Oral Leukoplakia Cell Research Articles

Abstract LB-171: Potential chemopreventative treatments in Fanconi anemia related HNSCC

Abstract Fanconi anemia (FA) is a DNA repair mechanism genetic disorder which leads to bone marrow failure and cancer. Risk of solid tumor malignancy in FA patients occurs decades earlier and at a rate several hundred-fold higher than the general population, necessitating regular cancer surveillance. Development of an adjunct therapy or preventative survival-enhancing therapy with a minimal side effect profile will benefit this population in which chemotherapy and radiation are particularly damaging. To this end, we established a cell line from a post bone marrow transplant FA patient with a T2N2bM0 oral SCC grown as an adherent monolayer culture. We tested pioglitazone, metformin, and N1-hexyl-N5-benzyl-biguanide (HBB), emerging chemopreventative therapies for head and neck squamous carcinoma, for growth inhibition in head and neck cancer cell lines, including our FA SCC cell line. Additional cell lines include UMSCC 11A (laryngeal squamous cell carcinoma), CA 9-22 (oral squamous cell carcinoma), Beas2B (SV40 immortalized normal bronchial epithelium), and MSK Leuk1 (oral leukoplakia cell line). All cell lines are HPV negative. MTT assays determined cell viability after 1, 2, and 3 days of treatment. Pioglitazone at all concentrations (5, 10, 20, 40 μM) decreases cell proliferation compared to solvent controls (P<0.0001). Metformin (1 μM - 1 mM) unexpectedly increased cell proliferation or was otherwise equal in growth with the controls, and at higher doses (10 mM - 100 mM) metformin decreased cell proliferation or resulted in cytotoxicity (P<0.0001). Pioglitazone (10 μM) and metformin (above 1 mM) resulted in larger, cell line dependent, decreases in cell proliferation than either agent alone. In the FA cell line, pioglitazone and HBB resulted in increased efficacy. HBB treatment resulted in decreases in proliferation and percent live cells at 20 μM equivalent to the effect of 10 to 100 mM metformin. Combining HBB with 10 μM pioglitazone decreased cell proliferation and percent live cells over pioglitazone alone. HBB also caused S phase blockade in a pilot FA cell cycle experiment. Pioglitazone combined with HBB appears to be a promising chemopreventative adjunct treatment for HNSCC in FA patients. Further studies are underway to evaluate the relative contribution of HBB to decreased cell proliferation versus apoptotic mechanisms. Recent studies of HBB in breast cancer indicate cytochrome P450 arachidonic acid epoxygenase activity as an HBB target and suggest further exploration of this target in head and neck squamous cell carcinoma. Citation Format: Kimberly A. Miller, Beverly R. Wuertz, Anna M. Haynes, David A. Potter, Frank G. Ondrey. Potential chemopreventative treatments in Fanconi anemia related HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-171.

Effects of Black Raspberry Extract and Berry Compounds on Repair of DNA Damage and Mutagenesis Induced by Chemical and Physical Agents in Human Oral Leukoplakia and Rat Oral Fibroblasts.

Black raspberries (BRB) have been shown to inhibit carcinogenesis in a number of systems, with most studies focusing on progression. Previously we reported that an anthocyanin-enriched black raspberry extract (BE) enhanced repair of dibenzo-[a,l]-pyrene dihydrodiol (DBP-diol)-induced DNA adducts and inhibited DBP-diol and DBP-diolepoxide (DBPDE)-induced mutagenesis in a lacI rat oral fibroblast cell line, suggesting a role for BRB in the inhibition of initiation of carcinogenesis. Here we extend this work to protection by BE against DNA adduct formation induced by dibenzo-[a,l]-pyrene (DBP) in a human oral leukoplakia cell line (MSK) and to a second carcinogen, UV light. Treatment of MSK cells with DBP and DBPDE led to a dose-dependent increase in DBP-DNA adducts. Treatment of MSK cells with BE after addition of DBP reduced levels of adducts relative to cells treated with DBP alone, and treatment of rat oral fibroblasts with BE after addition of DBPDE inhibited mutagenesis. These observations showed that BE affected repair of DNA adducts and not metabolism of DBP. As a proof of principle we also tested aglycones of two anthocyanins commonly found in berries, delphinidin chloride and pelargonidin chloride. Delphinidin chloride reduced DBP-DNA adduct levels in MSK cells, while PGA did not. These results suggested that certain anthocyanins can enhance repair of bulky DNA adducts. As DBP and its metabolites induced formation of bulky DNA adducts, we investigated the effects of BE on genotoxic effects of a second carcinogen that induces bulky DNA damage, UV light. UV irradiation produced a dose-dependent increase in cyclobutanepyrimidine dimer levels in MSK cells, and post-UV treatment with BE resulted in lower cyclobutanepyrimidine dimer levels. Post-UV treatment of the rat lacI cells with BE reduced UV-induced mutagenesis. Taken together, the results demonstrate that BE extract reduces bulky DNA damage and mutagenesis and support a role for BRB in the inhibition of initiation of carcinogenesis.

