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 with either amplification or over-expression of the mdr1 lathyrane skeletons were isolated from methanol extracts of gene, which encodes a cell-surface P-glycoprotein (P-gp). Hungarian Euphorbia species and evaluated for multidrug This protein acts as an energy-dependent efflux pump, and resistance (MDR)-reversing activity on a human colon cancer extrudes a variety of hydrophobic anti-tumour drugs out of cell line. MDR-reversing activity was tested by using a standard the tumour cells, thus P-gp inhibitors may therefore re- functional assay with Rhodamine 123 as a fluorescent sensitize MDR cells (2, 3). substrate analogue of epirubicin. In the model of combination Several studies have shown that most chemotherapeutic chemotherapy, the interactions between epirubicin and certain agents exert their anticancer activity by inducing apoptosis resistance modifiers were studied in vitro. Compound 8 proved or programmed cell death, which is an essential to be the most active, exhibiting a synergistic interaction. The physiological process required to eliminate abnormal cells. capacity of the most effective derivative to induce apoptosis It is caused by the activation of intracellular cystein was demonstrated by flow cytometric analysis and by staining proteases, known as caspases, which are responsible for the with ethidium bromide and acridine orange, using human morphological and biochemical events that characterize the mdr1 gene-transfected mouse lymphoma cells and a human apoptotic cell (4). Consequently, resistance to apoptosis may cervical adenocarcinoma cell line. The selected diterpene was be a major factor for the ineffectiveness of cancer able to induce moderate apoptosis in the tested cell lines. The treatment. Compounds which regulate and overcome the data presented here indicate that naturally occurring apoptosis deficiency of cancer cells are of great therapeutic Euphorbia diterpenes can be regarded as effective lead importance, and the development of apoptosis-modulating compounds for the reversal of MDR. agents has become an important approach for the discovery of new antitumor drugs (5). Prolonged cancer chemotherapy may lead to the selective Euphorbia species have been used in traditional medicine survival of multidrug-resistant (MDR) cells that exhibit in many countries to treat cancer and warts (6). Many simultaneous resistance to a wide spectrum of structurally chemical studies on Euphorbia species have focused on the and functionally unrelated chemotherapeutic agents. The occurrence of highly skin-irritant compounds, mainly with molecular changes contributing to the development of tigliane, ingenane or daphnane skeletons (7). These MDR include the up-regulation or activation of transporter compounds, jointly known as phorboids possess extremely proteins, detoxification systems and target repair strong pro-inflammatory and tumour-promoting effects, mechanisms, and the dysregulation of cell death pathways due to activation of the enzyme protein kinase C (8). (1). Despite multiple mechanisms being involved in MDR, Besides the presence of those toxic compounds, Euphorbia the most important MDR process is generally associated 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 Correspondence to: Prof. Joseph Molnár, Department of Medical non-phorboid jatrophane and lathyrane type, which have ó Microbiology, University of Szeged, D m tér l0, H-6720 Szeged, been considered to be potent modulators of MDR (9-10). Hungary. Tel: +36 62 545115, Fax: +36 62 545113, e-mail: [email protected] Our research group previously reported that extracts containing macrocyclic diterpenes may reverse MDR by Key Words: Euphorbiaceae, jatrophane diterpenes, multidrug inhibiting P-gp in human mdr1 gene-transfected mouse resistance, P-glycoprotein, apoptosis. lymphoma cells (11-14).

