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The American Journal of Surgery (2012) 204, 868–873

The Southwestern Surgical Congress: Best Poster Award

Honokiol induces cytotoxic and cytostatic effects inmalignant melanoma cancer cells

Gaurav Kaushik, Ph.D.a, Satish Ramalingam, Ph.D.b,c,Dharmalingam Subramaniam, Ph.D.b,c, Parthasarthy Rangarajanb,c,iero Protti, M.S.a, Prabhu Rammamoorthy, Ph.D.b,c, Shrikant Anant, Ph.D.b,c,*,oshua M.V. Mammen, M.D., Ph.D.a,b,c

aDepartment of Surgery, University of Kansas Medical Center, Kansas City, KS, USA; bDepartment of Molecular andIntegrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA; cUniversity of Kansas Cancer

Center, 3901 Rainbow Blvd., Mail Stop 3040, Kansas City, KS 66160, USA

AbstractBACKGROUND: Melanomas are aggressive neoplasms with limited therapeutic options. Therefore,

developing new therapies with low toxicity is of utmost importance. Honokiol is a natural compoundthat recently has shown promise as an effective anticancer agent.

METHODS: The effect of honokiol on melanoma cancer cells was assessed in vitro. Proliferation andphysiologic changes were determined using hexosaminidase assay and transmission electron micros-copy. Protein expression was assessed by immunoblotting.

RESULTS: Honokiol treatment inhibited cell proliferation and induced death. Electron microscopyshowed autophagosome formation. Reduced levels of cyclin D1 accompanied cell-cycle arrest. Hon-okiol also decreased phosphorylation of AKT (known as protein kinase B) and mammalian target ofrapamycin, and inhibited �-secretase activity by down-regulating the expression of �-secretase complexproteins, especially anterior pharynx-defective 1.

CONCLUSIONS: Honokiol is highly effective in inhibiting melanoma cancer cells by attenuatingAKT/mammalian target of rapamycin and Notch signaling. These studies warrant further clinicalevaluation for honokiol alone or with present chemotherapeutic regimens for the treatment ofmelanomas.© 2012 Elsevier Inc. All rights reserved.

KEYWORDS:Melanoma;AKT;Cyclin D1;Cell-cycle arrest;AKT;mTOR;�-secretase

ntcp

Malignant melanoma is an aggressive form of skin can-cer with an extremely poor survival rate and few therapeuticoptions. The incidence of melanoma in the United States hasincreased more rapidly in the past few decades than anyother malignancy.1,2 Approximately 40% to 60% of cuta-

No potential conflicts of interest were disclosed.* Corresponding author. Tel.: �1-913-945-6334; fax: �1-913-945-6327E-mail address: sanant@kumc.eduManuscript received July 12, 2012; revised manuscript September 6,

012

002-9610/$ - see front matter © 2012 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.amjsurg.2012.09.001

eous melanomas also may carry B-Raf mutations that leado the constitutive activation of downstream signaling cas-ade through the mitogen-activated protein kinase (MAPK)athway.3,4 Approximately 90% of these mutations result in

the substitution of glutamic acid for valine at codon 600(BRAF V600E) and renders B-RAF in a constitutively ac-tive form that confers mitogenic responses for the growth ofcancerous cells.5 Finally, alterations of the chromosome10q24 to 26 region are present in 30% to 50% of melanoma

cell lines and in 5% to 20% of uncultured melanomas.6 This

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869G. Kaushik et al. Honokiol and the AKT/mTOR pathway

region includes the lipid phosphatase and tensin homologuedeleted from chromosome 10 (PTEN), which functions bynegatively regulating the phosphatidylinositol 3’-kinase(PI3K)/protein kinase B (AKT) pathway. Loss of PTENfunction in melanoma needs further investigation.6 The

I3K/AKT and Raf/MEK (MAP kinase/ERK kinase)/extra-ellular signal-regulated kinase (ERK) pathways promoteumorigenesis by positively regulating cell survival, cell-ycle progression,7–9 tumor angiogenesis,10–12 and tumor

invasion.13–15 All these events together activate a multitudeof downstream signaling cascades including the Raf/MAPKkinase (MEK)/ERK and PI3K/AKT pathways. B-RAF mu-tations activate the MAPK cascade and loss of PTEN acti-vates PI3K; these 2 pathways are important mediators ofmelanoma downstream of Ras.