Ultrastructural Changes during the Life Cycle of Mycoplasma salivarium in Oral Biopsies from Patients with Oral Leukoplakia.

Bacteria in genus Mycoplasma spp. are the smallest and simplest form of freely replicating bacteria, with 16 species known to infect humans. In the mouth, M. salivarium is the most frequently identified species. Mycoplasma spp. are parasites with small genomes. Although most of the Mycoplasma spp. that infect humans remain attached to the host cell surface throughout their life cycle, we have previously reported the presence of Mycoplasma salivarium in the epithelial cells of oral leukoplakia and oral lichen planus. However, the mechanism underlying the pathogenicity of M. salivarium has remained unclear. Further studies are needed to identify the process of infection of human cells and the stages in the life cycle of M. salivarium. Electron microscopy (EM) is the method of choice for morphological investigation of Mycoplasma spp. in cells or tissues. This study was performed to clarify and detail the ultrastructure of M. salivarium in tissue biopsies of oral mucosal leukoplakia, using three EM methods: (1) a standard EM processing method; (2) an ultracryotomy and immunolabeling method; and (3) the LR White resin post-embedding and immunolabeling method. This study included five oral leukoplakia tissue samples showing hyperplasia and hyperkeratosis. Although there was some variation in ultrastructural appearances between the three EM methods used, there were four ultrastructural appearances that are believed to reflect the stages of the M. salivarium life cycle in the epithelial cells of the oral mucosa: (1) small, electron-dense cellular-like structures or elementary bodies of M. salivarium; (2) large structures of M. salivarium; (3) M. salivarium organisms in cell division; (4) the sequence of events in the life cycle of M. salivarium that includes: (a) elementary bodies of M. salivarium deep in the oral mucosal epithelium; (b) replication by binary fission and daughter cell division from the elementary bodies; (c) maturation or degeneration of M. salivarium in the epithelial cells mainly in the upper part of the epithelium; and (d) death of the organisms in the granular and/or keratinized layer. These ultrastructural images may provide a useful reference for the identification of M. salivarium in diagnostic cytology or biopsy material.

Abstract 503: Enhanced repair of bulky DNA adducts induced by a tobacco carcinogen and UV light in human oral epithelial cells by black raspberry extract

Abstract We were the first to report that an anthocyanin-enriched black raspberry extract (BE) inhibited mutagenesis, and reduced levels of DNA adducts induced by metabolites of the tobacco carcinogen dibenzo(a,l)pyrene (DBP), in a rat oral fibroblast cell line, by enhancing removal of DNA adducts (Guttenplan, et al., Cancer Prev Res; 9(8) August 2016). Here we extend these findings to the repair of DBP-induced DNA damage in a human oral leukoplakia cell line (MSK leuk1), and a reduction in the toxicity (assayed by dead cell numbers and cell survival) of DBP diolepoxide (DBPDE) in these cells. In addition, we examined the effect of BE on DNA damage and toxicity induced by UV light. Treatment of the MSK cells with DBP, and 2 of its metabolites, DBP-dihydrodiol and DBPDE led to a major adenine adduct, (-)-anti-trans-DB[a,l]PDE-dA (DBPDE-dA). The order of potency was DBPDE>DBP>DBP-dihydrodiol with one µM DBP, producing adduct levels of about 20/10E6 dA. BE in the range of 75-150 µg/ml significantly inhibited adduct formation when cells were pretreated with BE before addition of DBP. However, when cells were first treated with DBP and one day later BE was added, adduct levels were reduced by about 50% two days after treatment with DBP, indicating that the BE was enhancing DNA repair. This conclusion results from the fact that BE was not present during the metabolic activation of DBP to DBPDE, and hence couldn’t modulate the activation steps. We also tested whether BE, added 4 hr after treatment with the short-lived DBPDE, inhibited toxicity to MSK cells. Initial toxicity was measured by counting the numbers of dead cells in the medium 24 hr after addition of DBPDE; and the relative levels of surviving cells were determined using an MTT cell viability assay. It was found that 100ug/ml BE reduced toxicity induced by 25 - 200 nM DBPDE by about 50%, with a concomitant increase in survival in the BE-treated cells. As DBP produces bulky DNA adducts we also investigated the effects of BE on toxicity and levels of DNA adducts produced by UV light. Cells were irradiated for 30 - 120 seconds with 254 nm light and 15 minutes later, treated with 50 - 150 µg/ml BE. Toxicity was measured, as above, and a logarithmic decrease in cell death was observed with increasing concentration of BE. Similar to results with DBPDE, BE increased cell survival. The effects of BE on relative levels of the UV-light-induced cyclopyrimidine adducts were also determined, using an ELISA assay. BE at levels of 150 - 300 µg/ml reduced adduct levels by 30 - 60%. These results indicate that BE may provide chemopreventive effects on initiation of carcinogenesis by environmental agents that produce bulky DNA adducts - by enhancing DNA repair, likely via the nucleotide-excision repair pathway. Supported by NIH grant #CA173465. Note: This abstract was not presented at the meeting. Citation Format: Joseph B. Guttenplan, Kun-Ming Chen, Yuan-Wan Sun, Ross Teicher, Wieslawa Kosinska, Krishne Gowda, Shantu Amin, Gary D. Stoner, Karam El-Bayoumy. Enhanced repair of bulky DNA adducts induced by a tobacco carcinogen and UV light in human oral epithelial cells by black raspberry extract [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 503. doi:10.1158/1538-7445.AM2017-503

Immunohistochemical detection of Mycoplasma salivarium inoral lichen planus tissue.

