Original Paper 515 Induction of Mitochondrial-Dependent Apoptosis by an Essential Oil from Tanacetum gracile Author Monika Verma1, Shashank Kumar Singh1, Shashi Bhushan1, Harish C. Pal1, Surinder Kitchlu2, Maharaj K. Koul2, Rajender K. Thappa3, Ajit K. Saxena1 Affiliation 1 3 Key words " Tanacetum gracile ● " Asteraceae ● " apoptosis ● " essential oil ● " cytotoxicity ● " HL-60 ● Division of Pharmacology, Indian Institute of Integrative Medicine, Jammu-Tawi, India Division of Botany, Indian Institute of Integrative Medicine, Jammu-Tawi, India Division of chemistry, Indian Institute of Integrative Medicine, Jammu-Tawi, India Abstract ! The essential oil of Tanacetum gracile (Accession no. AT-01 termed AT-01 in the manuscript), a cold desert alpine highly aromatic herb, has 40 constituents including lavendulol (21.5 %), lavendulol acetate (1.7 %), α-pinene (11.2 %), 1,8-cineole (15.2 %), cis-β-ocimene (6.9 %), borneol (6.1 %), limonene (5.1 %) and chamazulene (3.7 %). AT-01 was evaluated for its anticancer activity. It inhibited HL-60 cell proliferation with an IC50 of 27 µg/ mL. Furthermore, AT-01 induced apoptosis in human leukemia HL-60 cells as measured by several biological end points. AT-01 induced apoptotic Introduction ! received August 7, 2007 revised March 1, 2008 accepted March 4, 2008 Bibliography DOI 10.1055/s-2008-1074503 Planta Med 2008; 74: 515–520 © Georg Thieme Verlag KG Stuttgart · New York Published online April 10, 2008 ISSN 0032-0943 Correspondence Shashank K. Singh Division of Pharmacology Indian Institute of Integrative Medicine Jammu-Tawi 180001 India Tel.: +91-191-2572002 extn 241 shashankksingh@gmail.com monikaverma24@rediffmail.com Tanacetum gracile Hook's F & Thomson, (Accession no. AT-01) a perennial aromatic plant of the family Asteraceae, grows wild in Ladakh (India), Pakistan and Afghanistan at an altitude of 2800 – 3600 meters in the alpine western Himalayas and blooms from July to September. Several species and chemotypes of Tanacetum have been explored in past for their chemistry and biological properties [1], [2], [3], [4]. Tanacetum vulgare and Tanacetum millefolium are two different species of same genus Tanacetum, which contain as main components β-thujone (91 %) and the sesquiterpenoid hydrocarbon γ-cadinene, respectively, [5], [6].The essential oil of T. gracile earlier has not been reported for any pharmacological activity. A recent study of the essential oil of T. gracile collected from Ladakh revealed the presence of 40 chemical constituents of which 40 % were monoterpenes [7]. Many of these monoterpenes have been identified to have anticancer and anticarcinogenic activities [8], [9], [10], [11]. Many anticancer drugs are derived from plant sources, e. g., taxol from Taxus brevifolia, camptothecin from Camptotheca acuminata and there body formation, enhanced annexinV-FITC binding of the cells, increased sub-G0 DNA fraction, loss of mitochondrial membrane potential (Δψmt) and release of cytochrome c from mitochondria, activated caspase-9 as well as caspase-3, and increased cleavage of PARP in HL-60 cells. Thus, AT-01 induced apoptosis through the mitochondrial dependant pathway in HL-60 cells. Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica continues to be a need for new anticancer drugs. We therefore assessed the anticancer potential of AT-01 and its mechanism of action of apoptosis in HL-60 cells. Deregulation of apoptosis is the hallmark of all cancer cells and agents that activate programmed cell death in cancer cells could be valuable anticancer therapeutics. There were two major pathways involved in the regulation of apoptosis. The drugs may kill cells either by activation of extrinsic or intrinsic apoptotic pathways. The extrinsic apoptotic pathway involves cell surface death receptors, such as Fas/CD95 and TNFR1 which, upon activation, regulate downstream signaling cascades leading to the activation of caspase-8. The intrinsic pathway is dependent on various cell stress stimuli leading to an altered ratio of Bcl-2 family members affecting cytochrome c and apoptotic protease activating factor-1 (Apaf-1) release leading to caspase-9 activation. The active forms of caspase-8 and caspase-9 may activate downstream effector caspases-3, -6, and 7, thus, enabling the cleavage of several intracellular polypeptides such as PARP as well as activation of DNAses leading to DNA fragmentation. Our studies for the first time provide the molecular mechanism of action of apop- Verma M et al. Induction of Mitochondrial-Dependent … Planta Med 2008; 74: 515 – 520 Downloaded by: Indian Institute Of Integrative Medicine. Copyrighted material. 