BBRC Biochemical and Biophysical Research Communications 315 (2004) 589–594 www.elsevier.com/locate/ybbrc
a-Bisabolol, a nontoxic natural compound, strongly induces apoptosis in glioma cells Elisabetta Cavalieri,a,1 Soﬁa Mariotto,a,1 Cinzia Fabrizi,b Alessandra Carcereri de Prati,a Rossella Gottardo,c Stefano Leone,b Luigi Valentino Berra,a Giuliana Maria Lauro,b Anna Rosa Ciampa,a and Hisanori Suzukia,* a
Department of Neuroscience and Vision, Section of Biochemistry, University of Verona, Verona, Italy b Department of Biology, University of Roma Tre, Italy c Department of Medicine and Public Health, Unit of Forensic Medicine, University of Verona, Italy Received 16 December 2003
Abstract In this study, a-bisabolol, a sesquiterpene alcohol present in natural essential oil, was found to have a strong time- and dosedependent cytotoxic eﬀect on human and rat glioma cells. After 24 h of treatment with 2.5–3.5 lM a-bisabolol, the viability of these cells was reduced by 50% with respect to untreated cells. Furthermore, the viability of normal rat glial cells was not aﬀected by treatment with a-bisabolol at the same concentrations as above. Glioma cells treated with high concentration of a-bisabolol (10 lM) resulted in a 100% cell death. Judging from hypo-G1 accumulation, poly(ADP-ribose) polymerase cleavage, and DNA ladder formation, the cytotoxicity triggered by a-bisabolol resulted from apoptosis induction. Moreover, the dissipation of mitochondrialinner transmembrane potential and the release of cytochrome c from mitochondria indicated that, in these glioma cells, apoptosis occurred through an intrinsic pathway. As pointed out by the experimental results, a-bisabolol may be considered a novel compound able to inhibit glioma cell growth and survival. Ó 2004 Elsevier Inc. All rights reserved. Keywords: a-Bisabolol; Glioma; Apoptosis; Human; Chemotherapy
Glioma is one of the most malignant human tumours  and, despite aggressive surgical resection and radiotherapy, the median survival in these patients does not normally exceed one year [2–4]. The use of systemic chemotherapy may improve the eﬃcacy of treatment, but its use is associated with signiﬁcant toxicity and the long-term prognosis remains poor . Carmustine, one of the more eﬀective anti-glioma drugs used in clinical therapy is, at a concentration corresponding to LD10 (13 mg/kg), not able to cause a 100% cell death of glioma cells in vitro . Numerous compounds from plants such as betulinic  and asiatic acids  have been reported to be potential anti-glioma agents, although
Corresponding author. Fax: +39-045-802-7170. E-mail address: [email protected]
(H. Suzuki). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.01.088
most of these compounds are no longer considered as possible treatment for human glioma. This is either due to their modiﬁcation in the liver or to their incapacity to pass through the blood–brain barrier. In the course of our research attempting to identify new natural compounds modulating inﬂammatory processes, we observed that a-bisabolol rapidly killed a number of human transformed cell lines, including highly malignant glioma cell lines. a-bisabolol (Fig. 1A) is a small oily sesquiterpene alcohol with molecular mass of 222.37 Da isolated from the essential oil of a variety of plants, shrubs, and trees. For example, the essential oil of Matricaria chamomilla contains up to 50% of a-bisabolol and this is the molecule considered to be the main component contributing to the mild anti-inﬂammatory eﬀect of chamomile [8,9]. Due to its intoxicity in animals (LD50 ¼ 13–14 g/kg ), it is widely used in cosmetic preparations. However, only a few scientiﬁc
E. Cavalieri et al. / Biochemical and Biophysical Research Communications 315 (2004) 589–594 Tetrachloro-1,10 ,3,30 -tetraethylbenzimidazol carbocyanine iodide (JC1) was obtained from Molecular Probes (Eugene, Oregon, USA). Cell culture Human and rat glioma cell lines were cultured in DMEM (BioWhittaker) supplemented with 10% fetal bovine serum (FBS) (BioWhittaker), 2 mM glutamine, and 40 lg/ml gentamicin, in a 5% CO2 atmosphere at 37 °C. The T67 cell line was obtained from a III WHO gemistocytic astrocytoma  and U87 and C6 cell lines were from ATCC. Liquid chromatography–mass spectrometry analysis (LC–MS analysis)
Fig. 1. (A) Molecular structure of a-bisabolol. (B) Amounts of a-bisabolol in the culture medium were estimated, at the time points indicated, by LC–MS analysis.
