journal of dermatological science -...

Journal of Dermatological Science 67 (2012) 120–129

Topically applied Melaleuca alternifolia (tea tree) oil causes direct anti-cancercytotoxicity in subcutaneous tumour bearing mice

Demelza J. Ireland a,1,*, Sara J. Greay a,1, Cornelia M. Hooper a, Haydn T. Kissick b, Pierre Filion c,Thomas V. Riley a,c, Manfred W. Beilharz a

a School of Pathology and Laboratory Medicine (M504), Faculty of Medicine, Dentistry and Health Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA

6009, Australiab Department of Surgery, Beth Israel Deaconess Medical Centre, Harvard Medical School, Boston, MA, USAc Microbiology and Infectious Diseases, PathWest Laboratory Medicine WA, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia

A R T I C L E I N F O

Article history:

Received 2 November 2011

Received in revised form 13 April 2012

Accepted 21 May 2012

Keywords:

Tea tree oil

Cancer

Immune response

Cytotoxicity

Penetration

A B S T R A C T

Background: Melaleuca alternifolia (tea tree) oil (TTO) applied topically in a dilute (10%) dimethyl

sulphoxide (DMSO) formulation exerts a rapid anti-cancer effect after a short treatment protocol.

Tumour clearance is associated with skin irritation mediated by neutrophils which quickly and

completely resolves upon treatment cessation.

Objective: To examine the mechanism of action underlying the anti-cancer activity of TTO.

Methods: Immune cell changes in subcutaneous tumour bearing mice in response to topically applied

TTO treatments were assessed by flow cytometry and immunohistochemistry. Direct cytotoxicity of TTO

on tumour cells in vivo was assessed by transmission electron microscopy.

Results: Neutrophils accumulate in the skin following topical 10% TTO/DMSO treatment but are not

required for tumour clearance as neutrophil depletion did not abrogate the anti-cancer effect. Topically

applied 10% TTO/DMSO, but not neat TTO, induces an accumulation and activation of dendritic cells and

an accumulation of T cells. Although topical application of 10% TTO/DMSO appears to activate an

immune response, anti-tumour efficacy is mediated by a direct effect on tumour cells in vivo. The direct

cytotoxicity of TTO in vivo appears to be associated with TTO penetration.

Conclusion: Future studies should focus on enhancing the direct cytotoxicity of TTO by increasing

penetration through skin to achieve a higher in situ terpene concentration. This coupled with boosting a

more specific anti-tumour immune response will likely result in long term clearance of tumours.

� 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights

reserved.

Contents lists available at SciVerse ScienceDirect

Journal of Dermatological Science

jou r nal h o mep ag e: w ww .e lsev ier . co m / jds

1. Introduction

Topicalchemotherapyrepresentsasafeandrelativelyinexpensivetreatmentapproachforskincancers.Preclinicaland clinical studies ofplant derived terpene agents, including ingenol mebutate derivedfromthesapofEuphorbiapeplus[1]andMelaleucaalternifolia(teatree)oil [2],havedemonstratedthepotential forthesetopicalchemothera-pies to exert a rapid anti-cancer effect after a relatively shorttreatment protocol. Long treatment regimes, incomplete clearancerates, poor cosmetic outcomes and systemic toxicity have unfortu-nately limited patient satisfaction with currently approved topicalchemotherapeutics such as imiquimod and 5-fluorouracil [3,4].

* Corresponding author. Tel.: +61 8 9346 2214; fax: +61 8 9346 2912.

E-mail addresses: [email protected] (D.J. Ireland),

[email protected] (S.J. Greay).1 D.J. Ireland and S.J. Greay contributed equally to this research.

0923-1811/$36.00 � 2012 Japanese Society for Investigative Dermatology. Published b

http://dx.doi.org/10.1016/j.jdermsci.2012.05.005

Tea tree oil (TTO), abundant in terpenes, has been wellcharacterised as an antimicrobial agent [5]. Its use as a topicaltreatment has been reported in numerous clinical trials and has beenrigorously examined for toxicity and safety. TTO formulationscontaining 5–100% oil have been patch tested for up to 21 days inlarge cohorts of patients with no or few irritancy reactions recorded.Those irritant reactions that do occur are significantly reduced withmore dilute preparations. Moreover, allergic reactions to topical TTOare negligible with fresh, and appropriately stored TTO (reviewed in[6]). More recently, TTO and some of its components have beenexamined for anti-tumour activity [7–10]. When applied topically ina specific formulation to fully immune-competent, subcutaneous,tumour-bearing mice, TTO showed in vivo anti-tumour activity [2].Four, daily, topical treatments of 10% TTO/DMSO caused subcuta-neous AE17 mesotheliomas in mice to regress for a period of 10 daysand significantly retarded the growth of subcutaneous B16-F10melanomas. We previously reported this anti-tumour effect oftopical 10% TTO/DMSO to be accompanied by skin irritation similar

y Elsevier Ireland Ltd. All rights reserved.


mailto:[email protected]

mailto:[email protected]

http://www.sciencedirect.com/science/journal/09231811


D.J. Ireland et al. / Journal of Dermatological Science 67 (2012) 120–129 121

to other topical chemotherapeutic agents but, unlike other approvedtopical chemotherapeutics, 10% TTO/DMSO induced skin irritationresolved quickly and completely. Furthermore, by histologicalanalysis we showed that topical 10% TTO/DMSO caused an influxof neutrophils into the epidermis and dermis with no evidence ofsystemic toxicity.

We originally proposed that local immune activation inresponse to topical treatment with 10% TTO/DMSO may be by aprocess similar to that of ingenol mebutate which induces aneutrophil mediated, antibody dependent, cellular cytotoxiceradication of residual tumour cells [11]. In this report, wedocument evidence suggesting that although topical application of10% TTO/DMSO activates a local immune response, our evidenceshows that the anti-tumour mechanism of action underlying theanti-tumour effect is a result of direct cytotoxicity.

2. Materials and methods

2.1. Cells and cell culture

AE17 murine mesothelioma cells were derived from asbestosinduced primary tumours of syngeneic C57BL/6J mice [12]. All cellswere maintained at 37 8C in 5% CO2 in 75 cm2 or 225 cm2 tissueculture flasks in supplemented RPMI-1640 media and subculturedas previously described [2,13].

