VERBENA OFFICINALIS ESSENTIAL OILAND ITS COMPONENT CITRALAS APOPTOTIC-INDUCING AGENT IN CHRONIC LYMPHOCYTIC LEUKEMIA

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1 INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY Vol. 22, no. 4, (2009) VERBENA OFFICINALIS ESSENTIAL OILAND ITS COMPONENT CITRALAS APOPTOTICINDUCING AGENT IN CHRONIC LYMPHOCYTIC LEUKEMIA L. DE MARTINO, G. D'ARENA l, M.M. MINERVINI!, S. DEAGLI02, B.M. FUSC0 3, N. CASCAVILLAI and V. DE FEO Department ofpharmaceutical Sciences, University ofsalerno, Salerno; 1Hematology and Stem Cell Transplantation Unit, "Casa Sollievo della Sofferenza" Hospital, San Giovanni Rotondo; 'Laboratory ofimmunogenetics, University oftorino, Torino; 3 San Raffaele Institute IRCCS Rome, Italy ReceivedApril 2, 2009 AcceptedJuly 2, 2009 We evaluated the proapoptotic activity of Verbena officinalis essential oil and of its main component citral, on lymphocytes collected from normal blood donors and patients with chronic lymphocytic leukemia (CLL). The number of apoptotic cells was greater in CLL patients than in healthy subjects at all different times of incubation (4, 8 and 24 hours) for samples treated with Verbena officinalis essential oil (A) and citral (B) as well vs controls at different concentrations (0.1% and 0.01%). The greater proapoptotic ability was showed by both essential oil of Verbena officinalis and citral at lower concentrations (after 4 h A 0.1%: 17.8% vs 37.1%; A 0.01%: 15.8% vs 52%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%; after 8 h A 0.1%: 23% vs 38%; A 0.01%: 22.2% vs 55%; B 0.1%: 32% vs 42.2%; B 0.01%: 22% vs 54.3%; after 24 h A 0.1%: 5% vs 20.7%; A 0.01%: 25.8% vs 47.2%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%). Patients carrying deletion 17p13 (p53 mutation) showed a reduced ability to undergo apoptosis with respect to patients with other genomic aberrations or normal karyotype. The proapoptotic activity of Verbena officinalis essential oil and citral is thought to be due to a direct procaspase 3 activation. These data further support evidence that indicate natural compounds as a possible lead structure to develop new therapeutic agents. Numerous bioactive compounds have been hypothesized to act as cancerpreventing agents by inhibiting the activation of procarcinogens, enhancing the detoxification of carcinogens, or impeding the progression of carcinogenesis (12). One of the most promising mechanisms capable of blocking the cancer progression is a selective induction of apoptosis of neoplastic cells. Preliminary evidence indicates that isoprenoids, a broad class of mevalonatederived phytochemicals.which are ubiquitous in the plant kingdom, may act, with great potency, by inhibiting tumor cell proliferation of human breast adenocarcinoma (MCF7), human leukemia (HL60) and human colon adenocarcinoma (CaCo 2) (34). Verbena officinalis L. (Verbenaceae), commonly known as vervain, is a medicinal plant whose essential oil is mainly constituted by isoprenoids. The plant and/or its essential oil has been widely used in different traditional medicines. The plant Key words: Verbena officinalis, citral, apoptosis, chronic lymphocytic leukemia, Mailing address: Prof. Vincenzo De Feo, Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Salerno, Via Ponte don Melillo n. 1, Fisciano (Sa), Italy Tel: Fax: defeo@unisa.it (2009) Copyright by BIOLlFE, s.a.s, This publication and/or article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties

2 1098 L. DE MARTINO ET AL. was approved as a herbal medicine and dietary supplement by several regulatory acts of many countries (5). Despite its widespread use, the mechanisms of pharmacological actions of the herb or the volatile oil are still unclear (5). Apoptosis is a physiologic process which normally occurs to maintain tissue homeostasis. In the early stage of apoptosis, which occurs at the cell surface, one of the plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Previously, our research group reported the proapoptotic effect of vervain oil and the citral on granulocytes collected from patients with chronic myeloid leukemia (6). The purpose of this study is to evaluate the proapoptotic activity of vervain essential oil and