reports describing the biological eﬀects of a-bisabolol are so far available in the literature [11,12]. In the present work, we intended to study in detail the cytotoxic eﬀect and the type of death induced by a-bisabolol in glioma cells. For this purpose, we examined as a human glioma cell model, the T67 cell line which was isolated and characterised by Lauro et al. , and the commercially available U87 cell line. As an animal model, we tested the rat glioma cell line C6. Our data indicate that a-bisabolol may be considered as a promising inducer of apoptosis in highly malignant glioma cells, since it is neither toxic in animals nor can it reduce the viability of normal astroglial cells.
Methods Materials a-Bisabolol was purchased from Fluka and Riedel-de Ha€en, Sigma Chemical (St. Louis MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was obtained from Sigma Chemical (St. Louis MO, USA). Anti-PARP and anti-cytochrome c antibodies were purchased from Zymed Laboratories (South San Francisco, CA, USA) and from Santa Cruz (Santa Cruz, CA, USA), respectively. Antimouse and anti-rabbit IgG peroxidase-conjugated secondary antibodies were purchased from Amersham (Buckinghamshire, UK). 5,50 ,6,60 -
Sample extraction. To 1.8 ml of DMEM containing 10% of FBS diﬀerent amounts of a-bisabolol (from a stock solution 5 mg/ml in ethanol) were added to obtain ﬁnal concentrations of 100 and 250 lM. The samples were mixed and centrifuged at 3500g for 10 min; 1 ml of hexane was added to 0.5 ml sample collected with a syringe from the bottom of each sample tube (polypropylene). After vigorous mixing for 1 min followed by centrifugation at 3500g for 10 min, the organic phase was collected and evaporated to dryness under air stream. The residue was ﬁnally reconstituted with 1 ml methanol and injected in the liquid chromatograph (injection volume: 0.3 ll). Analysis. All experiments were performed on a Series 1100 Liquid Chromatograph/Mass spectrometer from Agilent (Palo Alto, CA, USA). The system was composed of a gradient microchromatograph with a diode-array detector (DAD) and an ion trap mass spectrometer with an electrospray ionisation (ESI) source, operating in the positive mode. The chromatographic conditions were as follows: column, Zorbax SB C18 (150 0.5 mm i.d., 5 lm, Agilent); mobile phase, 20 mM formic acid, pH 3: methanol (15:85); ﬂow rate, 20 ll/min; and temperature, 25 °C. MS conditions were as follows: nebuliser gas, N2 (15 psi); drying gas N2 (4 ml/min, 325 °C); capillary voltage, 5000 V; fragmentator voltage, 40 V; ionisation mode, positive; and mass scan range, 50–300 m=z. The MS was operated in a selected ion monitoring mode (SIM). Quantiﬁcation was performed on the 205 m=z ion generated in the source from the parent ion (222.4 m=z) by water loss. A daughter fragment ion at m=z 149 was used for conﬁrmation (manuscript in preparation). Cell viability MTT assay. Cell viability was measured by MTT reduction essentially as described . Following 24 h treatments, 10 ll of MTT solution (5 mg/ml) was added to each well and the incubation was continued for 3 h. Lysis buﬀer was prepared by dissolving 40% (w/v) sodium dodecyl sulphate (SDS) in deionised water, after adding an equal volume of N ,N -dimethylformamide, the pH was adjusted to 4.7. After a 3 h incubation with MTT, 100 ll of the lysis buﬀer was added to each well and the absorbance was read at 570 nm on a Microplate Reader (Camberra Packard, Milan, Italy). Viability was reported relative to untreated control cells. Trypan blue assay. Cells (2 105 cells/wells) were plated onto 6-well plates in 1 ml culture medium. After 24 h, the cells were treated with abisabolol and with ethanol alone (control). Cell viability was determined with trypan blue exclusion assay. Counts were performed in duplicate wells. Cytometric analysis Cells were washed twice in a phosphate buﬀered saline (PBS) w/o Ca2þ and Mg2þ , gently resuspended in 1 ml ﬂuorochromic solution containing 0.05 mg/ml propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100, and then placed at 40 °C overnight in the dark.
