SAllylcysteine inhibits tumour progression and the epithelial– mesenchymal transition in a mouse xenograft model of oral cancer

S-allylcysteine inhibits tumor progression and the epithelial–

mesenchymal transition in a mouse xenograft model of oral cancer

Man-Hui Pai

, Yueh-Hsiung Kuo

, En-Pei Isabel Chiang

and Feng-Yao Tang

*

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Department of Anatomy, Taipei Medical University, 11031 Taipei, Taiwan ;

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Tsuzuki Institute for Traditional Medicine, Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung 40402,

Taiwan;

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Department of Food Science and Biotechnology, National Chung-Hsing

University, 402, Taichung, Taiwan ;

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Biomedical Science Laboratory, Department of Nutrition, China Medical University, 40402 Taichung, Taiwan

Key Words: S-allylcysteine: osteopontin: vimentin: cyclooxygenase-2:

human oral cancer cells 1

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*

Corresponding author:

Dr. Feng-Yao Tang

Biomedical Science Laboratory Department of Nutrition

China Medical University 91 Hsueh-Shih Road, Taichung, 40402

Taiwan, Republic of China Telephone: (886-4) 22060643 Facsimile: (886-4) 22062891

E-mail: vincenttang@mail.cmu.edu.tw

Running Title: Inhibitory effect of SAC on human oral cancer

Financial support: This material is based upon work supported, in part, by the National Science Council grant, under agreement No. NSC-97-2320-B-039- 043-MY3 , Department of Health grant under agreement No. DOH 100-TD-B- 111-004 and DOH-100-TD-C-111-005, and China Medical University (CMU)

grant under agreement No. CMU98-P-08 and CMU98-P-08-M.

Abstract

Oral cancer is prevalent worldwide. Studies have indicated that an increase in the osteopontin (OPN) plasma level is correlated with the 1

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progression of oral cancer. Our previous report showed that the aqueous garlic extract S-allylcysteine (SAC) inhibited the epithelial- mesenchymal transition (EMT) of human oral cancer CAL-27 cells in vitro. Therefore, the present study investigated whether SAC consumption would help prevent tumor growth and progression, including the EMT, in a mouse xenograft model of oral cancer. The results demonstrated that SAC dose-dependently inhibited the growth of oral cancer in tumor-bearing mice. The

histopathological and immunohistochemical staining results indicated that SAC was able to effectively suppress tumor growth and progression of oral cancer in vivo. The chemopreventive effect of SAC was associated with suppression of carcinogenesis factors such as N-Methylpurine-DNA glycosylase (MPG) and OPN. SAC significantly suppressed the

phosphorylation of Akt, mTOR, IBand ERK 1/2 in tumor tissues. The results demonstrated that the SAC-mediated suppression of cyclin D1 protein was associated with an augmented expression of the cell cycle inhibitor p16

. Furthermore, SAC inhibited the expression of cyclooxygenase-2 (COX-2), Vimentin and nuclear factor- B (NF-B) p65 (RelA). These results show that SAC has potential as an agent against tumor growth and

progression in a mouse xenograft model of oral cancer.

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Introduction

Oral cancer is one of the most prevalent types of cancer in the world today

. It is well known that oral cancer is characterized by the aberrant proliferation and invasion of malignant cells into the underlying connective tissues

. Recent studies have suggested a strong correlation between the 1

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OPN plasma level and oral carcinogenesis

. OPN activates a number of different signaling pathways, thus exerting an effect on the migration, proliferation and survival of cancer cells

. For example, the

phosphatidylinositol -3-kinase (PI3K)/Akt/mTOR and MAPK/ ERK signaling

cascades play important roles in tumor growth and progression

. The Akt/mTOR proteins regulate cell-cycle progression, growth-factor- mediated survival and tumor cell growth

. Upon the activation of the PI3K signaling pathway, the NF-B inhibitor protein (IB) is phosphorylated by IB

kinase (IKK) and then subjected to ubiquitin-mediated degradation

. The degradation of IB permits the translocation of activated NF-B from

cytoplasm into the nucleus, where it upregulates COX-2 gene expression and thus triggers the progression of oral cancer

. The COX-2 protein is

responsible for the production of prostaglandins and tumor-associated

inflammation

. Several studies have reported the expression of NF-B and

COX-2 proteins is associated with treatment resistance in oral cancer

. Overactivated MAPK/ERK signaling pathways are reportedly involved in the accelerated cell cycle progression and proliferation of cancer cells

. During the proliferation of oral cancer cells, cell cycle-related proteins, such as cyclin D1 and proliferating cell nuclear antigen (PCNA), function as major 1

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regulators of cell-cycle progression and DNA replication, respectively

. Recent studies have indicated that the p16

protein may serve as a cell- cycle inhibitor and suppress the activity of the cyclin D1 and PCNA proteins

(16,17)

. Taken together, the PI3K/Akt / mTOR, MAPK/ERK and NF-B signaling

pathways play crucial roles in both OPN-mediated tumor growth and the poor

prognosis associated with oral cancer.