BackgroundOral lichen planus (OLP) is a T‐cell‐mediated inflammatory disease; however, its exact etiology is unknown. Hyperkeratosis is often observed in OLP lesions. Previous studies have revealed the localization of Mycoplasma salivarium in the epithelial cells of oral leukoplakia with hyperkeratosis. Herein, we investigated the presence of M. salivarium in OLP tissue by immunohistochemistry to determine the causative factor of OLP.MethodsForty‐one formalin‐fixed, paraffin‐embedded samples obtained from 31 patients with OLP were examined. Ten samples of normal‐appearing oral mucosa were used as controls. Immunohistochemistry (IHC) was performed using anti‐M. salivarium monoclonal antibodies.Results and Conclusions Mycoplasma salivarium was detected in the epithelium and lymphocyte infiltrate area in 24 of 41 OLP samples (58.5%). The bacteria were intracellularly localized in epithelial cells, while it was unclear whether they were also localized in lymphocyte cells or in the extracellular spaces among the lymphocytes in the subepithelial lymphocyte infiltrate area. Little or no staining was observed in the epithelium in the normal‐appearing mucosa samples. Sawtooth rete ridge formation was observed in 21 OLP samples (51.2%), and a significant positive correlation between sawtooth rete ridge formation and IHC positivity was demonstrated. However, the role of M. salivarium in the epithelium and lamina propria of OLP tissue remains unknown.

Abstract 4063: Effects of electronic cigarettes (E-cigs) and hookah on the metabolic activation of the tobacco carcinogen, benzo(a)pyrene (BaP), and cellular proliferation in human oral leukoplakia cells

Abstract The possible carcinogenic effects of E-cigs on the oral cavity are uncertain because their recent availability and usage precludes long-term epidemiological studies. Also of concern is the use of hookah, which has recently increased in popularity. In addition, many, E-cig and hookah users are tobacco smokers or individuals trying to quit or cut down on smoking. Such individuals likely have precancerous cells and/or lesions in the oral cavity, as do former smokers or oral cancer survivors. The use of E-cigs and hookah then, may affect the initiation and promotion/progression stages of carcinogenesis, and be of particular concern to dual users. Here we have investigated the effects of E-cigs and to a lesser extent, hookah on the metabolism of BaP to its ultimate carcinogenic metabolite, BaP diol-epoxide (BPDE), and also the effects of E-cigs on proliferation, and expression of proliferation-related genes in leukoplakia cells. Tobacco-flavored E-cigs, and hookah were prepared from local products using a mechanical smoking device to simulated smoking, and the resulting vapor collected in a liquid N2-cooled trap to generate E-cig or hookah vapor condensates (VC). To determine the effects of E-cigs and hookah on BaP metabolism, cells from a leukoplakia-derived cell line, MSKLeuk1, were pretreated for 16 hr with E-cig or hookah VC at levels yielding nicotine concentrations of 0.5 - 8 uM (similar to plasma nicotine levels in smokers) and then treated with one uM BaP. After 8 hr the concentrations of BaP-tetrols released into the medium were measured using HPLC. BPDE hydrolyzes into BP tetrols, and their concentration serves as a surrogate for BPDE concentration. Cells pretreated with a tobacco smoke extract (TSE) from the reference cigarette, 2R4F, were included, as were untreated cells. Pretreatment with E-cig or hookah VC increased the concentration of BaP tetrols in the cell medium from 22 to 165 and 64 pM, resp. at the highest dose of each. For comparison, pretreatment with TSE at a nicotine level of 4uM increased the concentration BaP tetrols to 252 pM. The effects E-cigs on cell proliferation were assayed using an MTT assay. The cells were plated with and without E-cig VC and 24 hr later cell proliferation was assayed. Exposure to E-cigs significantly increased the rate of cell proliferation by 61%. The increase maximized at one uM nicotine. We also monitored the effects of 2 different E-cigs on the expression of the certain proteins related to cell proliferation-related genes (PCNA and pAKT) by immunoblotting. Both E-cigs increased the levels of these proteins to various extents. Our results demonstrate, for the first time, that E-cig or hookah VC enhanced metabolism of BaP to its ultimate carcinogen, and E-cig VC stimulated cellular proliferation. These results suggest that “vaping”, or hookah usage may contribute to oral cancer risk. (Support: CA173465). Citation Format: Joseph B. Guttenplan, Yuan-Wan Sun, Kun-Ming Chen, Wieslawa Kosinska, Karam El-Bayoumy. Effects of electronic cigarettes (E-cigs) and hookah on the metabolic activation of the tobacco carcinogen, benzo(a)pyrene (BaP), and cellular proliferation in human oral leukoplakia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4063.

