coumarins from Calophyllum bra-siliense Life Sci 75 1635ndash1647

Riviere C Goossens L Pommery N Fourneau C Delelis AHenichart J P (2006) Antiproliferative effects of isopentenylatedcoumarins isolated from Phellolophium madagascariense BakerNat Prod Res 20 909ndash916

Sovak M Seligson A L Konas M Hajduch M Dolezal MMachala M Nagourney R (2002) Herbal composition PC-SPESfor management of prostate cancer identification of active princi-ples J Natl Cancer Inst 94 1275ndash1281

Stevens P F (1980) A revision of the old world species of Calo-phyllum (Guttiferae) J Arnold Arb 61 117ndash171

Tsuda H Ohshima Y Nomoto H Fujita K Matsuda E Iigo MTakasuka N Moore M A (2004) Cancer prevention by naturalcompounds Drug Metab Pharmacokinet 19 245ndash263

Wang H Z Chang C H Lin C P Tsai M C (1996) UsingMTT viability assay to test the cytotoxicity of antibiotics and ster-oid to cultured porcine corneal endothelial cells J Ocul Pharma-col Ther 12 35ndash43

Yang H Protiva P Gil R R Jiang B Bagget T S Basile M JReynertson K A Westein I B Kennelly E J (2005) Antioxi-dant and cytotoxic isoprenylated coumarins from Mammea ameri-cana Planta Med 71 852ndash860



ABA ABB BBB COA COB BBA cyclo F BBB cyclo Fand isomammeigin (Reyes-Chilpa etal 2004) Recent studieshave shown that mammea ABA and ABB have high cytotoxicactivity against some tumour cell lines (Reyes-Chilpa etal 2004)

We have studied the biological effects of the antiprolifera-tive activity of mammea ABA and ABB isolated fromC brasiliense on the survival cell cycle and apoptosis of BMKcells We attempted to define their antitumour effects in mice

Chemicals

Vincristine was obtained from Lemery Laboratories (Mexico)DMEM fetal calf serum MTT ((3-(45-dimethyl-2-thia-zolyl)-25-diphenyl-2H-tetrazolium bromide) trypsin trypsininhibitor ribonuclease A (RNase) and propidium iodidewere obtained from Sigma Chemical Co (USA)

Extraction and isolation of mammea ABA and ABB from C brasiliense

C brasiliense (Clusiaceae) was collected at Ejido BenignoMendoza Sierra de Santa Marta Veracruz Mexico A voucherspecimen was deposited in the Herbarium of the Instituto deInvestigaciones Biologicas Universidad Veracruzana at XalapaMexico labelled number 435 Dried leaves (2075g) wereextracted at room temperature over one week with hexane ace-tone and methanol successively Compounds were isolated afterspontaneous crystallization or by column chromatography onSilica Gel-60 and individualized by their spectroscopy (1HNMR 13C NMR IR UV) EIMS and CIMS The results of theseexperiments were compared with Reyes-Chilpa etal (2004)

Hexane extracts (703 g) While in solution the extract showed spontaneous precipita-tion giving a white powder (198 g) Part of this material(51 g) was treated with CH2Cl2 The insoluble part was amixture (583 mg) of friedelin and canophyllol and the solu-ble part afforded a mixture (4 g) of coumarins of two mam-mea types mammea ABA and mammea ABB The extractwas then concentrated in-vacuo (353 g) and 94 g subjectedto column chromatography (200 g) Elution with hexane firstyielded friedelin (459 mg) then a mixture of coumarins(5 mg mammea ABA and mammea ABB)

Identification and chemical structure of mammea ABA and mammea ABB

The compounds isolated after spontaneous crystallization orcolumn chromatography were individually identified Thepurification of compounds (mp 122ndash124degC) was obtained byHPLC using an ODS-column and elution with 90 MeOHChemical structures are shown in Figure 1 The identificationof the mammeas was as follows

1H NMR (200 MHz CDCl3TMS) 756 m 3H (Ar)741 m 2H (Ar) 60 s 1H (H-3) 595 s 1H (OH-5) Isopre-nyl on C-6 509 tm J = 69 Hz 1H (CH) 329 d J = 69 Hz2H (CH2) 165 and 170 both s and 3H (2 CH3)

1 1461 s 1H (OH-7) R = 3-methylbutyryl 319 d J =67 Hz 2H (CH2) 232 m J = 67 Hz 1H (CH) 106 dJ = 66 Hz 6H (2 CH3)

2 1457 s 1H (OH-7) R = 2-methylbutyryl (ABB)395 m J = 66 Hz 1H (CH-CH3) 129 d J = 67 Hz 3H (CH-CH3) 194 m 2H (CH2) 101 t J = 72 Hz 3H (CH3)

EMIE 70 eV mz () 406 M+ (964) [C25H26O5]+363 (556) 351 (760) 293 (100) IR n max (KBr)3985 (OH) 2964 2932 2872 (C-H) 1729 (C= O) 1724(C=O) 1614 (C = C) 1556 (C = C) 1390 (C-O)