2 Original Paper tosis induction by the essential oil AT-01 in HL-60 cells. AT-01 induced apoptosis through the intrinsic apoptotic pathway by causing loss of mitochondrial membrane potential, caspases-9 and -3 activation, cytochrome c release, PARP cleavage and finally DNA fragmentation. nally 85 : 15. Fifty fractions of 10 mL each were collected and analyzed by TLC and GLC. Fractions having similar TLC pattern (silica gel plates) were pooled, the solvent was distilled off and subjected to 1H-NMR and GC analysis. The major compound lavandulol was confirmed in the fractions eluted with petroleum ether:ethyl acetate (90 : 10) through 1H-NMR and 13C-NMR assignments. Material and Methods ! Cell culture Chemicals The human cancer cell lines were obtained either from NCCS, Pune, India or NCI, Frederick, USA. The acute lymphoblastic leukemia (HL-60) cell line was grown and maintained in RPMI1640 medium, pH 7.4. The medium was supplemented with FCS (10 %), penicillin (100 Units/mL), streptomycin (100 µg/mL) and glutamine (2 mM) and cells were grown in a CO2 incubator (Heraeus) at 37 °C with 90 % humidity and 5 % CO2. Cells were treated with AT-01 dissolved in DMSO, while the untreated control cultures received an equivalent amount of DMSO only. The final concentration of DMSO in both treated and untreated cells was always less than 0.2 %. RPMI-1640, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ethidium bromide (∼95 %, HPLC), propidium iodide (PI) (≥ 95 %, HPLC), camptothecin (≥ 95 %, HPLC), adriamycin, rhodamine 123 (≥ 95 %, HPLC), staurosporine (≥ 98 %, HPLC) were purchased from Sigma Chemical. Proteinase K (∼95 %, HPLC), dimethyl sulphoxide (≥ 99 %, HPLC), DNase-free RNase-A (≥ 90, HPLC) were obtained from Banglore Genei. Annexin-V/FITC apoptosis detection kit, Cycle Test plus DNA reagent Kit and mouse anti-human antibodies to cytochrome c (#556 433) were from BD Bioscience. Caspase-3 and -9 colorimetric assay kits were from R & D Systems. Mouse anti-human antibodies to PARP-1 (#SC8007) and β-actin (#SC-47 778) were from Santa Cruz Biotechnology. All electrophoresis reagents, protein concentration measurement kit and protein markers were from Bio-Rad Laboratories. Hyper film and ECL Plus western blotting detection kit were from Amersham Biosciences. Plant material and isolation of essential oil Flowering shoots of Tanacetum gracile were collected and its essential oil AT-01 was extracted from 500 g by hydro-distillation in a round-bottomed flask fitted in a Clevenger type apparatus for 6 h. The blue colored oil (yield: 0.4 %) was dried over anhydrous sodium sulphate and stored at –5 °C until further analysis. The analysis of oil was performed by GC and components were identified by 1H-NMR, 13C-NMR and GC-MS. GC of the essential oil GC of the oil sample was performed on a Nucon-5765 fitted with a fused silica capillary column 25 m × 0.25 mm × 0.25 µm coated with methyl polysiloxane. Helium was the carrier gas at a flow rate of 1 mL/min and an injector temperature of 250 °C and FID detector at 260 °C. The programming was at 90 °C for 2 min followed by an increase of 7 °C/min to 220 °C/5 min. GC-MS was recorded on a QP-2000 Shimadzu model at 90 – 250 °C with a programming rate of 6 °C/min using helium as the carrier gas. The GC column was coupled directly to a quadrupole mass spectrometer using EI at 70 eV. Mass spectra were recorded at a scan speed of 9/s from m/z 700 to 10. Identification of the essential oil components was achieved by comparing the mass spectra with the library search programme on the mono- and sesquiterpene mass spectral database and by comparing the relative retention times with those of reference compounds. The main components identified by the combined chromatographic and spectroscopic technique are lavandulol (21.5 %), α-pinene (11.2 %), lavandulyl acetate (1.7 %), 1,8-cineole (15.2 %), cis-β-ocimene (6.4 %) borneol (6.1 %) and chamazulene (3.7 %), etc. Isolation of the components The essential oil (500 mg) was chromatographed over a 10 g silica gel glass column, 