In vitro Cytotoxicity of Norditerpenoid AlkaloidsConcepcion de Inesa, Matias Reinab, Jose A. Gavfnc, andAzucena Gonzalez-Colomaa,*a Centro de Ciencias Medioambientales, CSIC Serrano 115-dpdo., 28006 Madrid, Spain.

Fax: 34-915640800. E-mail: azu@ccma.csic.esb Instituto de Productos Naturales y Agrobiologia, CSIC, Avenidada Astrofisico Francisco

Sanchez 3, 38206 La Laguna, Tenerife, Spain ,C Instituto Universitario de Bio-Organica "Antonio Gonzalez'.!, Universidad de La Laguna,

Avenida Astroffsico Francisco Sanchez 2, 38206 La Laguna, Tenerife, Spain.* Author for correspondence and reprint requests

z. Natllrforsch. 61c, 11-18 (2006); received June 16/July 27, 2005

Forty-three norditerpenoid alkaloids isolated from Aconitum, Delphinium and Consolidaspecies have been evaluated for their cytotoxic effects on the tumor cell lines CT26 (murinecolon adenocarcinoma), SW480 (human colon adenocarcinoma), HeLa (human cervical ade-nocarcinoma), SkMel25 (human melanoma) and SkMel28 (human malignant melanoma)with several multidrug resistance mechanisms and the non-tumor cell line CHO (Chinesehamster ovary cells). Neoline (5), 8-0-methylcolumbianine (6), 1,14-diacetylcardiopetaline(9), 18-0-demethylpubescenine (13), 14-deacetylpubescenine (14), pubescenine (15), 14-de-acetylajadine (25), lycoctonine (26), browniine (28), delphatine (29), dehydrotakaosamine(34), and ajadelphinine (37) exhibited selective cytotoxicity to cancerous versus non-cancer-ous cells. Some of these compounds had an irrever~ible effect on SW480 (5, 15, 25, 26, and34), HeLa (15, 34, and 37) and SkMel25 (15 and 34) cell lines. In order to gain insights intothe mechanism of irreversible cytotoxic action o..these compounds we compared the cellviability by means of the 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(M1T) and the acid phosphatase (AP) methods. Our results suggest that the effects of thesecompounds could be related to the inhibition of ATP production.

Key words: Norditerpenoid Alkaloids, Cytotoxicity, Thmor Cells

Introduction

Plant species of the genera Aconitum, Delphin-ium, and Consolida are known sources of C19-norditerpene (NDAs) and Czo-diterpene alkaloids(DAs) and they are of pharmacological and eco-nomic importance (Atta-ur-Rahman and Choud-hary, 1999; Panter et al., 2002). NDAs act as potentnicotinic cholinergic receptor (nAcChR) agonistsand antagonists in invertebrates, including insects,and vertebrates (see Panter et al., 2002; Seitz andAmen, 1998). The insecticidal and antifeedant ac-tivity of NDAs (Jennings et al., 1986; Ulubelenetal., 2001; Gonzalez-Coloma etal., 2004a) suggesta plant defensive role played by these compounds.NDAs are well known pharmacologically for theiranti-inflammatory, analgesic, anti-arrythmia andantifungal actions (Atta-ur-Rahman and Choud-hary, 1999). The biological actions of DAs are lessknown. There are a few reports on their plant de-fensive and pharmacological properties, includingtheir effects on Trypanosoma cruzi epimastigoteforms and anti-leishmanial properties (Bessonova

and Shaidkhozaeva, 2000; Gonzalez-Coloma et at.,1998, 2004b; Gonzalez et at., 2005; Li et at., 2002a,b; Ulubelen et at., 2001), however, their neurotoxiceffects are unknown. The selective cytotoxic ef-fects of some of these structures indicate thatNDAs and DAs can act on biological targets otherthan neuroreceptors with strong molecularselect-'ivity (Gonzalez-Coloma et at., 2004a, b). However,little is known on their effects on human tumorcells.

