Doi:10.1016/j.ejphar.2006.07.020

European Journal of Pharmacology 548 (2006) 9 – 20 Phenylbenzopyrones structure-activity studies identify betuletol derivatives as potential antitumoral agents Sara Rubio a, José Quintana a, Mariana López b, José Luis Eiroa b, Jorge Triana b, Francisco Estévez a,⁎ a Department of Biochemistry, I.C.I.C., University of Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Spain b Department of Chemistry, I.C.I.C., University of Las Palmas de Gran Canaria, Campus Universitario de Tafira, 35017 Las Palmas de Gran Canaria, Spain Received 10 March 2006; received in revised form 20 June 2006; accepted 13 July 2006 Available online 22 July 2006 We have analyzed the cytotoxicity of 22 compounds with a phenylbenzo-γ-pirone core structure, most of them obtained from natural sources, in five human tumor cell lines (HL-60, A431, SK-OV-3, HeLa and HOS). Betuletol 3-methyl ether and its diacetate were the most cytotoxiccompounds. The HL-60 cell line was especially sensitive to these compounds, with IC50 values of approximately 1 μM. Treatment of HL-60 cellswith betuletol 3-methyl ether was associated with apoptosis induction which was prevented by a non-specific caspase inhibitor (z-VAD-fmk) andalso by a specific inhibitor of caspase-8 (z-IETD-fmk) indicating activation of the extrinsic apoptotic pathway. The results suggest that betuletol 3-methyl ether has potential as new anticancer agent.
2006 Elsevier B.V. All rights reserved.
Keywords: Apoptosis; Flavonoids; Cytotoxic activity; Caspases; DNA fragmentation; poly(ADP-ribose) polymerase activation of caspase-3 ).
The extrinsic pathway is initiated by activation of death Many anticancer drugs have been shown to cause the death receptors and thereby cleavage of procaspase-8 of sensitive cells through the induction of apoptosis. This ). Caspase-8 directly activates caspase- cellular suicide program is initiated by ligation of death 3 (). Caspase-8 also cleaves Bid-a Bcl-2 receptors, growth factor deprivation or various environmental family member— and thereby plays a role in the release of stresses which are mediated by molecular pathways that cytochrome c. However, it is becoming increasingly clear that culminate in the activation of a conserved family of aspartate- apoptosis can also occur independently of caspase activation specific cysteine proteases, known as the caspases, which orchestrate the dismantling and clearance of the dying cell.
Flavonoids comprise a vast array of biologically active Caspases themselves are present as proenzymes that are readily compounds ubiquitous in plants, many of which have been used cleaved and activated during apoptosis, providing the cell with a in traditional Eastern medicine for thousands of years. These means to rapidly amplify its apoptotic response ( polyphenolic compounds exert several biological functions such Two major pathways have as apoptosis-inducing activity, free radical scavenging activity, been identified for the induction of apoptosis. The intrinsic and anti-tumorigenic activity ().
pathway is activated by release of mitochondrial cytochrome c Here we have studied the effect of twenty two flavonoids, most of them isolated from endemic plants to the Canary Islands, on forms a complex with Apaf-1 and activates caspase-9 leading to cell viability of five human tumor cell lines. The betuletolderivatives, betuletol 3-methyl ether and betuletol 3-methyl ether diacetate were the most potent cytotoxic compounds in all Corresponding author. Tel.: +34 928 451443; fax: +34 928 451441.
E-mail address: (F. Estévez).
cell lines studied, but also the most potent apoptotic inducers on 0014-2999/$ - see front matter 2006 Elsevier B.V. All rights reserved.
S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 human myeloid leukemia HL-60 cells. Increased levels of anti- apoptotic or decreased levels of pro-apoptotic proteins can cause Vis λmax nm, (MeOH): 287, 323,329; (+NaOMe): 284, 325, acquired or intrinsic resistance against apoptosis induction by 393; (+ AlCl3): same as MeOH; (+AlCl3+ HCl): same as AlCl3; antineoplastic drugs thereby contributing to treatment failure (+ NaOAc): 287, 360; (+ NaOAc + H3BO3): 287,325. PMR and poor clinical prognosis. Thus, novel agents targeting (300 MHz, CDCl3): δ 8.06 (d, J = 8.8, H-2′, H-6′), 7.21 (d, aberrant apoptosis pathways or inducing alternative death J = 8.5, H-3′, H-5′), 6.89 (s, H-8), 3.94 (s, OMe), 3.76 (s, OMe), pathways may be suited to overcome treatment resistance 2.50 (s, OAc), 2.33 (s, OAc). EIMS m/z (rel. int.): 413 [M]+ (The present studies demonstrate (36.6), 372 (100), 330 (49.2), 315 (46.8), 312 (42.8), 287 (22.5), that betuletol 3-methyl ether induces apoptosis, at least in large 269 (10.6), 121 (14.9), 69 (15.6).
part, by activation of the extrinsic caspase-8 pathway of death.
Quercetin 4 (3,3′,4′,5,7-pentahydroxyflavone) was obtained from Sigma. Cryptostrobin 18 was isolated from Rumohra 2. Materials and methods adiantiformis. The structure of this compound was determinedby a combination of spectroscopic analysis and comparison with reported data Dihydroquercetin 7,3′-dimethyl ether 16 and dihydrokaemp- Most of the flavonoids used in this study were obtained from ferol 7,4′-dimethyl ether 17 were isolated from the aerial parts the Canary Islands' endemic plants and were isolated according of Pulicaria canariensis as recently described to published methods with minor modifications. Structural All other reagents were of analytical grade. Purity of all identities of flavonoids were determined spectroscopically compounds was 99.0% as judged by high-performance liquid (proton nuclear magnetic resonance and 13C nuclear magnetic chromatography. Stock solutions of 10 mM flavonoids were resonance, infrared and UV/Visible spectroscopy and mass made in dimethyl sulfoxide (DMSO), and aliquots were frozen spectrometry) as described previously: Betuletol 3-methyl ether at −20 °C. Polyvinylidene difluoride (PVDF) membranes were 1, quercetin 3,3′-dimethyl ether 7, kaempferol 3-methyl ether 9, purchased from Millipore. Antibodies for poly(ADP-ribose) 5,7-dihydroxy-3,3′,4′-trimethoxyflavone 10, 4′, 5, 7-trihy- polymerase (PARP), caspase-3, caspase-8 and caspase-9 were droxy-3,6-dimethoxyflavone 12 and eriodictyol 19 were purchased from Stressgen. Antibody for cytochrome c was isolated from the aerial parts of Allagopappus dichotomus as purchased from BD PharMingen. Secondary antibodies were described (Quercetin 3-methyl ether 5, from Amersham Pharmacia Biotech. The caspase inhibitors naringenin 21, eriodictyol 19 and kaempferol 3-methyl ether 9 were from Sigma. The caspase-3 and caspase-8 substrates were were isolated from Allagopappus viscosissimus as described from Sigma. The caspase-9 substrate was from AnaSpec.
