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Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect. However, chemoprevention is a plausible strategy and we had previously demonstrated that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) significantly inhibited tongue tumors-induced by the synthetic 4-nitroquinoline-N-oxide (not present in tobacco smoke). In this study, we demonstrated that p-XSC is capable of inhibiting B[a]P-DNA adduct formation, cell proliferation, cyclin D1 expression in human oral cells in vitro. In addition, we showed that dietary p-XSC inhibits B[a]P-DNA adduct formation, cell proliferation and cyclin D1 protein expression in the mouse tongue in vivo. The results of this study are encouraging to further evaluate the chemopreventive efficacy of p-XSC initially against B[a]P-induced tongue tumors in mice and ultimately in the clinic.
Tobacco smoking and alcohol consumption are major etiological factors in the development of oral cancer; these agents are responsible for about 75% of oral cancer in the USA . In the USA, over 30,000 cases and about 8,000 deaths from oral cancer occur annually . Oral cancer constitutes up to 25% of all types of cancer in developing countries, especially in southern Asia (e.g. India) as well as in China and certain regions of Brazil where the highest rates of oral cancer are observed [3; 4]. The oral cancer incidence and mortality rates have been increasing in the USA, Japan, Germany, and Scotland, especially among young males [3; 4]. Histologically, the most frequent cancers of the oral cavity are squamous cell carcinomas. Oral cancer is generally treated with surgery; however, surgery usually involves high degree of morbidity and high recurrence rates and does not address the development of multifocal neoplastic lesions in the entire epithelial field at risk . Prevention is obviously preferential to treatment, and preventive strategies can be tailored to the status of the individuals. An obvious means of preventing this disease would be to remove the etiological factors; however, this strategy is very difficult to implement due to the addictive power of etiological agents (tobacco products and alcohol).
The use of naturally-occurring or synthetic agents that can inhibit the appearance of clinically detectable tumors is an attractive and plausible approach to the prevention of cancer . Indeed, some success has been obtained with retinoids in the treatment of premalignant lesions of the oral cavity . An important goal in the development of cancer prevention strategies is the identification of molecular pathways by which normal cells progress to the first definable stage of cancer. Preclinical animal models of oral cancer can provide important mechanistic insights into the role of cancer chemopreventive agents [8; 9]. The induction of oral cancer in laboratory animals by the synthetic carcinogens 4-nitroquinoline-N-oxide (4-NQO) in rats and mice, and 7,12-dimethylbenz[a]anthracene (DMBA) in hamster have served as in vivo models to examine the etiology of the disease and to identify agents that suppress oral carcinogenesis . However, neither 4-NQO nor DMBA is found in the human environment. While it is not clear which compounds in tobacco and tobacco smoke are involved in the oral cancer development in smokers, polycyclic aromatic hydrocarbone (PAH) and tobacco-specific nitrosamine (TSNA) are recognized as potential etiological agents .
Epidemiologic, and preclinical studies, and some clinical intervention trials indicate that selenium-containing compounds is an important class of chemopreventive agents. Studies in our laboratories indicated that the organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC) is a highly effective chemopreventive agent in animal models including oral cancer . We showed that p-XSC, administered during the initiation or post-initiation phases of carcinogenesis, significantly reduced the incidence of carcinoma in the tongue of rats treated with 4-NQO . Recently, several potential mechanisms by which p-XSC inhibited 4-NQO-induced tongue tumors in the rats were proposed based on the effects of p-XSC in inhibiting DNA damage, mutagenesis, and p53 protein expression . However, to date, there are no studies of the effectiveness of this compound against oral carcinogenesis induced by a tobacco smoke relevant carcinogen. In a previous report, Culp et al.  showed that the human carcinogen, B[a]P (100 ppm in the diet, for two years) produced a 48% incidence in tongue tumors in B6C3F1 mice. In the present investigation, using a short-term B[a]P-induced bioassay in mice, and human oral cancer cells, we examined the effects of p-XSC on B[a]P-DNA adduct formation, cell proliferation, induction of apoptosis, expression of PCNA and cyclin D1. The results of the present investigation suggest that p-XSC is a promising chemopreventive candidate against the development of tongue tumors induced by B[a]P in mice.