Here we report on the cytotoxic effects of 43NDAs isolated from Aconitum, Detphinium andConsotida species (Figs. 1-4) against mammalianCHO cells and the tumor cell lines Cf26 (murinecolon adenocarcinoma), SW480 (human colonadenocarcinoma), HeLa (human cervical adeno-carcinoma), SkMel25 (human melanoma) andSkMe128 (human malignant melanoma). Thesecell lines express different resistance mechanismsincluding the multidrug resi.stance (MDR) pheno-type, due to the overexpression of any of theenergy-dependent drug efflux transmembraneproteins, such as the P-glycoprotein (Pgp), or the

0939-5075/2006/0100-0011 $ 06.00 @ 2006 Verlag der Zeitschrift flir Naturforschung, Thbingen . http://www.znaturforsch.com .D~

C.~ de Ines et al.. Cytotoxicity of Norditerpenoid Alkaloids

multidrug resistance protein (MRP1) (Higgins,1992; Ling, 1997; Cole and Deeley, 1998; Kleinet al., 1999), and the intracellular glutathione/glutathione S-transferase detoxification system(GSH/GST) which protects and detoxifies cellsfrom highly reactive free radicals and organic per-oxides and metabolizes xenobiotics (Zhang et al.,1998). Among the cell lines used, HeLa expressesintermediate levels of GSH-conjugate export ac-tivity; CT26 expresses low levels of Pgp and MRP1and murine GSTs; SkMe128 expresses GSHpx andGSTs and low levels of MRP1; SW480 has ele-vated levels of Pgp with low levels of MRP1 andthe y-glutamylcysteine synthetase (y-GCS) re-sponsible for de novo synthesis of GSH (for refer-ences see De Ines et al., 2004). The SkMe125 cellline was included in this study for comparativepurposes since these cells have a low invasive andmetastatic potential (Suter et al., 1985; Rass et al.,2001) as opposite- to SkMe128 cells. Additionally,in order to gain insight into the cytotoxic a~ion ofthe active compounds we compared the cell viabil-ity by means of the MTT [3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] and theAP (acid phosphatase) methods.

Cytotoxicity assaysCell viability was analyzed by means of an adap-

tation of the MTr colorimetric assay method(Mossman, 1983) as previously described (De Ineset al., 2004). In brief, cells in the logarithmicgrowth phase were added to 96-well flat-bottommicrotiter plates (Falcon) and incubated for 6 dwith different concentrations of the compoundsdissolved in absolute ethanol. For reversibility ex-periments, cells were incubated with the minimalcytotoxic concentration (MIC) of each compound,washed three times with fresh culture medium andcultured in compound-free medium for differentperiods of time. Three independent experimentswere carried out in duplicate.

Acid phosphatase (AP) method

In order to gain insights about the mechanismof action of the irreversibly cytotqxic compounds,cell viability was also measured by the AP methodwhich determines the cellular acid phosphatase ac-tivity (Martin and Clynes, 1991). Cells were incu-bated for 6 d according to De Ines et al. (2004).To validate the experiment, the sensitive SkMel25cells were incubated with Taxol@ (dissolved in ab-solute ethanol) and rotenone (dissolved in dime-thyl sulfoxide, DMSO). The viability of SkMel25cells treated under the same conditions with theresidual concentration of DMSO was ~ 95%.Three independent experiments were carried outin duplicate.

Materials and Methods

Materials

Compounds 1-43 (Figs.1-4) were isolatedfrom Aconitum, Consolida and Delphinium spe-cies (Gonzalez-Coloma et al., 2004a). RPMI 1640,fetal bovine serum (FBS), L-glutamine and peni-cillin/streptomycin were from GIBCO-BRL. Ro-tenone, M1T and p-nitrophenylphosphate werefrom Sigma-Aldrich. Taxol was a gift from Dr. L.Gunatilaka (University of Arizona, USA). Thecompounds were dissolved freshly and diluted inculture medium before their addition to the cellcultures.