(). Axillarin 11 and apigenin 22 wereisolated from the aerial parts of Tanacetum ferulaceum and Ta- 2.2. Cell culture nacetum ptarmiciflorumas described ). Acetyl derivatives of betuletol 3-methyl ether (com- HL-60 cells were cultured in RPMI 1640 medium containing pound 2), quercetin 3-methyl ether (compound 6), quercetin 10% (v/v) heat-inactivated fetal bovine serum and 100 units/ml 3,3′-dimethyl ether (compound 8) and eriodictyol (compound penicillin and 100 μg/ml streptomycin at 37 °C in a humidified 20) were obtained by treatment of the corresponding alcohols atmosphere containing 5% CO2. The cultures were passed twice with acetic anhydride (Ac2O) in pyridine for 12 h at room weekly exhibiting characteristic doubling times of ∼24 h. The cell temperature. Betuletol 3, 5, 7-trimethyl ether 3 was obtained by numbers were counted by a hematocytometer, and the viability was methylation of 4′,5,7-trihydroxy-3, 6-dimethoxyflavone 12.
always greater that 95% in all experiments as assayed by the Mono-di- and triacetyl derivatives (compounds 13, 14 and 15) 0.025% trypan blue exclusion method. Further dilutions of stock of 4′,5,7-trihydroxy-3, 6-dimethoxyflavone 12 were obtained by solutions of flavonoids were made in culture media just before use.
acetylation of this compound with acetic anhydride in pyridine In all experiments, the final concentration of DMSO did not exceed as above. The acetyl derivatives were purified by chromatog- 0.3% (v/v), a concentration which is non-toxic to the cells. The raphy on a silica gel column, eluted with hexane, and hexane- same concentration was present in control experiments. The A431 ethyl acetate mixtures (8:2). The mono and diacetyl derivatives, cell line was grown as monolayers in plastic tissue flasks containing 7-Acetoxy-4′,5-dihydroxy-3,6-dimethoxyflavone 13 and 5,7- Dulbecco's modified Eagle's medium (DMEM) supplemented diacetoxy-4′-hydroxy-3,6-dimethoxyflavone 14, are described with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml for the first time.
streptomycin. SK-OV-3 was grown as monolayers in DMEM. The human cervix carcinoma HeLa and human osteosarcoma HOS Vis λmax nm, (MeOH): 289, 316,330; (+ NaOMe): 286, 325, cells were grown in Eagle's minimum essential medium (EMEM) 398; (+ AlCl3): 294, 350, 396; (+ AlCl3+ HCl): same as AlCl3; supplemented with 2 mM glutamine, 1% Non essential amino acids (+ NaOAc): 290, 376; (+ NaOAc + H3BO3): same as NaOAc.
and 10% fetal bovine serum. Cells at logarithmic growth phase PMR (300 MHz, CDCl3): δ 8.11 (d, J = 8.8 Hz, H-2′, H-6′), were harvested using 0.25% trypsin-EDTA solution for 5 min, 7.23 (d, J = 8.8 Hz, H-3′, H-5′), 6.76 (s, H-8), 4.04 (s, OMe), pelleted by centrifugation at 500 ×g for 5 min, washed once with 3.87 (s, OMe), 2.35 (s, OAc). EIMS m/z (rel. int.): 372 [M]+ medium and resuspended at a concentration of 1 × 104 cells/ml.
(100), 357 (15.6), 330 (41.1), 329 (45.8), 287 (39.1), 269 (17.8), These cell lines were obtained from the European Collection of Cell 121 (22.9), 69 (66.0).
Cultures (Salisbury, UK).
S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 Human peripheral blood mononuclear cells (PBMC) were containing 50 μg/ml propidium iodide and 100 μg/ml RNase A isolated from heparin-anticoagulated blood of healthy volunteers and incubation for 1 h at 37 °C in the dark. The percentage of by centrifugation with Ficoll–Paque Plus (Amersham Bios- cells with decreased DNA staining, composed of apoptotic cells ciences). PBMCs were also stimulated with phytohemagglutinine resulting from either fragmentation or decreased chromatin, of a (PHA, 2 μg/ml) for 48 h before experimental treatment.
minimum of 10,000 cells per experimental condition wascounted. Cell debris was excluded from analysis by selective 2.3. Cytotoxicity of flavonoids on HL-60 cells, A431 cells, gating based on anterior and right angle scattering.
HeLa cells, SK-OV-3 cells and HOS cells 2.6. Analysis of DNA fragmentation The cytotoxicity of flavonoids in HL-60 cells was analyzed HL-60 cells were washed with PBS and incubated in 20 μl of tetrazolium bromide (MTT) assay. Briefly, 1 × 104 exponential- 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS and 1 μg/ ly growing cells were seeded in 96-well microculture plates μl RNase A (Sigma) at 37 °C for 1 h. Then, 10 μg/μl proteinase with various flavonoids concentrations. After the addition of K (Sigma) (2 μl) was added, and the mixture was incubated at MTT (0.5 mg/ml) cells were incubated at 37 °C for 4 h. Sodium 50 °C for 2 h more. DNA was extracted with 100 μl of phenol- dodecyl sulfate (SDS) (10% w/v) in 0.05 M HCl was added to chloroform-isoamyl alcohol (24:24:1) and mixed with 5 μl of the wells and then incubated at room temperature overnight loading solution (10 mM EDTA, pH 8.0, containing 1% (w/v) under dark conditions. The absorbance was measured at low melting-point agarose, 0.25% bromophenol blue and 40% 570 nm. Concentrations inducing a 50% inhibition of cell sucrose). Samples were separated by electrophoresis in 2% growth (IC50) were determined graphically for each experiment agarose gels and visualized by UV illumination after ethidium as described A431, SK-OV-3, HeLa and bromide staining.