A recent study also indicated that COX-2 expression is associated with the EMT in various types of human cancer

. Many studies have indicated that EMT is a critical cellular mechanism which plays an important role in tumor progression and metastasis in many types of cancer, including oral cancer

. E-cadherin complexes are major constituents of the epithelial junctions in the normal oral epithelium

. The loss of E-cadherin and

augmented expression of Vimentin are considered to be key steps in the EMT and tumor progression

. However, suppression of the ERK1/2 and

PI3K/Akt/NF-B signaling cascades induces the mesenchymal-to-epithelial reverting transition along with increasing E-cadherin expression in cancer cells

. Therefore, these results suggest that the MAPK/ERK, PI3K/Akt/NF-

B signaling pathways and COX-2 are associated with EMT process in human oral cancer. Clinical studies have indicated that increases in the OPN 1

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plasma levels over time are significantly associated with poor patient survival

(25)

. Therefore, any therapeutic application or nutritional intervention that resulted in a suppression of the COX-2 or OPN proteins might be an effective

approach to the treatment of oral cancer

.

Studies have shown that MPG, a DNA repair enzyme that repairs N- alkylpurine damage, is upregulated in cancer cell lines

. Overexpression of this enzyme contributes to chromatid exchange, chromosomal aberration and genetic mutation, possibly due to incomplete excision repair

. Therefore, MPG is considered a promoter of carcinogenesis

. In the course of considering the role of DNA lesions in mutagenesis and carcinogenesis, we

also investigated MPG expression.

Epidemiological studies have suggested that the consumption of garlic extracts exert a protective effect against various types of cancer, including prostate, colon and oral cancers

. Garlic contains certain lipid-soluble and water-soluble anticancer constituents

. The lipid-soluble garlic constituents include diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS). The water-soluble garlic constituents include SAC and S-

allylmercaptocysteine (SAMC). SAC is abundant in aged garlic extract (AGE).

AGE is produced by the immersion and extraction of raw garlic in aqueous 1

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ethanol for more than 10 months at room temperature. During the process, most of the orangosulfur compounds are changed naturally into more stable and bioavailable water-soluble compounds. Studies have shown that the active ingredients in garlic (Allium sativum) extracts, including DADS and DATS, effectively inhibit the proliferation of cancer cells

. SAC also reportedly suppresses the growth of several types of cancer

. Our previous study showed that SAC inhibited the proliferation of human oral cancer CAL-27 cells in vitro

. Moreover, SAC reportedly prevents the EMT and suppresses tumor progression in human oral squamous cancer cells in vitro

. However, the in vivo inhibitory effects of SAC on tumor growth and progression in oral cancer have not been demonstrated. The current study was undertaken to evaluate the in vivo anti-cancer effects of SAC, including the inhibition of tumor growth and progression. Immunodeficient nude mice with xenografted human oral cancer CAL-27 cells under the skin comprised the experimental model. It is demonstrated that the consumption of SAC significantly inhibited both the tumor growth and progression of oral cancer in this mouse xenograft model.

Materials and methods 1

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Reagents and Antibodies

SAC was purchased from LKT laboratories, Inc. (St. Paul, MN) . The anti- phospho-Akt, Anti-phospho- mTOR, Anti-phospho-IkB, Anti-phospho-ERK 1/2, anti-E-cadherin, anti- p16

, anti-cyclin D1, anti-NF-B p65 (RelA), anti- Vimentin, and anti-COX-2 monoclonal antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Anti--actin antibody was

purchased from Sigma (St Louis, MO). Anti-MPG and anti-PCNA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The human oral cancer CAL-27 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM)/ F12 medium was purchased from Invitrogen Inc.

(Carlsbad , CA). Tissue nuclear extraction reagent and tissue lysis kits were purchased from Pierce Biotechnology Inc. (Rockford, IL). Phosphate buffered saline (PBS), fluorescein isothiocyanate (FITC) and Rhodamine were

purchased from Invitrogen Inc. (Carlsbad, CA). The OPN ELISA kit was purchased from R&D Systems, Inc. (Minneapolis, MN). SAC was dissolved in distilled water at a concentration of 400 mM and stored at -20

C. Immediately before the experiment, various concentrations of SAC solution were freshly prepared and given to the experimental animals.

