Old age and other factors associated with salivary microbiome variation.

BACKGROUND: Many factors can contribute to the exact makeup of the salivary microbiome. Differences in the oral microbiome occur with old age, which may be due to oral conditions and diseases associated with old age, such as edentulism, as well as other unknown causes. METHODS: The salivary microbiome was sampled in patients from a large urban clinic. For all subjects age, gender, periodontal status, caries status, presence of edentulism, medications, and tobacco usage were recorded. Multifactor analysis was used to study variation in salivary microbiome profiles linked to these factors. RESULTS: In the population sampled, there were significantly higher numbers of edentulous subjects, and increased levels of polypharmacy found with aging. Large differences in alpha diversity and beta diversity of the salivary microbiome in the old age group were largely linked to edentulism. However, multivariable analysis revealed, even after adjusting for differences in edentulism, polypharmacy, tobacco usage, periodontal disease, caries level, and gender, that old age itself was associated with lower levels of taxa Porphyromonas endodontalis, Alloprevotella tannerae, Filifactor alocis, Treponema, Lautropia Mirabilis and Pseudopropionibacterium sp._HMT_194. Surprisingly, of these taxa, most were ones known to reside on or near tooth surfaces. CONCLUSIONS: Another factor or factors beyond edentulism, polypharmacy and periodontal disease play a role in the differences seen in oral microbiome with old age. The nature of this factor(s) is not known.

Salivary microbiome with gastroesophageal reflux disease and treatment.

The effect of oral microbial composition on periodontal health and on systemic health has been, and is being established. The oral microbiome, in turn, can be altered by local and systemic diseases and conditions. Gastroesophageal reflux disease (GERD), has been associated with increased acidity in the oral cavity resulting in dental erosion, and controversially a reduced risk of periodontal disease. We hypothesized that presence of GERD was linked to a modified microbial profile in untreated GERD patients and that the use of proton pump inhibitor (PPI) drugs: potent disruptors of gut microbiome, in GERD patients might result in a salivary microbiome that is further distinct. Untreated GERD patients showed multiple differences in salivary microbiome as compared to healthy controls. Taxa found at lower levels related to the presence of GERD not treated by PPI included: Prevotella melaninogenica, Prevotella pallens, Leptotrichia, and Solobacterium moorei and thirteen others. In contrast, GERD patients chronically using PPI showed minimal differences in salivary taxa compared to healthy controls not using PPI.

Gut/Oral Bacteria Variability May Explain the High Efficacy of Green Tea in Rodent Tumor Inhibition and Its Absence in Humans.

Consumption of green tea (GT) and GT polyphenols has prevented a range of cancers in rodents but has had mixed results in humans. Human subjects who drank GT for weeks showed changes in oral microbiome. However, GT-induced changes in RNA in oral epithelium were subject-specific, suggesting GT-induced changes of the oral epithelium occurred but differed across individuals. In contrast, studies in rodents consuming GT polyphenols revealed obvious changes in epithelial gene expression. GT polyphenols are poorly absorbed by digestive tract epithelium. Their metabolism by gut/oral microbial enzymes occurs and can alter absorption and function of these molecules and thus their bioactivity. This might explain the overall lack of consistency in oral epithelium RNA expression changes seen in human subjects who consumed GT. Each human has different gut/oral microbiomes, so they may have different levels of polyphenol-metabolizing bacteria. We speculate the similar gut/oral microbiomes in, for example, mice housed together are responsible for the minimal variance observed in tissue GT responses within a study. The consistency of the tissue response to GT within a rodent study eases the selection of a dose level that affects tumor rates. This leads to the theory that determination of optimal GT doses in a human requires knowledge about the gut/oral microbiome in that human.

Deep DNA metagenomic sequencing reveals oral microbiome divergence between monozygotic twins discordant for multiple sclerosis severity.

In contrast to gut, the oral microbiome of MS patients has not been characterized. Deep sequencing of saliva DNA from a pair of monozygotic twins (MSF1 with relapsing remitting MS; MSF2 with clinically isolated syndrome) identified 2036 bacterial species. Relative abundances of 3 phyla were higher, and 3 lower in MSF1 versus MSF2. Species diversity was greater in MSF2, and 20 abundant species differed at least 2-fold. Pathway analysis identified 116 functional hierarchies differing 50% or more. Although limited to one pair of twins, our data suggests that oral microbiome analysis may be useful for diagnosis or monitoring therapeutic efficacy.

A subtype of oral, laryngeal, esophageal, and lung, squamous cell carcinoma with high levels of TrkB-T1 neurotrophin receptor mRNA.