Aberrant activation of Notch signaling also has beenassociated with the development of skin cancer. In melano-

Figure 1 Effect of honokiol on melanoma cancer cells. (A) Cincreasing doses of honokiol (0–50 �mol/L) at different time interv

he honokiol treatment resulted in a significant dose- and time-dontrols (P � .05). (C) Transmission electron micrograph showinonokiol (30 �mol/L) treatment. All images were taken at 2,000�

mas, levels of Notch expression are associated with pro-

gression and metastasis.16 Notch signaling is initiated whenligand such as Jagged interacts with the Notch transmem-rane receptor, leading to translocation of notch intracellu-ar domain (NICD) and binding of 2 co-factors to transac-ivate target genes, such as those in the hairy and enhancerf split, related to the YRPW motif, and cyclin D1 pro-eins.16,17 The importance of Ras activation and the Notch

pathways in melanoma suggests that they may be effectivetargets for the prevention and treatment of melanomas. Thereal challenge in inhibiting these signaling cascades is thatthey regulate important growth and survival pathwaysshared by both neoplastic and normal cells. Therefore, it isof prime importance to develop a therapeutic strategy that isable to exert the maximum effect on cancer cells withminimum toxicity on normal cells.

Honokiol (Fig. 1A) is a biphenolic compound from Mag-nolia officinalis that is used in traditional Chinese and Jap-

l structure of honokiol. (B) B16-F10 cells were incubated with, 48, and 72 h) to determine the cell proliferation of B16-F10 cells.ent decrease in cell proliferation compared with their respectiveokiol-induced vacuole formation in B16-F10 cells at 24 hours ofnification.

hemicaals (24ependg hon

anese medicine for the treatment of various ailments includ-

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870 The American Journal of Surgery, Vol 204, No 6, December 2012

ing ulcers, allergies, and bacterial infections. It also is usedas a muscle relaxant and possesses antithrombotic activ-ity.18 Recent studies have shown that it has antitumor ac-tivity with low toxicity.18 In this article, we investigated theeffect of honokiol on melanoma cancer cells. This articlefocuses on defining nontoxic approaches to eradicate mel-anoma cancer cells.

Materials and Methods

Cell line and reagents

B16-F10 cells (American Type Culture Collection, Manas-sas, VA) were grown in Dulbecco’s modified Eagle mediumsupplemented with 10% fetal bovine serum (Sigma-Aldrich,St. Louis, MO) and antibiotic solution (Mediatech, Inc, Ma-nassas, VA) at 37°C in a humidified atmosphere containing 5%CO2. Cells used in this study were within 20 passages afterreceipt or resuscitation (approximately 3 months of noncontin-uous culturing). Growth medium was changed every 3 day andcells were split (1:3) when they reached 80% confluence. Forhonokiol treatment, stock solution of honokiol was prepared indimethyl sulfoxide, stored at �20°C in aliquots, and dilutedwith fresh medium immediately before use. Honokiol waspurchased from Sigma-Aldrich.

Cell viability assay

Assessment of cell proliferation after honokiol treatmentwas performed by using a hexosaminidase assay.19 Cellswere plated in a 96-well plate, grown overnight, and treatedthe next day with increasing concentrations of honokiol(0–50 �mol/L) for 24, 48, and 72 hours. Cell growth wasalculated as the percentage viability: [(A/B) � 100], where

and B are the absorbance of treated and control cells,espectively.

Immunoblot analysis

After honokiol treatment cell lysates were extracted andquantitated with a Pierce BCA protein assay kit (ThermoScientific, Rockford, IL). Cell lysates were subjected tosodium dodecyl sulfate–polyacrylamide gel electrophoresisand blotted onto nitrocellulose membrane (GE Healthcare,Pittsburgh, PA), and proteins of interest were detected byusing an enhanced chemiluminescence system (GE Health-care). Antibodies for immunoblotting were purchased fromCell Signaling Technology, Inc (Piscataway, NJ), Gen-Script, Inc (Piscataway, NJ), and Santa Cruz Biotechnology,Inc (Santa Cruz, CA). For equal loading of protein in eachwell, each blot was normalized with its respective internalcontrols (�-actin or �-tubulin, total AKT, and mammalian

target of rapamycin [mTOR]).

Statistical analysis

All experiments were performed in triplicate. Significantdifferences were analyzed using the Student t test. Datawere considered statistically significant if the P value wasless than .05. Data were expressed as the mean � standarddeviation between experiments in triplicate.