HOS cells (5 × 103 − 1 × 104) were inoculated in 96-well platesand were then incubated at 37 °C for 24 h to allow the 2.7. Immunoblotting of caspase-3 attachment to plates after which the culture media were changedand flavonoids were added and provided different final HL-60 cells (1 × 106) were treated with flavonoids at the concentrations. Cells were then incubated at 37 °C in a 5% indicated concentrations in RPMI 1640 medium. Cell pellets 95% air atmosphere for 72 h. Cell viability was were lysed in lysis buffer containing 125 mM Tris-HCl pH 6.8, determined as above using a colorimetric assay with the 2% SDS, 5% glycerol, and 1% β-mercaptoethanol, and boiled reduction of MTT.
for 5 min. The samples were separated on 15% SDS-polyacrylamide gel, electrotransferred to a PVDF membrane, 2.4. Quantitative fluorescent microscopy immunoblotted with anti-caspase-3 antibody [Stressgen; 1:1000dilution in Tris-buffered saline containing 0.1% Tween-20 Cells were harvested and fixed in 3% paraformaldehyde and (TBST) supplemented with 3% nonfat milk] overnight. After incubated at room temperature for 10 min. The fixative was washing and incubation with horseradish peroxidase-conjugat- removed and the cells were washed with PBS, resuspended in 30– ed anti-rabbit (Amersham Pharmacia Biotech), the antigen– 50 μl of PBS containing 20 μg/ml bis-benzimide trihydrochloride antibody complexes were visualized by enhanced chemilumi- (Hoechst 33258), and incubated at room temperature for 15 min.
nescence (ECL, Amersham Pharmacia Biotech) using the Ten-microliter aliquots of the cells were placed on glass slides, and triplicate samples of 500 cells each were counted and scoredfor the incidence of apoptotic chromatin condensation using a 2.8. Immunoblot analysis of poly(ADP-ribose) polymerase Zeiss fluorescent microscopy. Stained nuclei with condensed chromatin (supercondensed chromatin at the nuclear periphery),or nuclei that were fragmented into multiple smaller dense bodies Induction of apoptosis was also examined by proteolytic were considered as apoptotic. Nuclei with uncondensed and cleavage of poly(ADP-ribose) polymerase. After treatments, cells dispersed chromatin were considered as not apoptotic.
were pelleted by centrifugation, and resuspended in lysis buffercontaining 25 mM PBS, 0.1 mM phenylmethylsufonylfluoride 2.5. Quantification of apoptosis by flow cytometry and protease inhibitors leupeptin, aprotinin and pepstatin A (5 μg/ml each). After centrifugation, the pellet was resuspended in the To study changes in the cell DNA content, histogram loading buffer containing 125 mM Tris-HCl, pH 6.8, 2% SDS, measurements of hypodiploid DNA formation was performed 5% glycerol, and 1% β-mercaptoethanol. The mixture was by flow cytometry using a Coulter EPICS™ cytometer (Beck- sonicated for 30 s at 4 °C and then boiled to 100 °C for 3 min. The man Coulter). Histograms were analyzed with the Expo 32 ADC cell lysates were fractionated on a 7.5% polyacrylamide gel Software™. Cells were collected and centrifuged at 500 ×g, containing 0.1% SDS and proteins were electrophoretically washed with PBS and resuspended in 50 μl of PBS. Following transferred onto a PVDF membrane. Membrane was blocked with dropwise addition of 1 ml of ice-cold 75% ethanol, fixed cells 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 were stored at −20 °C for 1 h. Samples were then centrifuged at for 1 h, followed by incubation with anti-poly(ADP-ribose) 500 ×g and washed with PBS before resuspension in 1 ml of PBS polymerase polyclonal antibody (Stressgen; 1:3000 dilution in S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 TBST supplemented with 3% nonfat milk) overnight. After Antiproliferative studies on betuletol 3-methyl ether 1 indicate washing and incubation with anti-rabbit antibody conjugated to that this compound displays strong cytotoxic properties in all horseradish peroxidase (Amersham Pharmacia Biotech), the cell lines assayed, although HL-60 cells was especially sensitive antigen–antibody complexes were visualized by enhanced to this substance with an IC50 as low as 1.8 ± 0.9 μM chemiluminiscence (ECL, Amersham Pharmacia Biotech) using Since betuletol 3-methyl ether 1 has two hydroxyl groups on C5 the manufacturer's protocol. The appearance of an 85 kDa and C7, respectively, we next obtained the diacetate 2- and cleavage product was used as a measure of apoptosis.
dimethyl ether 3-derivatives of betuletol 3-methyl ether 1 inorder to know more about the structural requirement for the 2.9. Detection of cytochrome c release from mitochondria antiproliferative action of this compound. Therefore, thecytotoxic studies performed with the di-acetylated compound Release of cytochrome c from mitochondria was detected by indicate that theses changes have no impact on the IC50 in HL-60 Western blot analysis. After treatments, HL-60 cells were cells (1.8 ± 0.9 μM vs 1.3 ± 0.4 μM). However, in the washed twice with PBS and then suspended in ice-cold buffer dimethylated derivative a dramatic consequence on the growth [20 mM HEPES (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 1 mM inhibitory activity is observed. Contrary to betuletol 3-methyl EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenyl- ether 1 and betuletol 3-methyl ether diacetate 2, betuletol 3, 5, 7- methylsulfonylfluoride, and 5 μg/ml leupeptin, aprotinin, and trimethyl ether 3 is ineffective as an antiproliferative agent in all pepstatin A] containing 250 mM sucrose. After 15 min cell lines tested. Since the methoxy group is smaller than the incubation on ice, cells were lysed by pushing them several acetyl group, it seems clear that this is not a consequence of steric times through a 22-gauge needle and the lysate spun down at hindrance. Moreover, if the hydroxyl groups in the betuletol 3- 1000 ×g for 5 min at 4 °C. The supernatant fraction was methyl ether 1 interact by hydrogen bridges with vicinity groups centrifuged at 105,000 × g for 45 min at 4 °C and the resulting on the biological targets, then the presence of the keto groups on supernatant was used as the soluble cytosolic fraction. Cytosolic the di-acetyl derivative could be important. One group that proteins (50 μg) were resolved on an SDS/15% polyacrylamide seems to play a key role in determining the potency of these gel and electrotransferred onto a PVDF membrane. The compounds on cell viability is the methyl group on position 4′of membrane was probed with monoclonal anti-cytochrome c the B ring (2-phenyl group) since betuletol 3-methyl ether 1 is antibody (BD Transduction Laboratories) (1:250 dilution) and significantly more potent than 4′,5,7-trihydroxy-3,6-dimethox- then with secondary antibody conjugated to horseradish yflavone 12 in all cell lines studied.