of its main component, citral, on lymphocytes collected from normal blood donors and patients with untreated chronic lymphocytic leukemia (ell), a disease entity in which derailed apoptosis is still retained one of the primary pathogenic events causing the accumulation ofneoplastic Bvcells (79). MATERIALS AND METHODS ou isolation and analysis Verbena officinalis aerial parts were collected in July 2006 from plants growing at the Garden ofmedicinal and Aromatic Plants on the Campus of Salerno University. Five hundred grams of fresh aerial parts were submitted to hydrodistillation, in agreement with procedures of the European Pharmacopoeia (10). A pale yellow essential oil was recovered in a 0.39% yield. The chemical composition of the oil was obtained by Gas Chromotography and GCMass spectrometry methods. Analytical gas chromatography was carried out on a PerkinElmer Sigma 115 gas chromatograph fitted with an HP5 MS capillary column (30 m x 0.25 mm i.d.; 0.25 IJlIl film thickness). The carrier gas was Helium (I ml mini). Column temperature was initially kept at 40 C for 5 min, then gradually increased to 250 C at 2 C min:', held for 15 min and finally raised to 270 C at 10 C min:', Diluted samples (1/100 v/v, in nhexane) of 1 ul. were injected manually at 250 C, and in splitless mode. Flame ionization detector (FID) was performed at 280 C. GC MS analyses were performed on an Agilent 6850 Ser. II apparatus, fitted with a fused silica HPl capillary column (30 m x 0.25 mm; m film thickness), coupled to an Agilent Mass Selective Detector MSD 5973; ionization voltage 70 ev; electron multiplier energy 2000 V. Gas chromatographic conditions were as reported above; transfer line temperature, 295 C. Most constituents were identified through gas chromatography by comparing their retention indices to either those from the literature (1112) or with those ofauthentic compounds available in our laboratories. The retention indices were determined in relation to a homologous series ofnalkanes (C,C 24 ) under the same operating conditions. Further identification was made by comparing the mass spectra either to those stored in NIST 02 and Wiley 275 libraries or to the mass spectra from literature (11, 13) and our homemade library. Component relative concentrations were calculated based on GC peak areas without using correction factors. Citral was purchased by SigmaAldrich Co, Milan, Italy. Assessment ofapoptosis Peripheral blood samples were obtained from 13 patients (8 male, 5 female, median age 68 years, range 5576 years) with Bcell CLL and from 10 healthy donors (5 male, 5 female, median age 55 years, range 2870 years). Informed consent on the purpose and the modality of the study was obtained from all subjects. The individual patient characteristics at the study entry are given in Table I. Mononuclear cells were isolated from heparinized peripheral blood by density gradient centrifugation and washed twice with phosphate buffered saline (PBS). Cells were then incubated for up to 24 h at a density of.. 23 x 1O~/IlL in RPMI 1640 at 37 C, 5% C02, with a rrihcture of90 ill ofpbs and 0.1 ill ofvervain essential oil (A) and 9.9 ul, of distilled water (to obtain vervain essential oil diluted 1; 100) or 10 ul. of pure citral (B) at the concentration of 1.9 mm and with only 100 ul, of PBS to also assess spontaneous apoptosis. We used the same dilutions of vervain essential oil and citral tested in previous assays on chronic myeloid leukemia patients and published elsewhere (6). The proapoptotic effect of the compound was evaluated after three different times of incubation (4, 8, 24 h at room temperature in dark conditions), by adding to treated mononuclear cells, after having been washed in PBS, annexin V 5 ill and propidium iodide 5 ul, [Annexin VFITCApoptosis Detection Kit I (BD Pharmingen)] and CD19APCCy7 (Becton Dickinson) in flow cytometry (FACSCanto, Becton Dickinson). An acquisition gate was set according to side light scattering cell properties and fluorescence intensity in order to collect and compare only CD19positive Bcells ofboth patients and controls. More than 3,000 events were stored and analyzed. The software to acquire and elaborate data was CellDivaTM, Becton Dickinson.

