New MDR Modulators and Apoptosis Inducers from Euphorbia Species

ANTICANCER RESEARCH 27: 3451-3458 (2007)

New MDR Modulators and Apoptosis Inducers from Euphorbia Species HELGA ENGI1, ANDREA VASAS2, DÓRA RÉDEI2, JOSEPH MOLNÁR1 and JUDIT HOHMANN2 1Department

of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dóm tér 10, H-6720 Szeged; 2Department of Pharmacognosy, Faculty of Pharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary

Abstract. Several macrocyclic diterpenes with jatrophane or lathyrane skeletons were isolated from methanol extracts of Hungarian Euphorbia species and evaluated for multidrug resistance (MDR)-reversing activity on a human colon cancer cell line. MDR-reversing activity was tested by using a standard functional assay with Rhodamine 123 as a fluorescent substrate analogue of epirubicin. In the model of combination chemotherapy, the interactions between epirubicin and certain resistance modifiers were studied in vitro. Compound 8 proved to be the most active, exhibiting a synergistic interaction. The capacity of the most effective derivative to induce apoptosis was demonstrated by flow cytometric analysis and by staining with ethidium bromide and acridine orange, using human mdr1 gene-transfected mouse lymphoma cells and a human cervical adenocarcinoma cell line. The selected diterpene was able to induce moderate apoptosis in the tested cell lines. The data presented here indicate that naturally occurring Euphorbia diterpenes can be regarded as effective lead compounds for the reversal of MDR. Prolonged cancer chemotherapy may lead to the selective survival of multidrug-resistant (MDR) cells that exhibit simultaneous resistance to a wide spectrum of structurally and functionally unrelated chemotherapeutic agents. The molecular changes contributing to the development of MDR include the up-regulation or activation of transporter proteins, detoxification systems and target repair mechanisms, and the dysregulation of cell death pathways (1). Despite multiple mechanisms being involved in MDR, the most important MDR process is generally associated

Correspondence to: Prof. Joseph Molnár, Department of Medical Microbiology, University of Szeged, Dóm tér l0, H-6720 Szeged, Hungary. Tel: +36 62 545115, Fax: +36 62 545113, e-mail: [email protected] Key Words: Euphorbiaceae, jatrophane diterpenes, multidrug resistance, P-glycoprotein, apoptosis.

0250-7005/2007 $2.00+.40

with either amplification or over-expression of the mdr1 gene, which encodes a cell-surface P-glycoprotein (P-gp). This protein acts as an energy-dependent efflux pump, and extrudes a variety of hydrophobic anti-tumour drugs out of the tumour cells, thus P-gp inhibitors may therefore resensitize MDR cells (2, 3). Several studies have shown that most chemotherapeutic agents exert their anticancer activity by inducing apoptosis or programmed cell death, which is an essential physiological process required to eliminate abnormal cells. It is caused by the activation of intracellular cystein proteases, known as caspases, which are responsible for the morphological and biochemical events that characterize the apoptotic cell (4). Consequently, resistance to apoptosis may be a major factor for the ineffectiveness of cancer treatment. Compounds which regulate and overcome the apoptosis deficiency of cancer cells are of great therapeutic importance, and the development of apoptosis-modulating agents has become an important approach for the discovery of new antitumor drugs (5). Euphorbia species have been used in traditional medicine in many countries to treat cancer and warts (6). Many chemical studies on Euphorbia species have focused on the occurrence of highly skin-irritant compounds, mainly with tigliane, ingenane or daphnane skeletons (7). These compounds, jointly known as phorboids possess extremely strong pro-inflammatory and tumour-promoting effects, due to activation of the enzyme protein kinase C (8). Besides the presence of those toxic compounds, Euphorbia species are of further considerable interest owing to a large diversity of structurally unique and non-irritant macrocyclic diterpenoid constituents. Among these, considerable attention has recently been devoted to compounds of the non-phorboid jatrophane and lathyrane type, which have been considered to be potent modulators of MDR (9-10). Our research group previously reported that extracts containing macrocyclic diterpenes may reverse MDR by inhibiting P-gp in human mdr1 gene-transfected mouse lymphoma cells (11-14).

3451


Figure 1. Chemical structure of compounds 1-9. Compounds 1-5 and 7-9 are jatrophane diterpenes and compound 6 is a lathyrane diterpene.