Cell lines and culture conditions

Mammalian Chinese hamster ovary cells (CHO)(a gift from Dr. Pajares, ICB, CSIC, Spain), mu-rine colon adenocarcinoma (Cf26), human colonadenocarcinoma (SW480), human cervical adeno-carcinoma (HeLa), human melanoma (SkMel25)and human malignant melanoma (SkMel28) (fromDeutsches Krebsforschungszentrum, DKFZ, Hei-delberg, Germany) cells were grown as previouslydescribed (De Ines et al.. 2004).

Results and Discussion

Table I shows the minim~l inhibitory concentra-tion (MIC) of the test compounds required to pro-duce a cytotoxic effect on the different cell lines.CHO cells were sensitive to a few compounds ran-domly distributed among the chemical classes withMIC values ranking as follows: 36 > 30 > 35, 41 >Z4 > 11. Overall Cf26 and SW480 cells were sensi-tive to the largest number of compounds (33%)followed by SkMel25 (310;0), HeLa (24%) andSkMel28 (12%) cells. HeLa cells showed the low-est MIC value. The different cellular range ofaction of these compounds could be related to fac-tors ~uch as intracellular transportation, metabo-lism, inactivation and receptor geometry.

11ie cytotoxicity of the test alkaloids followeddifferent patterns for each chemical class. Amongthe aconitine-type alkaloids (Fig. 1), 5 was themost active comoound (Hp.T.A ~W4RO f'T')~

C. de Ines et al.' Cytotoxicity of Norditerpenoid Alkaloids

Table I. Minimal inhibitory concentration (MIC) of the active test compounds, classified by chemical type, on severalmammalian cell lines.

Compound8 MIC [jlgiml]

CHO CT26 SW480 SkMeI25 SkMe128

569

1113141524252629303435363741

> 100> 100> 100

100> 100> 100> 100

50> 100> 100> 100

12.50> 100

256.25

> 10025

Aconitine-type 25

50

100

100

> 100

> 100

100

50

> 100

50

> 100

12.

6.

50

12.

50

50

12.50

100100

> 100> 100

2550

10050

> 100506.

2512.25100

6> 100> 100> 100

25> 100

50> 100

50> 100

10050O.

2512.12.

> 100

2550

> 100100

505050

> 100100

> 100> 100

1006.25

252525

100

> 100> 100> 100

10050

> 100> 100

50> 100> 100> 100

10025

> 1006.25

> 100> 100

Lycoctonine-type

Gadesine-type

Miscellaneous type

a Compounds 1-4, 7, 8, 10, 12, 16-23, 27, 28, 31-33, 38-40, 42 and 43 had MIC values> 100 /lg/ml for all the celllines tested.

R5 8R

IIR6

[""'--- IIIII R'R~'\\l,., 4

I/-' HzR

Rl R2 R3 R4OMe OR OMe OAcOMe OAc OMe OAcOMe OR OMe OCR2CR3OMe OR OMe OCH2CR3OR ROMe OROR R ROMeOR R R OROR R R OROAc R R OR

R5 R6OH OBzOH OBzOH OBzOH OHH OHH OHH OHH OHH OAc

R7OHOHOHOHHHHHH

R8OMeOMeOMeOMeOMeOMeOMe

HH

R9OMeOMeOMeOMeOMeOHHHH

Aconitine (1)3-Acetylaconitine (2)8-0-Ethyl-14-benzoylaconine (3)8-0-Ethylaconine (4)Neoline (5)8-0-Methylcolumbianine (6)Karak:oline (7)Cardiopetaline (8)1,14-Diacetylcardiopetaline (9)

Fig. 1. AcoJiitine-type structures.

SkMel25) followed by 6 (CT26, SW480, SkMel25)and 9 with low-moderate activity (CT26, SW480).Methylation at C-6 plus hydroxylation/methyla-tion at C-8 (5 and 6) determined the activity ofthese compounds. Acetylation at C-l and C-14 re-sulted in a moderate-low increase of activity (9 incontrast to the inactive 7 and 8). Compound 9 wascytotoxic to insect Sf9 cells (Gonzalez-ColomaetaZ.,2004a).