B[a]P was obtained commercially (Sigma Chemicaal Co.), 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and N2-(7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl)-2′-deoxyguanosine (N2-BPDE-dG) were obtained from National Cancer Institute Chemical Carcinogen Reference Standard Repository at the Midwest Research Institute, Kansas City, MO. p-XSC was synthesized as reported previously .
The human oral squamous cell carcinoma SCC 1483 cells were maintained in a 1:1 mixture of DMEM/F12 supplemented with 10% FBS and penicillin/streptomycin (50 μg/mL). The MSK-Leuk1 cell line was established from a dysplastic leukoplakia lesion adjacent to a squamous cell carcinoma of the tongue . Squamous cell carcinoma SCC1483 or oral keratinocyte MSK Leuk1 cells (10,000/well) were seeded and grown in a 96-well plate for 24 h, and then treated with different doses of p-XSC for 24 h. Methylthiazoletetrazolium (MTT) (50 μg/100 μl) was added to the cells in each well for 4 h at 37°C. MTT was aspirated and 100 μl of DMSO was added to each well and absorbance at 570 nm was measured to quantify the number of surviving cells.
A cell death detection enzyme-linked immunosorbent assay kit (Roche Diagnostics, Indianapolis, IN) was used to quantitatively determine cytoplasmic histone-associated DNA oligonucleosome fragments associated with apoptotic cell death according to the manufacturer's manual. Briefly, after cells were lysed, and incubated for 30 min at room temperature, 20 μl of supernatant was transferred into the streptavidin-coated microtiter plate, and 80 μl of the immunoreagent was added to each well. After incubation at room temperature for 2 h, the solution was decanted, and each well was rinsed three times with incubation buffer. Color development was carried out by adding 100 μl of ABTS solution, and absorbency was measured at 405 nm in a microtiter plate reader against ABTS solution as a blank.
SCC1483 cells treated with various doses of p-XSC (2.5, 5 and 10 μM) for 1 hour, were harvested by scraping, and washed with phosphate buffered saline. Cellular proteins were isolated with cell lysis buffer purchased from Cell Signaling (Beverly, MA). Equal amounts of protein (30 mg) was taken, boiled for 5 min, electrophoresed on a 10% SDS-PAGE at 100 V for 110 min, then electro-transferred to a nitrocellulose membranes. Antibodies used for western blots were cyclin D1 and actin from Santa Cruz. Band expressions were developed using ECL reagents from Amersham, Piscataway, NJ, and analyzed by Quantity One Analysis Software v4.5.0 (Bio-Rad Laboratories, Richmond, CA)
Levels of BPDE-DNA adduct in SCC1483 cells that had been treated with B[a]P were measured by competitive ELISA using monoclonal antibody 5D11 according to a published method . Anti-BPDE-DNA adduct antibody 5D11 was obtained from Dr. Santella, Columbia University. Briefly, the genomic DNA isolated from cultured cells using the Qiagen genomic DNA isolation kit (Valencia, CA) was analyzed. Polyvinyl flexible flat bottom 96-well plates (BD Falcon, Franklin Lakes, NJ) were coated with 50 ng per well of the BPDE-DNA in 100 μL of 50 mM Tris-buffer (pH 7.5) at 4°C overnight. The plate was then washed with 200 μL of PBS containing 0.05% tween-20 and 0.02% NaN3 as washing buffer. Non-specific binding to the plate was minimized by incubating the plates with 200 μL of blocking buffer containing 1% FCS in washing buffer. BPDE-DNA standards and DNA samples (4 μg in 50μL/well) were mixed with 50 μL of 5D11 (1:100 dilution), and added into each duplicate well. Wells were incubated and washed before adding goat anti-mouse IgG alkaline phosphatase conjugate (100 μL, 1:100 dilution) , then the plate was incubated for 90 min at 37°C. The plate was washed and 200 μL p-nitrophenyl phosphate (1mg/mL in 1 M diethanolamine, pH 8.6) was then added. After incubation for 30 min, the color development at 405 nm was measured by microtiter plate reader.
Female 8-week old B6C3F1 mice were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). Mice were housed in an animal facility maintained on a 12-h light/dark cycle, at a constant temperature (22 ± 2°C) and relative humidity (55 ± 15%) controlled room. p-XSC was given to the animals in the diet (AIN-76A) at 5 and 20 ppm over the entire course of the experiment beginning two weeks prior to B[a]P exposure and during carcinogen exposure until termination. B[a]P in corn oil was administered in five doses over a two weeks period. Twenty-four hours after the last administration of B[a]P, the animals were sacrificed and their tongues were isolated. The tongue was sliced lengthwise; one half was used for DNA adduct analysis and the other half was used for PCNA and cyclin D1 examinations. DNA was isolated from the epithelial layer of the tongue by a modified Marmur method as described .