2.2. Mice

Female C57BL/6J mice between 6 and 8 weeks of age wereobtained from the Animal Resources Centre (Perth, Australia) andmaintained under SPF housing conditions (Animal Care Unit,University of Western Australia) in tinted Perspex cages with filterlids on a 12-h day/night cycle. All animal work was carried out inaccordance with the guidelines of The National Health and MedicalResearch Council of Australia and with the approval of theUniversity of Western Australia’s Animal Ethics Committee (AECRA/100/728).

2.3. Tumour cell implantation and growth monitoring

1 � 107 AE17 cells/100 ml PBS/mouse were implanted using a100 ml glass syringe (26½ gauge needle) sub-cutaneously (s.c.)onto the shaved, right hand flank of syngeneic C57BL/6J mice aspreviously described [13]. Tumour development was monitoredvisually and physically by palpating the tumour implantation site.Tumours were measured using a microcalliper to obtain twotumour diameters taken at right angles to each other; these weremultiplied to obtain a tumour area (mm2).

Simultaneously, animals were monitored daily for any loss ofoverall condition by checking for: hunching, ruffled coat, loss ofbody weight, bloating, slowed movement and seclusion. Humaneeuthanasia by methoxyfluorane inhalation followed by cervicaldislocation was carried out when tumour area reached 100 mm2 orat predetermined experimental endpoints.

2.4. TTO and topical treatments

TTO compliant with the International Standard ISO4730 waskindly provided by P. Guinane Pty. Ltd., Chinderah, NSW (BatchA352) [2]. A 10% solution of TTO was made by dissolving TTO inDMSO Hybri-max (Sigma Chemical Co., St Louis, MO). All TTOsolutions were kept in airtight containers in the dark, with regularstability testing to ensure normal levels of terpinen-4-ol and 1,8-cineole.

Mice with shaved flanks and established tumours measuring9 mm2 (approximately 12–14 days post tumour inoculation) were

treated topically with either 10% TTO/DMSO (50 ml), neat TTO(5 ml: an amount equivalent to that found in 10% TTO/DMSO), orvehicle control (50 ml: 10% H2O/DMSO) applied by pipetting therequired volume onto the tumour and surrounding skin and gentlerubbing [2]. Treatments were applied once daily (at the same timeeach day) for 4 consecutive days. Mice were monitored daily forskin irritation resulting from topical treatments and under our AECregulations were humanely euthanised if a severe skin irritationscore of >3 (average: dryness score + erythema score + escharformation score) was observed.

2.5. Antibody depletions

A total of 100 mg of Gr-1 mAb (RB6.8C5, Monoclonal AntibodyFacility, Western Australian Institute for Medical Research) wasadministered i.p. to mice on days �1, +1, +3 and +5 to depleteneutrophils (topical treatments were applied concurrently on days0–3). Control mice were administered with an isotype-matchedcontrol antibody. Samples of blood (1 ml) collected via a tail veinnick 1 day post Gr-1 mAb depletion were smeared onto glassmicroscope slides and stained with haematoxylin and eosin (H&E),as well as stained for Nimp-1 for flow cytometry, and the numbersof neutrophils were counted. As determined, >90% systemicneutrophil depletion was achieved.

2.6. Flow cytometry

Preparations of single cell suspensions were made from tumours(including overlying skin) and tumour draining lymph nodes(TDLNs). Tumour/skin and TDLNs were surgically removed andminced finely at room temperature in 500 ml of un-supplementedRPMI. Following the Indianapolis, IN addition of 1.4 U (12.5 ml)liberase blendzyme 3 (Roche, Indianapolis, IN) and 1000 kU(13.5 ml) DNase 1 (Sigma Chemical Co., St Louis, MO), sampleswere digested by mixing on a rotating wheel at room temperaturefor 20 min before centrifugation at 1500 � g for 3 min. Resulting cellhomogenates were pushed through fine wire mesh to break downany remaining clumps. The single cell suspension was washed twicewith 500 ml wash buffer (PBS containing 5% fetal calf serum (FCS)and 5 mM NaN3) and centrifuged at 1500 � g for 3 min beforestaining with fluorochrome-conjugated antibodies for flow cyto-metric analysis. Flow cytometric staining for cell surface markersMHCII, Ly-6G (Gr-1), CD4, CD8, CD11c, CD69, CD80, and CD86 (allpurchased from eBioScience, San Diego, CA), as well as Nimp-1(Abcam, Cambridge, MA) was analysed using a BD FACSCantoTM

benchtop flow cytometer (BD Biosciences, San Jose, CA), recording atleast 100,000 events per sample. Data were then analysed usingFlowJo software (Treestar Inc., San Carlos, CA).

2.7. Histology

For histology and immunohistochemistry, mice were euthanisedfollowing 1 or 3 topical treatments with either 10% TTO/DMSO orvehicle and embedded in O.C.T. compound (optical cutting tempera-ture, Thermo-Shandon, Pittsburgh, PA). Frozen cross-sections oftumour plus overlying skin were prepared at 10 mm thickness. Forlipid staining, frozen cross sections were rehydrated in PBS andstained for 20 min in a 0.5% Oil red-O (Merck, Darmstadt, Germany)solution in Dextrin (BDH, US). After rinsing, the sections werecounterstained withhaematoxylinfor 20–30 s andmounted withthesections analysed using a Digital Slide Scanner and ImageScopesoftware version 10.2.2.2352 (Aperio Technologies Inc., CA). Forvisualisation and localisation of neutrophils (Nimp-1), the frozensections were rehydrated and blocked with 1� PBS supplementedwith 1% BSA (bovine serum albumin) (Sigma Chemical Co., St Louis,MO), 0.5% Triton X-100 (Sigma Chemical Co., St Louis, MO), 2 mM

D.J. Ireland et al. / Journal of Dermatological Science 67 (2012) 120–129122

EDTA (Sigma Chemical Co., St Louis, MO) and 10% goat serum(Invitrogen, Frederick, MD).The blocked sectionswere incubated withNimp-1 (Abcam, Cambridge, MAA) and conjugated with secondarygoat anti-rabbit AlexaFluor488 antibody (Invitrogen, Eugene, OR).Tissue sections were washed in 1� PBS supplemented with 1% BSAand 2 mM EDTA and counterstained with 1 mg/ml Hoechst 33342(Sigma Chemical Co., St Louis, MO) for 1 min. The stained preparationswere cover slipped using ImmuMount (Thermo Shandon Ltd., UK) andimages were captured using an inverted AI confocal microscope andNIS-elements imaging software (Nikon, Japan).