For the pharmacological studies a mixture of coumarinsmammea ABA and mammea ABB at a ratio of 21 wasused The mix of mammeas (5 mg mLminus1) was prepared in01 dimethyl sulfoxide (DMSO) (Aldrich USA) at roomtemperature

Cell culture and pharmacological treatments

For in-vitro studies BMK cells were used derived from babymouse kidney These cells were kindly donated by ProfessorSophie Hallez from Brussels University Belgium Cells werecultured in minimal essential medium (Gibco-BRL IncGrand Island NY) supplemented with nonessential amino acids(Gibco-BRL Inc Grand Island NY) 10 fetal calf serum(Gibson-BRL Inc Grand Island NY) 2 mol Lminus1 L-glutamineand antibiotics Cells were plated in 100-mm culture dishes(106 cellsdish) and maintained at 37degC in an atmosphere of5 CO2 in humidified air Subcultures were obtained bytrypsinization (0025 trypsin solution containing 001NN-diethyldithiocarbamic acid sodium salt and EDTA)

For cytological investigation 105 cells were plated andallowed to adhere overnight The medium was then aspiratedand replaced with medium containing vehicle alone ormedium containing the mixture of mammeas (3 5 8 10 1620 and 24 mg mLminus1) and the cells were then incubated for24 h After incubation the cells were collected for furthercytological investigation

Assessment of cell viability

BMK cells were incubated in 96-well plates After 24 h themedium was removed the cells were washed twice withphosphate-buffered saline (PBS) and then incubated with themixture of coumarins (3 5 8 10 16 20 and 24 mg mLminus1)

Materials and Methods

Figure 1 Coumarins isolated from Calophyllum brasiliense leaves

Mammea ABA

HOHO

OHOH

6

78 1

OO

OO

O O

5 43

2 6

7 8 1

5 43

2

Mammea ABB




Mitotic activity


















Results



Cell cycle analysis











120

100

80

60

40

20

0

Surv

ival

(

of

con

tro

l)


lowastlowast

lowast

lowastlowast

Control 3 5 8 10 16 20 24



Normal cells ()

Apoptotic cells ()

Necrotic cells ()



Cel

l po

pu

lati

on

(

)

80

60

40

20

0


Control 3 5 8 10 16 20 24

G1SG2M

lowast



lowastlowast

lowast


6

5

4

3

2

1

0

Mit

oti

c in

dex

(

)

Control 3 5 8 10 16 20 24

lowastlowast

lowast

lowastlowastamp

ampamp

lowastlowast













Discussion


20

16

12

8

4

0


BMK cellsinjection

Mea

n t

um

ou

r d

iam

eter

(m

m)

Vincristine

Start

Coumarins

Killed

3 6 9 12 15 18 21 24 27 30 33 36 39 Time (day)






Conclusion

























References

























Mitotic activity


















Results



Cell cycle analysis











120

100

80

60

40

20

0

Surv

ival

(

of

con

tro

l)


lowastlowast

lowast

lowastlowast

Control 3 5 8 10 16 20 24



Normal cells ()

Apoptotic cells ()

Necrotic cells ()



Cel

l po

pu

lati

on

(

)

80

60

40

20

0


Control 3 5 8 10 16 20 24

G1SG2M

lowast



lowastlowast

lowast


6

5

4

3

2

1

0

Mit

oti

c in

dex

(

)

Control 3 5 8 10 16 20 24

lowastlowast

lowast

lowastlowastamp

ampamp

lowastlowast













Discussion


20

16

12

8

4

0


BMK cellsinjection

Mea

n t

um

ou

r d

iam

eter

(m

m)

Vincristine

Start

Coumarins

Killed

3 6 9 12 15 18 21 24 27 30 33 36 39 Time (day)






Conclusion

























References
























Cell cycle analysis











120

100

80

60

40

20

0

Surv

ival

(

of

con

tro

l)


lowastlowast

lowast

lowastlowast

Control 3 5 8 10 16 20 24



Normal cells ()

Apoptotic cells ()

Necrotic cells ()



Cel

l po

pu

lati

on

(

)

80

60

40

20

0


Control 3 5 8 10 16 20 24

G1SG2M

lowast



lowastlowast

lowast


6

5

4

3

2

1

0

Mit

oti

c in

dex

(

)

Control 3 5 8 10 16 20 24

lowastlowast

lowast

lowastlowastamp

ampamp

lowastlowast













Discussion


20

16

12

8

4

0


BMK cellsinjection

Mea

n t

um

ou

r d

iam

eter

(m

m)

Vincristine

Start

Coumarins

Killed

3 6 9 12 15 18 21 24 27 30 33 36 39 Time (day)






Conclusion

























References

































Discussion


20

16

12

8

4

0


BMK cellsinjection

Mea

n t

um

ou

r d

iam

eter

(m

m)

Vincristine

Start

Coumarins

Killed

3 6 9 12 15 18 21 24 27 30 33 36 39 Time (day)






Conclusion

























References



























Conclusion

























References












































Science

2007 antiproliferative, cytotoxic and antitumour activity