Abstract. Background: 2’-Hydroxycinnamaldehyde (HCA)exerts antitumor activity against several human cancer celllines. However, its antitumor activity in oral cancer has notbeen demonstrated. Materials and Methods: The antitumoractivity of HCA was assessed in oral cancer cell lines and ina rat oral tumor model. Results: Cell cycle analysisconfirmed that HCA showed anti-proliferative activity viacell cycle arrest at the G2/M-phase and increased thenumber of cells in the sub-G1 (apoptotic cells) phase in SCC-15 and HEp-2 oral cancer cells. Additionally, direct injectionof HCA into an RK3E-ras-Fluc-induced tumor significantlyinhibited growth of the tumor mass. Histological analysisshowed that HCA decreased tumor cell proliferation andinduced apoptosis in a rat tumor model. Conclusion: Takentogether, these observations suggest the potential value ofHCA as a candidate for the treatment of oral cancer.

Cinnamon, one of the oldest and most versatile spices in theworld, is a good source of polyphenol. Recent studies havesuggested that cinnamon extract exerts chemopreventiveproperties such as anti-proliferative and anti-oxidative effects(1, 2).

We purified 2’-hydroxycinnamaldehyde (HCA) from thestem bark of Cinnamomum cassia and synthesized a novelHCA derivative, 2-benzoyl-oxycinnamaldehyde (BCA).HCA and BCA exerted anti-angiogenic, anti-inflammatory

and anti-proliferative activities against human cancer celllines such as breast, leukemia, ovarian, lung and coloncancer cells (3-7).

Oral carcinomas are the world’s eleventh most commonform of human neoplasm and account for 3% of all newlydiagnosed cancer cases (8-10). Despite efforts to improveoverall outcomes, survival rates have not changed during thelast 20 years. In fact, the prognosis is very poor andapproximately 50-70% of patients die within 5 years (9).Late presentation, lack of suitable markers for early detectionand failure of advanced lesions to respond to chemotherapycontribute to the poor outcome of oral carcinomas.

Biologically and clinically relevant animal models areessential to investigate the progression of disease and theelaboration of diagnostic or therapeutic protocols. Recently,we developed a tumor animal model using k-ras-transformedRK3E cells in Sprague-Dawley rats (11). This model has theadvantage of allowing short-term screening of antitumoragents (12).

Although the antitumor activity of HCA has been widelystudied, previous studies have primarily been conducted invitro. Therefore, in the present study, the antitumor activity ofHCA was assessed in vitro and in vivo using a rat oral tumormodel.

Materials and Methods

Cell cultures. Two human oral squamous cell carcinoma cell lineswere used. SCC-15 was obtained from the Korean Cell Line Bank(Seoul, South Korea) and HEp-2 was obtained from the AmericanType Culture Collection (Manassas, VA. USA). Luciferase andgreen fluorescent protein-transformed RK3E-ras cells (RK3E-ras-Fluc) were described previously (12). The cells were grown inDMEM (GibcoBRL, Carlsbad, CA. USA) supplemented with 10%fetal bovine serum, 100 units/mL penicillin and 100 μg/mLstreptomycin (GibcoBRL). The cells were maintained at 37˚C in a5% CO2 humidified atmosphere.

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Correspondence to: Su-Hyung Hong, Department of DentalMicrobiology, School of Dentistry, Kyungpook National University,188-1 Samdeok-dong-2-ga, Jung-gu, Daegu 700-412, South Korea.Tel: +82 536606831, Fax: +82 534256025, e-mail: hongsu@knu.ac.kr

Key Words: Oral cancer, 2’-hydroxycinnamaldehyde, cell cycle,apoptosis, tumor model.