0250-7005/2007 $2.00+.40 3451 ANTICANCER RESEARCH 27: 3451-3458 (2007)

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 (compound 6) and E. serrulata (compound 7, 8 and 9). The plants from E. esula L., E. peplus L., E. villosa W. and K. or E. were collected in Hungary. The tested diterpenes were isolated serrulata Thuill. (9-10, 15-18), were investigated for their by means of multistep chromatographic purifications, and their structures were characterized by spectroscopic methods as antiproliferative and MDR-reversal effects on a human previously described (15-18). All compounds were dissolved in colon cancer cell line. An evaluation of the capacity of the dimethyl sulfoxide (DMSO; Sigma-Aldrich Ltd., Budapest, most effective resistance modifier, as an apoptosis inducer, Hungary). is also reported. Cell cultures. The L5178 Y mouse T-cell lymphoma cells [obtained Materials and Methods from Prof. Gottesmann, National Cancer Institute of Health (NCI) and Food & Drug Administration (FDA), USA] were transfected Compounds. Nine Euphorbiaceae diterpenes were involved in the with pHa MDR1/A retrovirus, as previously described (19). study (Figure 1). The compounds were isolated from the MDR1-expressing cell lines were selected by culturing the infected lipophilic phase of methanol extracts of Euphorbia esula cells with 60 ng/mL colchicine to maintain the expression of the (compounds 1, 2 and 3), E. peplus (compounds 4 and 5), E. villosa MDR phenotype. Cells were grown in McCoy's 5A medium