An Assessment of Serum Total Sialic Acid in Oral Leukoplakia and Oral Squamous Cell Carcinoma at Nadiad, Gujarat

Background and Objectives: Altered glycosylation of glycoconjugates is among the important molecular changes that accompany malignant transformation. Various studies in the past have shown the direct relation of Total Sialic acid in premalignant lesions and different malignancies. The purpose of this study is to investigate clinical usefulness of circulatory levels of Total Sialic acid in Leukoplakia, Squamous cell carcinoma and healthy controls of age and sex matched subjects. Method: Blood samples were collected from 25 untreated Leukoplakia, 25untreated Squamous cell carcinoma and 25 healthy subjects. Total serum Sialic acid were evaluated by the simplified quick method by G Sydow and measured spectrophotometrically at 525nm. Results: Serum levels of Total Sialic acid were significantly elevated (P

Establishment of a new OSCC cell line derived from OLK and identification of malignant transformation-related proteins by differential proteomics approach.

Oral squamous cell carcinoma (OSCC) is usually preceded by the oral premalignant lesions, mainly oral leukoplakia (OLK) after repeated insults of carcinogens, tobacco. B(a)P and DMBA are key carcinogens in tobacco smoke. In the present study, for the first time we established the cancerous cell line OSCC-BD induced by B(a)P/DMBA mixture and transformed from dysplastic oral leukoplakia cell line DOK. Cell morphology, proliferation ability, migration ability, colony formation, and tumorigenicity were studied and confirmed the malignant characteristics of OSCC-BD cells. We further identified the differential proteins between DOK and OSCC-BD cells by stable isotope dimethyl labeling based quantitative proteomic method, which showed 18 proteins up-regulated and 16 proteins down-regulated with RSD < 8%. Differential proteins are mainly related to cell cycle, cell proliferation, DNA replication, RNA splicing and apoptosis. Abberant binding function, catalysis activity and transportor activity of differential proteins might contribute to the malignant transformation of OLK. Of the 34 identified differential proteins with RSD < 8%, 13 novel cancer-related proteins were reported in the present study. This study might provide a new insight into the mechanism of OLK malignant transformation and the potent biomarkers for early diagnosis, meanwhile further facilitate the application of the quantification proteomics to carcinogenesis research.

Morphometry As a Diagnostic Tool for Potentially Malignant Lesions.

Though we are in 21(st) century with nano technology & tissue printing, there still exist many lacunae in the field of diagnosis. Not much is known about prognostic markers till now from literature to assess potentially malignant lesions. Lesions so called potentially malignant can be termed only after clinical & malignant changes have been developed and there are no means of predicting with certainty the risk of cancerous transformation. Our present study was undertaken to establish the morphometric parameters of the parabasal and spinous cells of normal oral epithelium with the changes occurring in cells of Oral Leukoplakia (OL), Oral Verrucous Carcinoma (OVC) and Oral Squamous Cell Carcinoma (OSCC). Total 40 patients were divided into Group I which includes patients with normal oral mucosa, group II oral leukoplakia patients, group III oral verrucous carcinoma patients and group IV includes oral squamous cell carcinoma patients. Tissue sections were taken and morphometric analysis of cell area, cell diameter, nuclear area, nuclear diameter, nuclear cytoplasmic ratio was done for parabasal and spinous layer cells. Statistical analysis was done using one-way ANOVA and T-test. Nuclear diameter, nuclear area, cell area, nuclear cytoplasmic ratio were significantly increased in OL, OVC, OSCC patients than normal oral mucosa, which was statistically significant. Cell diameter was decreased in OL, OVC, OSCC patients than with normal oral mucosa which was statistically significant. Cellular & nuclear parameters showed statistically significant changes in oral leukoplakia, oral verrucous carcinoma & oral squamous cell carcinoma in comparison with normal oral mucosa.

Comparative cytomorphometric analysis of oral mucosal cells in normal, tobacco users, oral leukoplakia and oral squamous cell carcinoma.