In the present study, nine Euphorbia diterpenes, isolated from E. esula L., E. peplus L., E. villosa W. and K. or E. serrulata Thuill. (9-10, 15-18), were investigated for their antiproliferative and MDR-reversal effects on a human colon cancer cell line. An evaluation of the capacity of the most effective resistance modifier, as an apoptosis inducer, is also reported.

Materials and Methods Compounds. Nine Euphorbiaceae diterpenes were involved in the study (Figure 1). The compounds were isolated from the lipophilic phase of methanol extracts of Euphorbia esula (compounds 1, 2 and 3), E. peplus (compounds 4 and 5), E. villosa

3452

(compound 6) and E. serrulata (compound 7, 8 and 9). The plants were collected in Hungary. The tested diterpenes were isolated by means of multistep chromatographic purifications, and their structures were characterized by spectroscopic methods as previously described (15-18). All compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Ltd., Budapest, Hungary). Cell cultures. The L5178 Y mouse T-cell lymphoma cells [obtained from Prof. Gottesmann, National Cancer Institute of Health (NCI) and Food & Drug Administration (FDA), USA] were transfected with pHa MDR1/A retrovirus, as previously described (19). MDR1-expressing cell lines were selected by culturing the infected cells with 60 ng/mL colchicine to maintain the expression of the MDR phenotype. Cells were grown in McCoy's 5A medium

Engi et al: MDR Modulators and Apoptosis Inducers from Euphorbia Species

supplemented with 10% heat-inactivated horse serum (Gibco, Auckland, New Zealand), L-glutamine and antibiotics. The human colon cancer cells (COLO320) were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated foetal bovine serum (JRH Biosci, Lenexa, KS, USA), 2 mM L-glutamine, 1 mM Na-pyruvate and 100 mM Hepes. The cell lines were incubated in a humified atmosphere (5% CO2 + 95% air) at 37ÆC. The semiadherent human colon cancer cells were detached with 0.25% trypsin and 0.02% EDTA for 5 min at 37ÆC. The human cervical adenocarcinoma cells (HeLa) were cultivated in MEM (Gibco BRL, Paisley, UK) supplemented with 10% heat-inactivated foetal bovine serum, 1% non-essential amino acids and an antibiotic-antimycotic mixture. Assay for the reversal of MDR in the COLO320 cell line. The cells were trypsinized and the cell concentration was adjusted to a density of 2x106/mL. The cells were suspended in serum-free RPMI 1640 medium and distributed in 0.5 mL aliquots into Eppendorf centrifuge tubes. The compounds to be tested were added (4 and 40 Ìg/mL) and the samples were incubated for 10 min at room temperature (25ÆC). Rhodamine 123 (R123; Sigma, St. Louis, MO, USA) was added (5.2 ÌM final concentration) to the samples and the cells were incubated for 20 min at 37ÆC. The cells were then centrifuged (2000 rpm, 2 min), washed twice in 0.5 mL phosphatebuffered saline (PBS; Sigma-Aldrich Ltd.) and finally resuspended in 0.5 mL PBS for assay. Verapamil (EGIS, Hungarian Pharmaceutical Company, Budapest, Hungary) a well-known MDR modifier was used as a positive control. The fluorescence of the cell population was measured with a Beckton Dickinson FACScan flow cytometer (Cell Sorter, Oxford, UK). The fluorescence activity ratio (FAR) was calculated (20), on the basis of the measured fluorescence intensities (FL-1): FAR = FL-1 treated cells/FL-1 untreated cells

(Equation 1)

Assay for antiproliferative effect. The effects of the chemotherapeutic agent, epirubicin hydrochloride (Farmitalia Carlo Erba, Milano, Italy), the compounds and their combinations on the proliferation of the COLO320 cell line were tested in 96-well microtitre plates. The compounds were diluted from high to low concentrations horizontally in the plates. Cells were seeded into each well (1x104) with the exception of the medium control wells. The plates were incubated at 37ÆC for 72 h and 15 ÌL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT solution; Sigma) was then added to each well. After incubation at 37ÆC for 4 h, 100 mL 10% sodium dodecylsulfate solution (Sigma) was added to each well. The plates were further incubated at 37ÆC overnight to allow the dissolution of formazan crystals produced by the mitochondrial enzymes of the living cells. The extent of inhibition of cell proliferation was determined by measuring the optical density (OD) of the chromogenic products at 550 nm (ref. 630 nm) with a Dynatech MRX vertical beam ELISA reader (Labsystems, Helsinki, Finland). The extent of inhibition of cell growth (as a percentage) was determined with the following formula, in which OD cell control means the untreated cells.