peroxidase. Proteins bands were detected by chemiluminescence The critical relationship of fruit and vegetable intake and (ECL, Amersham Pharmacia Biotech) as described above.
cancer prevention has been thoroughly documented Since quercetin 4 is the most abundant flavonoid in the 2.10. Assay of caspase activity human diet we have investigated the effect of a series ofstructurally related flavonoids. The results indicate that methyl- After treatments, cells were harvested by centrifugation at ation of hydroxyl group at position C3 of quercetin 4 yields 1000 ×g for 5 min at 4 °C and washed with PBS, and the cell quercetin 3-methyl ether 5, a compound with a higher pellets were kept on ice. The cells were resuspended in cell lysis antiproliferative activity. Cytotoxic effects were observed in all buffer (50 mM HEPES, pH 7.4, 1 mM dithiothreitol, 0.1 mM cancer cell lines tested except in HOS cells, which indicates that EDTA, 0.1% Chaps) and held on ice for 5 min. After the above chemical change in the quercetin molecule 4 affects cell centrifugation for 10 min at 17,000 ×g at 4 °C, the supernatants proliferation in a cell-type specific manner. However, the were analyzed for protein concentration by the Bradford dye- hydroxyl group located at the 3′ position seems to have little binding assay and stored at −20 °C until used to study caspase importance for the growth inhibitory activity of the above colorimetric enzymatic activity. Equal amounts of protein quercetin derivative since the additional methylation at this level (∼20 μg) from different treatments were used, and the assays to yield quercetin 3,3′-dimethyl ether 7 does not significantly were set up on ice. The net increase of absorbance at 405 nm change the IC50 value. The presence of a hydroxyl group in the 3′ after incubation at 37 °C was indicative of enzyme activity.
position of ring B has different consequences depending on the Specific labelled substrates for caspase-3, -8 and 9 activities configuration of molecule. For example, kaempferol 3-methyl were N-acetyl-Asp-Glu-Val-Asp- p-nitroaniline (DEVD- ether 9 lacks the 3′-hydroxyl group and displays similar IC50 to its 3′-hydroxylated counterpart, quercetin 3-methyl ether 5 in all cell lines assayed. Similarly, quercetin 3-methyl ether tetracetate 6 and quercetin 3,3′-dimethyl ether triacetate 8 differ only by thepresence of an acetyl- or methylether-group, respectively, at the 3′ position, but they showed similar potency. On the other hand, thepresence of the 3′ hydroxyl group improves the antiproliferative 3.1. Betuletol derivatives inhibit the growth and cell viability of activity of axillarin 11 as compared with the 3′-dehydroxylated human tumor cell lines counterpart 12 in all cell types tested except HL-60 cells. When amethoxy group is placed at C6 instead of hydrogen in quercetin 3- In the present study, we examined the effects of 22 flavonoids methyl ether 5, the impact on the IC50 is dependent on the cell type (), on the growth of five human tumor cell lines.
under study. The resulting compound (axillarin 11) is significantly S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 Fig. 1. Structures of the investigated flavonoids.
less cytotoxic on HL-60 (IC50 = 14.3 ± 4.6 μM versus 32.6± (90.4 ± 27.6 μM versus 9.4 ± 3.0). Unlike the HOS cells, the 8.8 μM) but significantly more cytotoxic on HOS cells remaining cell lines were resistant to these chemical changes.
(IC50 = 90.4 ± 27.6 μM versus 25.6± 9.6 μM). In contrast, the Also, an increase in the growth inhibitory activity was clearly presence of this methyl ether group does not change its cytotoxic observed when quercetin 3,3′-dimethyl ether 7 was acetylated at potency on A431, HeLa and SK-OV-3 cells.
positions 4′, 5 and 7 to produce quercetin 3,3′-dimethyl ether The influence of the hydroxyl group at position 4′ of the B ring triacetate 8. HL-60 cells, A431 cells and particularly HOS cells (2-phenyl group) on cell viability was also tested. A dramatic were susceptible to these chemicals substitutions.
decrease in the growth inhibitory activity was observed when Selected acetylations were also performed on the free quercetin 3,3′-dimethyl ether 7 was methylated in that position to hydroxyl groups of the 4′, 5, 7-trihydroxy-3, 6-dimethoxy- produce 5,7-dihydroxy-3,3′,4′-trimethoxyflavone 10. The IC50 flavone molecule 12. Acetylation of the hydroxyl group at increased from 12.2 ± 0.4 μM and 30.6± 7.3 μM to N100 μM, in position 7 yielded a monoacetylated derivative, 7-acetoxy-4′,5- HL-60 and HeLa cells respectively. As occurs with others dihydroxy-3,6-dimethoxyflavone 13, that was significantly substitutions, this chemical change affected the cell lines under more active on HL-60 cells. This effect is abolished when a study to different degrees. The HOS cell line was resistant to both second and a third acetyl group are sequentially introduced at quercetin 3,3′-dimethyl ether 7 and 5,7-dihydroxy-3,3′,4′- position 5 and at position 4′ to produce the derivative trimethoxyflavone 10. Thus, acetylation of all free hydroxyl groups locating at positions 3′, 4′, 5 and 7 in the quercetin 3- 14 and 4′,5,7-triacetoxy-3,6-dimethoxyflavone 15 respectively.
methyl ether 5 molecule to produce quercetin 3-methyl ether Another set of flavonoids are those related to the structure of tetracetate 6 greatly improved the cytotoxic activity on HOS cells.
the low cytotoxic compound naringenin 21. This flavanone In this cell line the IC50 decreased about one order of magnitude decreased the proliferation rate of the cell lines studied only at S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 3.2. Betuletol derivatives induce apoptosis on human myeloid Effects of phenylbenzo-γ-pirone derivatives on the growth of human tumor cell In order to evaluate whether betuletol derivatives decrease cell viability through apoptosis activation, morphological changes characteristic of apoptotic cells (condensed and fragmented chromatin) were analyzed and quantified by fluorescent micros- copy (A and B). Evaluation of nuclear morphology , indicates that the percentage of apoptotic cells increased from 6 ± 1% to 24 ± 2% (4 times increase) in betuletol 3-methyl ether treated cells, after 12 h exposure at a concentration as low as 10 μM A). Next, we examined whether these flavonoids induce poly(ADP-ribose) polymerase cleavage, a hallmark of apoptosis that indicates activation of caspase. Hydrolysis of the 116 kDa poly(ADP-ribose) polymerase protein to the 85 kDa fragment was detected in betuletol 3-methyl ether and betuletol 3- methyl ether diacetate treated cells after 12 h exposure at 10 μM , and increased in a dose-dependent manner (data not shown). These results indicate that poly(ADP-ribose) polymerase cleavage was involved in apoptosis induced by these compounds and, as expected (due to cytotoxic ineffectiveness), betuletol 3,5,7-trimethyl ether did not induce poly(ADP-ribose) polymer- ase cleavage C). When cells were incubated with these Cells were cultured for 72 h and the IC50 values were calculated as described inthe Experimental Section. The data shown represent the mean ± S.E.M. of 3–5independent experiments with three determinations in each.