Results

Honokiol affects viability of B16-F10melanoma cells

To study the effect of honokiol on melanoma cellgrowth, B16-F10 melanoma cells were treated with increas-ing concentrations of honokiol (0–50 �mol/L) for 24, 48,nd 72 hours and cell proliferation was performed by usingexosaminidase assay. Honokiol treatment significantly de-reased cell proliferation of melanoma cells in a dose- andime-dependent manner with respect to their controls (P �05). A decrease in cell proliferation was observed within 24ours of honokiol treatment, which was suppressed furtherver the next 72 hours in a dose-dependent manner (Fig.B). We further studied the morphologic changes in B16-10 by transmission electron microscopy. Honokiol treat-ent increased the autophagosomes such as vacuoles for-ation in B16-F10 cells (Fig. 1C). Together, these findings

ndicate that honokiol showed cytotoxic effects on mela-oma cancer cells.

Honokiol down-regulates the expression of cyclinD1 in melanoma cells

Honokiol effectively attenuated the levels of cyclin D1protein in B16-F10 cells in a time- and dose-dependent manner(Fig. 2A) and thus resulted in cell-cycle arrest of melanomacancer cells. Honokiol attenuation of cyclin D1 levels in B16-F10 cells further strengthens the fact that a significant decreasein cell growth of B16-F10 cells by honokiol was partly owingto inhibition of cell proliferation and induction of cell deathand partly owing to cell-cycle arrest. Altogether, these datashowed that honokiol has both cytostatic and cytotoxic prop-erties against melanoma cancer cells.

Signaling affected by honokiol in melanoma cells

Most of the chemotherapeutic agents against melanomaare partly or fully designed for targeting of malfunctioningRas and Raf signaling molecules in melanoma cancer cells.Therefore, we further tried to explore the pathways involvedin honokiol-induced cell death and growth inhibition ofmelanoma cells. We observed that honokiol significantlyaffected the Ras and Raf signaling in melanoma cells by

dephosphorylating the constitutively phosphorylated AKT

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871G. Kaushik et al. Honokiol and the AKT/mTOR pathway

and mTOR protein in B16-F10 cells in a dose- and -time-dependent manner (Fig. 2B and C). This substantiates thefact that honokiol inhibited Ras and Raf signaling in mela-noma cells and thus induced cell death, cell-cycle arrest, andinhibited the growth of aggressive melanoma cells. In ad-dition to conventional melanoma signaling, honokiol alsowas found to inhibit cancer stem cell–related signaling (ie,Notch signaling). Honokiol significantly attenuated the lev-els of anterior pharynx-defective 1 in B16-F10 cells in atime-dependent manner (Fig. 2D). This protein is part of the�-secretase enzyme complex. �-secretase is the key enzymenvolved in cleavage of NICD and this cleaved notch then isranslocated to the nucleus for transcriptional up-regulationf downstream client genes, especially cyclin D1.

Comments

Honokiol, because of its diverse antioxidative, anti-inflammatory, antitumor, and antimicrobial pharmaco-logic properties, is a putative candidate for both thera-peutic interventions and chemoprevention. Previousstudies have shown the efficacy of honokiol against sev-eral types of cancer, such as pancreatic, colon, and breast,without any toxicity.20 Unlike other polyphenolic agentsthat have been hindered by poor absorption and rapid

Figure 2 Honokiol attenuates AKT/mTOR and Notch signalincyclin D1 protein levels in B16-F10 cells. Cells were treated withstudy the effect of honokiol. For time-course effects, cells were treareatment, protein lysates were prepared and analyzed by immunobnd time dependent manner in response to honokiol treatment. (Bysates after honokiol treatment showed a significant reduction inctivities in a dose- and time-dependent manner. (D) Honokiol i

Honokiol showed a significant reduction in the expression of ante

excretion, honokiol shows significant systemic levels in v

blood in preclinical models, and it also can cross theblood-brain barrier.20 We have shown honokiol’s effecton inducing B16-F10 melanoma cancer cell death byusing a hexosaminidase assay and by electron micros-copy. Honokiol began to induce these changes within 24hours of treatment and the effect remained at 48 and 72hours of treatment. Honokiol-mediated cell death waspartly owing to the formation of autophagosomes such asvacuoles in B16-F10 cells, as indicated by arrow inFigure 1C. Additional studies are required to confirm thetypes of vacuoles in B16-F10 cells after honokiol treat-ment. Subsequently, we showed that honokiol also exertscytostatic effects by significantly down-regulating cyclinD1 levels in B16-F10 cells in a dose- and time-dependentmanner. Cyclin D1, often found to be overexpressed inhuman melanomas, is an important positive regulator ofthe G1–S cell-cycle transition through its binding andactivation of cyclin-dependent kinases 4/6. Activation ofcyclin-dependent kinases further inactivate retinoblas-toma protein, blocks its growth-inhibitory activity, andpromotes release of bound E2F, which finally leads tocell-cycle progression.21,22 Recent studies also have re-ported the honokiol-induced G0/G1 cell-cycle arrest incolon and lung cancer cells.18,23 Further studies are re-