(Equation 2)

Drug interactions. A checkerboard microplate method was applied to study the effects of drug interactions between the selected diterpenes and epirubicin on the human colon cancer cell line. The dilutions of epirubicin (A) were made in a horizontal direction, and the dilutions of the resistance modifiers (B) vertically in the microtitre plate in a volume of 100 mL. The cells were distributed into each well in 50 ÌL (1x104 cells), and incubated for 72 h at 37ÆC in a CO2 incubator. The cell growth rate was determined after MTT staining and the intensity of the blue colour was measured on a micro ELISA reader. Drug interactions were evaluated according to the following expressions: (Equation 3) FICA = ID50A in combination / ID50A alone (Equation 4) FICB = ID50B in combination / ID50B alone where ID is the inhibitory dose, and FIC is the fractional inhibitory concentration. The fractional inhibitory index, FIX = FICA + FICB, demonstrated the effect of the combination of the anticancer drug and the tested compound. It was accepted that, if the value of FIX was 0.51-1.00, it was an additive effect; if FIX was 2.00 indicated an antagonistic effect (21). Assay of induction of apoptosis. The cells were adjusted to a density of 2x105/mL and were distributed in 1.0 mL aliquots into microcentrifuge tubes. The apoptosis inducer 12Hbenzo[·]phenothiazine (M627) was added to the samples as a positive control at a final concentration of 5 or 25 Ìg/mL. The M627 was synthetized by Motohashi et al. (22). In the control cultures, 10 ÌL DMSO was added. The compound used for treatment was added to the samples at a final concentration of 2 or 10 Ìg/mL. After incubation for 24 h at 37ÆC, the cells were transferred from a 24-well plate into Eppendorf centrifuge tubes, centrifuged (2000 rpm, 2 min) and washed with PBS, and resuspended in 195 ÌL binding buffer. The samples were mixed and centrifuged (2000 rpm, 2 min) and the supernatant was removed from each tube. Five microlitres AnnexinV-FITC (AV; human recombinant-FITC; Alexis Biochemical, Grünberg, Germany) was added to the tubes. Controls without AV were also prepared. The samples and controls were incubated at room temperature for 10 min in the dark, then centrifuged (2000 rpm, 2 min), washed with PBS, and resuspended in 190 ÌL binding buffer. Before the measurement of fluorescence activity, 10 ÌL of 20 Ìg/mL propidium iodide (PI; Sigma-Aldrich Ltd.) was added to the samples. The fluorescence activity (FL-1 and FL-2) of the cells was measured and analysed by flow cytometry on a Beckton Dickinson FACScan instrument. In each analysis, 10.000 events were recorded, and the percentages of the cells in the different states were calculated by square analysis using Windows Multiple Document Interface for Flow Cytometry (winMDI2.8) (23, 24). Ethidium bromide and acridine orange (EB/AO) staining. Staining with EB/AO (Sigma-Aldrich Ltd.) was carried out in a 96-well plate format after 24 h of treatment in order to visualize the morphological events (25). Plates were centrifuged at 1000 rpm 2 min, and 8 ÌL of staining solution (0.1 mg/mL for both AO and EB in PBS) was added to each well. After 10 min, the cells were washed with PBS, and the cells were viewed with a Nikon Eclipse inverted microscope at 200x magnification with a 500/20 nm excitation filter, a cut-on 515 nm LP dichromatic mirror, and a 520 nm LP barrier filter (Chroma Technology, Rockingham, VT, USA). Pictures were taken with a Nikon Coopix 4500 digital camera (Nikon, Tokyo, Japan).

3453

ANTICANCER RESEARCH 27: 3451-3458 (2007) Table I. Antiproliferative effects of diterpenes on human colon cancer cells.

Table II. Antiproliferative effects of selected diterpenes in combination with epirubicin on human colon cell line.