very high concentrations (IC N 100 μM) but the introduction of a hydroxyl group at position 3′ (eriodictyol 19) greatlyincreases its antiproliferative activity although only on HL-60and A431 cells. The 3′, 4′, 5 and 7 tetracetylated derivative,eriodictyol tetracetate 20, is even more active with IC50 valuesas low as 4.5 ± 1.3 μM and 10.8 ± 1.3 μM on HL-60 and A431,respectively. A moderate increase in the cytotoxicity ofnaringenin 21 is also observed on HL-60 and A431 cellswhen the hydroxyl group placed at the 4′ position on the B ring(2-phenyl group) is absent and the molecule has a methyl groupon C8 (cryptostrobin 18). Another structural requirement toimprove the cytotoxic activity of naringenin 21 is theintroduction of a double bond between C2 and C3. Theresulting flavone (apigenin 22) showed antiproliferative activityin all cell lines tested although HL-60 cell line was the mostsusceptible to this chemical change (IC50 = 13.1 ± 1.0 μM).
A detailed inspection of reveals that among the five tumor cell lines used in this study the growth of promyelocytic Fig. 2. Effects of betuletol 3-methyl ether and betuletol 3-methyl ether diacetate HL-60 cells were, in general, most susceptible to the cytotoxicity on apoptosis on HL-60 cells. (A) Cells were stained with bisbenzimide induced by flavonoids. Interestingly, quercetin 4, a flavonoid trihydrochloride after treatment with 10 μM of betuletol 3-methyl ether for 12 h, widely studied due to abundance in the fruit and vegetable and apoptotic cells were determined by fluorescence microscopy. Valuesrepresent means ± S.E. This histogram is representative of two independent usually consumed by humans, displayed significantly cytotoxic experiments each performed in duplicate. ⁎P b 0.05, significantly different from properties for HL-60 cells alone (IC50 = 27.8 ± 4.1 μM). The the untreated control. (B) Photomicrographs of representative fields of HL-60 remaining lines were insensitive to quercetin 4 effects on cell cells treated as above and stained with bisbenzimide trihydrochloride to evaluate nuclear chromatin condensation (i.e. apoptosis). (C) Analysis for the cleavage of Since HL-60 cell line was highly sensitive to the anti- poly(ADP-ribose) polymerase (PARP) by immunoblotting. (D) Cells wereincubated as above and DNA extracted. Laddered electrophoretic patterns of proliferative effect of betuletol 3-methyl ether 1 we decided to oligonucleosomal DNA fragments were resolved by conventional agarose gel determine whether this natural flavonoid displays its cytotoxic electrophoresis, stained with ethidium bromide and visualized under UV light.
action through activation of the apoptotic pathway.
Control lane refers to untreated cells.
S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 flavonoids, the DNA showed the typical fragmentation patterns 3.4. Betuletol derivatives-induced apoptosis involves caspase-3 formed by internucleosomal hydrolysis of chromatin thus confirming the apoptosis-inducing effects. As expected, betuletol3,5,7-trimethyl ether was unable to induce DNA fragmentation To determine whether caspase-3 is activated in HL-60 cells by . Taken together, these results indicate that both betuletol the betuletol 3-methyl ether, dose-response experiments were derivatives, betuletol 3-methyl ether and betuletol 3-methyl ether performed and cell lysates assayed for cleavage of the tetrapeptide diacetate, are potent inducers of human myeloid leukemia HL-60 cells apoptosis and that both compounds are equally active.
pNA). The results A), demonstrate that caspase-3 activityincreased 1.5- and 2.3-fold in betuletol 3-methyl ether treated 3.3. A general caspase inhibitor abolished betuletol cells with 3- or 10-μM of compound, respectively, for 12 h.
derivatives-induced poly(ADP-ribose) polymerase cleavage Hydrolysis of procaspase-3 to the active enzyme, caspase-3, by and DNA fragmentation betuletol 3-methyl ether and betuletol 3-methyl ether diacetatewas also determined by immunobloting. The results indicate that To confirm that betuletol derivatives-triggered apoptosis these compounds at concentrations as low as 3 μM promote requires the activation of caspases, HL-60 cells were pretreated cleavage of pro-caspase-3 into active caspase-3 B). At high with the broad-spectrum caspase inhibitor z-VAD-fmk (benzy- concentrations (100 μM) of a specific caspase-3 inhibitor, loxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone). The results demonstrate that this inhibitor completely blocked tone (z-DEVD-fmk), the percentage of apoptotic cells and DNA betuletol derivatives-induced DNA fragmentation (A).
laddering induced by betuletol 3-methyl ether was significantly The effect of z-VAD-fmk on poly(ADP-ribose) polymerase decreased. In contrast, there was not any effect when the cleavage by betuletol 3-methyl ether was also analyzed. As concentration of the inhibitor used was lowered to a minimum observed (B), pretreatment of HL-60 cells with this of 10 μM (results not shown).
inhibitor completely abolished the generation of the 85 kDafragment. Taken together, these results indicate that these events 3.5. Effects of betuletol derivatives on mitochondrial cytochrome are caspase dependent.
To determine whether betuletol 3-methyl ether and betuletol 3-methyl ether diacetate-induced apoptosis on HL-60 cellsinvolves the release of cytochrome c from mitochondria tocytosol, dose-response experiments were performed and cyto-solic preparations were analyzed by immunoblotting. Asdemonstrated A), a low concentration of both compounds(3 μM) induce cytochrome c release at the same proportion andno difference was observed with higher concentrations ofcompounds. The effect of these compounds on caspase-9formation was also analyzed. The results indicate that bothbetuletol derivatives stimulate cleavage of inactive pro-caspase-9 to the active 37 kDa fragment B). To determine whetherthis fragment was associated with caspase-9 activity, lysatesfrom treated cells were assayed for cleavage of the specificsubstrate N-acetyl-Leu-Glu-His-Asp-p-nitroaniline (LEHD-pNA). The results indicated that compound 1 (10 μM, 12 h)also increases caspase-9 activity In order to determinethe contribution of caspase-9 to the betuletol derivatives-inducedapoptosis we examined the impact of the irreversible caspase-9inhibitor benzyloxycarbonyl-Leu-Glu-His-Asp(OMe)-fluoro-methyl ketone (z-LEHD-fmk). As shown the inhibitorwas completely ineffective at blocking betuletol 3-methyl ether-induced DNA nucleosomal fragmentation typical of apoptoticcell death.