The epithelial cells from the tongue of mice were carefully removed and placed into phosphate-buffered saline, washed and fixed with 1.0% paraformaldehyde. The primary immunohistochemical staining of cells was analyzed for levels of PCNA and cyclin D1 (AbCam, Cambridge, MA, USA). A rabbit anti-mouse secondary antibody (Zymed; San Francisco, CA) with a fluoresceinated isothiocyanate conjugate (FITC) was used to detect the primary antibody. The primary antibody was added for 1 h and washed at least twice with 1× PBS and a secondary detection antibody was added and washed at least twice with 1× PBS. Cells were analyzed using a laser scanning cytometer purchased from CompuCyte (Cambridge, MA, USA). The fluorescence was detected at 480 nm using an argon laser. The laser scans the total slide area which is gated in the instrument.
Levels of B[a]P-DNA adduct in the tongue of mouse that had been treated with B[a]P was analyzed by the 32P-postlabeling assay . DNA isolated from tissue was hydrolyzed to nucleoside 3′-monophosphates with micrococcal endonuclease and spleen phosphodiesterase, followed by nuclease P1 treatment to convert only the unmodified nucleotides to nucleosides. The B[a]P-modified nucleotides were radiolabeled with [γ-32P] ATP and polynucleotide kinase. The 3′,5′-bisphosphates were separated by Macherey-Nagel polyethyleneimine cellulose TLC Plate (Alltech Associates, Deerfield, IL) using N2-BPDE-dG as standard, and exposed to Cronex X-ray film (Dupont, Wilmington, DE) for visualization of adducts.
We observed that p-XSC can inhibit cell survival in both squamous cell carcinoma SCC1483 cells and oral leukoplakia keratinocyte MSK Leuk1 cells in a dose dependent fashion (Fig. 1A, B). The IC50 for p-XSC is approximately 2.5 μM for both cell lines. The induction of apoptosis by p-XSC is shown (Fig. 1C) in SCC 1483 cells. Compared to control with no p-XSC added, increasing the concentration to 10 μM resulted in more than double the rates of apoptosis. The inhibitory effect of p-XSC on the expression of cyclin D1 was also observed in SCC 1483 cells. The level of cyclin D1 was found to be decreased after 1 hour treatment of cells with 2.5, 5 and 10 μM of p-XSC (Fig. 2); however, the effect was not dose-dependent.
To determine whether p-XSC could affect cell proliferation of the oral tissue in vivo, the expression of PCNA was analyzed by immunohistochemistry. p-XSC at both doses (5 and 20 ppm in diet) significantly inhibited the expression of PCNA (P<0.01, 2-tailed T-test) in B[a]P treated female B6C3F1 mice (Fig. 3A). The expression of cyclin D1 in the tongue of mice treated with B[a]P and p-XSC was also determined; p-XSC at both doses significantly inhibited the expression of cyclin D1 (P<0.01, 2-tailed t-test) (Fig. 3B), but the effect was not dose dependent.
In SCC 1483 cells, B[a]P treatment resulted in the formation of BPDE-DNA adducts in a dose dependent fashion. p-XSC treatment was found to decrease the level of BPDE-DNA adducts induced by B[a]P treatment (Fig.4A); however, the effects was not significant. Figs 4B-F show autoradiogram of a thin layer chromatogram of 32P-labeled nucleotides from DNA of tongues of mice treated with B[a]P. Consistent with our observations in SCC1483 cells, dietary p-XSC at 20 ppm (10 ppm as selenium) significantly inhibited the formation of N2-BPDE-dG adduct induced by B[a]P treatment in mouse tongue (P<0.05). No significant inhibition of adduct formation was observed at the lower dose of p-XSC (5 ppm). Figure 4F shows a representative quantitative comparison of the autoradiogram with and without 20 ppm p-XSC.