Among the lycoctonine-type alkaloids (Fig. 2),compound 11, acetylated at C-l and bearing OAc/Hgroups at C-14/C-18, moderately affected CHO,CT26, SW480, SkMe125 and SkMe128 cells, in con-trast to the inactive compound 10, hydroxylated atC-l and C-14. The C-6a epimers 13, 14, and 15,with OAc/OH, OMe/OH, OMe/OMe C-14/C-18combinations, affected a lower number. of cell lines(CT26.15: SW480.15.1l: HeLa. 13. 15: SkMe125.

5025

50

50

25

50

.25

,40

,5050

14 C. de Ines et ai.' Cytotoxicity of Norditerpenoid Alkaloids

R5OHOAcOHOAcOHOAcOMeOR

R6HHOMeOMeOMeOMeOMeOMe

R7HHHOHOMeOMeOMeOBz

OHOHOHOHOH'OWOMeOMeOMeOMeOMeOMeOMe

popopopopopopopopopopopopo

0000000000000

OMeOHOHOHOHOHOHOHOHOHOHOHOH

OAcOAcOMeOROMeOROAcOROMeOAcOROMeOMe

OMeOMeOMeOMeOMeOMeOMeOMeOMeOMeOMeOMeOMe

Cardiopetalidine (10)1,14-tJ-Acetylcardiopetalidine (U)8-0-Methylconsolarine (12)18-0-Demethylpubescenine (13)14-Deacetylpubescenine (14)Pubescenine (15)Consolidine (16)18-0- Benzoyl-18-0-demethyl-14-0-deacetylpubescenine (17)14-0-Acetyldeltatsine (18)14-0-Acetyldelcosine (19)Delsoline (20)Takaosamine (21)Gigactonine (22)Delcosine (23)Ajadine (24)14-Deacetylajadine (25)Lycoctonine (26)14-0-Acetyldelectinine (27)Browniine (28)Delphatine (29)Methyllycaconitine (30)

OMeOMeOMeOHOHOMeOCOPhNHAcOCOPhNHAcOHOHOMeOMe

"Vn OJ

poFig. 2. Lycoctonine-type structures.

13, 14, 15 and SkMe128, 13) with varying potencies.Compounds 24, 25, 26, 29 and 30, methylated atC-l, fJ-methylated at C-6 and bearing OAc, OH/OCOPhNHAc, OMe/OMe and OMe/methylsuc-cinylantranoyl C-14/C-18 combinations, affectedall the cell lines (CHO, 24, 26; Cf26 and SW480,30, 24, 26; HeLa and SkMel25, 25, 30 andSKMeI28, 24, 30) with varying potencies. Amongthese lycoctonine-type alkaloids, 10, 13, 14, 19 and23 resulted cytotoxic to insect Sf9 cells (Gonzalez-Coloma et al., 2004a), indicating cell-dependentselectivity of action for these compounds exceptfor 13 and 14.

The most active alkaloids were found among thegadesine-type (Fig. 3). All the cell lines respondedto 34, 36 and 35 with varying potencies. Com-pound 36 was the most cytotoxic to CHO andSkMel28 while 34 was the most cytotoxic to Cf26,

SW480, HeLa and SkMe125 cells, indicating a se-lective structure-dependent cytotoxicity for thischemical class of compounds. Hydroxylation atboth C-14/C-18 determined a strong cytotoxic ef-fect (34) while complete or partial methylation atC-14/C-18 (36 or 35) reduced the potency of thiseffect for the sensitive cell lines. Compound 36 wasalso cytotoxic to Sf9 cells (Gonzalez-Coloma etal., 2004a:J:

Among the miscellaneous compounds (Fig. 4),all the tumor cell lines, except SkMe128, respondedto 37, with. a 7,8-methylenedioxy group, followedby 41 with a 19-oxo and C-l/C-18 diacetyl group(the structurally related 20 and 22 were not cyto-toxic); this compound was also toxic to Sf9 cells(Gonzalez-Coloma et al., 2004a).

The selective cytotoxic effects of some struc-tures indicate that these compounds can act on

MeMeMeMeMeMeMeMeMeMeMeMeMe

'HHHHHHHHHHHHH

, ~ ..