ANTICANCER RESEARCH 30: 489-494 (2010)

2’-Hydroxycinnamaldehyde Shows Antitumor Activity againstOral Cancer In Vitro and In Vivo in a Rat Tumor Model

SOO-A KIM1, YOUNG-KWAN SUNG2, BYOUNG-MOG KWON3, JUNG-HOON YOON4, HAEAHM LEE5, SANG-GUN AHN4 and SU-HYUNG HONG5

1Department of Biochemistry, Dongguk University College of Oriental Medicine, Gyeongju 780-714, South Korea; 2Department of Immunology, School of Medicine, Kyungpook National University, Daegu 700-422, South Korea;

3Korea Research Institute of Bioscience and Biotechnology, Taejeon 305-806, South Korea; 4Department of Pathology, Chosun University College of Dentistry, Gwangju 501-759, South Korea;

5Department of Dental Microbiology, School of Dentistry, Kyungpook National University, Daegu 700-412, South Korea

0250-7005/2010 $2.00+.40

Cell proliferation assays. The cells (5,000 cells/well) were seededinto 96-well plates, which were subsequently incubated for 24 h.The cells were then replenished with fresh complete mediumcontaining HCA or BCA (dissolved in 0.1% DMSO), after whichthey were incubated for an additional 24 h. The cell proliferationwas then evaluated by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays as describedpreviously (13).

Cell cycle analysis. The cells were harvested and fixed with ice cold70% ethanol overnight. The fixed cells were washed with PBScontaining 1% fetal bovine serum and then incubated with 100μg/mL RNase A at 37˚C for 30 min. Propidium iodide was added toa final concentration of 50 μg/mL for DNA staining, after which thefixed cells were analyzed by flow cytometry using a FACScalibur(BD Biosciences, San Jose, CA. USA).

Western blot analysis. The cells were treated with HCA or BCA for24 h. The cells were then washed twice with ice-cold PBS and lysedin RIPA buffer (0.01 M Tris-HCl [pH 7.4], 0.15 M NaCl, 1%sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecylsulfate [SDS], 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride).Next, the protein contents of the cell extracts were determined by aBradford assay (Bio-Rad, Hercules, CA. USA). The proteins werethen resolved by SDS-polyacrylamide gel electrophoresis,electrotransferred onto a polyvinylidene fluoride (PVDF) membraneand then immunoblotted with anti-poly (ADP-ribose) polymerase(PARP) antibody, anti-caspase-3 antibody (Cell SignalingTechnology, Danver, MA. USA), or anti-actin antibody (Santa CruzBiotechnology, Santa Cruz, CA. USA).

Animals and tumor growth. Three-week-old male Sprague-Dawleyrats (Samtaco, Osan, South Korea) were kept under standardhousing conditions. RK3E-ras-Fluc cells were harvested bytrypsinization, centrifuged and then resuspended in DMEM withoutserum at a density of 2.5×107/mL. Two hundred μL of cellsuspension (total 5×106 cells) were then injected into the oralmucosa of control and treated rat groups; each group consisted offive rats. HCA treatment began on the 5th day after oral injection

of RK3E-ras-Fluc cells. HCA (50 mg/kg) or an equal volume of thevehicle, DMSO, was directly injected into the tumor every other dayfor five times. On the next day after the final treatment, the animalswere sacrificed and the solid tumors were excised for furtheranalysis. The tumor growth was evaluated using a caliper and thetumor volume (V) was calculated using the following formuladescribed by Carlsson: V=(ab2)/2, where ‘a’ is the longest diameterand ‘b’ is the shortest diameter of the tumor (14). The excised solidtumors were immediately fixed in 10% buffered formalin and thenembedded in paraffin. Body weight was regularly checked todetermine cinnamaldehyde toxicity. All the experiments wereconducted following protocols approved by the Animal Care andUse Committee at Chosun University College of Dentistry(Gwangju, South Korea).

Bioluminescence imaging. Luciferin (Molecular Probes, Palo Alto,CA. USA) was injected i.p. at a dose of 80 mg/kg of body weightwith xylazine/ketamine anesthesia. The bioluminescent signalsemitted from the tumor of the rats were then evaluated using aXenogen IVIS-100 Imaging System (Xenogen Co., Alameda, CA.USA) equipped with a CCD camera system for emitted lightacquisition. The Living Image software (Xenogen) was used for dataanalysis.

Histopathology and immunohistochemistry. For light microscopicexamination, 4-μm sectioned tissues were stained with hematoxylin-eosin (H-E). Immunohistochemical staining was performed onsimilar sections by the avidin-biotin peroxidase complex (ABC)method using anti-proliferating cell nuclear antigen (PCNA)antibody (Dako, Glostrup, Denmark). Immune reactions werevisualized with 3,3’-diaminobenzidine (DAB) and counterstainedwith Mayer’s hematoxylin.