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Cell Culture

Human oral cancer CAL-27 cells were cultured in a 37

C humidified incubator with 5% CO

and grown to confluency using fetal bovine serum (FBS)

supplemented DMEM/ F12 media. DMEM/ F12 medium was supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine and 1.5 g/L sodium bicarbonate in the absence of antibiotics.

Xenograft Implantation of Tumor Cells

Human oral cancer CAL-27 cells were maintained at 37

C in a 5% CO

incubator and grown to confluency using 10% FBS and 0.15% (w/v) sodium bicarbonate DMEM/ F12 media. To establish the mouse xenograft model, subconfluent cultures of oral cancer CAL-27 cells were given fresh medium 24h before being harvested by a brief treatment with 0.25% trypsin and 0.02%

ethylenediaminetetraacetic acid (EDTA). Trypsinization was stopped with medium containing 10% FBS, and the cells were washed twice and resuspended in serum-free medium. Only single-cell suspensions with a viability of > 90% were used for the injections.

Animals, Diet, and SAC Supplementation 1

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Adult (3-4 week old) BALB/C AnN-Foxn1 nude mice (22-25 g) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Mice were maintained under specific pathogen-free conditions in facilities approved by the National Laboratory Animal Center in accordance with current regulations and standards (animal protocol no. 97-5-D). During the entire experimental period, mice were fed a standard Lab 5010 diet purchased from LabDiet Inc.

(St. Louis, MO). The standard diet contained crude fat (13.5% total diet energy), protein (27.5% total diet energy ) and carbohydrate (59% total diet energy ), and had no detectable amounts of SAC, as indicated by the

supplier.

Mice which had been anesthetized with inhaled isofluorane were placed in a supine position. The mice were subcutaneously (s.c.) injected with ~ 1 million human oral cancer CAL-27 cells into the right flank of each BALB/C AnN- Foxn1 nude mouse. A well-localized bleb was considered to be a sign of a technically satisfactory injection. After the inoculation, mice were divided into three subgroups. SAC was dissolved in distilled water and given to

experimental animals by gavage once a day at a total volume 0.15 mL. One group (low dose SAC) received a daily oral consumption dose of SAC dissolved in distilled water at 5 mg / kg of body weight (BW) once per day.

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The other group (high dose SAC) received SAC at an oral dose of 40 mg / kg of BW once per day. The tumor control group did not receive any SAC

supplementation. To measure the OPN plasma levels, tumor-free mice were used as the normal control group. Both the normal control group and the tumor control group received only distilled water instead of treatment. Tumor volume was calculated by the formula: 0.524 L

(L

)

, where L

represents the long axis and L

represents the short axis of the tumor. Body weight was determined once weekly. At the end of the experimental period, animals were euthanized with CO

inhalation; tumor tissues were then excised, weighed,

and frozen for further experiments.

The remaining tissues of liver, lung, spleen, pancreas and intestine were also frozen immediately, sectioned and stained with Mayer’s hematoxylin –eosin (H&E) for light microscopy. Blood samples were collected from the heart in a 1-ml vacutainer tube containing heparin and centrifuged for 10 min at 1000g to obtain plasma.

Histopathological, Immunohistochemical and Immunofluorescent Staining of

Tumor Tissues

Frozen tumor tissues were cut in 5 m sections and immediately fixed with 1

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4% paraformaldehyde. Sections were stained with Mayer’s H&E for light microscopy. Negative controls did not exhibit any staining. In a blinded manner, three hot-spots were examined per tumor section (high power fields X 400) of six different tumors in each group. Images of tumor sections were acquired on an Olympus BX-51 microscope using an Olympus DP-71 digital

camera and imaging system.

For immunohistochemical staining, frozen tissue sections were treated with 0.3% hydrogen peroxide to block the endogenous peroxide activity. Non- specific protein bindings were blocked with 10% normal goat serum (NGS) for 1 hr followed by incubation with an anti-MPG primary antibody (1:300). Tissue sections were washed with 0.1M PBS and incubated with biotinyated

immunoglobin G (1:300 secondary antibody) at room temperature for 1hr.

Tissue sections were stained with Avidin-Biotin complex (ABC),

diaminobenzidine (DAB) and hydrogen peroxide. Cell nuclei were stained with hematoxilin. Imaging was performed at 200X and 400X magnification. Images of tumor sections were acquired on an Olympus BX-51 microscope using an

Olympus DP-71 digital camera and imaging system.

For immunofluorescent staining, primary oral cancer tissues were frozen, sectioned and subjected to anti-PCNA, anti-Vimentin, and anti-COX-2 1

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antibodies. The sectioned tissues which were probed with anti- PCNA antibody or anti-Vimentin antibody were further subjected to a secondary antibody with an anti-IgG conjugated FITC label. The sectioned tissues which were probed with anti- COX-2 antibody were further subjected to a secondary antibody with a Rhodamine label. Cell nuclei were stained with 4,6-Diamidino- 2-phenyindole (DAPI). Imaging was performed at 400X magnification. Images of the tumor sections were acquired on an Olympus BX-51 microscope using an Olympus DP-71 digital camera and imaging system.