2.8. Transmission electron microscopy

For transmission electron microscopy (TEM), tumours wereexcised and manually sectioned, avoiding the necrotic tumour core.Tumour sections were fixed overnight in 1 ml of 2.5% glutaraldehydein caco buffer (0.05 M, pH 7.0) and prepared for TEM as previouslydescribed [13]. Tumour samples were examined on a CM10transmission electron microscope (Phillips, Netherlands) fitted withMegaView III Soft Imaging System (Olympus, Japan). All figures wereprepared in Photoshop Elements 9 (Adobe Systems Incorporated,San Jose, CA).

2.9. Statistics

All data fulfilling criteria for normal distribution are presentedas mean � SD, or mean � SE for pooled independent experiments.Significant differences as indicated were calculated using theStudent’s t tests (p < 0.05), and two-way ANOVA test (p < 0.01 orp < 0.001). Skewed data, not following normal distribution, ispresented using the median and range. For significance, the two-tailed Mann Whitney test (p < 0.05) and the ANOVA equivalentKrusWallis test were used. All statistical tests and figures wereprepared using the Graph Pad Prism software version 5 (GraphPadSoftware, San Diego, CA).

3. Results

3.1. Neutrophil accumulation in the skin following topical 10% TTO/

DMSO treatment

Flow cytometry of single cell suspensions of the skin/tumour ofmice bearing established s.c. AE17 tumours treated topically with50 ml of 10% TTO/DMSO for 4 consecutive days demonstrated asignificant 200-fold (p < 0.001) increase in the number of neu-trophils by comparison of medians with vehicle control treatedtumours. A 230-fold (p < 0.01) increase in neutrophils was observedfollowing 10% TTO/DMSO compared to neat TTO treated tumours oruntreated tumours at days 3 and 6 post treatment initiation (Fig. 1A).The peak accumulation of neutrophils at day 3, confirmed in 2further independent experiments (Fig. 1B), was followed thereafterby a decrease in most animals as indicated by the increase invariance in neutrophil numbers on day 6. This increase in neutrophilnumber correlated well with the peak in skin irritation and thepreviously reported regression of AE17 s.c. tumours [2]. By day 10post treatment, neutrophil numbers returned to normal, pre-treatment levels. This also coincided with the previously observedresumption of AE17 s.c. tumour growth/tumour relapse followingcessation of topical 10% TTO/DMSO treatment [2].

In situ analysis of the populations of neutrophils in topical 10%TTO/DMSO treated skin and s.c. AE17 tumours was performed byconfocal fluorescence microscopy of cross-sections of tumour/skinharvested from mice 3 days post treatment initiation. Visualisationrevealed the presence of high numbers of neutrophils (Nimp-1+) inthe skin and the upper dermal layer but not infiltrating thetopical 10% TTO/DMSO treated s.c. tumour (Fig. 1C). Very few

neutrophils were evident in the vehicle control treated tumour orskin overlying the tumour.

3.2. Topical treatment with 10% TTO/DMSO increases dendritic cell

(DC) and T cell numbers in TDLNs

Based on the dynamics of the acute neutrophil response, the DCand T cell response to topically applied 10% TTO/DMSO was alsoinvestigated in TDLNs of mice bearing established s.c. AE17tumours. TDLNs were harvested and analysed for changes in bothDC number and activation (defined by an increase in expression ofthe CD80 and CD86 co-stimulatory molecules). Throughout thetreatment period, and up to 6 days post treatment initiation withtopical 10% TTO/DMSO, the number of DCs in the TDLN of s.c.tumour bearing mice was significantly higher than that found inneat TTO, vehicle-control and untreated tumour bearing mice(Fig. 2A and C). A similar increase in the number of DCs 3 days postTTO treatment initiation was also observed in non-tumour bearingnaı̈ve mice (data not shown). In addition, topical 10% TTO/DMSOsignificantly increased the CD80 expression by DCs in TDLNsduring and up to 6 days post treatment initiation, with CD80expression returning to normal levels by day 10 (Fig. 2B and D). Asimilar trend was also observed for the DC activation marker CD86(data not shown).

T cell activation by DCs is an immune event required for T cellmediated tumour clearance and the creation of immunologicalmemory; we therefore examined the number of CD4+ and CD8+ Tcells in TDLNs 3 days post topical 10% TTO/DMSO treatmentinitiation. Whilst topical treatment with 10% TTO/DMSO increasedthe numbers of both CD4+ and CD8+ T cells in TDLNs of AE17 s.c.tumour bearing mice (Fig. 2E and F), neither the CD4+ nor the CD8+

T cell populations appeared to show increased expression of theactivation marker CD69 (data not shown). A T cell mediatedclearance of tumours was also deemed unlikely as a doubledepletion of CD4+ and CD8+ T cells did not abrogate the anti-cancereffect of topically applied 10% TTO/DMSO (data not shown).

3.3. Neutrophils appear to be involved in skin irritation but not

tumour regression after topical 10% TTO/DMSO treatment

The functional role of neutrophils in DC activation and T cellchanges were examined next. Immunocompetent mice weredepleted of neutrophils using a monoclonal antibody against Gr-1 administered i.p. every second day, starting 1 day prior to theinitiation of topical 10% TTO/DMSO treatment. Systemic depletionof neutrophils was assessed over the treatment period in theperipheral blood by both microscopy (Fig. 3F) and flow cytometry(data not shown) as well as in the tumour and overlying skin byboth flow cytometry (data not shown) and immunohistochemistryusing the neutrophil marker, Nimp-1 (Fig. 3F). Neutrophils wereabsent in the blood and in the epidermis of neutrophil (Gr-1+)-depleted animals confirming a neutrophil depletion of >90% wasachieved throughout the monitored time frame.