TUNEL assay. A TUNEL assay was performed using an ApopTagPlus Peroxidase In Situ Apoptosis Detection kit (Intergen, Purchase,NY, USA) according to the manufacturer’s instructions. Briefly, thetumor sections, mounted on slides were deparaffinized andincubated with 20 μg/mL proteinase K at 37˚C for 15 min, thenimmersed in 3% hydrogen peroxide and incubated with terminal

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Figure 1. Anti-proliferative effects of HCA and BCA in oral cancer cells. SCC-15 (A) and HEp-2 (B) cells were treated with HCA or BCA for 24 hor vehicle (0.1% DMSO) alone. Cell viability was determined by MTT assay. Means±SD of separate experiments.

deoxynucleotidyl transferase containing reaction buffer at 37˚C for1 h. Finally, the sections were incubated with peroxidase-conjugatedanti-digoxigenin antibody for 30 min, after which the reactionproducts were visualized with 0.03% DAB solution containing 2 mM/L hydrogen peroxide. Counterstaining was performed using0.5% methyl green.

Statistical analysis. The differences in mean values among groupswere evaluated and the values were expressed as the means±SD.All the statistical calculations were conducted using MicrosoftExcel.

Results

Effect of HCA on cell proliferation and the number of cellsin the sub-G1-phase. As shown in Figure 1, HCA stronglyinhibited the growth of SCC-15 and HEp-2 cells in a dose-dependent manner with IC50 values of 20.2 and 40.5 μmol/L,respectively. BCA, one of the HCA derivatives, also showedsimilar growth inhibitory activity against these two cell lines.

Because the growth-inhibitory effects of BCA did notdiffer from those of HCA, the following experiments were

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Figure 2. Effect of HCA on cell cycle distribution. SCC-15 (A) or HEp-2 (B) cells were treated with HCA for 48 h, stained with propidium iodideand subjected to FACScalibur analysis. (C) Percentage of cells in each cycle phase.

performed using HCA. The cell cycle distribution wasanalyzed under the HCA-induced growth inhibitoryconditions using flow cytometry. When treated with HCA for48 h, the SCC-15 cells were arrested in the G2/M-phasedepending on the HCA concentrations (Figures 2A and 2C).Specifically, the proportion of cells arrested in the G2/M-phase increased from 27.97% in the untreated control cellsto 40.88% in the 30 μM HCA-treated SCC-15 cells. Moreimportantly, the number of cells in the sub-G1 phase(apoptotic cells) also increased markedly, from 5.41% in theuntreated control cells to 21.97% in the 30 μM HCA-treatedSCC-15 cells (Figure 2A and 2C). The number of HEp-2cells arrested in the Sub-G1 phase also increased from 3.64%to 22.32% on HCA treatment (Figure 2B and 2C).

Effect of HCA on apoptosis. Because HCA treatmentincreased the population of apoptotic cells, the mechanism ofapoptosis induction was investigated. The SCC-15 or HEp-2cells were treated with various concentrations of HCA orBCA for various times. The levels of cleaved PARP and

cleaved caspase-3 in the total cell lysates were determined bywestern blot analysis. As shown in Figure 3, treatment withHCA led to increased levels of cleaved PARP and caspase-3in a time- and dose-dependent manner. Treatment with BCAalso led to increased levels of PARP and caspase-3 cleavagein the oral carcinoma cells (Figure 3).

Effect of HCA on the growth of oral tumors in vivo. Both thegross appearance and bioluminescence imaging showedsignificant inhibition of tumor growth after 10 days oftreatment with HCA (Figures 4A, 4B and 4C). Tumor masseswere reduced to 23.9±8.8% upon HCA treatment comparedto the controls (Figure 4D) (p<0.05). Furthermore, nodifference in the body weight loss between groups treatedwith HCA and the vehicle controls was evident for 14 days(data not shown).