Compounds

Treatment

1 2 3 4 5 6 7 8 9 Epirubicin

ID50±S.E.M*(Ìg/mL) 7.54±0.36 6.41±0.05 10.16±0.08 10.65±0.51 3.29±0.30 13.27±0.87 17.46±0.77 10.04±0.55 2.61±0.88

1 3 5 6 7 8 9

FIX±S.E.M*

Interaction

1.05±0.12 0.68±0.07 1.12±0.19 0.54±0.08 0.64±0.06 0.25±0.03 1.15±0.07

no interaction additive no interaction additive additive synergism no interaction

*FIX-fractional inhibitory index; S.E.M values were derived from the standard errors of the mean of at least three FIX values.

0.10±0.03

*All values are expressed as means±S.E.M from parallel experiments (n=2-4).

Table III. Effects of diterpenes on Rhodamine 123 accumulation of human colon cancer cells. Compounds

Statistical analysis. Statistical analysis of the data was performed with Graphpad Prism 2.01 (Graph Pad Software, San Diego, CA, USA).

Results Assay for antiproliferative effect. The antiproliferative effects of the investigated compounds on the human colon cancer cells are listed in Table I. A majority of the compounds exhibited a moderate antiproliferative effect (ID50=6.4117.46 Ìg/mL), the jatrophane diterpenes 5 and 9 were the most effective (ID502.00 at 40 Ìg/mL). For compound 7, the effect was almost the same at the two concentrations, meaning that both of the applied concentrations were in the saturation zone. Compounds 1-5 were moderately effective in modulating the P-gp on the resistant human colon cell line.

jatrophane diterpene 8 was noted in the applied concentration range (2 and 10 Ìg/mL) as compared with the positive control M627.

Assay of induction of apoptosis. Compound 8 was selected for apoptosis induction on resistant mouse lymphoma cells, based on its synergistic effect with epirubicin (Table IV and Figure 3). A moderate apoptosis-inducing effect of the

Ethidium bromide and acridine orange (EB/AO) staining. The EB/AO staining of the HeLa cells allowed the identification of live, apoptotic and necrotic cells. AO permeates all cells and makes the nuclei appear green. EB is taken up by cells

3454


Figure 2. Interaction of compound 8 with epirubicin on human colon cancer cell line (The curves relate to different concentrations of compound 8 in combination with decreasing amounts of epirubicin; symbols represented different concentrations of compound 8).

only when the cytoplasmic membrane integrity has been lost; it stains the nucleus red. Even at a lower concentration of compound (2 Ìg/mL), treatment of HeLa cells with compound 8 led to the typical morphological features of apoptosis, including increased cell membrane permeability, cellular shrinkage and granulation in the nucleus (Figure 4).

Discussion The fluorescent activities of the compounds indicated that a majority of the Euphorbia derivatives enhanced drug retention, as can be observed in Tables I-III. Three of the tested diterpene derivatives (6, 8 and 9) displayed a significant concentration-dependent effect in inhibiting the efflux pump activity on the COLO320 cell line. The most effective synergistic effect was found between compound 8 and epirubicin. The strong activity of this derivative can be explained by its high lipophilicity, but other parameters, such as the presence of functional groups, may also be involved in the synergistic effect and in the interaction with P-gp. The number of double bonds, pseudorotation of the ester groups and transannular interactions of the substituents have significant effects on the stereostructure and the activity of the molecules. It is known that the conformational flexibility of the twelve-membered ring of the jatrophanes is strongly influenced by the steric interactions of the substituents. On comparison of the efficacies of compounds 7 and 8, it can be presumed that

Table IV. Apoptosis induction by compound 8 on mdr1 gene-transfected mouse lymphoma cell line. Sample

Conc. Early Total Cell (Ìg/mL) apoptosis apoptosis death (%) (%) (%)

Cell control wihout staininga Cell control + AVb Cell control + PIc Cell control double stainingd 1% DMSO control 12H-benzo (·)-phenothiazine Compound 8

5 25 2 10

1.14 8.05 0.31 4.83 1.20 77.67 26.25 4.04 3.02

1.22 11.43 1.18 6.93 3.05 99.74 99.55 5.80 6.14

0.06 1.97 2.91 2.98 2.93 0.04 0.03 3.36 3.02

aAnnexin-V-negative/propidium

iodide-negative samples = intact viable cells (98-100%). bAnnexin-V-positive/propidium iodidenegative=apoptotic cells. cAnnexin-V-negative/propidium iodidepositive = necrotic cells. dAnnexin-V-positive/propidium iodidepositive = apoptotic/necrotic cells.