E. Cavalieri et al. / Biochemical and Biophysical Research Communications 315 (2004) 589–594 The ﬂuorescence of nuclei was measured with Galaxy (DAKO) ﬂow cytometer and the percentage of apoptotic nuclei was calculated using WinMDI v2.8 software. Western blot analysis Cells were homogenised at 4 °C in 50 mM Tris–HCl, pH 8, containing 0.1% Nonidet-P40 (NP-40), 200 mM KCl, 2 mM MgCl2 , 50 lM ZnCl2 , 2 mM DTT, and protease inhibitors (1 mM PMSF, 1 mg/ml leupeptin, and 1 mg/ml antipain). An aliquot of the homogenates (40 lg proteins/lane) was loaded onto 7.5% polyacrylamide gels. The run was performed at 100 V with a running buﬀer, containing 0.25 M Tris–HCl, pH 8.3, 1.92 M glycine, and 1% SDS and proteins were blotted to a PVDF membrane (Immobilon P, Millipore). Membranes were incubated with an anti-poly(ADP-ribose) polymerase (PARP) monoclonal antibody and, after washing, with an anti-mouse IgG peroxidase conjugate. Blots were successively incubated with enhanced chemiluminescent detection reagents (ECL kits, Amersham Life Science) and proteins were detected by chemiluminescence, exposing blots to Kodak X-AR ﬁlm. DNA ladder For the internucleosomal DNA laddering, 3 106 cells, resuspended in 0.3 ml of culture medium with 10% FBS, were incubated for 45 min at 65 °C and then overnight at 37 °C in the presence of 0.4 M NaCl, 5 mM Tris–HCl, pH 8, 2 mM EDTA, 4% SDS, and 2 mg/ml proteinase K. The lysate, supplemented with 1.58 M NaCl, was centrifuged twice for 10 min at 6000g to separate the DNA fragments from intact DNA. The DNA soluble fraction was precipitated and the DNA pellet was resuspended in 10 mM Tris–HCl, pH 7.4, 1 mM EDTA, and 1 mg/ml ribonuclease A and then loaded onto 1% agarose gel in the presence of ethidium bromide. After electrophoresis, the DNA was visualised by UV light. JC-1 estimation of inner transmembrane potential (Dwm) in living cells 5,50 ,6,60 -Tetrachloro-1,10 ,3,30 -tetraethylbenzimidazol carbocyanine iodide (JC-1) is lipophilic, cationic dye that enters into mitochondria and forms red ﬂuorescent J-aggregates (590 nm) at the high membrane potential. However at lower potential JC-1 exists as a green ﬂuorescent monomer (527 nm) . T67 and U87 cells were grown on coverslips and treated with a-bisabolol for 15, 30, 60, and 90 min. After these incubation times, the medium was removed, and a modiﬁed medium containing 2 mg/ml JC-1 in prewarmed DMEM was added. Cells were placed back into the incubator (37 °C, 5% CO2 , and 100% humidity) for 30 min, and then washed twice with PBS in order to remove unbound dye and ﬁxed using 4% paraformaldehyde. Fluorescence was analysed using confocal laser scanning microscope (Axioplan 2, LSM 510, Carl Zeiss, G€ ottingen, Germany) equipped with argon (488) and helium/neon (543) excitation beams.