Background:Squamous cell carcinoma (SCC) is the third most common cause of oral morbidity in India despite the numerous advances made in the treatment protocol.Aim:To compare the cytomorphometric changes of oral mucosal cells in normal subjects (Group I) with that of tobacco users without any lesion (Group II), tobacco users with oral leukoplakia (Group III), and tobacco users with oral SCC (Group IV) through a semi-automated image analysis system.Materials and Methods:Oral mucosal cells collected from study subjects (n = 100) stained using rapid Papanicolaou stain. Photomicrograph of 50 nonoverlapping cells captured at 50× magnification with a digital image capture system. Cytomorphometric analysis of cells in the captured images was performed with Image-Pro image analysis software. Image analysis was performed to obtain cell diameter (CD), cytoplasmic area (CyA), nuclear diameter (ND), nuclear area (NA), and nuclear-to-cytoplasmic ratio. These values were statistically compared among the groups using one-way analysis of variance (ANOVA) and Mann-Whitney U test.Results:The ND, NA, and nuclear-to-cytoplasmic ratio values were found to be increased in the samples collected from leukoplakia and oral SCC. The CD and CyA decreased compared to the normal mucosa in oral SCC samples.Conclusion:The cytomorphometric changes observed in samples from oral SCC and oral leukoplakia were consistent with the current diagnostic features. Hence, the semi-automated cytomorphometric analysis of oral mucosal cells can be used as an objective adjunct diagnostic tool in the diagnosis of these lesions.

Physiologic Behavioural Changes of Precancerous Leukoplakia Cell lines Exerted to Extremely Low Frequency Electric Field

Objectives: Oral galvanism arising from the presence of two different metallic fillings in the mouth that may reach up to 16V/m, induces several changes in oral environment such as gingival swelling and erythema, mucosal pain, lichenoid reactions and leukoplakia. Study design: To investigate in vitro the physiologic reactions of oral leukoplakia caused by electrostatic corrosion potentials, human leukoplakia cell lines (MSK-LEUK1), cultivated in KGM-2 supplemented with bullet kit in 5% CO2 humidified air at 37°C, were exposed to electric field strength of 1-20 V/m for 24 hours in a pseudo realistic apparatus called “Pulse chamber”. Following this, the cells were analysed for proliferation using BrdU assay, and for apoptosis using TUNEL assay. Findings were assessed utilizing fluorescent microscopy. Ultra structural changes were studied by TEM. Data was evaluated statistically using non parametric Chi-square test. Results: Electric field strength of 1-10V/m led to upregulation of cell proliferation rate between 10.64% and 44.06% (p=0.0001). The apoptotic index increased significantly (p=0.0001) from 20.03% at 1V/m to 46.56% at 10V/m. Leukoplakia cells treated with 16V/m show individual cell keratinization. Conclusion: Oral galvanism induces subcellular changes in oral precancerous leukoplakia cells in vitro that resemble some of the histopathologic features of oral squamous cell carcinoma cells in vivo.

Cellular differentiation regulator BLIMP1 induces Epstein-Barr virus lytic reactivation in epithelial and B cells by activating transcription from both the R and Z promoters.

Epstein-Barr virus (EBV) maintains a lifelong latent infection within a subset of its host's memory B cells, while lytic EBV replication takes place in plasma cells and differentiated epithelial cells. Therefore, cellular transcription factors, such as BLIMP1, that are key mediators of differentiation likely contribute to the EBV latent-to-lytic switch. Previous reports showed that ectopic BLIMP1 expression induces reactivation in some EBV-positive (EBV(+)) B-cell lines and transcription from Zp, with all Z(+) cells in oral hairy leukoplakia being BLIMP1(+). Here, we examined BLIMP1's role in inducing EBV lytic gene expression in numerous EBV(+) epithelial and B-cell lines and activating transcription from Rp. BLIMP1 addition was sufficient to induce reactivation in latently infected epithelial cells derived from gastric cancers, nasopharyngeal carcinomas, and normal oral keratinocytes (NOK) as well as some, but not all B-cell lines. BLIMP1 strongly induced transcription from Rp as well as Zp, with there being three or more synergistically acting BLIMP1-responsive elements (BRE) within Rp. BLIMP1's DNA-binding domain was required for reactivation, but BLIMP1 did not directly bind the nucleotide (nt) -660 Rp BRE. siRNA knockdown of BLIMP1 inhibited 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced lytic reactivation in NOK-Akata cells, cells that can be reactivated by R, but not Z. Thus, we conclude that BLIMP1 expression is both necessary and sufficient to induce EBV lytic replication in many (possibly all) EBV(+) epithelial-cell types, but in only a subset of EBV(+) B-cell types; it does so, at least in part, by strongly activating expression of both EBV immediately early genes, BZLF1 and BRLF1. This study is the first one to show that the cellular transcription factor BLIMP1, a key player in both epithelial and B-cell differentiation, induces reactivation of the oncogenic herpesvirus Epstein-Barr virus (EBV) out of latency into lytic replication in a variety of cancerous epithelial cell types as well as in some, but not all, B-cell types that contain this virus in a dormant state. The mechanism by which BLIMP1 does so involves strongly turning on expression of both of the immediate early genes of the virus, probably by directly acting upon the promoters as part of protein complexes or indirectly by altering the expression or activities of some cellular transcription factors and signaling pathways. The fact that EBV(+) cancers usually contain mostly undifferentiated cells may be due in part to these cells dying from lytic EBV infection when they differentiate and express wild-type BLIMP1.

Differential prooxidative effects of the green tea polyphenol, (-)-epigallocatechin-3-gallate, in normal and oral cancer cells are related to differences in sirtuin 3 signaling.

