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Supporting informationDetailed 1H- and 13C‑NMR spectral data of 1 are available as Sup-porting Information.

Acknowledgements!

This studywas supported in part by the Inter-Institutional Collab-orationResearchProgramunder theKoreaResearchCouncil for In-dustrial Science & Technology (KOCI) as well as by the Forest Sci-ence & Technology Projects (project no. S120808L1101104) pro-vided by the Korea Forest Service.

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received April 1, 2010revised May 7, 2010accepted May 19, 2010

BibliographyDOI http://dx.doi.org/10.1055/s-0030-1250049Published online June 17, 2010Planta Med 2011; 77: 52–54© Georg Thieme Verlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceDr. Sang Un ChoiBio-organic Science DivisionKorea Research Institute of Chemical TechnologyP.O. Box 107Sinseongno 19305–600 DaejeonKoreaPhone: + 82428607545Fax: + 82428614246suchoi@krict.re.krsuchoi4@hotmail.com

Bozzuto G et al. Tea Tree Oil… Planta Med 2011; 77: 54–56

Tea Tree Oil Might Combat Melanoma

Giuseppina Bozzuto, Marisa Colone, Laura Toccacieli,Annarita Stringaro, Agnese MolinariDepartment of Technology and Health, Istituto Superiore di Sanità,Rome, Italy

Abstract!

In this study we present new data from experiments focused onthe antitumor activity of tea tree oil (TTO), an essential oil dis-tilled fromMelaleuca alternifolia. TTO proved to be capable of in-hibiting the growth of melanoma cells and of overcoming multi-drug resistance (MDR), as we reported in our previous study.Moreover, the survival role of the MDR-marker P-glycoproteinappears to be involved in the mechanism of invasion of melano-ma cells. The results reported herein indicate that TTO and itsmain active component, terpinen-4-ol, can also interfere withthe migration and invasion processes of drug-sensitive anddrug-resistant melanoma cells.

Key wordsMelaleuca alternifolia · Myrtaceae · human melanoma cells ·migration and invasion · drug resistance

Abbreviations!

ADR: adriamycinERK: extracellular signal-regulated kinaseERM: ezrin, radixin, moesinF4: anti-CD44 monoclonal antibodyMAPK: mitogen-activated protein kinaseMMP: matrix metalloproteinaseMM4.17: anti-P-glycoprotein monoclonal antibodyMDR: multidrug resistancePD: PD 098059; 2-(2-amino-3-methoxyphenyl)

-4H‑1-benzopyran-4-oneP‑gp: P-glycoproteinSB: SB 203580; 4-(4-fluorophenyl)-2-

(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imida-zole

TTO: tea tree oil

Supporting information available online athttp://www.thieme-connect.de/ejournals/toc/plantamedica

Humanmelanoma is a highly invasive andmetastatic tumor that,to date, is one of the most difficult cancers to fight with currenttreatments, due to its intrinsic resistance to chemotherapy andradiotherapy. The basis for drug resistance in melanoma is mostlikely dysregulation of apoptosis, although mechanisms such asdrug transport, detoxification, and enhanced DNA repair may al-so play a role [1].Numerous experimental data and other clinical-pathologicalstudies have shown that the drug-resistant phenotype is oftenassociated with more aggressive behavior in tumors of differenthistological derivation. In particular, the overexpression of thexenobiotic transporter P-glycoprotein (P‑gp) often correlateswith a phenotype with a high invasive and metastatic potential

Fig. 1 Analysis performed by transwell chamberinvasion assay of migration (a, c) and invasion (b, d)potential of M14 WT and M14 ADR cells, in the ab-sence or in the presence of either TTO or terpinen-4-ol. SB: SB203580, p38MAPK inhibitor; PD:PD098059, ERK-1/2 inhibitor; F4: anti-CD44 MAb;MM4.17: anti-P‑gp MAb.

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[2]. Our previous study showed that humanmelanoma cells (M14WT) grown in the presence of the antitumor drug adriamycin(M14 ADR) expressed the multidrug transporter P‑gp. This over-expression conferred to M14 ADR cells a cross-resistance toadriamycin and vincristine and an increased resistance to cas-pase-dependent apoptosis (Fas-mediated, serum deprivation)compared with M14 WT parental cells [3].We previously demonstrated that the essential oil steam (tea treeoil, TTO) distilled from Melaleuca alternifolia (Myrtaceae), a spe-cies of northern New South Wales, Australia, was able to reversethe resistance of M14 ADR cells to P‑gp−mediated caspase-de-pendent apoptosis [3]. TTO is an aboriginal Australian traditionalmedicine for bruises, insect bites, and skin infections. It was re-discovered in the 1920s as a topical antiseptic with more effec-tive activity than phenol. The effect of TTO onmelanoma cells ap-pears to be mediated by its interaction with the lipid bilayer ofthe plasma membrane, as delineated by a biophysical and ultra-structural study conducted by our group [4].As previously demonstrated by our studies, multidrug-resistantM14 ADR cells displayed a more invasive phenotype comparedwith their parental counterparts M14 WT. This phenotype wasaccomplished by a different migration strategy adopted by resis-tant cells (“chain collective”), which was already described in tu-mor cells with high metastatic capacity [5].In the present study the effect of TTO and of its main active com-ponent, terpinen-4-ol, on the migratory and invasive potential ofM14WT and M14 ADR cells was evaluated. To this aim the trans-well chamber invasion assay was employed. The assay was per-formed in the absence or in the presence of TTO and terpinen-4-ol (see Supporting Information).Chemical components of TTO were identified by comparing theirGC retention times, Kovats indices, and GC/MS spectrawith thoseof the reference substances. Quantitative data were based onpeak-area normalization without using a correction factor.The oil contained 42.35% terpinen-4-ol, 20.65% γ-terpinene,9.76% α-terpinene, 3.71% terpinolene, 3.57% 1,8-cineole, 3.09%