Fig. 3. Involvement of activation of caspases in the induction of apoptosis. HL- 3.6. Caspase-8 is activated by betuletol derivatives 60 cells were incubated with the indicated doses of compounds for 12 h inabsence or presence of broad-spectrum caspase inhibitor z-VAD-fmk (100 μM).
Since the intrinsic pathway does not seem to contribute DNA fragmentation (A) and proteolysis of poly(ADP-ribose) polymerase (B) significantly to betuletol derivatives-induced apoptosis, we were then analyzed. The migration positions of full-length poly(ADP-ribose) decided to determine the contribution of the extrinsic pathway.
polymerase and the 85 kDa cleavage product are indicated. The results arerepresentative of three independent experiments.
Therefore, HL-60 cells were treated with increasing doses of S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 percentage of apoptotic cells from 6% (control) to 24% and 30%with 10 μM and 30 μM of product, respectively, as determinedby flow cytometry (C). The effect of betuletol 3-methylether was, however, completely abolished when the cells werepretreated with z-IETD-fmk. No impact on basal apoptosis level Fig. 4. Activation of caspase-3 in HL-60 cells. (A) The cells were incubated inthe presence of the indicated concentrations of betuletol 3-methyl ether for 12 h,and total cell lysate was assayed for caspase-3 activity using the DEVD-pNAcolorimetric substrate. The results are expressed as fold-increase in caspaseactivity compared with control. Values represent means ± S.E. This histogram isrepresentative of two independent experiments each performed in triplicate.
⁎Pb0.05, significantly different from the untreated control. (B) Western blotanalysis for the cleavage of caspase-3. The cells were treated with differentconcentrations of betuletol 3-methyl ether and betuletol 3-methyl ether diacetateand whole cell lysates were subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) followed by blotting with an anti-caspase-3antibody. β-Actin was used as loading control.
betuletol 3-methyl ether and the lysates were then assayed forcleavage of the tetrapeptide N-acetyl-Ile-Glu-Thr-Asp-p-nitroani-line (IETD-pNA) as a specific substrate for caspase-8. As shown(, this compound induced caspase-8 activation in a dose-dependent manner. The enzymatic activity increased to 2.4- and3.5-fold over control with 3 μM and 10 μM of betuletol 3-methylether, respectively. Also, we subjected cell lysates to immunoblotanalysis with an antibody which recognizes the inactiveprocaspase-8 and the proteolytic (32 kDa and 42–44 kDa) Fig. 5. Effects on the intrinsic pathway of apoptosis. (A) HL-60 cells were fragments. The results clearly demonstrate that incubated with the indicated concentrations of compounds 1 and 2 for 12 h, concentrations of betuletol 3-methyl ether as low as 3 μM cytosolic extracts were separated by sodium dodecyl sulfate-polyacrylamide gel significantly promotes procaspase-8 hydrolysis. A dramatic electrophoresis (SDS-PAGE) and cytochrome c release was detected byimmunoblotting. As loading control β-actin was also analyzed. (B) Western increase in the hydrolysis of procaspase-8 (visualized as 32 kDa blot analysis for the cleavage of procaspase-9. Cells were treated as above and and 42–44 kDa fragments) was observed with the higher whole cell lysates were subjected to SDS-PAGE followed by immunoblotting concentration tested (10 μM) while the lowest concentration with an anti-procaspase-9 antibody that also recognizes the proteolytic used (1 μM) had no impact on the cleavage at the time assayed fragments generated. (C) Cells were incubated in the presence or absence of (12 h). Proteolytic cleavage of the initiator caspase-8, which is betuletol 3-methyl ether for 12 h, and total cell lysates were assayed for caspase-9 activity using the LEHD-pNA colorimetric substrate. The results are expressed demonstrated in response to betuletol 3-methyl ether, typically as fold-increase in caspase activity compared with control. Values represent occurs after triggering cell surface death receptors like the CD95 means ± S.E. This histogram is representative of two independent experiments each performed in triplicate. ⁎P b 0.05, significantly different from the untreated To determine whether caspase-8 is implicated in the betuletol control. (D) Lack of effect of z-LEHD-fmk treatment on betuletol 3-methyl 3-methyl ether-induced apoptosis on HL-60 cells, we examined ether-induced DNA fragmentation. Cells were preincubated in the presence orabsence of the caspase-9 inhibitor z-LEHD-fmk (100 μM) for 1 h and then the impact of the irreversible caspase-8 inhibitor benzylox- followed by treatment with betuletol 3-methyl ether for 12 h. DNA fragmentation was analyzed by agarose gel electrophoresis as described in IETD-fmk). Therefore, betuletol 3-methyl ether increased the Materials and methods.
S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 showed no similar effect. Low concentrations (10 μM) of eachspecific caspase inhibitor (z-LEHD-fmk for caspase-9 and z-IETD-fmk for caspase-8) were ineffective in decreasing thepercentage of apoptotic cells and on DNA laddering induced bybetuletol 3-methyl ether (results not shown).
To define the temporal relationships among the betuletol 3- methyl ether-induced events, HL-60 cells were treated with thiscompound and then harvested at various intervals. Induction ofboth caspase-8 (3-fold) and caspase-3 (3.8-fold) activities wassignificantly detectable at 8 h of treatment. By contrast, theincrease in caspase-9 activity (3-fold) was not detectable until12 h These findings indicated that betuletol 3-methylether induces caspase-8 activation and thereby direct ofcleavage of caspase-3.