We have previously reported that the green tea catechin, (-)-epigallocatechin-3-gallate (EGCG), can induce oxidative stress in oral cancer cells but exerts antioxidant effects in normal cells. Here, we report that these differential prooxidative effects are associated with sirtuin 3 (SIRT3), an important mitochondrial redox modulator. EGCG rapidly induced mitochondria-localized reactive oxygen species in human oral squamous carcinoma cells (SCC-25, SCC-9) and premalignant leukoplakia cells (MSK-Leuk1), but not in normal human gingival fibroblast cells (HGF-1). EGCG suppressed SIRT3 mRNA and protein expression, as well as, SIRT3 activity in SCC-25 cells, whereas it increased SIRT3 activity in HGF-1 cells. EGCG selectively decreased the nuclear localization of the estrogen-related receptor α (ERRα), the transcription factor regulating SIRT3 expression, in SCC-25 cells. This indicates that EGCG may regulate SIRT3 transcription in oral cancer cells via ERRα. EGCG also differentially modulated the mRNA expressions of SIRT3-associated downstream targets including glutathione peroxidase 1 and superoxide dismutase 2 in normal and oral cancer cells. SIRT3 represents a novel potential target through which EGCG exerts differential prooxidant effects in cancer and normal cells. Our results provide new biomarkers to be further explored in animal studies.

Abstract 1582: Comparative transforming effects of ultra low tar (ULT) and full flavor low tar (FFLT) cigarette smoke particulate extracts on human oral epithelial cells

Abstract ULT cigarettes produce much lower levels of tars and particulate matter than FFLT cigarettes, but are generally not safer alternatives with respect to carcinogenesis, possibly because of compensatory smoking behavior. In a previous study (P.G. Sacks, et al., Food Chem Toxicol 49 (2011) 2348-2355) we reported that tobacco smoke particulate (TSP) extracts from FF, FFLT and ULT cigarettes exerted similar effects on the induction of phase I and II proteins and the activation of the tobacco carcinogen, benzo(a)pyrene (BaP) to genotoxic metabolites in primary oral epithelial cells when compared on a concentration basis, although ULT cigarettes were less effective on a per cigarette basis. Here we report that both a FFLT and an ULT TSP extract transform a human oral epithelial cell line to growth independence in soft agar, and enhance the transforming effects of BaP. At equal concentrations the ULT extract was more effective than the FFLT extract. A human oral leukoplakia cell line, MSKleuk1, was employed in these studies. Cells were treated twice over a two week period or 3 times over a 3 week period with: a) one uM BaP, b) 4ug/ml TSP extract, c) TSP extract + BaP, or d) vehicle control. One week after the last treatment cells were seeded on soft agar and were assayed either by counting colonies 3 weeks after seeding, or 8 days after seeding using an anchorage-independent growth assay (CytoSelect 96-Well Cell Transformation Assay Kit, Cell Biolabs). Results in initial experiments for the two week treatment were assayed by counting colonies, and the fraction of growth-independent cells for groups a-d resp. were: a, 5.5 +/- 0.2; b, 6.0 +/- 0.6; c, 6.6 +/- 0.3 and d, 0.78 +/- 0.3 (all in units of 10E-4). For the three week treatment the results were: a, 9.1 +/- 0.5; b, 9.2 +/- 1.2; c, 11.9, +/- 2.5; d, 1.4 +/- 0.7. The TSP extract was from a reference filtered cigarette, 2RF4. A further soft agar assay was carried out using the cell transformation assay; and TSP extracts from a commercial ULT and 2RF4 cigarettes were compared, in the presence and absence of BaP. The groups were a) 1 uM BaP, b) 4ug/ml 2RF4 extract, c) 4ug/ml 2RF4 extract + 1 uM BaP, d) 4ug/ml ULT extract, e) 4ug/ml ULT extract + 1uM BaP and f) vehicle control. All groups were treated 2x as described above. Results are expressed relative to the vehicle control which is set as 1. a, 1.9 +/- 0.4; b, 1.4 +/- 0.3; c, 2.2 +/- 0.4; d, 2.1 +/ 0.3; e, 2.6 +/- 0.6.The results of these show: 1) human oral epithelial cells can be transformed by BaP, and TSP extracts from FFLT and ULT cigarettes, 2) the effects of BaP and TSP extracts are approximately additive, 3) when compared on a concentration of particulate basis, ULT TSP extracts were more effective than FFLT TSP extracts. Unlike previous studies relating primarily to carcinogenesis initiation, ULT TSP extracts appear to more effective at transformation than FFLT TSP extracts. Citation Format: Tianzhen Han, Peter Sacks, Joseph B. Guttenplan. Comparative transforming effects of ultra low tar (ULT) and full flavor low tar (FFLT) cigarette smoke particulate extracts on human oral epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1582. doi:10.1158/1538-7445.AM2014-1582

Cytomorphometric analysis of oral submucous fibrosis and leukoplakia using methyl green-pyronin Y, Feulgen staining and exfoliative brush cytology