Histologically, the oral tumors were anaplasticundifferentiated carcinoma with characteristic mitotic figures,multifocal necrosis and hemorrhage. However, HCA treatmentled to extensive tumor cell death surrounded by granulomatous

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Figure 3. Effect of HCA on induction of apoptosis. SCC-15 or HEp-2 cells were treated with 30 μM HCA for the indicated time periods (A), or theindicated concentrations of HCA or BCA for 24 h (B). Cleaved PARP and cleaved caspase-3 in total cell lysates were determined by Western blotanalysis. Actin was used as loading control.

inflammation with calcification (Figure 5, top panel). Inaddition, the immunohistochemistry of the tumor cellsrevealed that nearly all the cells in the control tumors werePCNA positive, which suggested a high proliferation rate.However, treatment of the tumor with HCA resulted in anotably decreased number of PCNA-positive cells (Figure 5,middle panel). Conversely, the number of TUNEL-positivecells increased greatly in response to HCA treatment, whichsuggested that HCA inhibited tumor growth by inhibiting cellproliferation and activating apoptosis (Figure 5, bottom panel).

Discussion

Previous study showed that HCA induced apoptosis inhuman colon and breast cancer cells through the elevation ofreactive oxygen species (ROS), caspase-3 activation, anddegradation of PARP (15). It was also reported that HCA-induced apoptosis was associated with proteasome inhibitionthat led to the increase of ER stress and mitochondrialperturbation, which are representative events associated withapoptosis induced by proteasome inhibitors (16). In thisstudy, HCA showed strong anti-proliferative activity towardthe human oral squamous cell carcinoma cell lines, SCC-15and HEp-2 by inducing cell cycle arrest at the G2/M phaseand apoptosis (Figures 2 and 3). Furthermore, direct

intratumoral injection of HCA successfully inhibited growthof tumor mass via inhibition of cell proliferation andinduction of apoptosis (Figures 4 and 5).

As a part of preclinical evaluations, plasma pharmaco-kinetics and metabolism of BCA have been characterizedrecently (17). BCA was not detected in the plasma because ofits rapid conversion to HCA. In addition, HCA was convertedto its inactive metabolite o-coumaric acid (t1/2=2.1±0.1) in aquantitative manner. The biotransformation of HCA to o-coumaric acid is probably mediated by aldehyde oxidase.Therefore, the biotransformation process of HCA could bepartially inhibited in the hypoxic conditions of a solid tumor(18). Solid tumors are inherently resistant to some types ofchemotherapy due to hypoxia and to target this resistantpopulation, bioreductive drugs that are preferentially toxic tothe tumor cells in a hypoxic environment are being evaluated inclinical trials (19). HCA, from this point of view, wouldtherefore be an effective and valuable antitumor agent for oralcancer treatment, since oral cancer is easily accessible forintratumoral injection. Furthermore, the hypoxic environmentwould become more pronounced upon HCA treatment, becauseof its anti-angiogenic effect (4).

In general, proteasome inhibitors induce apoptosis incancer cells that are resistant to apoptosis due to mutations insome components of the apoptotic machinery, such as p53(20, 21). The SCC-15 and HEp-2 cells used in this studycarried p53 mutations, which suggest that HCA induces p53-independent apoptosis in cancer cells. According to our

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Figure 4. Antitumor effect of HCA in an oral tumor model. A: Grossappearance. B: in vivo bioluminescence image. C: Excised tumormasses on day 14. D: Tumor volume mean±SD (n=5).

Figure 5. Histological and immunohistochemical appearance of tumorsfrom RK3E-ras-Fluc cell-injected rats. H & E staining (top panel),immunohistochemical staining for PCNA (middle panel) and TUNELassay (bottom panel) of sections from the tumors.

unpublished data, HCA induces potent apoptosis in cancercells, while normal cells show much less sensitivity to HCA.The mechanisms for the different responses of HCA innormal cells and tumor cells are now under investigation.

HCA shows antitumor activity in SCC-15 and HEp-2 oralcarcinoma cells and inhibits cell proliferation by inducingcell cycle arrest and apoptosis. More importantly, HCAeffectively arrests tumor growth by inhibiting cellproliferation and inducing apoptosis in a rat oral tumormodel. These results suggest the potential value of HCA as acandidate for oral cancer therapies.

Acknowledgements

Su-Hyung Hong was supported by BioMedical Research Institutegrant, Kyungpook National University Hospital (2007).

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Received August 27, 2009Accepted December 11, 2009

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