The flow cytometric analysis of DC populations in the TDLNs ofneutrophil depleted AE17 s.c. tumour bearing mice treated withtopical 10% TTO/DMSO revealed that the increase in the number ofDCs (above vehicle treated AE17 s.c. tumour bearing mice) and theincrease in CD80 expression by DCs in TDLNS occurred equally andindependently of neutrophils (Fig. 3A and B). In contrast, thepreviously observed CD4+ and CD8+ T cell accumulation in TDLNsof immuno competent AE17 s.c. tumour bearing mice in responseto topical 10% TTO/DMSO treatment was less obvious whenneutrophils were depleted (Fig. 3C and D). Finally, as shown inFig. 3E, the depletion of neutrophils did not abrogate the anti-tumour efficacy of topically applied 10% TTO/DMSO, as tumoursregressed independent of the presence of neutrophils. Analysis

Fig. 2. Topical 10% TTO/DMSO causes DC and T cell accumulation in tumour draining lymph nodes of AE17 s.c. tumour bearing mice. C57BL/6J mice with established (9 mm2)

subcutaneous AE17 tumours treated topically daily for 4 days with 50 ml of 10% TTO/DMSO ( ), 5 ml neat TTO ( ), 50 ml vehicle control 10%H20/DMSO (-~-) and no

treatment (-&-). On days 1, 3, 6 and 10 after the initiation of treatment, the (A) number of CD11c+/MHCIIhigh cells (DCs) per TDLN and (B) fold increase in the CD80 mean

fluorescence intensity (MFI) of DCs was assessed by flow cytometry. (C) DC number, (D) DC activation, (E) CD4+ T cell and (F) CD8+ T cell numbers measured in TDLNs 3 days

post treatment initiation with daily topical 10% TTO/DMSO or the vehicle control. Data are presented as the median � range of �3 mice per treatment group from 3 (DC) and 2 (T

cells) independent experiments (*p < 0.05, **p < 0.01, ***p < 0.0001 by two-tailed Mann–Whitney test).

Fig. 1. Topical 10% TTO/DMSO treatment induces an accumulation of neutrophils in the skin overlying s.c. AE17 tumours. (A) The number of neutrophils in the s.c. AE17

tumour and skin above the tumour (pooled for analysis) following daily, topical treatment for 4 days with 10% TTO/DMSO (50 ml ), neat TTO (5 ml ), vehicle control

10% H2O/DMSO (50 ml -~-) or no treatment (-&-), on days 1, 3, 6 and 10 post treatment initiation in C57BL/6J mice bearing established (9 mm2) s.c. AE17 tumours. The data

are shown as median � range of the number of neutrophils per gram skin/tumour analysed by flow cytometry (n = 5 mice). * Indicates statistically significant (**p < 0.01,

***p < 0.001) from vehicle control by two-way ANOVA (KrusWallis) test. (B) For increased statistical confidence, the number of neutrophils was compared between the s.c. AE17

tumour and the skin overlying the tumour following daily, topical treatment for 3 days with 10% TTO/DMSO or the vehicle control (10% H2O/DMSO) using 12 mice from 3

independent experiments. The data are shown as median and **** indicates a p-value <0.0001 by two-tailed Mann–Whitney test. (C) C57BL/6J mice bearing established s.c. AE17

tumours were treated topically with 10% H2O/DMSO (left) and 10% TTO/DMSO (right) for three consecutive days with cross-sections of tumour/skin stained for neutrophils using

Nimp-1 (green). Tissue sections were counter stained with Hoechst 33342 (blue).


Fig. 3. Neutrophil depletion does not abrogate the anti-tumour efficacy of topical 10%TTO/DMSO on established AE17 subcutaneous tumours. C57BL/6J mice with

subcutaneous AE17 tumours (9 mm2) were treated with 100 mg of anti-Gr-1 (neutrophil depleting antibody) on days �1, 1, 3, 5 and 7 and then treated topically, daily for 4

days with 50 ml of 10% TTO/DMSO and compared to isotype treated controls. (A) DC numbers in TDLNs of neutrophil depleted mice, (B) DC activation (CD80 expression) in

TDLNs of neutrophil depleted mice, (C) CD4+ T cells and (D) CD8+ T cells in TDLNS of neutrophil depleted mice. (E) AE17 tumour growth following 4, topical, daily 10% TTO/

DMSO treatments of neutrophil depleted mice ( ) and isotype matched control treated mice (-~-) or immuno competent mice treated with 10% H2O/DMSO ( ). The

data are represented as the mean � SD of at least 2 independent experiments (3 mice per treatment group). (F) Blood smears from immuno competent and neutrophil depleted

(anti-Gr-1 mAb) mice were stained for haematoxylin and eosin and assessed for neutrophils in circulating blood by microscopy. Neutrophil depleted C57BL/6J mice bearing

established s.c. AE17 tumours were treated topically with 10% H2O/DMSO for three consecutive days with cross-sections of tumour/skin stained for neutrophils using Nimp-1

(green). Tissue sections were counter stained with Hoechst 33342 (blue). (G) Representative photographs of 10%TTO/DMSO treated murine skin of immunocompetent and

neutrophil depleted mice.


of skin irritation during this experiment revealed a less pro-nounced scabbing of the skin in neutrophil depleted animals after10% TTO/DMSO (Fig. 3G) plus a shorter healing time.