3.8. Lack of cytotoxicity of betuletol derivatives on normalhuman lymphocytes Since an ideal anti-cancer agent should have no effect on normal, non-tumoral cells, we investigated whether betuletolderivatives 1 and 2 were also cytotoxic for human peripheralblood mononuclear cells (PBMC). No cytotoxicity (up 10 μM) toeither fresh or proliferating PBMC growth was observed. As a Fig. 6. Activation of caspase-8. (A) HL-60 cells were treated with the indicatedconcentrations of betuletol 3-methyl ether for 12 h and cell lysates were thenassayed for caspase-8 activity. (B) The cells were treated as described in (A) andactivation of caspase-8 was determined by immunoblot analysis. (C) HL-60cells were incubated with the indicated concentrations of betuletol 3-methylether for 12 h, in absence or presence of the caspase-8 inhibitor z-IETD-fmk(100 μM). Apoptotic cells (i.e. hypodiploid DNA content) was determined andquantified by flow cytometry after staining with propidium iodide. (D) Cellswere treated as above and genomic DNA was extracted, separated on an agarosegel and visualized under UV light by ethidium bromide staining. Valuesrepresent means ± S.E. Histograms are representative of two independentexperiments each performed in triplicate. ⁎P b 0.05, significantly differentfrom the untreated control.
was detected in cells incubated with z-IETD-fmk. The effect ofcaspase-8 inhibitor on DNA fragmentation induced by betuletol3-methyl ether was also analyzed. As shown D), in thepresence of z-IETD-fmk no DNA-laddering was observed inHL-60 cells treated with 10 μM of betuletol 3-methyl ether.
These results demonstrate that caspase-8 plays a central role inthe caspase-dependent apoptosis induced by betuletol 3-methylether.
3.7. Inhibition of caspase-3 activation in the presence of thecaspase-8 inhibitor but no effect by the caspase-9 inhibitor Fig. 7. (A) Western blot analysis for the effects of z-IETD-fmk (caspase-8 specific inhibitor) and z-LEHD-fmk (caspase-9 specific inhibitor) on betuletol3-methyl ether-induced cleavage of procaspase-3 or poly(ADP-ribose) poly- To further improve the demonstration of the key role played merase. (B) Kinetics of caspase-3, caspase-8 and caspase-9 activation in by caspase-8 in betuletol derivative-induced apoptosis, we response to betuletol 3-methyl ether. HL-60 cells were treated with 10 μM performed additional experiments.
betuletol 3-methyl ether and harvested at the indicated times. Cell lysates were Pretreatment of HL-60 cells with the irreversible caspase- assayed for caspase-3, caspase-8 and caspase-9 activities. Results are expressedas fold-increase in caspase activity compared with control. Values represent 8 inhibitor z-IETD-fmk fully blocked the procaspase-3 and poly means ± S.E. This histogram is representative of two independent experiments (ADP-ribose) polymerase (a typical substrate for caspase-3) each performed in triplicate. ⁎Pb0.05, significantly different from the untreated cleavage (A), but the caspase-9 inhibitor z-LEHD-fmk S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 because betuletol 3-methyl ether 1 is much more potent than 4′,5,7-trihydroxy-3,6-dimethoxyflavone 12 in all cell lines studied.
A study on structure-antiproliferative activity reported relationships between several polymethoxylated flavonoids,indicating that the C2–C3 double bond and the 3-hydroxylgroup of the C ring are important structural factors Our finding that naringenin 21 and dihydroquercetin 7,3′-dimethyl ether 16 are not effective antiproliferative agentsconfirmed that the C2–C3 double bond may be important. Therole of the 3′-hydroxyl group is deduced when comparing theantiproliferative activities of naringenin 21 and eriodictyol 19.
The only structural difference in both flavonoids is the presenceof a 3′-hydroxyl group in the eriodictyol molecule 19, whichclearly increases its cytotoxic capabilities. These results must beinterpreted as cell-type specific since only HL-60 and A431 cellswere susceptible to eriodictyol 19.
Some authors have suggested that the growth-suppressive activity of polymethoxylated flavonoids may, in part, beattributed due to their chemical stability (It has been demonstrated that quercetin 4 may undergoautoxidation and can also be oxidatively degraded, while Fig. 8. Differential effect of betuletol derivatives on proliferation of normal methylation of phenolic groups, as in the case of quercetin 3,3′- peripheral blood mononuclear cells (PBMC) vs. HL-60 cells. Proliferation of dimethyl ether 7 would be expected to confer greater stability to HL-60 cells, quiescent PBMC and phytohemagglutinine (PHA)-activated this flavonoid ). Moreover, addition of healthy human PBMC cultured in presence of indicated concentrations ofcompounds 1 and 2 for 24 h. Values represent means ± S.E. These histograms are ascorbic acid at low concentrations increases the antiprolifera- representative of two independent experiments each performed in triplicate.
tive activity of quercetin 4 against the HTB 43 squamous cell ⁎Pb0.05, significantly different from the untreated control.
carcinoma which seems to be related to the ability of ascorbicacid to inhibit the oxidative degradation of the polyhydroxy- positive control, HL-60 cells were also included in the experiment lated flavonoids The finding of and, as expected, there was an important reduction in the significantly higher cytotoxic activity of quercetin 3,3′- proliferation of these cells ).
dimethyl ether 7 than quercetin 4 in most cell lines studied,concords with this. Unlike quercetin 4, methylation of quercetin 3,3′-dimethyl ether 7 to yield quercetin 3,3′,4′-trimethyl ether10 decreases its cytotoxic potency. Similarly, when betuletol 3- Flavonoids are naturally occurring phenylbenzo-γ-pyrones methyl ether 1 is methylated to betuletol 3,5,7-trimethyl ether 3, found in abundance in diets rich in fruits, vegetables and plant- its cytotoxic activity is completely abolished. Although there derived beverages (These polyphenolic have been no studies on the chemical stability of betuletol and compounds are of great current interest due to their possible its methoxylated derivatives, this result cannot be explained by anticancer activities ( an increase in stability, but rather the loss of essential hydroxyl In this study, we have tested five human tumor cell lines for their group/s. Although there is much evidence that the position and sensitivity to twenty two different natural and semisynthetic number of the hydroxyl groups on A and B rings strongly phenylbenzo-γ-pyrones derivatives. Our data indicate that betu- influence the conformation of molecule and modulate their letol 3-methyl ether 1 and its diacetyl derivative 2 are potent growth inhibitory effect, it is generally accepted that the growth inhibitors of proliferation and display high cytotoxic activities on inhibition effect of flavonoids cannot be predicted on the basis human myeloid leukemia HL-60 cells and other cell lines, such as of their chemical composition and structure ( A431 human epidermoid carcinoma and HOS osteosarcoma cells.