C. de IDes et ai.. Cytotoxicity of Norditerpenoid Alkaloids 15

RS

. /''--, .- --

/', 3, ,, ,

" y" . 2 R

Rl ,R2OMe OHOMe OHOMe OHOMe OHOMe OHOMe OH

0

R3OHOHOHOHOHOH

R4OROBzOMeOROROMe

R5OMeOMeOMeOMeOMeOMe

R6HHOHOHOMeOMe

Gadesine (31)14-0-Benzoylgadesine (32)18-Hydroxy-14-0-methylgadesine (33)Dehydrotakaosamine (34)18-0-Methoxygadesine (35)Dehydrodelsoline (36)Fig. 3. Gadesine-type structures.

OMe OMe

~:Jl..If

RlOROROMe

R2 .OMeOHOH

AiadelDhinine (37) Thguaconitine (38)14-Demethyltuguaconitine (39)14-Demethyldelboxine (40)

OMeOMe

'ORhu

Ac~ 0--~1~~~1'= ~r:;- /1', I I ,

, iF' , " "'-C~/N

'- HIf - ~££

0 Ac OMe

1,18-0-Diacetyl-19-oxo-gigactonine (41)

-OMe

I. 1II ,"I

OMe

Olividine (42); R1 = OMe; R2 = OAcOlivimine (43); Rl = OR; R2 = OMe

Fig. 4. Miscellaneous structures.

biological targets other than neuroreceptors withstrong molecular selectivity as previously demon-strated for several alkaloids belonging to differentchemical classes (Wink et al., 1998). The cytotoxicactivity of the compounds studied here did not fol-low the expected structure-activity relationshipfrom their reported receptor binding activity(Kukel and Jennings, 1994; Hardick et al., 1996;Dobelis et al., 1999; Panter et al., 2002). The C-14

benzoyl group of nAcChR agonists 1 and 2 andrelated compounds 3, 31 and 32 resulted in nullcytotoxicity. The C-18 methylsuccinylanthranoylsubstituent in the antagonist methyllycaconitine(30) resulted in a more potent cytotoxic actionthan that of the C-18 benzoyl (24 or 25).

To determine if the cytotoxic effects of the se-lective compounds (cytotoxic to tumoral cells vs.CHO cells) were reversible. the recoverv of sensi-

16 C. de Ines et ai.. Cytotoxicity of Norditerpenoid Alkaloids

Table II. Reversibility of the cytotoxic effect of selective compounds on cell viability. Cells were incubated withtheir respective MIC value for each compound (Table I).

Reversibility (%)8

03603603603603603603603603036036036

21

331983

26193

1513384893

246

40

813

151174

-

20:t: 298:t: 3

20:t: 114:t: 068:t: 0

6

9

1655

113

2108

646

38104

165989

56

233

1340

14

25

26

28

')0

6191

1045572

111100

94

4198

4560895480

6078

106

59106

7497

6793

34

,,~

12106

13115

638

100

~

20114

22100

17109

2179

a Percentage cell viability (percent absorbance of the respective untreated control cells). Represented are meanvalues :t SE.

(Table III). As a positive control, the sensitiveSkMel25 cell line was incubateq with rotenone andTaxol@ at MIC values of 0.01 fig/mI. Rotenone in-terrupts mitochondrial electron transfer at theNADH dehydrogenase-ubiquinone junction of therespiratory chain (Palmer et al., 1968) and Taxol@binds specifically to microtubules and blocks nor-mal microtubule dynamics and cell division (Schiffand Horwitz, 1980). The viability of SkMe125 cellsincubated with Taxol@ was similar when measuredby both methoqs, as expected for a compound thathas no effect on cellular respiration and ATP gen-eration. However, incubation of SkMe125 cells

tive tumoral cells was studied (Table II). Com-pound 15 had irreversible effects on all treated celllines followed by 34 which affected 3 of 5 cell lines,with SW480 being the most sensitive of all. Alka-loids 25, 26 and 5 had a selective strong effect onthe recovery of SW480 cells with 25 being the mostpotent. Alkaloid 37 selectively acted on HeLa cellswith moderate potency.