the presence of a hydroxy group instead of peracylation is favourable as concerns the antiproliferative activity in combination with epirubicin. Several studies have indicated that diterpene derivatives exert apoptosis-inducing activity (13, 26-27). The apoptosisinducing potential rather than necrosis induction is accepted as a key feature of a potential anti-tumour drug. A change

3455


Figure 3. Jatrophane diterpene 8-induced apoptosis in human mdr1 gene-transfected mouse lymphoma cell line. The samples were analysed for green fluorescence (Annexin V-FITC) and red fluorescence (PI) by flow cytometry. The assay gives information about the numbers of vital (quadrant R1), early-apoptotic (quadrant R2), late-apoptotic (quadrant R3) and necrotic cells (quadrant R4). The subpopulation of cells stained with Annexin V-FITC only are those cells in the apoptotic pathway, while those that stain with both Annexin V-FITC and PI are either necrotic or are in transition from the apoptotic to the necrotic state.

in the architecture of the plasma membrane during apoptosis involves the redistribution of various phospholipid species between the two leaflets of the membrane. The most pronounced feature of this asymmetry is the almost complete absence of phosphatidylserine in the outer leaflet of the plasma membrane. Early in apoptosis, this phospholipid is rapidly exposed in the outer surface of the cells. AV, the Ca2+-dependent endogenous human protein, has a high affinity for membrane-bound phosphatidylserine, so this protein has been labelled with fluorescein and has

3456

been used to detect apoptosis in vitro (28). AV appears to be a potent discriminator between viable and apoptotic cells (23). Compound 8 was able to induce moderate apoptosis in the mdr1 gene-transfected mouse lymphoma cell line. Together with the previous data, this allows the conclusion that jatrophane and lathyrane derivatives can be regarded not only as effective anti-MDR agents, but also as apoptosis inducers. These compounds may be promising lead compounds for natural product-based drug development.


Figure 4. Fluorescent microscopic pictures of diterpene 8-treated HeLa cells after a 24-h incubation. Initial magnification: 200x. Treatment with 2 Ìg/mL diterpene 8. "Apo" indicates cellular shrinkage and nuclear granulation characteristic of apoptosis; "Nec" indicates necrosis evidence by fluorescence of ethidium bromide (EB). Cells were double stained with the membrane-permeable fluorochrome acridine orange and the DNA-binding fluorochrome EB, which is impermeable to the normal plasma membrane, but stains the nuclei of necrotic and late-apoptotic cells. An increase in cell membrane permeability was observed, as evidenced by the red fluorescence of EB in the nucleus.

Acknowledgements This study was supported by the Szeged Foundation for Cancer Research (Szegedi Rákkutatásért Alapítvány). The authors are also grateful to Dr. Imre Ocsovszki for the flow cytometry measurements and to Anikó Váradi Vigyikán for skilful technical assistance.

References 1 Filipits M: Mechanisms of cancer: multidrug resistance. Drug Discovery Today: Disease Mechanisms/Cancer 2: 229-234, 2004. 2 Higgins CF and Linton KJ: The ATP swich model for ABC transporters. Nat Struct Mol Biol 11: 918-926, 2004. 3 Hennessy M and Spiers JP: A primer on the mechanics of Pglycoprotein the multidrug transporter. Pharmac Research 55: 1-15, 2007. 4 Thornberry N and Lazabenik Y: Caspases: enemies within. Science 281: 1312-1316, 1998. 5 Reed JC: Apoptosis-regulating proteins as targets for drug discovery. Trends Mol Med 7: 314-319, 2001. 6 Hartwell JL: Plants used against cancer. A survey. J Nat Prod 62: 153-205, 1969. 7 Schmidt RJ and Evans FJ: Skin irritants of the sun spurge (Euphorbia helioscopia L.). Contact Dermatitis 6: 204-210, 1980. 8 Evans JF and Taylor SE: Pro-inflammatory, tumour-promoting and anti-tumour diterpenes of the plant families Euphorbiaceae and Thymelaeaceae. Prog Chem Org Nat Prod 44: 1-99, 1983.