The incidence of potentially malignant oral pathology such as leukoplakia, oral submucous fibrosis and squamous cell carcinoma has increased in India. We investigated whether cytoplasmic diameter, nuclear diameter and nucleus:cytoplasm ratio in exfoliative cytology are reliable indicators of potentially malignant lesions. We also investigated methyl green-pyronin Y and Feulgen staining as simple time saving and cost effective staining techniques for diagnostic exfoliative cytology. Cell and nuclear diameters of squamous cells of normal buccal mucosa, oral leukoplakia and oral submucous fibrosis were measured using an ocular micrometer disc. The nucleus:cytoplasm ratios in pathological cells were compared to age, sex and site matched controls. We found a significant reduction in the mean cytoplasmic and nuclear diameter in the experimental groups compared to normal controls. Methyl green-pyronin Y stained smears were clearer than Feulgen stained cells. We suggest that a decreased mean cytoplasmic diameter of exfoliated buccal mucosal cells could serve as an early indicator of dysplastic change in lesions that otherwise appear benign. Methyl green-pyronin Y may be useful for identifying premalignant and malignant transformations before a lesion is visible. The simplicity of the technique makes its routine use feasible.

Abstract 2580: Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells.

Abstract Oral leukoplakia is a precancerous lesion that may progress to squamous cell carcinoma of the head and neck (HNSCC). The long latency period to invasive cancer provides an opportunity for intervention at the precancerous stage. The inability of surgical resection to prevent the recurrence of oral leukoplakia speaks clearly to the need to develop an efficacious regimen for the chemoprevention of this disease. Tobacco smoke constituents and ethanol, the major risk factors for oral leukoplakia and HNSCC, are metabolized by cytochrome P450 1B1 (CYP1B1) to form potentially carcinogenic species. Our previous data indicate that knockdown of CYP1B1 in human oral leukoplakia cells (MSK-Leuk1) by shRNA decreases cell motility and proliferation, relative to control cells overexpressing empty vector. Homoeriodictyol (HED), a flavonoid found in citrus fruits, is a selective inhibitor of CYP1B1 (IC50 0.24 μM). As a next step, we hypothesized that pharmacologic inhibition of CYP1B1 by HED would attenuate cell motility and proliferation in a manner similar to genetic knockdown. Because HED is commonly found in our diet, its toxicity is expected to be low, making it a promising agent for preventive or therapeutic intervention. The ability of HED to inhibit CYP1B1 activity in MSK-Leuk1 cells was established by incubating cells with either vehicle (0.01% ethanol) or HED (5-10 μM) for 72 hrs and quantifying 7-ethoxyresorufin o-deethylation (EROD), a measure of the activity of CYP1B1 and other enzymes of the CYP1 subfamily. HED decreased EROD activity in a dose-dependent manner, with 40% inhibition observed at 10 μM, as compared to vehicle-treated controls. Western blot analyses revealed that CYP1B1 levels were unaltered following HED treatment. The effect of CYP1B1 inhibition on cell motility was examined by exposing MSK-Leuk1 cells to HED (0-10 μM) for 96 hrs followed by a wound-healing assay, in which the ability of cell monolayers to close a gap was monitored for 23 hrs. HED inhibited cell motility in a concentration dependent manner, with maximum inhibition of gap closure reached at 10 μM (6.7 fold relative to vehicle). To assess the effect of CYP1B1 inhibition on cell growth, MSK-Leuk1 cells were exposed to HED (0-10 μM) for 5 days and proliferation was analyzed (Fluorescent DNA Quantitation kit). A direct association was observed between HED dose and inhibition of proliferation, with a 33% decrease observed at 10 μM. In summary, inhibition of CYP1B1 activity by HED resulted in attenuation of the motility and proliferation of cultured MSK-Leuk1 cells. These data confirm that CYP1B1 is a promising molecular target for intervention in HNSCC and provide strong support for the further development of CYP1B1 inhibitors as efficacious agents for the prevention of HNSCC in patients with oral leukoplakia. (Supported by NCI fellowship 1 F32 CA144199-01). Citation Format: Ekaterina G. Shatalova, Margie L. Clapper. Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2580. doi:10.1158/1538-7445.AM2013-2580

Expression of P53 Protein in Premalignancies and Squamous Cell Carcinoma of the Oral Cavity

Introduction: Expression of p53 gene is one of the common findings in premalignancies and malignancies of the oral cavity. The aim of the study was to know whether P53 protein is overexpressed in histological sections of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. The aim of the present study was to determine the overexpression of P53 protein in histological sections of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. Materials and methods: Eighty-five histopathologically diagnosed. formalin-fixed, paraffin embedded tissue samples were divided into study group and control group. The study group was further subdivided into three groups: Group 1 consisted of 25 samples of oral submucous fibrosis: group 2 consisted of 25 samples of oral leukoplakia and group 3 consisted of 25 samples of oral squamous cell carcinoma. The control group consisted of 10 normal oral mucous membrane tissue samples. The samples were subjected to immunohistochemisty using indirect immunoenzyme Labelled StreptAvidin Biotin (LSAB) method. The results were analyzed statistically by Chi-square (X 2 ) test of significance Results: 8125 samples of group 1.5;25 samples of group 2. 8125 samples of group 3 and none of the samples of the control group were positive for P53 protein. P53 staining was seen mostly in the basal layer of the samples of group I while in the samples of groups 2 and 3 the staining was more diffuse. Conclusion: Among P53 may be considered a tumor marker as a definite number of samples were positive for P53