In order to gain insights about the mechanismof action of the irreversibly cytotoxic compounds,.the sensitive cells were incubated with the activealkaloids for 6 d and then the viability was deter-mined by using the M1T assay and the AP method

:to:to:t 1:t 1:to

:to:t6:to

:to:t 1:to:t 1:to:to:t 1:t 1:t 1:t 1:t 5

:1:1:1:0:1:0:to:1:0:1:0

:!::!::!:

:!::!:

:!::!::!:0:!::I:

:!::!::I::!::!::!::!::!::!:

11238

02

201

35

68

1000101

10

:t 4:t 9:t 0:t 12:t 4:t 0:t 10

:t 0

:t 6:t 0

:t 6:t 3:t 0:t 8:t 9

:t 3:t 5

:t 0

:t 4:t 1

:t 8:t 0

:t 1:t 1

:!: 1:!: ~5

~ 1:!: 5

:!: 0:!: 5:!: 1

-

:!: 0:!: 15

:!: 4:!: 8

:!: 2:!: 13

:!: 2:!: 5

C. de Ines et al.' Cytotoxicity of Norditerpenoid Alkaloids

Compound MIC Cell line Table III. Comparative cytotoxicityof the irreversible compounds onthe sensitive cell lines, determinedby the AP and MY[ methods.

MTT AP

Taxoll!>Rotenone

51525263437

SkMel25SkMel25SW480SW480SW480SW480SW480HeLa

3 :!: 0

10:!: 1

5 :!: 0

10:!: 1

na

7 :!: 2

'5:!: 0

4:!:0

a Percentage cell viability (percentabsorbance of the respective un-treated control cells). Representedare mean values + SE.

na, not enough compound available.

with rotenone resulted in significantly different re-:suIts for cell viability when measured by bothmethods.

The incubation of the sensitive lines with 5, 15,25, 26, 34 and 37 gave higher cell viability valueswhen measured with the AP method (Table III).Therefore, the mode of action of these compoundscould be related to the inhibition of ATP produc-tion. A decreased ATP production could compro-mise the tumor cell metabolism because of thehigh demand of energy needed for tumor growthand drug resistance. This will explain why SW480(Pgp+) cells, with higher energy demand relatedto their resistance mechanism, were the most sen-sitive to most of these compounds (5, 15, 25, 26and 34). HeLa and SkMel25 were the followingmore sensitive lines, suggesting that thes~ cellshave a high ATP demand maybe related to theirresistance mechanism (HeLa with intermediatelevels of GSH-conjugate export activity) and/ormetabolism. '

Cervical cancer is the second most commoncancer among women world wide in developingcountries (Parkin et al., 1988; Leminen et al.,1990). Colon cancer is a leading cause of death inthe Western world and one of the most untreata-ble and therapy-resistant cancers (Gryfe et al.,1997). Melanoma is a major medical problemcharacteriz~d by both rapidly rising incidence and

Aiyar V. N., Benn M. H., Hanna T., Jacyno J., Roth S. H.,and Wilkens J. L. (1979), The principal toxin of Del-phinium brownii Rybd., and its mode of action. Expe-rientia 35,1367-1368.

Atta-ur-RahInan M. and Choudhary M. I. (1999), Diter-penoid and steroidal alkaloids. Nat. Prod. Rep. 16,619-635.

Bessonova I. A. and Shaidkhodzaeva S. A. (2000), Heti-sane-type diterpene alkaloids. Chern. Nat. Compo 36,419-477-

Cole S. P. and Deeley R. G. (1998), Multidrug resistancemediated by the ATP-binding casette transporter pro-tein MRP. Bioassays 20, 931-940.

De Ines C., Argandofia V. R., Rovirosa J., San-MartinA., Diaz-Marrero A. R., Cueto M., and .GonzaIez-Coloma A. (2004), Cytotoxic activity of halogenatedmonoterpenes from Plocamium cartilagineum. Z.Naturforsch 59c, 339-344.