Results Estimation of the concentration of a-bisabolol dissolved in the culture medium Since a-bisabolol is a highly lipophylic molecule, we ﬁrst evaluated the dose-dependent solubilisation in the culture medium. a-bisabolol, dissolved in ethanol, was added to the culture medium to obtain ﬁnal concentrations of 100 and 250 lM. Thereafter, a-bisabolol dissolved in the culture medium was measured at various time points up to 24 h with LC–MS analysis. As shown in Fig. 1B, the amounts of a-bisabolol in the culture medium increased time-dependently, achieving the plateau after 15 h, although from these time points only 2.5% of the initial amounts of a-bisabolol was detected. Accordingly, in the present study, the indicated a-bisabolol concentrations are referred, unless otherwise described, to as the soluble fraction of a-bisabolol measured in each experiment. Eﬀect of a-bisabolol on glioma cell viability The eﬀect of a-bisabolol on the viability of T67, U87, and C6 cells was examined by MTT assay. As shown in Fig. 2A, a-bisabolol induced dose-dependently a decrease in cell viability in T67 and U87 cell lines after 24 h of treatment. The same eﬀect was also observed in abisabolol-treated C6 cells (Fig. 2B). After 24 h of treatment with 2.5 lM a-bisabolol, a 50% cell death of T67 and C6 cells was observed. Although the U87 cell line appeared to be more resistant to a-bisabolol treatment
Measurement of cytochrome c release T67 and U87 cells were washed twice with ice-cold PBS and scraped oﬀ the plates. Cells were collected by centrifugation at 500g for 5 min. Cell pellets were suspended in 1 ml of solution containing 10 mM NaCl, 1.5 mM MgCl2 , 10 mM Tris–HCl, pH 7.5, 1 mM sodium orthovanadate, and complete EDTA-free protease inhibitor cocktail (Boehringer–Mannheim GmbH). Cells were then chilled on ice for 10 min and gently lysed by adding 0.3% (v/v) NP-40. In order to restore an isotonic environment, a solution containing 525 mM mannitol, 175 mM sucrose, 12.5 mM Tris–HCl, pH 7.5, 2.5 mM EDTA, and protease inhibitor cocktail was added. Lysates were centrifuged at 17,000g for 30 min at 4 °C. The cytosols so obtained were separated on a 15% SDS–PAGE and probed using an anti-cytochrome c polyclonal antibody as described above.
Fig. 2. T67, U87 cell lines (A), rat astroglial cells and C6 cell line (B) were treated with diﬀerent concentrations of a-bisabolol for 24 h. Cell viability was determined by MTT assay and is reported as the percentage of viable cells. The results represent the mean value ( SD) of six independent experiments.
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than T67 and C6 cells, 5 lM a-bisabolol was suﬃcient to cause 50% cell death of this line after 24 h. To examine the capacity of a-bisabolol to annihilate glioma cells, we ﬁrst treated the U87 and T67 cell lines with 6 lM a-bisabolol for 24 h. After 24 h treatment remaining cells proliferated quickly if they were cultivated in a fresh medium without a-bisabolol, reaching conﬂuency in the successive 48 h. However, if these cells were cultivated in the medium containing freshly added a-bisabolol (6 lM), they were all killed in the successive 24 h, as determined either by MTT or trypan blue exclusion assays (data not shown). Furthermore, U87 and T67 cells were annihilated in 24 h in the presence of 10 lM a-bisabolol, indicating that this eﬀect is also timeand dose-dependent. We successively treated rat astroglial cells with a-bisabolol to ﬁnd out whether this compound is also toxic for normal cells. Absolutely no eﬀect on the viability was observed when normal astroglial cells were incubated for 24 h with increasing amounts of a-bisabolol up to 10 lM (Fig. 2B).