25 cm long and 1.5 cm wide. The column was eluted with light petrol, and petroleum ether: ethyl acetate in the ratios 99 : 1, 98 : 2, 97: 3, 96 : 4, 95 : 5, 94 : 6, 90 : 10 and fi- Cell proliferation assay Inhibition of cell proliferation by AT-01 was measured with the MTT assay [12]. Briefly, HL-60 cells (2 × 104/200 µL well) seeded in 96 well cell tissue culture plates were exposed to the indicated concentrations of AT-01 for 48 h, after which 20 µL of MTT solution (2.5 mg/mL) was added to each well followed by incubation at 37 °C for 2 h. The plates were centrifuged and the supernatant was discarded while the MTT-formazan crystals were dissolved in 100 µL DMSO. The OD measured at 570 nm with reference wavelength of 620 nm and IC50 value was determined. Morphological observation HL-60 cells (1 × 106 cells/1.5 mL) were seeded in 12 well plates and treated with AT-01 at concentrations of 10, 30, 50 and 100 µg/mL for 24 h. After that cells were observed under a phase contrast inverted microscope (Olympus 1 × 70). Cell morphology was assessed and photographed (30 × ). Flow cytometric analysis of apoptosis and necrosis The extent of apoptosis was measured using the Annexin V-FITC apoptosis detection kit as described by the manufacturer. Briefly, HL-60 cells (1 × 106/1.5 mL/well) were seeded in 12 well plates and treated with AT-01 at 10, 30 and 50 µg/mL for 24 h. Cells were collected, washed with PBS twice, gently resuspended in annexin-V binding buffer, incubated with annexin-V FITC/PI in the dark for 15 min and analyzed by flow cytometery using cell quest software (BD Biosciences). The fraction of cell populations in different quadrants was analyzed using quadrant statistics. Cells in the lower right quadrant represented apoptosis and in the upper right quadrant represent necrosis or post-apoptotic necrosis [13]. Cell cycle phase distribution The effect of AT-01 on the DNA content was assessed by cell cycle phase distribution analysis in HL-60 cells. HL-60 cells (1 × 106 / 1.5 mL/well) in 12-well plates were incubated with AT-01 at 10, 30, 50 and 100 µg/mL for 24 h, washed twice with ice-cold PBS, harvested, fixed with ice-cold 70 % ethanol and stored at –20 °C for 30 min. After fixation, the cells were incubated with RNase (0.1 mg/mL) at 37 °C for 30 min, stained with propidium iodide (50 µg/mL) for 30 min in dark. Cells were analyzed immediately Verma M et al. Induction of Mitochondrial-Dependent … Planta Med 2008; 74: 515 – 520 Downloaded by: Indian Institute Of Integrative Medicine. Copyrighted material. 516 Original Paper Assessment of the change in mitochondrial membrane potential (ΔΨmt) Changes in the mitochondrial transmembrane potential (Ψmt) as a result of mitochondrial perturbation were measured after staining with rhodamine-123 [13]. Cells (1 × 106/1.5 mL/well) in 12 well plates were treated with AT-01 at 10, 30, 50 µg/mL and staurosporine at 5 µM for 24 h. Rhodamine-123 (10 nM) was added 1 h before termination of the experiment. Cells were washed in PBS and suspended in sheath fluid. The intensity of fluorescence from 10,000 events was analyzed in FL-1 channel vs. counts on a BD-SLR flow cytometer (Becton Dickinson). Caspase-3/-9 assay Activation of caspases-3 and 9 was measured using caspase-3 & -9 colorimetric kits from R & D Systems. HL-60 cells (2 × 106/3 mL/ well of 6-well plates) were incubated with AT-01 at 10 µg/mL for 6, 12 and 24 h using staurosporine (1 µM) as a reference. Cells were collected and washed with PBS, lysed in cell lysis buffer (50 µL) and the supernatant was collected and added into 96well plates. 50 µL final reaction buffer (1 mL reaction buffer + 10 µL DTT) and 5 µL caspase-3/9 colorimetric substrate (DEVDpNA/LEHD-pNA) were added into 96-well pates. Plates were incubated at 37 °C for 2 h and optical density was measured at 405 nm with an ELISA micro-plate reader. Western blot Cells (2 × 106/3 mL/well of 6-well plate) were treated with 1, 10, 30 µg/mL of AT-01 for 24 h and washed twice with PBS. Whole cell lysates were prepared by incubating cells with lysis buffer containing (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 % v/v Nonidet P-40, 1 mM PMSF and 0.01 % v/v protease inhibitor cocktail) for 15 min on ice [16]. Cells were centrifuged at 12 000 × g and the supernatant was collected and analyzed for PARP level. Cytosolic fractions