Dobelis P., Madl J. E., Pfister J. A., Manners G. D., andWalrond J. P. (1999), Effects of Delphinium alkaloids

. +1) -

~:f:l- +2)-

':f:2

.na

I:f: 1

1:f:4

~:f: 1

growing lifetime risk (Marks, 1995). Therefore, theirreversible and selective cytotoxic compounds 5,15, 25, 26, 34, and 37 could be considered candi-date leads for the design of new drugs combatingchemoresistant human colon, cervical and mela-noma cancers.,In summary, the present results indicate that

neoline (5), 8-0-methylcoltimbianine (6), 1,14-di-~cetylcardiopetaline (9), 18-0-demethylpubesce-nine (13), 14-deacetylpubescenine (14), pubesce-nine (15), 14-deacetylajadine (25), lycoctonine(26), browniine (28), delphatine (29), dehydrota-kaosamine (34), and ajadelphinine (37) havegreater cytotoxic activity to cancerous versus non-cancerous cells. Considering the cell viability dataobtained from the MTf and AP analysis as wellas the non-neural cell lines used in this study, themode of action of these cytotoxic compoundscould be related to low ATP levels. To our knowl-edge, this is the first report on the potential anti-tumor activity of such norditerpenoid alkaloids.

AcknowledgementsThis work was supported by grants MCYT

(BQU2001-1505), CAM (O7M/OO73/2002), and apostdoctoral fellowship (Comunidad de Madrid,Spain) to Concepcion de Ines. We gratefullyacknowledge S. Carlin for language revision.

C. de Ines et al.. Cytotoxicity of Norditerpenoid Alkaloids18

Ling V. (1997), Multidrug resistance: molecular mecha-nisms and clinical relevance. Cancer Chemother.Pharmacol. 40 (Suppl.), S3-S8.

Marks R. (1995), An overview of skin cancers. Incidenceand causation. Cancer 75, 607 -612.

Martin A. and Clynes M. (1991), Acid phosphatase: end-point for in vitro tests. In vitro Cell Dev. BioI. 27 A,183-184.

Mossman T. (1983), Rapid colorimetric assay for cellu-lar growth and survival: application to proliferationand cytotoxicity assays. J. Immunol. Methods 65,55-63.

Palmer G., Horgan D. J., Tisdale H., Singer T. P., andBeinert H. (1968), Studies of the respiratory chain-linked reduced nicotamide adenine dinucleotide de-hydrogenase. XIV. Location of the sites of inhibitionof rotenone, barbiturates, and piericidin by means ofelectron paramagnetic resonance spectroscopy. J. BioI.Chem. 243, 844-847.

Panter K. E., Manners G. D., Stegelmeier B. L., GardnerD. R., Ralphs M. H., Pfister J. A., and James L. F.(2002), Larkspur poisoning: toxicology and alkaloidstructure-activity relationships. Biochem. Syst. Ecol.30,113-128.

Parkin D. M., Laara E., and Muir C. S. (1988), Estimatesof the world wide frequency of sixteen major cancerin 1980. Int. J. Cancer 41, 184-197.

Rass K., Gutwein P., Welter C., Meineke V., Tilgen W.,and Reichrath J. (2001), DNA mismatch repair en-zyme hMSH2 in malignant melanoma: Increased im-munoreactivity as compared to acquired melanocyticnevi and strong mRNA expression in melanoma celllines. Histochem. J. 33, 459-467. .

Schiff P. B. and Horwitz S. B. (1980), Taxol stabilizes mi-crotubules in mouse fibroblast cells. Proc. Natl. Acad.Sci. USA 77,1561-1565.

Seitz U. and Ameri A. (1998), Different effects of [3H]noradrenaline uptake of the aconitum alkaloids aconi-tine, 3-acetylaconitine, lappaconitine, and N-desace-tyllappaconitine in rat hippocampus. Biochem. Phar-macol. 55, 883-888.

Suter L., Bruggen J., Brocker E. B., and Sorg C. (1985),A tumor-associated antigen expressed in melanomacells with lower malignant potential. Int. J. Cancer 35,787-791.

Ulubelen A., Mericli A., Kilincer N., Ferizli A. G.,Emecki M., and Pelletier W. (2001), Insect repellentactivity of diterpenoid alkaloids. Phytother. Res. 15,170-171.