3.4. The anti-tumour efficacy of topical 10% TTO/DMSO is mediated by

a direct effect of TTO on tumour cells in vivo

Based on these functional studies there was no evidence toshow that TTO induced a T cell specific response against the AE17tumours nor that neutrophils contributed to more than skininflammation observed at the treatment site. This led to theinvestigation of the direct effect of TTO on tumour cells in vivo. Ourgroup has previously demonstrated that TTO exerts directcytotoxic effects on AE17 and B16-F10 tumour cells causing lowlevel apoptosis and primary necrotic cell death in vitro, atconcentrations non-cytotoxic to non-tumour fibroblasts [13]. Inorder to test whether TTO exerts direct cytotoxicity on tumourcells in vivo, topical 10% TTO/DMSO treated AE17 tumours werecross-sectioned on days 1 and 3 post treatment initiation andanalysed by TEM. AE17 tumour cells in topical 10% TTO/DMSOtreated mice revealed changes in both the gross tumour tissueorganisation as well as the individual cell ultrastructure when

compared to the spatially well organised vehicle control treatedtumour cells (Fig. 4A). After topical 10% TTO/DMSO treatment s.c.AE17 tumours progressively lost cellular organisation marked byan increase in intercellular spaces and an accumulation of celldebris, lipids (L) and membrane micelles (Fig. 4A). Moreover, thisloss of cellular organisation following TTO/DMSO treatment wasmore prominent in the upper tumour regions, proximal to the skin(Fig. 4B). Similar to our in vitro observations, fibroblasts adjacent todamaged tumour cells appeared healthy following 10% TTO/DMSOtreatment (Fig. 4C). This was also true for lymphocytes identifiedwithin tumour sections and skeletal muscle fibres adjacent totumours (data not shown).

AE17 tumour cells displayed signs of damage and death as earlyas 1 day post topical 10% TTO/DMSO treatment (Fig. 5). Thisincluded the compression of nuclei and contraction of chromatinaccumulating on nuclear membranes. Mitochondria showed signsof swelling with increasing disruption of mitochondrial cristae andmembranes together with a loss of protein organisation and anoccasional fusing with lysosomes. Remnant mitochondriaappeared pale and ruptured by day 3 post topical 10% TTO/DMSOtreatment initiation. A clear loss of the highly interconnectedorganisation of ER structures was also visible with ER appearing

Fig. 4. Topical 10% TTO/DMSO treatment causes cellular organisational changes within AE17 s.c. tumours. Established (9 mm2) tumours were topically treated daily for 3 days

with 10% TTO/DMSO (50 ml) or 10% H2O/DMSO (vehicle control, 50 ml) with tumours cross sectioned 1 and 3 days post treatment initiation and viewed by TEM. (A)

Progressive impact of TTO treatment over treatment time on tissue integrity is shown by progressive loss of cell–cell organisation, mitochondrial bloating (arrows),

accumulation of lipids (L) and nuclei compression (N) (magnification 2500�). (B) Increased effect of TTO treatment on day 3 in upper tumour region proximal to the treated

skin is displayed in comparison to lower tumour regions more distant from the treatment site (magnification 2500�). (C) Increased impact of TTO treatment on ultrastructure

in an AE17 tumour cell in comparison to neighbouring fibroblast is shown by mitochondrial bloating (M, arrows) and general loosening of intracellular membrane systems in

AE17 cells but not fibroblasts (magnification 8000�); L = lipid, N = nucleus, M = mitochondria; images are representative of 2 tumour sections from at least 2 mice.


dilated just 1 day after treatment initiation. The ER had almostcompletely dissolved by day 3 with ribosomes mostly detached.Similarly, cell membranes became less defined with increasingtopical 10% TTO/DMSO treatment duration. AE17 cell bodiesappeared distorted and ruptured with cellular constituents leakinginto the intercellular space. The cytoplasm and mitochondria of thes.c. AE17 tumour cells treated with topical 10% TTO/DMSO becameincreasingly more electron lucent with repeated exposure to TTOand appeared to contain little metabolites.

The differential tumour cell damage seen in the upper versus thelower regions of tumours (most distant from the treatment site)suggested that the ability of TTO in this formulation to penetratedeep into tumours may be the determining factor in the success ofthis topical chemotherapy. In order to investigate the localisationof TTO within the skin and s.c. AE17 tumours after topicalapplication of the 10% TTO/DMSO formulation, tumour/skin cross-sections of topical 10% TTO/DMSO treated (Fig. 6A right and B top)and vehicle treated (Fig. 6A left and B bottom) AE17 s.c. tumours

Fig. 5. Topical 10% TTO/DMSO selectively disrupts the ultrastructure of AE17 tumour cells in vivo. C57BL/6J mice bearing established AE17 tumours (9 mm2) were treated

topically with 10% TTO/DMSO daily for 3 days. Tumours were cross sectioned and viewed by TEM. Top panel shows representative images of tumour sections treated with 10%

H2O/DMSO (control) for 3 days. Tumour sections treated with 10% TTO/DMSO for 1 day (middle panel) and 3 days (bottom panel) showing cell membranes, mitochondria, ER

and chromatin/nuclei (columns from left to right). Arrows indicate progressive loss of membrane integrity (left panel) as well as loss of parallel alignment and integrity of ER (third

panel from left). Magnification as indicated by scale bars in each image), N = nucleus, M = mitochondria, ER = endoplasmic reticulum.


were stained for visualisation of lipophile compounds using Oilred-O stain and light microscopy. Lower magnification (Fig. 6A)revealed strong staining of adipose tissue (AdTi) and thethickened stratum corneum (SC) of AE17 s.c. tumours treatedwith topical 10% TTO/DMSO. An investigation of the localisation ofoils (red) at higher magnification (Fig. 6B) revealed that the viableepidermis (Ep) of mice bearing s.c. AE17 tumours treated topicallywith 10% TTO/DMSO contained more lipid droplets (LiD) withsome droplets penetrating into the sub-epithelial layers ascompared to the vehicle control treated tumours. The presenceof LiD below the stratum basale of mice treated topically with 10%TTO/DMSO suggested that TTO diffused into the dermal cell layersand s.c. AE17 tumour tissue but that the concentration of TTOwithin the actual tumour still appeared very low and wasundetectable by this method.

4. Discussion

Current topical chemotherapy options for actinic keratoses(precancerous skin lesions/sun spots), and superficial non-melanoma skin cancers, include imiquimod and 5-fluorouracil.Their respective modes of action are via (i) TLR mediatedimmune killing of tumours, in addition to apoptosis in sometumour cell types [14] and (ii) interference with RNA and DNAsynthesis leading to cell death in abnormal cells [15,16]. The

recently US Food and Drug Authority (FDA) approved ingenolmebutate may offer an alternative as current chemotherapiesare often plagued by limitations including long treatmentregimes and debilitating skin reactions. After a shortertreatment regime, ingenol mebutate induces primary necrotictumour cell death together with neutrophil mediated antibodydependent cellular cytotoxicity (ADCC) to prevent tumourrelapse [11].