However, the cytotoxicity of quercetin 4 was greatly improved Previous studies have shown that human ovarian adenocarcinoma by acetylation and methylation of the free hydroxyl groups in all SK-OV-3 cells are resistant to TRAIL (Tumor Necrosis factor- cell lines tested. In HL-60 the cytotoxic potency increased ten related Apoptosis-Inducing Ligand). The low sensitivity of SK- times. Since acetylated/methylated derivatives compounds can OV-3 cells to the tested compounds could be explained by the high pass through biological membranes more easily than quercetin, a levels of HER-2/neu protein and high levels of Akt (an anti- possible explanation of the difference in IC50 could be the higher apoptotic serine/threonine kinase that mediates survival signals) intracellular concentration reached. However, there are probably activity in this cell line ). However, the human additional factors that play important roles to determine the final myeloid leukemia HL-60 cell line was especially sensitive to both biological response in a particular tumor cell line. For example, betuletol derivatives (betuletol 3-methyl ether 1 and its diacetyl although the acetylated derivative of eriodictyol 20 greatly derivative 2). The introduction of a methyl group on position 4′ of improved the antiproliferative activity in most cell lines (HL-60, the B ring (2-phenyl group) likely contributes to this cytotoxicity A431, HeLa and SK-OV-3), the increase in lypophilicity does not S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20 explain its ineffectiveness in HOS cells. Also, among the 22 of cytochrome c release in time course experiments after compounds presented in betuletol 3-methyl ether 1 and betuletol derivatives treatments. The cytochrome c release was its diacetate-derivative 2 exhibited the highest cytotoxic activities not an early event in the apoptotic cell death induced by both against all human cancer cell lines evaluated. In most cell lines the betuletol derivatives (results not shown). Although the exposure IC50 values for these betuletol derivatives were lower than 10 μM, of HL-60 cells to both betuletol derivatives resulted in increased although in HL-60 cells these values were about 1 μM.
cytosolic cytochrome c, this response was not detected until As for all of the agents used or developed for cancer after 12 h of treatment. Consistently with this finding, the treatment, selectivity toward cancer cells is an important activation of caspase-9 (as determined by a colorimetric assay criterion. We therefore compared the effects of compounds 1 and western blot), was not observed until the same time period.
and 2 between HL-60 cells and human peripheral blood Furthermore, there have been ample examples of cross-talk mononuclear cells (PBMC). Interestingly, dose-response stud- between cell death receptor-mediated signaling through the ies revealed that quiescent PBMC and proliferating PBMC were caspase-8 and the apoptosome cascade. Caspase-8 directly resistant toward both betuletol derivatives 1 and 2.
activates downstream effector caspases and also cleaves Bid and In this work, we demonstrate that the exposure of HL-60 triggers mitochondrial damage that in turn leads to cytochrome cells to betuletol 3-methyl ether and its diacetyl derivative c release. Studies have demonstrated that the intrinsic pathway exerts a strong antiproliferative effect and both compounds for apoptosis initiation is controlled by members of the Bcl-2 displayed similar IC50 values. Among the number of pathways family ), which are that can be exploited for cancer prevention and treatment, crucial regulators of apoptosis in mammalian cells. However, apoptosis is a physiological process by which cells are removed we were unable to demonstrate alterations in the expression or when an agent damages their DNA, therefore compounds that in the mobility of Bcl-2 on sodium dodecyl sulfate-polyacryl- can induce apoptosis may be useful in management and therapy amide gel electrophoresis, a phenomenon that generally of cancer. Although previous studies have documented the (although not invariably) accompanies perturbations in phos- induction of apoptotic cell death and DNA fragmentation by phorylation state (data not shown).
phenylbenzo-γ-pirones derivatives in the human myeloid Previous studies have shown that apoptosis caused by anti- leukemia HL-60 cells (betuletol derivatives cancer drugs may be mediated via the CD95 system have yet to be assessed. Our results show that betuletol 3-methyl although many drugs seem to initiate the mitochondrial ether and its diacetyl derivative induced apoptosis in the pathway directly. An ample variety of mechanisms of resistance hematopoietic cell line HL-60.
to apoptosis that interfere at different levels of apoptosis Both betuletol derivatives promote the formation of apoptotic signalling have been described ).
bodies and the internucleosomal degradation of DNA, resulting in Thus a search for novel agents that target aberrant apoptosis the formation and eventual release of oligonucleosomal DNA pathways or induce alternative death pathways may be suited to fragments. The apoptosis induction was accompanied by poly overcome treatment resistance.
(ADP-ribose) polymerase cleavage which is considered to be one Using irreversible inhibitors for selected caspases, we of the hallmarks of apoptosis attempted to further delineate the pathways of caspase activation We report in the present study that z-VAD-fmk, a broad- and their relationship to poly(ADP-ribose) polymerase cleavage spectrum caspase inhibitor, completely abrogated the morpho- and DNA fragmentation induced by betuletol derivatives. The logical changes associated with apoptosis, DNA fragmentation effective blockage of apoptosis induction by a cell-permeable and the cleavage of poly(ADP-ribose) polymerase, which inhibitor of caspase-8, z-IETD-fmk, but not by a cell-permeable indicated that caspases are essential components in betuletol inhibitor of caspase-9, z-LEHD-fmk (also confirmed by kinetic 3-methyl ether-induced apoptosis.
studies of caspases activation) supports a model in which Caspase-3 is the most active effector caspase, in both the betuletol 3-methyl ether induces apoptosis by a caspase- intrinsic and extrinsic pathways where it is processed and 8 mediated mechanism.
activated by caspase-9 and caspase-8 respectively. Caspase-3has been found to be involved in leukemia-cell apoptosis induced by cytotoxic agents such as 1-β-D-arabinofuranosylcytosine,mitoxantrone, etoposide and CPT-11 ). A Our results indicate that betuletol 3-methyl ether inhibits high level of caspase-3 activation and processing was found after human HL-60 cell growth and induces apoptotic cell death treatment of HL-60 cells with betuletol derivatives 1 and 2.
involving the extrinsic pathway and could be useful in the Previous studies have already demonstrated that most of development of novel anticancer agents.
cytotoxic agents induce the release of mitochondrial cyto-chrome c (and this protein triggers a caspase-dependent assembly of the apoptosome ). Although we demonstrate that betuletol This work was supported by Grants from the Ministerio de derivatives initiated redistribution of cytochrome c into the Educación y Ciencia of Spain and FEDER (SAF2004-07928), cytosol and procaspase-9 processing, our results indicate that from Consejería de Educación, Cultura y Deportes (Canary the role of caspase-9 in the betuletol induced-apoptosis overall Islands Government) and FEDER (GRUP2004/44) and Insti- is, at most, minimal. Moreover, we analyzed the kinetic patterns tuto Canario de Investigación del Cáncer (08/2004). We thank J.
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