3452 Engi et al: MDR Modulators and Apoptosis Inducers from Euphorbia Species supplemented with 10% heat-inactivated horse serum (Gibco, Drug interactions. A checkerboard microplate method was applied Auckland, New Zealand), L-glutamine and antibiotics. to study the effects of drug interactions between the selected The human colon cancer cells (COLO320) were cultured in diterpenes and epirubicin on the human colon cancer cell line. The RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) dilutions of epirubicin (A) were made in a horizontal direction, and supplemented with 10% heat-inactivated foetal bovine serum (JRH the dilutions of the resistance modifiers (B) vertically in the Biosci, Lenexa, KS, USA), 2 mM L-glutamine, 1 mM Na-pyruvate microtitre plate in a volume of 100 mL. The cells were distributed and 100 mM Hepes. The cell lines were incubated in a humified into each well in 50 ÌL (1x104 cells), and incubated for 72 h at 37ÆC atmosphere (5% CO2 + 95% air) at 37ÆC. The semiadherent in a CO2 incubator. The cell growth rate was determined after human colon cancer cells were detached with 0.25% trypsin and MTT staining and the intensity of the blue colour was measured 0.02% EDTA for 5 min at 37ÆC. on a micro ELISA reader. Drug interactions were evaluated The human cervical adenocarcinoma cells (HeLa) were according to the following expressions: cultivated in MEM (Gibco BRL, Paisley, UK) supplemented with FICA = ID50A in combination / ID50A alone (Equation 3) 10% heat-inactivated foetal bovine serum, 1% non-essential amino FICB = ID50B in combination / ID50B alone (Equation 4) acids and an antibiotic-antimycotic mixture. where ID is the inhibitory dose, and FIC is the fractional inhibitory concentration. Assay for the reversal of MDR in the COLO320 cell line. The cells The fractional inhibitory index, FIX = FICA + FICB, demonstrated were trypsinized and the cell concentration was adjusted to a the effect of the combination of the anticancer drug and the tested density of 2x106/mL. The cells were suspended in serum-free RPMI compound. It was accepted that, if the value of FIX was 0.51-1.00, it 1640 medium and distributed in 0.5 mL aliquots into Eppendorf was an additive effect; if FIX was <0.50, it was a synergistic one; a FIX centrifuge tubes. The compounds to be tested were added (4 and value in the interval 1.00-2.00 was considered an indifferent effect, 40 Ìg/mL) and the samples were incubated for 10 min at room while a value >2.00 indicated an antagonistic effect (21). temperature (25ÆC). Rhodamine 123 (R123; Sigma, St. Louis, MO, USA) was added (5.2 ÌM final concentration) to the samples and Assay of induction of apoptosis. The cells were adjusted to a density the cells were incubated for 20 min at 37ÆC. The cells were then of 2x105/mL and were distributed in 1.0 mL aliquots into centrifuged (2000 rpm, 2 min), washed twice in 0.5 mL phosphate- microcentrifuge tubes. The apoptosis inducer 12H- buffered saline (PBS; Sigma-Aldrich Ltd.) and finally resuspended benzo[·]phenothiazine (M627) was added to the samples as a in 0.5 mL PBS for assay. Verapamil (EGIS, Hungarian positive control at a final concentration of 5 or 25 Ìg/mL. The M627 Pharmaceutical Company, Budapest, Hungary) a well-known MDR was synthetized by Motohashi et al. (22). In the control cultures, 10 modifier was used as a positive control. The fluorescence of the cell ÌL DMSO was added. The compound used for treatment was added population was measured with a Beckton Dickinson FACScan flow to the samples at a final concentration of 2 or 10 Ìg/mL. After cytometer (Cell Sorter, Oxford, UK). The fluorescence activity incubation for 24 h at 37ÆC, the cells were transferred from a 24-well ratio (FAR) was calculated (20), on the basis of the measured plate into Eppendorf centrifuge tubes, centrifuged (2000 rpm, 2 min) fluorescence intensities (FL-1): and washed with PBS, and resuspended in 195 ÌL binding buffer. The samples were mixed and centrifuged (2000 rpm, 2 min) and the FAR = FL-1 treated cells/FL-1 untreated cells (Equation 1) supernatant was removed from each tube. Five microlitres Annexin- V-FITC (AV; human recombinant-FITC; Alexis Biochemical, Assay for antiproliferative effect. The effects of the Grünberg, Germany) was added to the tubes. Controls without AV chemotherapeutic agent, epirubicin hydrochloride (Farmitalia were also prepared. The samples and controls were incubated at Carlo Erba, Milano, Italy), the compounds and their room temperature for 10 min in the dark, then centrifuged (2000 combinations on the proliferation of the COLO320 cell line were rpm, 2 min), washed with PBS, and resuspended in 190 ÌL binding tested in 96-well microtitre plates. The compounds were diluted buffer. Before the measurement of fluorescence activity, 10 ÌL of 20 from high to low concentrations horizontally in the plates. Cells Ìg/mL propidium iodide (PI; Sigma-Aldrich Ltd.) was added to the were seeded into each well (1x104) with the exception of the samples. The fluorescence activity (FL-1 and FL-2) of the cells was medium control wells. The plates were incubated at 37ÆC for 72 measured and analysed by flow cytometry on a Beckton Dickinson h and 15 ÌL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5- FACScan instrument. In each analysis, 10.000 events were recorded, diphenyltetrazolium bromide (MTT solution; Sigma) was then and the percentages of the cells in the different states were added to each well. After incubation at 37ÆC for 4 h, 100 mL 10% calculated by square analysis using Windows Multiple Document sodium dodecylsulfate solution (Sigma) was added to each well. Interface for Flow Cytometry (winMDI2.8) (23, 24). The plates were further incubated at 37ÆC overnight to allow the dissolution of formazan crystals produced by the mitochondrial Ethidium bromide and acridine orange (EB/AO) staining. Staining with enzymes of the living cells. The extent of inhibition of cell EB/AO (Sigma-Aldrich Ltd.) was carried out in a 96-well plate proliferation was determined by measuring the optical density format after 24 h of treatment in order to visualize the morphological (OD) of the chromogenic products at 550 nm (ref. 630 nm) with events (25). Plates were centrifuged at 1000 rpm 2 min, and 8 ÌL of a Dynatech MRX vertical beam ELISA reader (Labsystems, staining solution (0.1 mg/mL for both AO and EB in PBS) was added Helsinki, Finland). The extent of inhibition of cell growth (as a to each well. After 10 min, the cells were washed with PBS, and the percentage) was determined with the following formula, in which cells were viewed with a Nikon Eclipse inverted microscope at 200x OD cell control means the untreated cells. 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 (Equation 2) 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 Table II. Antiproliferative effects of selected diterpenes in combination cells. with epirubicin on human colon cell line.