Similar to the history of ingenol mebutate [1], there wasanecdotal evidence to suggest that TTO may have anti-cancerefficacy. Over the last 4 years, communications from the generalpublic have included reports that home remedies of topical TTOmixtures are effective at clearing both actinic keratoses and skintumours; including basal cell (BCC) and squamous cell (SCC)carcinomas. We have thus worked to scientifically validate thisanecdotal evidence and have successfully demonstrated both in

vitro and in vivo (murine models) that TTO can in fact inhibittumour cell growth. There are several novel findings in thiscurrent preclinical study of the mechanism of action underlyingthe anti-cancer activity of topically applied TTO in a DMSOformulation. Specifically, it is now clear that topically applied 10%TTO/DMSO causes a direct cytotoxic effect on subcutaneous AE17tumour cells coupled with an apparent non-tumour specificactivation of a local immune response including, at least,neutrophils, DCs and T cells.

Fig. 6. TTO localises predominantly to the epidermis following topically applied 10% TTO/DMSO. Representative microscopy images of C57BL/6J mice bearing established s.c.

AE17 tumours treated topically, daily for 3 days with 10% TTO/DMSO or vehicle control. (A) Vehicle control (left) and 10% TTO/DMSO (right) treated skin and s.c. AE17 tumour

sections on day 3 post treatment initiation. (B) Vehicle control (bottom) and 10% TTO/DMSO treated (top) skin sections at higher magnification. Ep = epidermis, AdTi = adipose

tissue, SeGl = sebaceous gland, SkMu = skeletal muscle, LiD = lipid droplet, SC = stratum corneum.


Little is known of the effect of topically applied essential oils,especially TTO, with respect to the induction of inflammation orimmune infiltration in the skin. Topically applied TTO can suppressboth the oedema induced by intradermal injection of histamine [17]and neutrophil accumulation [18]. We noted that skin inflammationis similar in both tumour bearing and non-tumour bearing micetreated with 10% TTO/DMSO and is confined to the upper epidermallayers, specifically the stratum corneum which undergoes thicken-ing and shedding in response to treatment (data not shown). Thereduced skin irritation in neutrophil depleted mice suggested theaccumulation of neutrophils was related to skin this inflammationand associated with the combination of DMSO with TTO. Skininflammation and damage to epithelial tissues is known to liberatepro-inflammatory cytokines (e.g., TNF-a and IL-1b) that activateendothelial cells to produce neutrophil attractants (e.g., IL-8) [19,20]with neutrophil granule-derived components, such as azuricidinand defensins known to be chemotactic for activated T cells andimmature DCs [21] to inflamed tissues [22]. In this study, topical 10%TTO/DMSO treatment was shown to induce the highest increase inneutrophil numbers on day 3 post treatment initiation; the specifictimepoint at which the most significant tumour regression isobserved together with the peak in DC accumulation and activationin TDLNs and the increase in T cell numbers in TDLNs. Takentogether, it was proposed that similar to the anti-cancer effect ofingenol mebutate, neutrophils likely played a role in our AE17 s.c.tumour model to influence the adaptive immune system and thusmediate 10% TTO/DMSO induced tumour regression. However,neutrophil depletion experiments demonstrated that the anti-tumour efficacy of topically applied 10% TTO/DMSO was notdependent on neutrophils; DC numbers still increased and DCswere still activated, suggesting an involvement of alternativeimmune cells and/or other mechanisms of tumour regression. Theincrease in neutrophils at the treatment site in response to topical10% TTO/DMSO treatment, the accumulation and activation of DCsin TDLNs together with an increase in both CD4+ and CD8+ T cellnumbers confirmed a local immune cell activation but did notdemonstrate an antigen-specific, T cell mediated clearance oftumours; akin to the non-tumour specific mechanism of action of 5-fluorouracil. In fact, a CD4 and CD8 co-depletion study demonstrated

that 10% TTO/DMSO cleared established tumours even in a state ofreduced T cell function.

Tumour cell death does not however go unnoticed by theinnate and adaptive immune responses; in fact, depending on thecell death inducer, some types of tumour cell death have beenshown to induce an anti-tumour immune response capable ofovercoming tumour-induced immune-suppression [23]. Tumourcell death induced by some chemotherapies and radiotherapiescan induce an ‘‘eat me’’ signal associated with the exposure ofcalreticulin on the cell surface [24]. The secretion of the high-mobility-group box 1 (HMGB1) alarmin protein as a second‘‘danger’’ signal is likely required for an efficient anti-tumourimmune response [25]. It is proposed that in some cases, andpotentially in this study in response to topical 10% TTO/DMSOtreatment, that there is still some signalling between dyingtumour cells and DCs. It is possible that TTO induced direct celldeath resulting in secretion of death signals and the activation ofDCs thereby explaining the local immune infiltration andactivation, including neutrophils [26], seen in response to topicalapplication of 10% TTO/DMSO.

Studies presented here have clearly shown that topicallyapplied 10% TTO/DMSO is directly, and selectively, cytotoxic toAE17 tumour cells in vivo. Our observations suggest that TTO/DMSO causes primary necrosis of AE17 tumour cells in vivo andconfirms our previous in vitro study demonstrating that TTO had adose dependent effect in AE17 cells by inducing low level apoptosisand primary necrotic cell death [13]. TEM demonstrated that in

vitro TTO treatment of murine tumour cells targeted primarilymitochondria resulting in mitochondrial membrane disruption,gross swelling and dissolution of internal structures. In vivo wehave now shown by TEM a significant impact of topical 10% TTO/DMSO treatment on the integrity of s.c. AE17 tumour cellmembranes and membrane-bound cell organelles. The changesin the plasma membrane, cell organelle swelling and disruption ofthe mitochondrial cristae are suggestive that AE17 cells underwentprimary necrosis. Similar changes in cell eukaryotic membranesfollowing TTO treatment have been observed previously in otherhuman and murine tumour cells [13,27] and in AE17 cells in vitro