Wink M., Schmeller T., and Latz-Bruning B. (1998),Modes of action of allelochemical alkaloids: interac-tion with neuroreceptors, DNA and other moleculartargets. J. Chem. Ecol. 24, 1881-1937.

Zhang K., Mack P., and Wong K. P. (1998), Glutathione-related mechanisms in cellular resistance to anticancerdrugs. Int. J. Oncol. 12, 871-882.

on neuromuscular transmission. J. Pharm. Exp. Ther.291, 538-546.

Gonzalez P., Marin C., Rodriguez-Gonzalez I., Hitos A.,Rosales M. J., Reina M., Diaz J. G., Gonzalez-ColomaA., and Sanchez-Moreno M. (2005), In vitro activity ofCzo diterpenoid alkaloid derivatives on promastigotesand intracellular amastigotes of Leishmania infantum.Int. J. Antimicrobial Agents 25,136-141.

Gonzalez-Coloma A., Guadafio A., Gutierrez C., Ca-brera R., De la Pella E., De la Fuente G., and ReinaM. (1998), Antifeedant Delphinium diterpene alka-loids. Structure-activity relationships. J. Agric. FoodChern. 46, 286-290.

Gonzalez-Coloma A., Reina M., Medinaveitia A., Gua-dafio A., Santana 0., Martinez-Diaz R., Ruiz-MesiaL., Alva A., Grandez M., Diaz R., Gavin J. A., andDe la Fuente G. (2004a), Structural diyers;ity anddefensive properties of norditerpenoid alkaloids. J.Chern. Ecol. 30, 1393-1408.

Gonzalez-Coloma A., Reina M., Guadafio A., Martinez-Diaz R., Diaz J. G., Garcia-Rodriguez J., and De laFuente G. (2004b), Antifeedant ~o diterpene alka-loids. Chern. Biodiv. 1, 1327 -1335.

Gryfe R., Swallow C., Bapat B., Redston M., GallingerS., and Couture J. (1997), Molecular biology of calo-rectal cancer. Curr. Probl. Cancer 21, 233-300. ~

Hardick D. J., Blagbrough I. S., Cooper G., Potter B. V.,Critchley T., and Wonnacott S. (1996), Nudicaulineand elatine as potent norditerpenoid ligands at ratneuronal alpha-bungarotoxin binding sites: Impor-tance of the (methylsuccinimido) benzoyl moiety forneuronal nicotinic acetylcholine receptor binding. J.Med. Chern. 39, 4860-4866.

Higgins C. F. (1992), ABC transporters: from microor-ganisms to man. Annu. Rev. Cell BioI. 8, 67 -113.

Jennings K. R., Brown D. G., and Wright D. P. J. (1986),Methyllycaconitine, a naturally occuring insecticidewith a high affinity for the insect cholinergic receptor.Experientia 42, 611-613.

Klein ~., Sarkadi B., and Varadi A. (1999), An inventoryof the human ABC proteins. Biochim. Biophys. Acta1461,237-262.

Kukel C. F. and Jennings K. R. (1994), Delphinium alka-loids as inhibitors of a-bungarotoxin binding to ratand insect neural membranes. Can. J. Physiol. Phar-macol. 72, 104-107.

Leminen A., Paavonen J., Forss M., Wahlstrom T., andVesterinen E. (1990), Adenocarcinoma of the uterinecervix. Cancer 65,53-59.

Li L., Shen Y. M., Yang X. S., Zuo G. Y., Shen Z. Q.,Chen Z. H., and Hao X. J. (2002a), Antiplatelet ag-gregation activity of diterpene alkaloids from Spiraeajaponica. Eur. J. Pharmacol. 449, 23-28.

Li L., Shen Y. M., Yang X. S., Wu W. L., Wang B. G.,Chen Z. H., and Hao X. J. (2002b), Effects of spiram-ine T on antioxidant enzymatic activities and nitricoxide production in cerebral ischemia-reperfusiongerbils. Brain Res. 944,205-209.