for cytochrome c expression were made by incubating the cells with lysis buffer containing (75 mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mM EDTA, 350 µg/mL digitonin and 0.01 % v/v protease inhibitor cocktail). The lysates were centrifuged at 12,000 g and the supernatant was collected [17]. For the immunoblotting, 60 µg of protein were run on 12 % SDSpolyacrylamide gels and then transferred to PVDF membranes. Non-specific binding was blocked by 5 % non-fat milk in Trisbuffered saline containing 0.1 % Tween 20 (TBST). The blots were probed with primary antibodies against cytochrome c, PARP (Santa Cruz 1 : 1000) and then incubated with respective horseradish peroxide conjugated secondary antibodies. Signals were detected using ECL plus chemiluminescence kit on radiography film in dark. Statistical analysis Data are mean ± S.D. from three similar experiments or represent one of three similar experiments. Comparisons were made between control and AT-01 by using Bonferroni test (post hoc test) and the mean difference is considered significant * at the 0.05 level. Supporting information Additional data regarding the effect of AT-01 on cell proliferation and the chemical composition of the essential oil AT-01 are available as Supporting Information. Results ! Treatment of cells with AT-01 for 48 h produced concentration dependent inhibition of cell proliferation of HL-60 cells with an IC50 value of approx. 27 µg/mL (Fig. 1S, Supporting Information). AT-01 caused significant morphological changes in HL-60 cells, including formation of apoptotic bodies, chromatin condensation, shrinkage of cells, bleb formation, resulting in internucleso" Fig. 1). Exmal DNA fragmentation and sub G1 accumulation (● posure of phosphotidyl serine (PS) on the surface of cells is an early event in the onset of apoptosis, which has strong binding affinity for annexinV in the presence of calcium. HL-60 cells were incubated with different concentrations of AT-01 and cells were stained with annexinV-FITC and PI to assess the apoptotic " Fig. 2). AT-01 produced a doseand necrotic cell population (● Fig. 1 Influence of AT-01 on cell morphology and apoptotic bodies formation in HL-60 cells. The cells (1 × 106/1.5 mL/12-well plates) were treated with the indicated doses of AT-01 for 24 h and analyzed by phase contrast. Indicated arrows show the formation of apoptotic bodies, chromatin condensation, cell shrinkage and bleb formation. Data are representative of one of three similar experiments and magnification of the pictures was 30 × . Verma M et al. Induction of Mitochondrial-Dependent … Planta Med 2008; 74: 515 – 520 Downloaded by: Indian Institute Of Integrative Medicine. Copyrighted material. on a BD-SLR flow-cytometer (Becton Dickinson). Data were collected in list mode on 10,000 events for FL2-A vs. FL2-W [14], [15]. The fluorescence intensity of the sub-G0 cell fraction represents the apoptotic cell population. 517 Original Paper Fig. 2 Flow cytometric analysis of AT-01 induced apoptosis in HL-60 cells using annexin V-FITC/PI. Cells (1 × 106/1.5 mL/12-well plates) were incubated with the indicated concentrations of AT-01 for 24 h and stained with annexin V-FITC/PI to analyze apoptotic and necrotic cell populations. Data are representative of one of three similar experiments. Fig. 3 DNA content analyses in AT-01 treated HL60 cells. Cells (1 × 106/1.5 mL/12-well plates) were exposed to the indicate concentrations of AT-01 for 24 h and stained with PI to determine DNA fluorescence and cell cycle phase distribution as described in Materials and Methods. The fraction of cells for hypo-diploid (sub-G0, <2n DNA) population analyzed from FL2-A vs. cell counts is shown ( %). Data are representative of one of three similar experiments. Fig. 4 AT-01 induced loss of mitochondrial membrane potential in HL-60 cells. Cells (1 × 106/1.5 mL/ 12-well plates) were incubated with the indicated doses of AT-01 for 24 h. Thereafter, cells were stained with rhodamine-123 (10 nM) for 1 h and analyzed in FL1-H vs. counts on flow cytometer. Data are representative of one of three similar experiments. Verma M et al. Induction of Mitochondrial-Dependent … Planta Med 2008; 74: 515 – 520 Downloaded by: Indian Institute Of Integrative Medicine. Copyrighted material. 