[2]. Scanning electron microscopy of migrating melanoma cells


showed them developing chasms in the plasma membrane withsurface blebbing and the release of blebs in response to treatmentwith TTO [7]. TTO and its single terpenes integrate into membranesincreasing their fluidity and making them less stable [28–30]. Thisappears to be concentration dependent and may cause membranerupture in cancer cells if a critical single or combined terpeneconcentration is reached; normal fibroblasts and immune cellsseem to show a higher tolerance. The upper area of a tumour,proximal to the skin, shows increased tumour cell destruction ascompared to the lower parts. This differential effect, and thepresence of residual tumour cells, likely explains the 100% tumourrelapse seen post cessation of 10% TTO/DMSO treatment [2]. DMSOis also well known to exert similar membrane thinning effects byincorporating into the lipid tails of the bilayer structure ofmembranes increasing membrane fluidity [31]. This inducestransient water pores and, above critical concentrations, adisintegration of cell membranes [32,33]. As topical treatmentwith DMSO vehicle or neat TTO did not affect tumour growth [2] orimmune activation, it is thought that in this case DMSO enhancespenetration of TTO through the stratum corneum into theepidermis by altering the cohesive lipid packing [34]. Consideringthat topical treatment with neat TTO or DMSO vehicle did notdemonstrate any visible membrane disruption in this study, it isvery likely that the membrane-based cytotoxic effect observed in

vivo is based on an interactive or accumulative effect of DMSO andthe terpenes of TTO.

It is clear from this research that topically applied dilute TTO ina DMSO formulation induces both local immune activation and adirect cytotoxic effect on tumour cells. Though the exact cascade ofevents leading to the clearance of s.c. AE17 tumours still remains tobe elucidated, it is clear that TTO is directly cytotoxic to thesetumour cells. DMSO is used in these studies as a penetrationenhancer with penetration shown to correlate with treatmentefficacy. Enhancing the direct cytotoxicity of TTO by increasingpenetration and in situ terpene concentration is the focus of currentresearch with the formulation of TTO into a more clinicallyacceptable vehicle also a key priority. The AE17 tumour modelused in these studies is particularly aggressive and known to besubject to significant immunosuppression potentially explainingthe lack of T cell mediated clearance of tumours [35–37] followingTTO treatment. Overcoming this immunosuppression has been thefocus of much research in our laboratory and has proved difficultuntil recently [38]. Topical TTO treatments coupled with such animmune boosting strategy will likely result in long term clearanceof tumours. The future demonstration of complete and long termtumour regression will facilitate the translation of topical TTOtreatment to the clinic for the treatment of human skin tumours.

Acknowledgements

The authors acknowledge the facilities, scientific and technicalassistance of the Australian Microscopy & Microanalysis ResearchFacility at the Centre for Microscopy, Characterisation & Analysis,The University of Western Australia, a facility funded by theUniversity, State and Commonwealth Governments. The authorsalso acknowledge the facilities, scientific and technical assistanceof CELLCentral, School of Anatomy and Human Biology, TheUniversity of Western Australia, and The M Block Animal CareUnit, The University of Western Australia. The authors wouldlike to thank Dr. Christine Carson for stimulating discussionsand UWA Honours students and Sharnice Koek and VincentKuek for technical assistance. This work was financiallysupported by the Rural Industries Research and DevelopmentCorporation, ACT, Australia (PRJ-002395) together with NovaselAustralia Pty. Ltd., Mudgeeraba, QLD, Australia.

References

[1] Ramsay JR, Suhrbier A, Aylward JH, Ogbourne S, Cozzi SJ, Poulsen MG, et al. Thesap from Euphorbia peplus is effective against human nonmelanoma skincancers. Brit J Dermatol 2011;164:633–6.

[2] Greay SJ, Ireland DJ, Kissick HT, Heenan PJ, Carson CF, Riley TV, et al.Inhibition of established subcutaneous murine tumour growth with topicalMelaleuca alternifolia (tea tree) oil. Cancer Chemother Pharmacol2010;66:1095–102.

[3] Gupta AK, Davey V, McPhail H. Evaluation of the effectiveness of imiquimodand 5-fluorouracil for the treatment of actinic keratosis: critical review andmeta-analysis of efficacy studies. J Cutan Med Surg 2005;9:209–14.

[4] Love WE, Bernhard JD, Bordeaux JS. Topical imiquimod or fluorouracil therapyfor basal and squamous cell carcinoma: a systematic review. Arch Dermatol2009;145:1431–8.

[5] Carson CF, Hammer KA, Riley TV. Melaleuca alternifolia (tea tree) oil: a review ofantimicrobial and other medicinal properties. Clin Microbiol Rev 2006;19:50–62.

[6] Hammer KA, Carson CF, Riley TV, Nielsen JB. A review of the toxicity ofMelaleuca alternifolia (tea tree) oil. Food Chem Toxicolgy 2006;44:616–25.

[7] Bozzuto G, Colone M, Toccacieli L, Stringaro A, Molinari A. Tea tree oil mightcombat melanoma. Planta Med 2011;77:54–6.

[8] Hayes AJ, Leach DN, Markham JL, Markovic B. In vitro cytotoxicity of Australiantea tree oil using human cancer cell lines. J Essent Oil Res 1997;9:575–82.

[9] Moteki H, Hibasami H, Yamada Y, Katsuzaki H, Imai K, Komiya T. Specificinduction of apoptosis by 1,8-cineole in two human leukemia cell lines, but nota in human stomach cancer cell line. Oncol Rep 2002;9:757–60.

[10] Wu CS, Chen YJ, Chen JJ, Shieh JJ, Huang CH, Lin PS, et al. Terpinen-4-ol inducesapoptosis in human nonsmall cell lung cancer in vitro and in vivo. Evid BasedComplement Altern Med 2012;2012. 818261.

[11] Challacombe JM, Suhrbier A, Parsons PG, Jones B, Hampson P, KavanaghD, et al. Neutrophils are a key component of the antitumorefficacy of topical chemotherapy with ingenol-3-angelate. J Immunol2006;177:8123–32.