Compounds ID50±S.E.M*(Ìg/mL) Treatment FIX±S.E.M* Interaction

1 7.54±0.36 1 1.05±0.12 no interaction 2 6.41±0.05 3 0.68±0.07 additive 3 10.16±0.08 5 1.12±0.19 no interaction 4 10.65±0.51 6 0.54±0.08 additive 5 3.29±0.30 7 0.64±0.06 additive 6 13.27±0.87 8 0.25±0.03 synergism 7 17.46±0.77 9 1.15±0.07 no interaction 8 10.04±0.55 9 2.61±0.88 *FIX-fractional inhibitory index; S.E.M values were derived from the standard errors of the mean of at least three FIX values. Epirubicin 0.10±0.03

*All values are expressed as means±S.E.M from parallel experiments Table III. Effects of diterpenes on Rhodamine 123 accumulation of (n=2-4). human colon cancer cells.

Compounds Conc. FSCa SSCb FL-1c FARsd (Ìg/mL) Statistical analysis. Statistical analysis of the data was performed with Graphpad Prism 2.01 (Graph Pad Software, San Diego, COLO320+R123e - 566.33 251.77 362.75 - CA, USA). Verapamil 10 575.21 253.14 991.73 2.73 1 4 564.75 256.88 485.97 1.33 Results 40 537.72 256.84 619.41 1.70 2 4 570.13 252.81 447.81 1.23 40 527.90 282.81 447.91 1.23 Assay for antiproliferative effect. The antiproliferative effects 3 4 538.46 254.37 260.29 0.71 40 532.09 269.35 405.53 1.11 of the investigated compounds on the human colon cancer 4 4 574.65 252.89 216.42 0.59 cells are listed in Table I. A majority of the compounds 40 533.00 269.96 378.48 1.04 exhibited a moderate antiproliferative effect (ID50=6.41- 5 4 555.18 262.78 389.04 1.07 17.46 Ìg/mL), the jatrophane diterpenes 5 and 9 were the 40 530.03 246.92 624.76 1.72 most effective (ID <3.5 Ìg/mL). 6 4 547.06 250.94 541.85 1.49 50 40 532.24 260.57 897.12 2.47 In further experiments, some selected compounds in 7 4 539.18 246.74 744.02 2.05 combination with epirubicin were examined. Table II 40 543.63 248.79 738.33 2.03 reveals that a synergistic interaction (FIX=0.25) was found 8 4 565.38 253.45 552.37 1.52 between epirubicin and compound 8 (Figure 2). Diterpenes 40 538.15 261.04 908.48 2.50 3, 6 and 7 had only marginal additive effects. In contrast, 5 9 4 542.66 248.22 783.75 2.16 40 533.61 255.74 936.24 2.58 and 9 did not enhance the antiproliferative effect of the DMSO contol 20 ÌL 530.22 264.50 54.54 0.15 anticancer drug when applied in combination to the COLO320-R123f - 538.14 246.65 3.38 - COLO320 cell line. aFSC, forward scatter count; bSSC, side scatter count; cFL-1, mean d Assay for the reversal of MDR in the COLO320 cell line. The fluorescence intensity of the cells; FARs, fluorescence activity ratio values were calculated by using Equation 1 given in the Material and results are summarized in Table III. Compounds 6, 7, 8 Methods section; eCOLO320+R123, cell control with Rhodamine 123; and 9 were found to be very strong inhibitors (FAR>2.00 fCOLO320-R123, cell control without Rhodamine 123. 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. jatrophane diterpene 8 was noted in the applied Compounds 1-5 were moderately effective in modulating concentration range (2 and 10 Ìg/mL) as compared with the the P-gp on the resistant human colon cell line. positive control M627.