Protein Extraction

Briefly, animal tissues were prepared using a Tissue Nuclear Extract Reagent Kit containing protease and phosphatase inhibitors according to the manufacturer’s instructions. After centrifugation for 10 minutes at 12000g to remove cell debris, the supernatants were further separated and retained as cytoplasmic and nuclear fraction extracts, respectively. Cross contamination between the nuclear and cytoplasmic fractions was not found (data not shown).

Western Blotting Analysis 1

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The cytoplasmic and nuclear fractions of tissue proteins (60g) were fractionated on 10% SDS-PAGE, transferred to a nitrocellulose membrane and blotted with an anti-phosphorylation Akt monoclonal antibody, according to the manufacturer’s instructions. The blots were stripped and reprobed with a -actin antibody as the loading control. The levels of phosphorylated mTOR, phosphorylated IB, phosphorylated ERK 1/2 and the total E-cadherin proteins in the tumor tissues were measured with the same procedure as described above. The levels of p16

, cyclin D1 and NF-B p65 (RelA) in the nuclear fractions of the tumor tissues were measured using a similar

procedure as described above. The blots were stripped and reprobed with a lamin A/C antibody as the loading control.

Detection of Plasma OPN by Enzyme-Linked Immunosorbent Assay (ELISA)

The OPN plasma level was measured by ELISA according to the

manufacturer’s instructions (R&D Systems). Briefly, a 100 L diluted plasma sample (1:100 dilution) from each group (tumor free mice, tumor control mice, Low_SAC mice and High_SAC mice) was added to each well and analyzed.

Upon completion of the ELISA process, the plate was read at 450/570 nm wavelength using a microplate reader.

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Statistical Analysis

Quantitative analysis was used to determine whether there were differences in the tumor weight or volume among the Low_SAC, High_SAC and tumor control groups (n=6 for each) in human oral cancer bearing mice. Statistical analyses of the differences in tumor weight or volume among the

experimental and control conditions were performed using SYSTAT software.

Confirmation of a difference in tumor weight or volume as statistically

significant required a rejection of the null hypothesis of no difference between the mean weight or volume obtained from the different sets of experimental and control groups at the p=0.05 level using one-way ANOVA. The

Bonferroni post hoc test was used to determine differences between the groups.

Results

SAC inhibited tumor growth and progression of oral cancer in a mouse xenograft model

Our previous study demonstrated that SAC inhibited the proliferation of human oral cancer CAL-27 cells in vitro. We therefore extended our

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the in vitro findings. The inhibitory effects of SAC on the growth of oral cancer cells in a mouse xenograft tumor model were investigated. A mouse xenograft tumor model was established by subcutaneously inoculating human oral cancer CAL-27 cells into the right flank of each nude mouse. The results showed that SAC consumption significantly suppressed the growth of oral cancer in tumor-bearing mice(p<0.05). By the end of the study (4th week), the tumor volume per mouse had decreased from 177 ± 18 mm

in the tumor control group to 126 ± 25 mm

and 47± 6 mm

in the 5 mg SAC/kg of BW (Low_SAC) and 40 mg SAC/kg of BW (High_SAC)- fed groups, respectively, accounting for a 30 % and 74 % inhibition in tumor growth (p<0.05) (Fig. 1A).

The tumor weight results at the end of the study further supported these findings. Compared with the tumor control group , which had a tumor weight of 0.28 ± 0.02 g/mouse, the Low_SAC and High_SAC- fed mice had 0.2 ± 0.01 g and 0.08 ± 0.02 g /mouse tumor weight, accounting for a 27% and 70%

decrease, respectively (p<0.05) (Fig. 1B). Both the Low_SAC and High_SAC doses significantly inhibited oral cancer tumor growth in this mouse xenograft

model (p<0.05).

Furthermore, SAC significantly inhibited the protein levels of PCNA in tumor- bearing mice (p<0.05) (Fig. 1C). The H&E staining results also demonstrated 1

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that SAC inhibited tumor progression in this mouse xenograft model (Fig. 1D).

Immunohistochemical staining showed that SAC suppressed the nuclear levels of MPG in tumor tissues (Fig. 1E; b,c vs. a & e,f vs. d). Compared to the tumor control group, SAC at both the low and high dose suppressed tumor tissue MPG levels by up to 34% and 71%, respectively. These results

demonstrated that SAC significantly suppressed both tumor growth and progression (p<0.05), providing evidence for the chemopreventive effects of SAC. The mechanism of action appears to be associated with the

suppression of nuclear PCNA and MPG.