Detailed studies aimed at identification of critical biochemical and molecular markers during the progression from normal, hyperplasic lesions, through dysplasia and carcinoma  could lead to improved diagnostic techniques for cancer prevention. In this study we have demonstrated that p-XSC can inhibit many of these critical steps in cancer development in a relevant in vivo animal model using a carcinogen found in tobacco smoke.. We showed that following a short-term treatment of mice with B[a]P resulted in the formation of B[a]P-induced DNA adduct in mouse tongue. Our results appear to be consistent with those reported previously that B[a]P is capable of inducing DNA adducts in human oral epithelial SCC-9 cells .
Consistent with other model systems, we found that p-XSC decreased B[a]P-induced DNA adduct formation using competitive ELISA assay in vitro and 32P-postlabeling assay in vivo. In our previous studies, we found that total GST enzyme activity, as well as GST- π and GST-μ enzyme activities were significantly induced by p-XSC in both mouse lung and liver ; p-XSC also induced glutathione peroxidase activity in the mouse lung but not in the liver . Although similar studies in the mouse oral tissues have not been performed, we have shown that dietary p-XSC induced GST activity and inhibited cell proliferation in the tongue of F344 rats . Together, these results obtained in the liver, lung and tongue reinforce the suggestion that the induction of GST by p-XSC may, in part, account for the reduction of B[a]P-DNA adduct formation in both SCC 1483 cells and mouse tongue observed in the present investigation.
A major characteristic of cancer cells is uncontrolled cell growth which is due to the loss of cell cycle control. Additionally, cell proliferation (DNA replication) is necessary for the conversion of DNA damage to mutations. Overexpression of cyclin D1 has been reported in a variety of human tumors, including breast carcinomas, mantle cell lymphomas, and squamous cell carcinomas derived from the oral cavity, larynx, and esophagus as well as from other sites . Cyclin D1 was amplified in approximately 30% of HNSCC . Immunohistochemical analysis of cyclin D1 performed by Nakahara et al.  on 78 oral SCC, 46 leukoplakia, and 20 normal mucosa samples found that the overexpression of cyclin D1 was not observed in normal mucosa and was observed in 36 % of SCC. These results clearly indicate that alteration in gene expression varies according to the stage of disease. However, for cancer prevention of HNSCC, it is critically important to elucidate molecular changes in addition to cyclin D1 in well-defined and well-controlled animal models.
The effect of p-XSC on cell proliferation in the B[a]P-treated tongue of mice was assayed by PCNA as a proliferative marker, and cyclin D1 as a key factor in cell cycle control. Consistent with our previous study using 4-NQO in the rats ; we demonstrated that p-XSC can inhibit cell proliferation in cells in culture, and dietary p-XSC is also capable of inhibiting cell proliferation induced by B[a]P in the mouse tongue. Our results also demonstrate that p-XSC inhibits cyclin D1 protein expression, which may be, in part, responsible for its inhibitory effects on cell proliferation.
Recently, p-XSC was found to reduce the expression of NF-κB and several target genes regulated by NF-κB including cyclin D1 in non-small cell lung cancer cells (NSCLC) . Furthermore, we have shown that p-XSC can ihhibit the NF-κB-DNA binding in both oral squamous cell carcinoma (SCC 1483) and non-small cell lung cancer NCI-H460 ; and by using MALDI-TOF/TOF and computer modeling studies, we have demonstrated that the Cys62 residue in the active site of NF-κB (p50) protein was modified by the organoselenocyanates, (e.g. p-XSC, benzyl selenocyanate) through the formation of a selenium-sulfur bond, which would subsequently hinder binding of the DNA substrate . Control of cell growth and differentiation by transcriptional regulation of cyclin D1 via NF-κB has been reported by Guttridge et al . Based on these reports, p-XSC may inhibit the activation of NF-κB, and lead to down-regulation of cyclin D1 expression, thereby inhibiting cell proliferation.
In conclusion, the results of this study together with those reported in the literature by us and by others, suggest several potential mechanisms by which p-XSC would inhibit B[a]P-induced oral carcinogenesis. The mechanisms are based on the ability of the synthetic organoselenium compound of a thin layer chromatogram of 32P-labeled nucleotides from DNA of tongues of mice treated with B[a]P to inhibit one or more of the following processes: Inhibition of B[a]P-DNA adduct formation, inhibition of cell proliferation, and induction of apoptosis. Our results encourage future studies examine the chemopreventive activity of p-XSC against B[a]P induced tongue tumors in mice.
Grant Supuport: NCI grant R01-CA100924
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