uired to study the transition of the cells through the

16-F10 melanoma cells. (A) Immunoblotting was performed forasing concentrations of honokiol (0–40 �mol/L) for 24 hours toth 30 �mol/L of honokiol for 12, 24, and 48 hours. After honokiolfor cyclin D1 protein. Cyclin D1 decreased in both a concentrationHonokiol affects AKT/mTOR signaling. Immunoblot analyses ofhorylation of AKT and mTOR phosphorylation and hence theirNotch signaling by inhibiting the �-secretase complex proteins.arynx-defective 1 (APH1).

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ted wilottingand C)

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arious phases of the cell cycle are along with the check-

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872 The American Journal of Surgery, Vol 204, No 6, December 2012

point-related markers like Bax, Bcl-2, and Ki-6 in orderto confirm this phenomenon.

We further explored the honokiol-attenuated molecularpathways involved in cell death and cystatic effects inB16-F10 cells. Melanomas often consist of mutant BRAF orN-RAS, and consequently increased ERK-MAPK and AKTactivities.24 Preclinical studies have shown a close and com-plex interconnection of the RAS-RAF-MEK-ERK and thePI3K-AKT-mTOR signaling pathways, which are involvedin growth, progression, and drug resistance of melanomas.AKT has a central role in regulating apoptosis and tumorregression.25 In BRAF V600E mutant cells, AKT activation

as required for initiation of melanoma, showing the interde-endence of these 2 pathways in melanoma. Our data revealhat honokiol significantly inhibited the AKT and mTOR phos-horylation in a dose- and time-dependent manner in B16-F10elanoma cells. This supports the position that honokiol acts

n the PI3K-AKT-mTOR signaling pathway. Supporting themportance of this pathway is that others have shown thatopical treatment of melanomas with a specific PI3K inhibitorLy294002) results in a significant reduction of TPras (mutateduman Ha-ras) transgenic melanoma growth in severe com-ined immunodeficient mice.26 In addition, inhibition of eitherhe Raf/MEK/ERK pathway by the MEK1/2-specific inhibitor0126 or PI3K leads to a reduction in tumor invasion and

ngiogenesis.26–28

The role of Notch expression has been reported in theprogression and metastasis of melanomas.16 Notch signal-ing is 10- to 30-fold higher in stem cells when comparedwith other cell types.29 In this study, we determined thathonokiol inhibited Notch signaling by inhibiting the essen-tial members of the �-secretase complex, the critical en-zyme that cleaves and releases the NICD from the mem-brane. Honokiol decreased the anterior pharynx-defective 1protein levels in a dose-dependent manner. Therefore, ho-nokiol-mediated inhibition of melanoma cancer cell growthalso partly is mediated via inactivation of the Notch-1 sig-naling cascade. A recent study suggested a role of Notch1 inthe prevention of cancer cells from cytotoxic insults. Furthersupporting this role for Notch was that the ectopic expres-sion of the Notch intracellular domain partially rescuing thecolon and breast cancer cells from cytotoxic compound’seffect.18,30 Another recent study also showed that honokiolrrests the cell cycle, induces apoptosis, and potentiates theytotoxic effect of gemcitabine by affecting nuclear factor-

�B.31,32 Recent studies have proposed that honokiol aloneor in combination with radiotherapy or chemotherapy re-sulted in growth inhibition of colon, pancreatic, and gliomacells in vitro and in vivo.18,32,33 In addition, the honokiolsignificantly suppressed Notch-1 activation. Taken together,these data suggest that honokiol to target cancer stem cellsis an attractive novel potential agent for the treatment andprevention of various cancers. Further studies are warrantedto show the efficacy of honokiol in the clinical setting. It

also would be interesting to determine whether there are

other client proteins for the �-secretase complex and therole of these client proteins in cancer stem cell biogenesis.

Acknowledgments

Supported by grants from the National Institutes ofHealth (S.A.) and from the Department of Surgery at theUniversity of Kansas Medical Center (J.M.V.M.).

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