a-bisabolol. Thus, biochemical features strictly correlated to apoptosis such as PARP cleavage and DNA ladder formation were successively examined in abisabolol-treated U87 and T67 cells. Eﬀect of a-bisabolol on poly(ADP-ribose) polymerase cleavage The integrity of the PARP molecule in glioma cell lines was examined by Western blot analysis of the cell homogenates. As shown in Fig. 4A, in both T67 and U87 cell lines there was no evidence of PARP cleavage before treatment with a-bisabolol. In T67 cell line, 4.5 lM a-bisabolol induced the appearance of a band corresponding to 85 kDa after 2 h of treatment and total disappearance of the intact band, corresponding to 116 kDa, after 16 h. In U87 cells, total cleavage of PARP
Eﬀect of a-bisabolol on hypodiploid DNA peak measured by cytometric analysis Flow cytometric analysis was performed in order to evaluate the eﬀect of a-bisabolol on the appearance of apoptotic nuclei in T67 and U87 cells after 4 and 24 h of treatment, respectively. As shown in Fig. 3, in both cell lines a-bisabolol induced the appearance of a subdiploid peak (from 2% of the control to 28% in T67 cells and from 7 to 30% in U87 cells), indicating the apoptotic nature of the death of glioma cells triggered by
Fig. 3. DNA ﬂuorescence histograms presented in log scale of propidium iodide-stained glioma cells after 4 h incubation for T67 cell line or 24 h incubation for U87 cell line in the absence or presence of a-bisabolol. The percentage of sub-G0/G1 peaks in treated and untreated cells is shown.
Fig. 4. (A) PARP cleavage was assessed by Western blotting with an anti-PARP antibody, which detects intact (116 kDa) and cleaved (85 kDa) products. T67 and U87 cell lines were treated with 4.5 lM abisabolol for 2 (lane 2), 5 (lane 3), and 16 h (lane 4). Control cells are shown in lane 1. Intact PARP (116 kDa) and cleaved fragment (85 kDa) are indicated with arrows. (B) T67 and U87 cells were treated with 4.5 lM a-bisabolol for 5 h.
E. Cavalieri et al. / Biochemical and Biophysical Research Communications 315 (2004) 589–594
was observed with 6 lM a-bisabolol after 16 h of treatment. Eﬀect of a-bisabolol on the formation of DNA ladder Finally, cell extracts were analysed by electrophoresis on agarose gel to observe the formation of DNA ladder. As shown in Fig. 4B, after 5 h treatment, 4.5 lM a-bisabolol induced the formation of DNA ladder in both cell lines, although no evidence of DNA ladder was present before treatment.
Fig. 6. Eﬀect of a-bisabolol on cytosolic cytochrome c release. T67 and U87 cell lines were treated with 250 lM a-bisabolol (administrated concentration) for 15, 30, 60, and 90 min. The soluble fractions of abisabolol in culture medium may be extrapolated from Fig. 1B. After treatment the cytosolic extracts were analysed by Western blotting with an anti-cytochrome c antibody.
Analysis of extrinsic pathway To evaluate whether a-bisabolol-induced apoptosis is mediated by an extrinsic pathway, U87 and T67 cells were treated for 5 h with 4.5 lM a-bisabolol, both in the presence of the antagonist anti-Fas antibody (B-D29) and the caspase-8 inhibitor, Z-IETD-FMK (Calbiochem). None of these treatments reverted a-bisabololinduced glioma cell death (data not shown). Eﬀect of a-bisabolol on mitochondrial permeability transition The analysis by confocal microscopy demonstrated a time-dependent decrease of Dw (Fig. 5) and, at the same time, the release of cytochrome c from the outer mitochondrial membrane (Fig. 6) in U87 and T67 cells treated by 1.5 lM a-bisabolol.
Fig. 5. Loss of mitochondrial Dwm. U87 and T67 cells stained with JC1 show the loss of red J-aggregate ﬂuorescence and cytoplasmic diffusion of green monomer ﬂuorescence after 15 min of induction with 1.5 lM a-bisabolol.