SAC decreased the OPN plasma level in tumor- bearing mice

To further investigate the chemopreventive effects of SAC on tumor

progression in oral cancer, we measured the OPN plasma level using ELISA analysis. As shown in Fig. 2, mice inoculated with oral cancer CAL-27 cells had high plasma levels of OPN. However, SAC at either a low dose (5 mg/kg of BW) or a high dose (40 mg/kg of BW) significantly decreased the plasma OPN protein level (p<0.05). By the end of the study, the basal OPN plasma levels in tumor-free mice were approximately 21 ± 5 ng/mL. The OPN plasma levels decreased from 115 ± 8 ng/mL in the tumor control group to 84 ± 3 1

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ng/mL and 43 ± 5 ng/mL in the Low_SAC and High_SAC –fed groups,

respectively. These results suggested that the chemopreventive effect of SAC are associated with decreases in the OPN plasma level in tumor-bearing mice.

SAC inhibited the PI3K/Akt and MAPK/ERK signaling pathways in tumor- bearing mice

To further validate the importance of SAC, we analyzed the inhibitory effects of SAC on different elements of the PI3K/Akt/mTOR and MAPK/ERK signaling pathways. As shown in Fig. 3A, increases in the phosphorylation levels of Akt and mTOR proteins were widely observed in tumor tissues. Moreover, SAC at either of the two different doses (5 and 40 mg/kg of BW) significantly

decreased the phosphorylation levels of Akt and mTOR proteins in a dose- dependent manner (Fig. 3B). SAC also significantly suppress the

phosphorylation level of the IB protein in tumor tissues (Fig. 3A). These quantitative results suggested that the consumption of SAC was able to inhibit the activation of the Akt/mTOR signaling pathways and NF-B in this mouse xenograft tumor model (Fig. 3B).

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In the current study, we further investigated whether SAC consumption would suppress MAPK/ERK signaling pathway activation in a mouse xenograft model. As shown in Fig. 3C, SAC (at a dose of 5 or 40 mg/kg of BW)

significantly inhibited the phosphorylation levels of ERK1/2 proteins in tumor- bearing mice. Moreover, SAC induced the expression of E-cadherin proteins in tumor tissues. These results show that the inhibitory effects of SAC on tumor growth and progression, including the EMT, are associated with a suppression of the MAPK/ERK signalling pathway in tumor-bearing mice (Fig.

3D). Together, these observations strongly suggest that SAC impairs PI3K/Akt/mTOR and MAPK/ERK pathways as broad effects in oral cancer CAL-27 cells in a mouse xenograft model.

SAC significantly suppressed the expression of cyclin D1 and NF- B in the

mouse xenograft tumor model

Previous investigation had indicated that the cyclin D1 protein plays an important role in the regulation of cell proliferation. Moreover, the NF-B mediated expression of COX-2 is strongly correlated with tumor progression.

Therefore, we investigated whether SAC would suppress the expression of the cyclinD1 proteins in vivo. As shown in Fig 4A, SAC significantly induced 1

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the expression of the cell cycle inhibitor p16

, and suppressed the nuclear levels of the cyclin D1 and NF-B p65 (RelA) proteins in tumor-bearing mice.

The quantitative results demonstrated that SAC (at 5 and 40 mg/kg of BW ) even suppressed the expression of NF-B by up to 60 % and 80%,

respectively (Fig. 4B). These results suggest that the inhibitory effects of SAC on tumor growth and progression in oral cancer were associated with the suppression of cyclin D1 and NF-B p65 (RelA). Therefore, SAC has potential as a chemopreventive agent for tumor growth and progression in oral cancer, as demonstrated here in this mouse xenograft tumor model.

Immunofluorescent staining indicated that SAC significantly blocked the EMT step and inflammation in oral cancer in these tumor-bearing mice

Due to the important roles of the MAPK/ERK and PI3K/Akt/NF-B signaling pathways in tumor progression and EMT, we examined the inhibitory effects of SAC on the expression of biomarkers such as Vimentin and COX-2 in a mouse xenograft tumor model. The immunofluorescent staining results showed that SAC significantly inhibited Vimentin expression in tumor-bearing mice (Fig 5A). SAC also significantly suppressed the expression of COX-2 at 1

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Vimentin and COX-2 indicated both the induction of EMT and inflammation in the tumor tissues. These results show that SAC minimized the inflammatory response and prevented EMT progression in tumor-bearing mice (Fig 5B).