[12] Jackaman C, Bundell CS, Kinnear BF, Smith AM, Filion P, van Hagen D, et al.IL-2 intratumoral immunotherapy enhances CD8+T cells that mediatedestruction of tumor cells and tumor-associated vasculature: a novelmechanism for IL-2. J Immunol 2003;171:5051–63.

[13] Greay SJ, Ireland DJ, Kissick HT, Levy A, Beilharz MW, Riley TV, et al. Inductionof necrosis and cell cycle arrest in murine cancer cell lines by Melaleucaalternifolia (tea tree) oil and terpinen-4-ol. Cancer Chemother Pharmacol2010;65:877–88.

[14] De Giorgi V, Salvini C, Chiarugi A, Paglierani M, Maio V, Nicoletti P, et al. In vivocharacterization of the inflammatory infiltrate and apoptotic status in imi-quimod-treated basal cell carcinoma. Int J Dermatol 2009;48:312–21.

[15] Wilgus TA, Breza Jr TS, Tober KL, Oberyszyn TM. Treatment with 5-fluorouraciland celecoxib displays synergistic regression of ultraviolet light B-inducedskin tumors. J Invest Dermatol 2004;122:1488–94.

[16] Jury CS, Ramraka-Jones VS, Gudi V, Herd RM. A randomized trial of topical 5%5-fluorouracil (Efudix cream) in the treatment of actinic keratoses comparingdaily with weekly treatment. Brit J Dermatol 2005;153:808–10.

[17] Koh KJ, Pearce AL, Marshman G, Finlay-Jones JJ, Hart PH. Tea tree oilreduces histamine-induced skin inflammation. Brit J Dermatol2002;147:1212–7.

[18] Maruyama N, Sekimoto Y, Ishibashi H, Inouye S, Oshima H, Yamaguchi H, et al.Suppression of neutrophil accumulation in mice by cutaneous application ofgeranium essential oil. J Inflamm (Lond) 2005;2:1.

[19] Delves PJ, Roitt IM. The immune system. Second of two parts. N Engl J Med2000;343:108–17.

[20] Delves PJ, Roitt IM. The immune system. First of two parts. N Engl J Med2000;343:37–49.

[21] Yang D, Chen Q, Chertov O, Oppenheim JJ. Human neutrophil defensinsselectively chemoattract naive T and immature dendritic cells. J Leukoc Biol2000;68:9–14.

[22] Chertov O, Yang D, Howard OM, Oppenheim JJ. Leukocyte granule proteinsmobilize innate host defenses and adaptive immune responses. Immunol Rev2000;177:68–78.

[23] Casares N, Pequignot MO, Tesniere A, Ghiringhelli F, Roux S, Chaput N, et al.Caspase-dependent immunogenicity of doxorubicin-induced tumor celldeath. J Exp Med 2005;202:1691–701.

[24] Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al.Calreticulin exposure dictates the immunogenicity of cancer cell death. NatMed 2007;13:54–61.

[25] Apetoh L, Ghiringhelli F, Tesniere A, Criollo A, Ortiz C, Lidereau R, et al. Theinteraction between HMGB1 and TLR4 dictates the outcome of anticancerchemotherapy and radiotherapy. Immunol Rev 2007;220:47–59.

[26] Abraham E, Arcaroli J, Carmody A, Wang H, Tracey KJ. HMG-1 as a mediator ofacute lung inflammation. J Immunol 2000;165:2950–4.

[27] Calcabrini A, Stringaro A, Toccacieli L, Meschini S, Marra M, Colone M, et al.Terpinen-4-ol, the main component of Melaleuca alternifolia (tea tree) oilinhibits the in vitro growth of human melanoma cells. J Invest Dermatol2004;122:349–60.

[28] Sikkema J, de Bont JA, Poolman B. Interactions of cyclic hydrocarbons withbiological membranes. J Biol Chem 1994;269:8022–8.

[29] Sikkema J, de Bont JA, Poolman B. Mechanisms of membrane toxicity ofhydrocarbons. Microbiol Rev 1995;59:201–22.


[30] Giordani C, Molinari A, Toccacieli L, Calcabrini A, Stringaro A, Chistolini P, et al.Interaction of tea tree oil with model and cellular membranes. J Med Chem2006;49:4581–8.

[31] Witzke S, Duelund L, Kongsted J, Petersen M, Mouritsen OG, Khandelia H.Inclusion of terpenoid plant extracts in lipid bilayers investigated by molecu-lar dynamics simulations. J Phys Chem B 2010;114:15825–31.

[32] Gurtovenko AA, Anwar J. Modulating the structure and properties of cellmembranes: the molecular mechanism of action of dimethyl sulfoxide. J PhysChem B 2007;111:10453–60.

[33] Chen X, Huang Z, Hua W, Castada H, Allen HC. Reorganization and caging ofDPPC, DPPE, DPPG, and DPPS monolayers caused by dimethylsulfoxide ob-served using Brewster angle microscopy. Langmuir 2010;26:18902–08.

[34] Kwak S, Lafleur M. Effect of dimethyl sulfoxide on the phase behavior of modelstratum corneum lipid mixtures. Chem Phys Lipids 2009;161:11–21.

[35] Needham DJ, Lee JX, Beilharz MW. Intra-tumoural regulatory T cells: apotential new target in cancer immunotherapy. Biochem Biophys Res Com-mun 2006;343:684–91.

[36] Kissick HT, Ireland DJ, Beilharz MW. Combined intratumoral regulatory T-celldepletion and transforming growth factor-beta neutralization induces regres-sion of established AE17 murine mesothelioma tumors. J Interferon CytokineRes 2009;29:209–15.

[37] Kissick HT, Ireland DJ, Greay SJ, Beilharz MW. Mechanisms ofimmune suppression exerted by regulatory T-cells in subcutaneousAE17 murine mesothelioma. J Interferon Cytokine Res 2010;30:829–34.

[38] Kissick HT, Ireland DJ, Krishnan S, Madondo M, Beilharz MW. Tumour eradi-cation and induction of memory against murine mesothelioma by combinedimmunotherapy. Immunol Cell Biol 2012.

journal of dermatological science -...

Documents