9 Hohmann J, Vass A, Günther G, Dombi G, Blazsó G, Falkay G, Máthé I and Jerkovich G: Jatrophane diterpenoids from Euphorbia peplus. Phytochemistry 51: 673-677, 1999. 10 Hohmann J, Molnár J, Rédei D, Evanics F, Forgó P, Kálmán A, Argay G and Szabó P: Discovery and biological evaluation of a new family of potent modulators of multidrug resistance: reversal of multidrug resistance of mouse lymphoma cells by new natural jatrophane diterpenoids isolated from Euphorbia species. J Med Chem 45: 2425-2431, 2002. 11 Valente C, Ferreira MJU, Abreu PM, Gyémánt N, Ugocsai K, Hohmann J and Molnár J: Pubescenes, jatrophane diterpenes from Euphorbia pubescens with multidrug resistance reversing activity on mouse lymphoma cells. Planta Med 70: 81-84, 2004. 12 Madureira AM, Gyémánt N, Ugocsai K, Ascenso JR, Abreu PM, Hohmann J, Molnár J and Ferreira MJU: Rearranged jatrophanetype diterpenes from Euphorbia species. Evaluation of their effect on the reversal of multidrug resistance. Planta Med 70: 45-49, 2004. 13 Duarte N, Varga A, Cherepnov G, Radics R, Molnár J and Ferreire MJ: Apoptosis induction and modulation of Pglycoprotein mediated multidrug resistance by new macrocyclic lathyrane-type diterpenoids. Bioorg Med Chem 15: 546-554, 2007. 14 Hohmann J and Molnár J: Euphorbiaceae diterpenes: plant toxins or promising molecules for the therapy? Acta Pharm Hung 74: 149-157, 2004. 15 Hohmann J, Vasas A, Günther G, Máthé I, Evanics F, Dombi G and Jerkovich G: Macrocyclic diterpene polyesters of the jatrophane type from Euphorbia esula. J Nat Prod 60: 331335, 1997.

3457

ANTICANCER RESEARCH 27: 3451-3458 (2007) 16 Günther G, Martinek T, Dombi G, Hohmann J and Vasas A: Structural characterisation and dynamic NMR studies of a new peracylated macrocyclic diterpene. Magn Reson Chem 37: 365370, 1999. 17 Vasas A, Hohmann J, Forgó P and Szabó P: New tri- and tetracyclic diterpenes from Euphorbia villosa. Tetrahedron 60: 5025-5030, 2004. 18 Hohmann J, Rédei D, Evanics F, Kálmán A, Argay G and Bartók T: Serrulatin A and B, new diterpene polyesters from Euphorbia serrulata. Tetrahedron 56: 3619-3623, 2000. 19 Cornwell MM, Pastan I and Gottesmann M: Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein. J Biol Chem 262: 2166-2170, 1987. 20 Gruber A, Peterson C and Reizenstein P: D-verapamil and Lverapamil are equally effective in increasing vincristine accumulation in leukemic cells in vitro. Int J Cancer 41: 224226, 1988. 21 Eliopoulos GM and Moellering RC: Antimicrobial combinations. In: Antibiotics in Laboratory Medicine. Lorian V (ed.). Williams and Wilkins, USA, pp. 432-433, 1980. 22 Motohashi N, Kurihara T, Satoh K, Sakagami H, Mucsi I, Pusztai R, Szabó M and Molnár J: Anti-tumour activity of benzo[·]phenothiazines. Anticancer Res 19: 1837-1842, 1999.

3458

23 Vermes I, Haanen C and Reutelingsperger C: Flow cytometry of apoptotic cell death. J Immunol Methods 243: 167-190, 2000. 24 Bilyy R and Stoika R: Search for novel cell surface markers of apoptosis cells. Autoimmunity 40: 249-253, 2007. 25 Ribble D, Goldstein NB, Norris DA and Shellman YG: A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnology 5: 12, 2005. 26 Block S, Gerkens P, Peulen O, Jolois O, Mingeot-Leclercq MP, De Pauw-Gillet MC and Quetin-Leclercq J: Induction of apoptosis in human promyelocytic leukemia cells by a natural trachylobane diterpene. Anticancer Res 25: 363-368, 2005. 27 Freire AC, da Silva Melo P, Aoyama H, Haun M, Duran N and Ferreira CV: Cytotoxic effect of the diterpene lactone dehydrocrotonin from Croton cajucara on human promyelocytic leukemia cells. Planta Med 69: 67-69, 2003. 28 Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST and van Oers MH: Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415-1420, 1994.

Received May 21, 2007 Revised August 1, 2007 Accepted August 14, 2007