Discussion

Many studies have suggested that the phytochemicals in fruits and vegetables might exert anti-cancer effects. The consumption of garlic has been associated with a reduced risk in the occurrence of cancer at different sites, including the liver, breast and colon

. A previous study indicated that administration of SAC increased the levels of both reduced glutathione and glutathione-dependent enzymes

. Our previous investigation

demonstrated that SAC inhibited the cell proliferation and EMT of human oral cancer cells in vitro

. We also demonstrated that SAC consumption inhibited the tumor growth of human non-small-cell lung carcinoma independent of its antioxidant activities

. SAC, a water soluble garlic constituent, is

characterized by its high bioavailability and is found in high concentrations in

AGE

. AGE contains much higher levels of antioxidants than raw or

cooked garlic. The present study was performed to determine whether SAC would suppress the tumor progression and EMT process.

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As shown in Fig. 1, the results show that SAC at 5 mg/kg of BW (low dosage) and 40 mg/kg of BW (high dosage) dose-dependently inhibited the growth of oral cancer in tumor- bearing mice without any apparent untoward toxicity (data not shown). SAC is therefore safe at the studied doses. There was no difference in food intake or body weight in the present study between the animal groups. A previous study showed that there were no toxic

symptoms at SAC doses between 250 and 2000 mg/kg of body weight. The H&E staining results suggested that SAC blocks oral cancer tumor

progression in tumor-bearing mice. Previous studies demonstrated that PCNA is a trimeric complex with an essential role in DNA replication. PCNA makes up the platform required for the activity of the DNA polymerases  and  during DNA replication. The present study shows that SAC also inhibits the expression of PCNA in tumor tissues. Immunofluorescent staining indicates that SAC is able to effectively block the proliferation of human oral cancer cells in nude mice. The results suggest that SAC inhibits tumor growth

through a suppression of DNA replication.

N-Methylpurine-DNA glycosylase (MPG), a carcinogenesis promoter, has been shown to be upregulated in several cancer cell lines

. Overexpression of this enzyme was found to contribute to the formation of chromatid

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exchanges, chromosomal aberration, and gene mutations

. In considering the role of MPG in carcinogenesis, we also investigated the expression of MPG protein in tumor tissues. Interestingly, our results demonstrated that MPG proteins were highly expressed in the nuclei of the tumor tissues in the tumor control group. However, consumption of SAC at both of the low and high doses prevented the nuclear translocalization of MPG proteins. Most of the MPG proteins were diminished in the nuclei and retained in the cytoplasm of the tumor tissues in both the Low_SAC and High_SAC –fed groups (Fig.

1E).

These results comprise novel evidence of chemopreventive effects and demonstrate that SAC is able to suppress the nuclear levels of MPG proteins in tumor tissues. Investigation into the effects of SAC on the formation of chromatid exchanges and chromosomal aberration will be undertaken in the near future. Previous study indicated that an overexpression of OPN is associated with certain activities related to tumor growth and progression, including angiogenesis, invasion and metastasis in oral cancer. The expression of OPN (a prognostic biomarker of human oral cancer) is

consistently associated with transformed epithelium in pre-malignant lesions and invasive squamous cell carcinoma. Secretion of OPN into the local tumor 1

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microenvironment may promote tumor cell proliferation, migration and angiogenesis by binding cell surface receptors such as the v3 integrin proteins. The results here show that SAC suppressed the expression and secretion of OPN in this mouse xenograft tumor model (Fig. 2). This indicates that the inhibitory effects of SAC on tumor progression of oral cancer were significantly associated with the suppression of OPN expression and decreases in nuclear MPG proteins in this mouse xenograft tumor model.

There is thus a potentially beneficial role of SAC in the chemoprevention of

oral cancer.

Many studies have demonstrated that the PI3K /Akt/mTOR signalling pathway plays an important role in the regulation of tumor growth and progression oral cancer

. The activated Akt/mTOR signaling pathway has been observed in oral cancer cells and an essential role suggested in the control of gene expression and protein translation, which exerts an impact on cell proliferation and inflammation during tumor development. Previous studies indicated that there was a PI3K- dependent phosphorylation of Akt or NF-B molecules from among a panel of oral cancer cell lines, which effect was correlated with resistance to therapy

. The mTOR inhibitor Rapamycin was shown to prevent tumorigenesis and to render oral cancer cell lines with 1

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highly activated Akt/mTOR more responsive to growth inhibition

. Collectively, the evidence shows a significant role of the activated

PI3K/Akt/mTOR pathway in oral cancer. In the present study, SAC effectively inhibited the phosphorylation of Akt, mTOR and I-B proteins (Fig. 3A). SAC, even at a low dosage (5 mg/kg BW), and thereby inhibited the activation of ERK 1/2 signaling pathway in tumor tissues (Fig. 3C). Moreover, SAC

consumption induced the expression of E-cadherin proteins in tumor tissues.