3 Int. J. Immunopathol. Pharmacol Table I. Individual patient characteristics at the study entry. UPN Age Sex Stage Karyotype Lymphocytes Status (years) (RaiIBinet) (109/L) 1 76 M OIA Normal Stable disease 2 73 M OIA Normal Stable disease 3 74 M OIA 13q Stable disease 4 60 F OIA Normal Progression disease 5 63 F IVIC Progression disease 6 75 F OIA Stable disease 7 61 M OIA Stable disease 8 72 M IVIC Normal Progression disease 9 64 M IIIB Progression disease M OIA 13q Stable disease F OIA Stable disease F OIA Normal Stable disease M IVIC 17p Progression disease Statistical analysis The values of apoptotic control cells and leukemic cells were compared by using Student t test for unpaired data. RESULTS Table II reports the percentage composition ofthe essential oil of Verbena ojficinalis. Forty components were identified, accounting for 94.9% of the total oil. The oil is mainly constituted by monoterpenes (92.0%), ofwhich oxygenatedcompounds constitute 87.0%; citral is the main constituent (45.5%). Dudai and coworkers reported that the citral is an inducer of caspase3 in Tumor Cell Lines (14). For this reason, we submitted to bioassays both the essential oil ofverbena ojficinalis (A) and its main constituent, citral (B). In all patients with CLLthe number ofbcellswas found 2: 80% of all lymphocytes. Nine patients had Rai OlBinet A clinical stage, the remaining 3 patients a less favorable stage (1 patient IIIB and 2 patients IVIC). Five patients showed a normal karyotype as detected.by fluorescence in situ hybridization (FISH), 3 patients a trisomy 12 as single abnormality, 2 patients a deletion 13ql4 and 2 patients a deletion 17p13, while one patient carried both trisomy 12 and deletion 17p13 (Fig. 1). Fig. 2 shows the proapoptotic activities on lymphocytes collected from normal blood donors and CLL patients. The cells of both controls and leukemic patients did not show significant relevant spontaneous apoptosis (data not shown) thus we focused analysis of data only on treated cells. The number of apoptotic cells was greater in CLL patients than in healthy subjects. The difference was statistically different at all different times of incubation for samples treated with Verbena officinalis essential oil and also citral (Fig. 2 AC) vs controls. The greater proapoptotic ability was retained by both essential oil of Verbena ojficinalis and citral at lower concentrations. The percentage of necrotic cells steadily increased with time peaking at 24 hours in all samples. At the same time we analyzed T cells in both patients and controls. No significant proapoptotic effect ofverbena essential oil and citral was found (data not shown). Two patients (cases n. 7 and 13 in Table I) showed a lower proapoptotic response to Verbena ojficinalis essential oil and citral compared to the others. Both patients carried deletion 17p 13. The same genomic aberration was found in a third patient (case n. 9 in Table I) but only in 10% oflymphocytes while in 50% ofcells trisomy 12 was found. This patient displayed a proapoptotic

4 1100 L. DE MARTINO ET AL. Table II. Percentage composition ofverbena officinalis essential oil. Ki Comoound Identification" %b 938 apinene RI, MS, CoGC Sabinene RI, MS, CoGC Heoten3one RI,MS Pinene RI, MS, CoGC T 1012 uteroinene RI,MS T 1020 ocvmene RI, MS, CoGC pphellandrene RI, MS, CoGC Limonene RI, MS, CoGC ,8Cineole RI, MS, CoGC (Z)130cimene RI, MS, CoGC T 1049 (E)130cimene RI, MS, CoGC yterpinene RI, MS, CoGC Terpinolene RI,MS T 1097 Linalol RI, MS, CoGC transpinocarveol RI,MS T 1153 Iso Pinocamnhone RI,MS trans Pinocamphone RI,MS T 1165 Pinocarvone RI,MS T 1167 Borneol RI, MS, CoGC Terpinen4o1 RI,MS pcymen8o1 RI,MS T 1189 aterpineol RI, MS, CoGC Isobornyl formate RI,MS cisanethole RI, MS, CoGC Bornvl acetate RI,MS T 1270 (E)Citral RI,MS Isobornyl acetate RI,MS T 1369 Eugenol methyl ester RI,MS T 1377 uconaene RI,MS Isoledene RI,MS Elemene RI,MS Lonzifolene RI,MS T Caryoohyllene RI,MS Cedrene RI,MS uhumulene RI,MS alloaromadendrene RI,MS rgurjunene RI,MS T 1491 