In situ gelatinolytic activity and immunoexpression of metalloproteinases 2 and 9 in oral leukoplakia and oral squamous cell carcinomas

In situ gelatinolytic activity and immunoexpression of metalloproteinases 2 and 9 in oral leukoplakia and oral squamous cell carcinomas

Abstract 1588: Synergistic apoptosis by combination of natural compound EGCG and resveratrol in head and neck cancer: Potential role for AKT-dependent signaling

Abstract Natural dietary agents have drawn great attention for cancer prevention because of their wide safety margin and ready availability. Green tea is one such agent that is widely used for chemoprevention (Amin et al. 2009, Kim et al., 2010). Recent clinical trials suggest that green tea alone might not be sufficient for chemoprevention of head and neck cancer (HNC) (Tsao et al., 2009). Therefore, identification of compounds which exhibit synergistic effects with green tea is warranted. In the current study, we investigated combination of green tea component EGCG and resveratrol (a major constituent of red wines and grapes), and found that their combination strongly induced apoptosis as measured by annexin-V staining and supported by cleavage of PARP and caspase 3. The combination of resveratrol (15 and 20 μM, which alone induce 10-15% apoptosis) with EGCG (30-80 μM, which also induce &amp;lt;10% apoptosis) strongly increased apoptosis (to 60-80%) in multiple HNC cell lines. Data analysis for the combination using CalcuSyn software suggests that the combination of EGCG and resveratrol has synergistic apoptotic effects with combination index between 0.4-0.7. Interestingly, a premalignant oral leukoplakia cell line was sensitive to relatively lower doses of the combination (5 and 10 μM resveratrol, 10 and 15 μM EGCG) than fully transformed cancer cell lines, suggesting that the combination of EGCG and resveratrol might be a suitable combination for prevention of HNC. To further elucidate the mechanism of synergistic apoptosis, we found that combination of EGCG and resveratrol strongly inhibited AKT and ERK phosphorylation along with its several downstream targets including p-mTOR, p-S6 and p-4EBP1. Retroviral transduction of constitutively active AKT significantly inhibited apoptosis induced by the combination (p=.003). We also found that the combination of EGCG and resveratrol strongly inhibited p-AMPK, a kinase that regulates autophagy, a protective mechanism during energy stress. The fact that inhibition of autophagy triggers apoptosis (Xu Y et al., 2011) suggests that inhibition of p-AMPK might also play a role in EGCG- and resveratrol-induced apoptosis. Furthermore, strong inhibition of anti-apoptotic Mcl-1 and survivin by the combination was observed as a consequence. Taken together, our studies identify a novel combination of two natural dietary agents, EGCG and resveratrol, which induces synergistic anti-tumor effects in both premalignant and fully transformed HNC cells by targeting multiple signal transduction pathways. The signaling pathways modulated by the combination of EGCG and resveratrol are critical for development of HNC, thus our study provides an important rationale for future preclinical and clinical development. (Supported by U01CA101244, R01CA112643, and P50CA128613 to DMS) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1588. doi:1538-7445.AM2012-1588

Induction of apoptosis and up-regulation of cellular proliferation in oral leukoplakia cell lines inside electric field

In dentistry, metallic alloys are used for dentures, restorative materials, and orthodontic devices. Electric voltages up to 950 mV may occur between different dental alloys in the oral cavity. This study aimed to investigate physiologic reactions of oral leukoplakia cells in vitro to electric fields. A human leukoplakia cell line (MSK-LEUK1), cultivated in keratinocyte growth medium (KGM-2) supplemented with growth factors in 5% CO(2) humidified air at 37°C, was exposed to electric field strength of 1-20 V/m for 24 hours in a custom-made pulse chamber. The cells were then analyzed for proliferation with the use of BrdU assay and for apoptosis with the use of TUNEL assay. Findings were assessed with the use of fluorescent microscopy. Ultrastructural changes were studied by transmission electron microscopy. Electric field strength of 1-10 V/m led to up-regulation of cell proliferation rate from 10.64% to 44.06% (P = .0001). The apoptotic index increased significantly (P = .0001) from 20.03% at 1 V/m to 46.56% at 10 V/m. Individual cell keratinization was seen in leukoplakia cells treated with 16 V/m. Oral galvanism induces subcellular changes in oral precancer cells in vitro that closely simulate some of the morphologic features of oral squamous cell carcinoma cells in vivo.