To examine whether SAC suppressed tumor growth and progression in oral cancer, we investigated the effects of SAC on the expression of cyclin D1 and NF-B. Our results demonstrated that SAC significantly suppressed the expression of the cyclin D1 and NF-B proteins in tumor tissues. Furthermore, we investigated the effects of SAC on the expression of the cell cycle inhibitor p16

. The consumption of SAC induced the expression of p16

(Fig. 4), which it was taken to be related to the finding that SAC inhibited tumor growth

and progression in tumor-bearing mice.

During the course of tumor progression, oral carcinoma cells typically lose their cell-cell adhesion capacity and become detached from neighboring cells. The poor prognosis of oral cancer is frequently associated with this detachment along with inflammation and the EMT . The inflammatory 1

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response is characterized by increased COX-2 activity and prostaglandin expression. EMT progression features the loss of E-cadherin and the overexpression of Vimentin. We previously demonstrated that SAC

consumption helped augment the expression of E-cadherin proteins

. To further investigate the effects of SAC on tumor progression and the EMT in oral cancer, we further identified the effects of SAC on the expression of COX-2 and Vimentin. To investigate the correlation between inflammation and EMT progression in the mouse xenograft model, we determined the

expression of these biomarkers using immunofluorescent staining. As shown in Fig. 5, SAC effectively suppressed the expression of Vimentin and COX-2 in tumor-bearing mice. The results also demonstrated a co-ordination

between inflammation and the EMT biomarkers. These results suggest a strong correlation between inflammation and EMT occurring in the oral carcinoma which developed in this mouse xenograft model. Consumption of SAC inhibited inflammation and tumor progression, including the EMT. The results from the current study are consistent with our previously reported

findings

.

In the present study, SAC consumption (at concentrations of 5 and 40 mg/ kg of BW per day) inhibited the activation of PI3K/Akt/mTOR and 1

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MAPK/ERK signaling pathways and suppressed the expression of the cyclin D1 and NF-B p65 (Rel A) proteins. Furthermore, SAC inhibited the

expression of the Vimentin and COX-2 proteins in tumor-bearing mice. In conclusion, SAC significantly suppressed oral cancer tumor growth and progression, including the EMT step, in this mouse xenograft tumor model of oral cancer. To the best of our knowledge, no in vivo experimental evidence has been previously reported regarding SAC-mediated suppression of tumor growth and progression, including the EMT. This is the first in vivo evidence for the chemopreventive effects of SAC.

Figure Legends

Figure 1.

SAC inhibited tumor growth and progression of oral cancer in a mouse

xenograft model

(A) Xenograft nude mice (n=6 for each group) were divided into three groups (tumor, tumor with low SAC, tumor with high SAC) and given SAC (0, 5 and 40 mg/kg of BW/day) for 4 weeks. The extent of tumor growth was evaluated by measurement of the tumor volume. The data on the tumor volume

represent the proliferation index in primary tumor tissues. At each time point, 1

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different letters are used to represent a statistically significant difference, i.e.

p<0.05.

(B) Data represent the change in tumor weight among the control, Low_SAC (5 mg/kg of BW/day) and High_SAC (40 mg/kg of BW/day) groups. The letters

represent a statistically significant difference, p<0.05. (p<0.05 at week 4).

(C) Samples from the different groups of oral cancer tissue were frozen, sectioned and subjected to anti-PCNA antibody by the immunofluorescent staining described in Material and Methods. Imaging was documented at 400X magnification. The green fluorescence area represented the distribution of the PCNA protein in CAL-27 cells stained with monoclonal antibody. The blue fluorescence area represented the location of cell nuclei stained with DAPI. The mean integrated fluorescence of the PCNA protein is shown in the bottom panel. The letters represent a statistically significant difference, i.e.

p<0.05. The results presented are representative of six different experiments.

(D) The oral cancer tissues were formalin-fixed, embedded in paraffin,

sectioned, and subjected to H&E staining. Imaging was documented at 400X magnification. The blue spots indicated with the green arrows represent the nuclei stained with hematoxylin. The red spots are the cytoplasm stained with eosin.

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antibody by the immunohistochemical staining described in Material and Methods. Imaging was performed at 200X (a-c) and 400X (d-f) magnification.

The dark brown spots indicated with the green arrows represent the distribution pattern and levels of the MPG proteins in the oral cancer cells stained with a monoclonal antibody. The blue area indicates the location of the cell nuclei stained with hematoxylin. The mean integrated MPG protein intensities are shown in the bottom panel. The letters represent a statistically significant difference at p<0.05.

Figure 2.