Bicyclogermacrene RI,MS cismuurola4(14),5diene RI,MS a7episelinene RI,MS 0.1 Q: RI = retention index identical to bibliography; MS = identification based on comparison ofmass spectra; CoGC = retention time identical to authentic compounds. b: T = trace < 0.05% response to in vitro stimulation as the other patients without deletion 17p13. DISCUSSION Preliminary literature evidence indicates that isoprenoids, a broad class of mevalonatederived phytochemicals which are ubiquitous in the plant kingdom, may suppress, with great potency, the proliferation of tumor cells such as human breast adenocarcinoma (MCF7), human leukemia (HL60) and human colon adenocarcinoma (CaCo2) (34). Verbena officinalis L. (Verbenaceae), commonly known as vervain, is a medicinal plant which grows

5 1101 Int. J. Immunopathol. Pharmacal. del17p Fig. 1. Patient with CLL carrying deletion 17p13 in 10% ofcells and trisomy 12 in 50% ofcells. Left panel: the single red signal indicates the deletion at p53 locus (BoutyTechnogenetics CGI p53 17p13 Red). Right panel: three orange (red) signals show the presence ofa supernumerary chromosome 12 (CEP 12 s.o. rysis Int.). A B % 80 P < P < ~ P n o P P < r 15.8±3.6 AO.1% ±2.1 ::r= :j A 0.01% B 0.1% ~ f22.0t5.2 ~ 10 0 AO.1 % I f 20 f 22.2± ~ B 0.01% 42.2±14.6 F ~O 9 P ± ~ P I~ p ± ~O 2 7,4= 37.1± % f A 0.01 % B 0.1% f... B 0.01 % 4 hour s C % 8 hours P P < e ~ P < ± , P ±7.0 ~ ~ AO.1% 25~... ~ i3.4 t...f B 0.1% A 0.01% 24 hours 25~ I f B 0.01% Fig. 2. AC) Verbena officinalis essential oil and citral inducedapoptosis in CLL patients and controls at different times. Data are reported as mean percentage (± standard deviation) of apoptotic Bcells at different times (A: 4 hours; B: 8 hours; C: 24 hours) of incubation with Verbena officinalis essential oil (A) and citral (B) at different concentrations (0.1% and 0.01%, respectively).

6 1102 L. DE MARTINO ET AL. Ql ~C'f:." 4 hours...,......,'" 8 hours 24 hours Annexin VFITC Fig. 3. One representative experiment. Typical contourdiagram ofannexin VFITC/Propidium Iodide (PI) flow cytometry of cells from mononuclear Bcells (CDi9positive) from a patient with CLL (left panels) and a healthy subjects (right panels) treated with Verbena officinalis essential oil andcitral evaluatedat different times (4, 8 and24 hours) from cultures. The lower left quadrants ofeach panels show the viable cells (PI exclusion and annexin VFITC negative binding); the upper right quadrants contain the nonviable (necrotic) cells (Pi uptake and annexin VFITC positive binding); the lower right quadrants (arrows) represent the apoptotic cells (Pi exclusion and annexin VFITC positive binding). wild everywhere and is used for several medical purposes without a clear mechanism of action. In this study, the essential oil and the citral, which is its main constituent, were tested on 10 normal adult healthy subjects and 13 untreated patients suffering from CLL. Both vervain essential oil and pure citral induced a significant apoptosis in CLL samples compared to controls. Only two patients showed a lower percentage of apoptotic cells with respect to controls. Significantly, both carried the deletion 17p13 as genomic aberration, The same aberration was only detected in a third patient who had a greater in vitro response to oil stimulation. In this case, however, the deletion 17p13 was found only in 10% of cells while trisomy 12 was found in 50% of cells, which was not found in the previous cases. Genomic aberrations were detected in up to 90% of the patients with CLL. The most common