Discussion The present study has indicated that a-bisabolol (Fig. 1A), a small oily sesquiterpene alcohol, has a strong time- and dose-dependent cytotoxic eﬀect on highly malignant human and rat glioma cell lines (EC50 ¼ 2 lM, Fig. 2). Another striking feature of a-bisabolol is its ability to annihilate glioma cell lines (U87 and T67) at a concentration of 10 lM in a 24 h treatment (Fig. 2A). Also, at lower concentration (6 lM), these glioma cells were completely killed in 48 h if a-bisabolol was added every 24 h. It should be stressed that one of the most eﬀective clinically used anti-glioma drugs, carmustine, is not able to annihilate glioma cells at concentration corresponding to LD10 (13 mg/kg) [5,16]. Despite its devastating eﬀect on malignant cells, a-bisabolol failed to induce any cytotoxicity in normal rat astroglial cells even under highly drastic conditions (up to 10 lM of a-bisabolol for 24 h of treatment). That being in line with its documented non-toxicity in animals  (LD50 was estimated to be around 15 g/kg, in oral administration to rats and mice). Judging from increase in hypo-G1 fraction (Fig. 3), PARP cleavage (Fig. 4A), and DNA ladder formation (Fig. 4B) in U87 and T67 cell lines, a-bisabolol-induced cytotoxicity in glioma cells may result from the induction of apoptosis . It is widely accepted that apoptosis is preferred to necrosis as a mechanism of tumour cell killing, since it enhances no inﬂammatory processes . Apoptosis is a physiological, energy requiring process  which is characterised by the formation of apoptotic bodies inside cells and seems to be genetically programmed. Tumour cells may be resistant to apoptosis, presumably due to defects in apoptosis pathways. Two major routes, extrinsic and intrinsic, have been identiﬁed through which cytotoxic drugs induce apoptosis. The ﬁrst pathway is mediated by death receptors, such as Fas or tumour necrosis factor receptor, and procaspase-8 is cleaved to the active form with subsequent activation of downstream caspases (caspase-3, -6, and -7) . In the second pathway, mitochondria play
E. Cavalieri et al. / Biochemical and Biophysical Research Communications 315 (2004) 589–594
essential roles through the mitochondrial permeability transition (PT). Induction of the mitochondrial PT leads to dissipation of the inner transmembrane potential (Dwm) and is one of the putative mechanisms triggering cytochrome c translocation . In the present study, we ﬁrst investigated if a-bisabolol induces apoptosis through the extrinsic pathway. Neither the antagonist anti-Fas antibody nor the caspase-8 inhibitor blocked cell death. Then, we evaluated mitochondrial involvement in a-bisabolol-induced apoptosis using determination of Dwm, a hallmark of PT, and cytochrome c translocation. We demonstrated both the loss of Dwm and the release of cytochrome c and noticed that these events occurred rapidly (Figs. 5 and 6). Altogether, these data indicate that a-bisabolol probably is able to trigger apoptosis in a Fas receptor independent manner, through the intrinsic pathway. Virtually, all previously examined compounds, that resulted to be highly cytotoxic to glioma cells, have been discarded as promising anti-glioma drugs mainly due to their fast elimination by the body and/or to their poor permeability through the blood–brain barrier. a-bisabolol bears a highly stable structure and its reported toxicity in animals is very low as already described . A pharmacokinetic study has indicated that in a rat receiving 120 mg/kg a-bisabolol, its concentration in brain exceeds 50 lM at 24 h after administration without any toxic eﬀect on the animal (manuscript in preparation). This feature further makes a-bisabolol highly suitable for the study of its eﬀect in an animal model of glioma. In conclusion, a-bisabolol, the main component of the essential oil of chamomile and other plants, was found to have a strikingly eﬃcient and potent cytotoxic eﬀect on human and rat malignant glioma cell lines, rapidly inducing apoptosis through the mitochondrial pathway with no toxic eﬀect on normal glial cells. Since glioma is among the worst tumours against which no eﬃcient and non-toxic treatments have so far been reported, the results obtained with a-bisabolol, i.e., no toxicity observed in animals and its fast accumulation in the brain, make the use of this substance very promising for the clinical treatment of this highly malignant tumour. Acknowledgments This work was supported by Italian MURST COFIN Project 2000, 2002 to H.S. and MURST COFIN 2001 to Guido Palladini, Department of Neuroscience, University La Sapienza, Rome, Italy. We thank Darcie Baynes for editorial assistance.
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