518 dependent increase in the apoptotic cell population. The basal apoptotic population in untreated culture was 1 %, which increased to 28 % at 50 µg/mL. Apoptosis thus appeared to be the primary mode of cell death induced by AT-01. The effect of AT-01 on the DNA content by cell cycle phase distribution was assessed by using human leukemia HL-60 cells. The percentage of the hypo diploid sub-G0 DNA fraction (<2n DNA) of cells treated with 10, 30, 50, 100 µg/mL of AT-01for 24 h was 15 %, 26 %, 57 %, 82 % respectively, while in control cultures it was only " Fig. 3). Further, the cell cycle G /M phase was not affected 3 % (● 2 indicating that AT-01 does not produce any mitotic block or cause delay in the cell cycle. It was clear that AT-01 induced early PS exposure, a sign of apoptosis. To confirm this finding genomic DNA was isolated from the treated and untreated HL-60cells. DNA laddering was diffuse interspersed with a smear indicative of some post-apoptotic necrosis when cells were treated with higher concentration of AT01. The loss of mitochondrial membrane potential (Δψmt) causes the release of cytochrome c and other pro apoptotic proteins from mitochondria to the cytosol. AT-01 caused concentration dependent mitochondrial damage and loss of mitochondrial " Fig. 4) AT-01 at 50 µg membrane potential in HL-60 cells (● caused nearly complete loss of mitochondrial membrane potential by 97 %. This suggests that AT-01primarily is active via mitochondrial cell death. Caspases are cysteine proteases because they require a cysteine residue in their target proteins, at the aspartic acid residue. Caspases are involved in different phases of apoptosis causing DNA fragmentation, chromatin condensation and apoptotic body formation. Therefore, we then assessed activation of caspases-3 and -9 activities induced by AT-01 at 10 µg/mL after 6, 12 and 24 hours. AT-01 increased caspase-3 to three times the level measured in controls, while the level of caspase-9 was increased 12 " Fig. 5 and ● " Fig. 6). Thus, times after 24 h in HL-60 cells (● strong caspase-9 activation by AT-01 further suggestes that AT- Fig. 6 AT-01 induced differential activation of caspase-9 in HL-60 cells. Caspase-9 activities were determined colorimetrically by the using caspase-9 colorimetric assay kit from R & D Systems as described in the Materials and Methods. All assays were performed according to the instructions provided by the supplier. Comparisons were made between control and 10 µg/mL of AT-01 at 6, 12, 24 h by using the Bonferroni test (post hoc test) and the mean difference is considered significant * at the 0.05 level. Fig. 7 Influence of AT-01 on the expression of important proteins involved in the initiation of apoptosis. Specific antibodies raised against human were used for detection of cytochrome c and PARP in AT-01 treated and untreated HL-60 cells as described in the Material and Methods. β-actin was used as an internal control to represent the same amount of proteins applied to SDS-PAGE. Data are representative of one of three similar experiments. 01 induces apoptosis through the intrinsic/ mitochondria dependent pathway. AT-01 also induced the release of cytochrome c from mitochon" Fig. 7). The dria in a dose-dependent manner in HL-60 cells (● released cytochrome c can further activate caspase-9 and caspase-3. PARP, an enzyme involved in DNA repair, is a preferential substrate for caspase-3, so we then assessed PARP cleavage in AT-01 treated cells. AT-01 caused the cleavage of PARP in a " Fig. 7). dose-dependent manner in HL-60 cells (● Fig. 5 AT-01 induced differential activation of caspase-3 in HL-60 cells. HL-60 cells (2 × 106/3 mL/6-well plates) were exposed to 10 µg/mL AT-01 for the indicated time periods. Caspase-3 activities were determined colorimetrically by using caspase-3 colorimetric assay kit from R & D Systems as described in the Materials and Methods. All assays were performed according to the instructions provided by the supplier. Data are mean ± S.D. from three similar experiments. Comparisons were made between control and 10 µg/mL AT-01 at 6, 12, 24 h by using the Bonferroni test (post hoc test) and the mean difference is considered significant * at the 0.05 level. Discussion ! Plant-derived drugs are a major focus in the development of novel anticancer therapeutics. Anticancer drugs having low side effects, inducing apoptosis and having specific