Assay of induction of apoptosis. Compound 8 was selected Ethidium bromide and acridine orange (EB/AO) staining. The for apoptosis induction on resistant mouse lymphoma cells, EB/AO staining of the HeLa cells allowed the identification based on its synergistic effect with epirubicin (Table IV and of live, apoptotic and necrotic cells. AO permeates all cells Figure 3). A moderate apoptosis-inducing effect of the and makes the nuclei appear green. EB is taken up by cells

3454 Engi et al: MDR Modulators and Apoptosis Inducers from Euphorbia Species

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 Table IV. Apoptosis induction by compound 8 on mdr1 gene-transfected lost; it stains the nucleus red. Even at a lower concentration mouse lymphoma cell line. of compound (2 Ìg/mL), treatment of HeLa cells with Sample Conc. Early Total Cell compound 8 led to the typical morphological features of (Ìg/mL) apoptosis apoptosis death apoptosis, including increased cell membrane permeability, (%) (%) (%) cellular shrinkage and granulation in the nucleus (Figure 4). Cell control wihout staininga - 1.14 1.22 0.06 Discussion Cell control + AVb - 8.05 11.43 1.97 Cell control + PIc - 0.31 1.18 2.91 Cell control double stainingd - 4.83 6.93 2.98 The fluorescent activities of the compounds indicated that 1% DMSO control - 1.20 3.05 2.93 a majority of the Euphorbia derivatives enhanced drug 12H-benzo (·)-phenothiazine 5 77.67 99.74 0.04 retention, as can be observed in Tables I-III. Three of the 25 26.25 99.55 0.03 tested diterpene derivatives (6, 8 and 9) displayed a Compound 8 2 4.04 5.80 3.36 10 3.02 6.14 3.02 significant concentration-dependent effect in inhibiting the efflux pump activity on the COLO320 cell line. The most aAnnexin-V-negative/propidium iodide-negative samples = intact effective synergistic effect was found between compound 8 viable cells (98-100%). bAnnexin-V-positive/propidium iodide- and epirubicin. The strong activity of this derivative can be negative=apoptotic cells. cAnnexin-V-negative/propidium iodide- d explained by its high lipophilicity, but other parameters, positive = necrotic cells. Annexin-V-positive/propidium iodide- positive = apoptotic/necrotic cells. 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 the presence of a hydroxy group instead of peracylation is substituents have significant effects on the stereostructure favourable as concerns the antiproliferative activity in and the activity of the molecules. It is known that the combination with epirubicin. conformational flexibility of the twelve-membered ring of Several studies have indicated that diterpene derivatives the jatrophanes is strongly influenced by the steric exert apoptosis-inducing activity (13, 26-27). The apoptosis- interactions of the substituents. On comparison of the inducing potential rather than necrosis induction is accepted efficacies of compounds 7 and 8, it can be presumed that as a key feature of a potential anti-tumour drug. A change

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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 been used to detect apoptosis in vitro (28). AV appears to apoptosis involves the redistribution of various phospholipid be a potent discriminator between viable and apoptotic cells species between the two leaflets of the membrane. The most (23). Compound 8 was able to induce moderate apoptosis pronounced feature of this asymmetry is the almost in the mdr1 gene-transfected mouse lymphoma cell line. complete absence of phosphatidylserine in the outer leaflet Together with the previous data, this allows the of the plasma membrane. Early in apoptosis, this conclusion that jatrophane and lathyrane derivatives can phospholipid is rapidly exposed in the outer surface of the be regarded not only as effective anti-MDR agents, but cells. AV, the Ca2+-dependent endogenous human protein, also as apoptosis inducers. These compounds may be has a high affinity for membrane-bound phosphatidylserine, promising lead compounds for natural product-based drug so this protein has been labelled with fluorescein and has development.

3456 Engi et al: MDR Modulators and Apoptosis Inducers from Euphorbia Species

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.

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