7 Int. J. ImmunopathoI. PharmacoI cytogenetic aberration is the deletion 13q14 as single abnormality, and patients with such a deletion have an excellent prognosis (1516). On the contrary, patients with deletion llq2223, involving ATM, and particularlythose with deletion 17p13, involving p53, have a worse prognosis and usually do not respond to conventional therapy, such as fludarabine (1718). The chromosomal site of p53 gene is located in band 17p13 and the disruption of one allele by point mutation together with deletion ofthe second allele is the hallmark of the inactivation of a recessively acting tumor suppressor gene such as p53. However, the possible relationship between p53 inactivation and a reduced proapoptotic response to Verbena officinalis essential oil and citral remains to be better elucidated. The data of this study are concordant to the study which reported the effect on cell cycle and apoptosis of some essential oil components in cancer cellular lines (1920). The molecular mechanism which is at the basis of this process is still unclear. The apoptotic effect which is induced by Verbena officinalis essential oil and citral may be related to activation of the caspase 3. Dudai and coworkers showed that treatments of human and mouse leukemic cell lines for 424 hours with citral resulted in activation of the enzymatic activity of caspase 3 (6). In fact, the proapoptotic activity of citral is thought to be due to a direct procaspase 3 activation. REFERENCES I. Hursting SD, Fisher SM, Wargovich MJ, Digiovanni J. Nutritional modulation of the carcinogenesis process. In: Nutritional Oncology, Heber D, Blackburn G (eds.). Academic Press: San Diego CA; p Wattenberg LW. Inhibition of carcinogenesis by minor dietary constituents. Cancer Res 1992; 52: Mo H, Elson CEo Apoptosis and cellcycle arrest in human and murine tumor cells are initiated by isoprenoids. J Nutr 1999; 129: Tatman D, Mo H. Volatile isoprenoid constituents of fruits, vegetables and herbs cumulatively suppress the proliferation of murine B16 melanoma and human HL60 leukemia cells. Cancer Lett 2002; 175: SauWan L, ManShan Y,WaiHung Y,Chang R cc Novel neuroprotective effects ofthe aqueous extracts from Verbena officinalis Linn. Neuropharmacology 2006; 50: De Martino L, Iorio M, Coppola G, Campana A, SavastanoC, Fusco BM, De Feo V. Verbena officinalis essential oil and citral as apoptotic inductors in leukocytes of healthy subjects and chronic myeloid leukemic patients. Pharmacologyonline 2008; 2: Reed JC, Kitada S. Apoptosis dysregulation in chronic lymphocytic leukemia In: Chronic lymphoid leukemias. Ed. Cheson B. Marcel Dekker, Inc., NY, USA. p ChiorazziN, Rai KR, FerrariniM. Chroniclymphocytic leukemia.n Engl J Med 2005; 352: Dighiero G, Hamblin TJ. Chronic lymphocytic leukemia. Lancet 2008; 371: Europeian Pharmacopoeia, 2004, (5th Edition): Council ofeurope, Strasbourg Cedex, France , p II. Jennings W, Shibamoto T Qualitative Analysis offlavour and Fragrance Volatilesby Glass Capillary Gas Chromatography. Academic Press: New York. 12. Davies NW. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. J Chromatogr 1990; 503: Adams RP Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing: Carol Stream, IL. 14. Dudai N, Weinstein Y, Krup M, Rabinski T, Ofir R. Citral is a new inducer of caspase3 in tumor cell lines. Planta Med 2005; 71: Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger L, Dohner K, Bentz M, Lichter P. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 2000; 343: Seiler T, Dohner H, Stilgenbauer S. Risk stratification in chronic lymphocytic leukemia. Semin Oncol 2006; 33: Sturm I, Bosanquest AG, Hermann S, Guner D, et al. Mutation of p53 and consecutive selective drug resistance in BCLL occurs as a consequence ofprior

8 1104 L. DE MERTINO ET AL. DNAdamaging chemotherapy. Cell Death Differ 2003; 10: Stilgenbauer S, Krober A, Busch R, Eichorst B, et al. 17p deletion predicts for shorter overall survival after fludarbinebased first line therapy in chronic lymphocytic leukemia: first analysis ofthe CLL trial of the GCLLSG. Onkologie 2005; 28(S): Camesecchi S, Schneider Y, Ceraline J, Duranton B, Gosse F, Seiler N, Raul F. Geraniol, a component of plant essential oils inhibits growth and polyamine biosynthesis in human colon cancer cells. J Pharmacol Exp Ther 2001; 298: Bardon S, Picard K, Martel P. Monoterpenes inhibit cell growth, cell cycle progression and cyclin D1 gene expression in human breast cancer cell lines. NutrCancer 1998; 32:17.