cytotoxicity to cancer cells are the drugs of choice. Keeping this in mind we investigated the cytotoxic potential of the essential oil AT-01 from Tanacetum gracile, family Asteraceae, from Ladakh Himalayas. The essential oil of many species of the Asteraceae family is com- Verma M et al. Induction of Mitochondrial-Dependent … Planta Med 2008; 74: 515 – 520 519 Downloaded by: Indian Institute Of Integrative Medicine. Copyrighted material. Original Paper Original Paper posed of mono- and sesquiterpenes, which have different pharmacological activities [18], [19]. The results of the present study describe the cytoxicity and proapoptotic activity of AT-01 on human leukemia HL-60 cells. AT01 significantly inhibited cell proliferation of HL-60 cells. Considering the potential that AT-01 offers in its development as anticancer agent, we further sought to understand the mechanism of apoptotic cell death in HL-60 cells. Apoptosis is a major form of cell death, characterized by a series of stereotypic morphological changes such as formation of apoptotic bodies, chromatin condensation, shrinkage of cells and bleb formation, internuclesomal DNA fragmentation and subG0 DNA accumulation [20]. DNA fragmentation is a relatively late event and hallmark of apoptosis. To confirm that apoptosis was induced by AT-01, we employed flow cytometery to determine the extent and causes of apoptosis. AT-01 significantly induced DNA fragmentation, apoptotic body formation, annexin-V binding and an increase in hypodiploid sub- G0 DNA fraction (< 2n DNA) of HL-60 cells. Mitochondria play a pivotal role in the intrinsic apoptotic pathway. The key element in the mitochondrial pathway is the efflux of cytochrome c from the mitochondria to the cytosol. The released cytochrome c together with Apaf-1 and procaspase-9 form a wheel-like structure known as the apoptosome, which activates caspase-9 and then execution of caspase-3 [21]. Loss of mitochondrial membrane potential (Δψmt) is largely due to the activation of mitochondrial permeability transition pores (PTP) and translocation of Bax from the cytosol to mitochondria, which allows leakage of cytochrome c and activation of caspases-9 and -3 [22]. Caspases are a ubiquitous family of cysteine proteases that include both upstream (initiator) and downstream (effector) caspases. Activation of caspase-3 is an important step in the execution phase of apoptosis and its inhibition blocks cell apoptosis. An elevated level of caspase-3 could utilize PARP a DNA repair enzyme as its substrate [23], [24]. Our data demonstrate a dose-dependent loss of mitochondrial membrane potential upon AT-01 treatment, overexpression of cytochrome c, along with an increase in the activation of caspases-3 and -9 and an increase in PARP cleavage. Thus, we conclude that the essential oil AT-01 from Tanacetum gracile exhibits potential cytotoxic and apoptotic activity in human leukemia HL-60 cells. We suggest that AT-01 induces loss of mitochondrial membrane potential, which causes release of cytochrome c from the mitochondria, further activating procaspase-9 to caspase 9 and the execution of caspase-3 ultimately leading to PARP cleavage and DNA fragmentation. So we can say that AT-01 induces apoptosis through the intrinsic or mitochondrial-dependent apoptotic pathway in HL-60 cells. These studies nevertheless provide important information about the pro-apoptotic nature of AT-01 promoting this candidate for development into a potential anticancer therapeutic and for also having the potential of targeting mitochondria in apoptosis. In vivo and safety studies of AT-01 are currently in progress. All these results provide the basis for further in-depth drug-targeted studies for the potential use of AT01 as an anticancer therapeutic. Acknowledgement ! References 1 Beauchamp P, Dev V, Kashyap T, Melkani A, Mathela C, Bottini AT. Composition of essential oil of Tanacetum nubigenum Wallich ex DC. J Essent Oil Res 2001; 13: 319 – 23 2 Nano GM, Bicchi C, Frattini C, Gallino M. Wild Piedmontese plants. II. A rare chemotype of Tanacetum vulgare L., abundant in Piedmont. Planta Med 1979; 35: 270 – 4 3 Tetenyl P, Kaposi P, Hethelyi E. Variation in essential oils of Tanacetum vulgare. Photochemistry 1975; 14: 1539 – 44 4 Abad MJ, Bermej P, Valverde S, Villar A. 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