SAC decreased the OPN plasma level in tumor- bearing mice

The plasma levels of OPN were quantified with an ELISA Kit (R&D systems).

Briefly, an equal amount of a diluted plasma sample (100 L) from each group (tumor free mice, tumor control mice, Low_SAC mice and High_SAC mice) was added to each well and reacted with the primary antibody against OPN according to the manufacturer’s instructions. Upon completion of the ELISA process, fluorescence intensities were read using a wavelength of 450/570 nm. These results are representative of six different experiments. The letters represent statistically significant difference at p<0.05.

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Figure 3.

SAC inhibited the PI3K/Akt and MAPK/ERK signaling pathways in tumor-

bearing mice

(A) The preparation of the cell lysates from animal tissues was briefly

described in the Materials and Methods Section. Cell lysates were blotted with anti-phosphorylation Akt, anti-phosphorylation mTOR and anti- phosphorylation IB monoclonal antibodies. The levels of detection in the cell lysate represent the amount of the phosphorylated Akt, mTOR and IB proteins in the tumor tissues. The blots were stripped and reprobed with anti--actin antibody as the loading control. The results are representative of six different experiments. The immunoreactive bands 1

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are noted with the arrow.

(B) The integrated densities of the p-Akt, p-mTOR and p-I  B proteins adjusted with the internal control protein (-actin) are shown in the panel.

The letters represent a statistically significant difference at p<0.05.

(C) Cell lysates were blotted with anti-phosphorylation ERK 1/2 and anti-E- cadherin monoclonal antibodies, as described in Materials and Methods. The detection levels in the cell lysate represent the amount of the phosphorylated ERK 1/2 proteins and total E-cadherin proteins in the human oral cancer cells.

The blots were stripped and reprobed with an anti--actin antibody as the loading control. The results are representative of six different experiments.

The immunoreactive bands are noted with the arrow.

(D) The integrated densities of the p- ERK 1/2 proteins and total E-cadherin proteins adjusted with the internal control protein (-actin) are shown in the panel. The letters represent a statistically significant difference at p<0.05.

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Figure 4.

SAC significantly suppressed the expression of cyclin D1 and NF-B in

the mouse xenograft tumor model

(A) Preparation of the nuclear fraction cell lysates from animal tissues was briefly described in the Materials and Methods Section. Nuclear lysates were blotted with anti- p16

, anti-cyclin D1 and anti-NF-B p65 (Rel A)

monoclonal antibodies, as described in Materials and Methods. The levels of detection in the cell lysate represented the amount of p16

, cyclin D1 and NF-B p65 (Rel A) in the tumor tissues. The blots were stripped and reprobed with anti-lamin A/C antibody as the loading control. The results presented are representative of six different experiments. The immunoreactive bands are noted with the arrow. (B) The integrated densities of the p16

, cyclin D1 and NF-B p65 (Rel A) proteins adjusted with the internal control protein (lamin 1

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A/C) are shown in the panel. The letters represent a statistically significant difference at p<0.05.

Figure 5.

Immunofluorescent staining indicated that SAC significantly blocked the EMT step and inflammation in oral cancer in these tumor-bearing mice

(A) Tumor tissues were frozen, sectioned, and subjected to anti-Vimentin and anti-COX2 antibodies by immunofluorescent staining, as described in

Materials and Methods. Imaging was performed at 400X magnification. The green fluorescence area indicated with the yellow arrows represents the distribution of the Vimentin protein in CAL-27 cells stained with the

monoclonal antibody. The red fluorescence area indicated with the green arrows represents the COX-2 protein in CAL-27 cells stained with monoclonal antibody. The yellow fluorescence area indicated with the orange arrows in the merged imaging represents the co-localization of the Vimentin and COX-2 proteins in the tumor tissues. The blue fluorescence area represents the 1

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location of the cell nuclei stained with DAPI. These results are representative

of six different experiments.

(B) The mean integrated fluorescence of Vimentin and COX-2 is shown. The letters indicate a statistically significant difference at p<0.05.

Acknowledgements

This material is based upon work supported, in part, by the National Science Council grant, under agreement No. NSC-97-2320-B-039-043-MY3,

Department of Health grant under agreement No. DOH 100-TD-B-111-004 and DOH-100-TD-C-111-005, and China Medical University (CMU) grant under agreement No. CMU98-P-08 and CMU98-P-08-M. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the National Science Council, Department of Health, and China Medical University. Dr.

M.H. Pai conducted part of the research. Dr. Y.H. Kuo provided assistance for analytical chemistry. Dr. E.P. Chiang performed data analysis for this study.

Dr. F.Y. Tang designed the experiment, conducted part of the research work, analyzed data and prepared the manuscript for this research project. The authors state that they have no conflicts of interest.

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