α-terpineol, 2.82% p-cimene, 2.42% α-pinene, 1.75% limonene,1.05% δ-cadinene, 0.94% α-thujene, 0.94% aromadendrene,0.87% myrcene, 0.73% β-pinene, 0.40% sabinene, and 0.34% α-phellandrene. The oil was observed to be of terpinen-4-ol typeaccording to International Standard ISO 4730 (1996).Whereas migration and invasion of M14WT cells were not inhib-ited by the essential oil (l" Fig. 1a,c), a significant decrease in thepercentage of area occupied by resistant M14 ADR cells, migratedin the presence of TTO and its active component terpinen-4-ol,was revealed (l" Fig. 1). The major inhibitory effect was foundafter treatment with 0.01% terpinen-4-ol, which reduced thepercentage of occupied area by about 60% in the migration pro-cess (l" Fig. 1b) and by 50% in the invasion process (l" Fig. 1d).The effect of TTO was compared with that of the kinase inhibitorsSB203580 (SB; p38MAPK inhibitor, purity > 99%) and PD098059(PD; ERK-1/2 inhibitor, purity > 99%), which are capable of inter-fering with the migration and invasion processes of drug-resis-tant melanoma cells, as previously reported [5]. As biological in-ducers of migration and invasion, F4 [6] and MM4.17 MAbs [5,7]were employed.Imaging by scanning electron microscopy showed that migratingmelanoma cells were dramatically affected by both TTO (data notshown) and terpinen-4-ol (l" Fig. 2). The observations were per-formed on both the upper (l" Fig. 2a,c) and the lower side(l" Fig. 2b,d) of the migration membrane. In sensitive cells, ad-herent to the filter, typical surface blebbing occurred, due to in-teraction of terpinen-4-ol with the plasma membrane (data notshown). The effect of the active component was more dramaticin the resistant cells that showed real chasms of the plasmamembrane (l" Fig. 2c). After the treatment, the front edge of cellsthat migrated on the lower side of the filter displayed profoundalterations, with a number of swollen blebs often released neigh-boring the cell body (l" Fig. 2d).Strikingly, under migratory stimulus the treatment with ter-pinen-4-ol also interfered with MAPK signaling of P‑gp–positivemelanoma cells. Terpinen-4-ol induced in migrating M14 ADR

Bozzuto G et al. Tea Tree Oil… Planta Med 2011; 77: 54–56

Fig. 2 Scanning electron microscopy observations performed on both theupper side (a, c) and the lower side (b, d) of the filter. Panels e and f: anal-ysis performed by Western blotting of the intracellular signaling. Lysatesfrom cells, both untreated and treated with 0.01% terpinen-4-ol, either be-fore or after migratory stimulus. * Cells under migratory stimulus.

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cells, but not in parental cells, the inhibition of ERM phosphory-lation (l" Fig. 2e). Similarly, under migratory stimulus terpinen-4-ol inhibited ERK phosphorylation in resistant cells but not insensitive cells (l" Fig. 2 f). As previously reported [3], the treat-ment of M14 ADR cells with TTO or terpinen-4-ol did not inter-fere with either the expression or the function of P‑gp. As dem-onstrated by our data [5], the P‑gp molecule, after stimulationwith specific antibodies, appeared to cooperate with CD44through the activation of ERK1/2 and p38MAPK proteins. This ac-tivation leads to an increase in metalloproteinase (MMP-2, MMP-3, and MMP-9) m-RNAs, and proteolytic activities, which are as-sociated with increased invasive behavior. RNA interference ex-periments further have demonstrated the involvement of P‑gpin migration and invasion of resistant melanoma cells. A link be-tween the MDR transporter P‑gp, MAPK signaling, and invasionwas pointed out. Intracellular signaling also included the activa-tion of ezrin, radixin, and moesin (ERM) proteins, which are in-volved in P‑gp function [8].The results reported herein demonstrate that TTO and terpinen-4-ol, in addition to overcoming the resistance to apoptosis ofMDR melanoma cells, are able to affect the aggressive behaviorof drug-resistant melanoma cells by inhibiting the intracellularsignaling stimulated by the MDR transporter. The lipophilic na-ture of the oil enables it to penetrate the skin, suggesting that itmay be suitable for topical therapeutic use in the treatment offungal mucosal and cutaneous diseases. This experimental evi-dence is suggestive of a new candidate to enlist in the fightagainst melanoma.

Bozzuto G et al. Tea Tree Oil… Planta Med 2011; 77: 54–56

Supporting informationDetails on materials and methods used are available as Support-ing Information.

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received April 8, 2010revised May 17, 2010accepted May 21, 2010

BibliographyDOI http://dx.doi.org/10.1055/s-0030-1250055Published online June 17, 2010Planta Med 2011; 77: 54–56© Georg Thieme Verlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceAgnese MolinariDepartment of Technology and HealthIstituto Superiore di SanitàViale Regina Elena 29900161 RomeItalyPhone: + 39649903406Fax: + 39649903563agnese.molinari@iss.it