BACKGROUND: The NTRK2 genetic locus encodes neurotrophin membrane receptors that play an important role in normal neural tissue plasticity, growth, and survival. One NTRK2-encoded protein is TrkB-FL, which can regulate multiple pathways relevant to cancer. A second NTRK2 gene mRNA isoform encodes TrkB-T1, a receptor that has a different cytoplasmic domain encoded in a mRNA with a unique 3' terminal exon. METHOD: Tumors from The Cancer Genome Atlas (TCGA) and other studies were classified according to the expression of a single form of NTRK2 mRNA, TrkB-T1, identified by its unique 3' terminal exon. Analysis of differentially expressed genes in TrkB-T1 high expressers was done to determine if tumors enriched for TrkB-T1 mRNA were a uniform group independent of anatomic site. RESULTS: The mRNA for TrkB-T1 is the most abundant NTRK2 gene mRNA in all squamous cell carcinomas (SCCs) in the TCGA database. Comparison of larynx SCC high TrkB-T1 RNA expressers to low expressers (n = 96) revealed gene expression differences consistent with the high TrkB-T1 tumors being more neural-like. The upregulated genes in the TrkB-T1 RNA high expressers also showed enrichment of pathways involved in retinol metabolism, hedgehog signaling, and the Nfe2l2 response, among other pathways. An examination of oral, esophagus, and lung SCCs (n = 284, 97, 501) showed induction of the same pathways among tumors that expressed high levels of TrkB-T1 mRNA. Proteins associated with regulation of the sonic hedgehog pathway, and the Nfe2l2 response, Tp63, and Keap1 and p62/SQSTM1 proteins, showed differential expression in larynx, oral and lung high TrkB1-T1 expresser SCCs. Unexpectantly, the relationship of high level TrkB-T1 expression to patient outcomes was SCC anatomic site specific. High TrkB-T1 mRNA levels in laryngeal SCC correlated with poor survival, but the opposite was true for lung SCC. This may be because pathways enriched in the TrkB high expressers, like those involving oncogenes NFE2L2, PIK3CA, and SOX2, are known to have SCC anatomic site-specific effects on progression. CONCLUSIONS: High level TrkB-T1 mRNA is a marker of a distinct SCC subtype enriched for at least 3 pathways relevant to tumor progression: Nfe2l2 response, retinol metabolism, and hedgehog signaling.

Diversity of the oral microbiome between dentate and edentulous individuals.

BACKGROUND: Measurement of saliva microbes is promoted as a way to detect oral and systemic disease, yet there is a multitude of factors that affect the oral microbiome. The salivary microbiome is influenced by oral biofilm of shedding (epithelial) and non-shedding (tooth) surfaces. METHODS: To gauge the ability of salivary microbial analytics to distinguish between edentulous and dentate oral conditions, we looked for differences in the saliva microbiome of subjects with and without teeth. Fifty-two dentate and 49 edentulous subjects provided stimulated saliva samples. 16S rRNA gene sequencing, QIIME-based data processing, and statistical analysis were done using several different analytical approaches to detect differences in the salivary microbiome between the two groups. RESULTS: Bacteria diversity was lower in the edentulous group. Remarkably, all 31 of the most significant differences in taxa were deficits that occur in the edentulous group. As one might expect many of these taxa are attributed to dental plaque and gingival sulcus associated bacteria. CONCLUSION: In sum, the measurement of 16S rRNA genes in the bacteria of the saliva can be used to reproducibly measure differences in the oral microbiome that occur with edentulism, mainly the lack of tooth and tooth-related structures.

Effects of green tea on miRNA and microbiome of oral epithelium.

Consumption of green tea (GT) extracts or purified catechins has shown the ability to prevent oral and other cancers and inhibit cancer progression in rodent models, but the evidence for this in humans is mixed. Working with humans, we sought to understand the source of variable responses to GT by examining its effects on oral epithelium. Lingual epithelial RNA and lingual and gingival microbiota were measured before and after 4 weeks of exposure in tobacco smokers, whom are at high risk of oral cancer. GT consumption had on average inconsistent effects on miRNA expression in the oral epithelium. Only analysis that examined paired miRNAs, showing changed and coordinated expression with GT exposure, provided evidence for a GT effect on miRNAs, identifying miRNAs co-expressed with two hubs, miR-181a-5p and 301a-3p. An examination of the microbiome on cancer prone lingual mucosa, in contrast, showed clear shifts in the relative abundance of Streptococcus and Staphylococcus, and other genera after GT exposure. These data support the idea that tea consumption can consistently change oral bacteria in humans, which may affect carcinogenesis, but argue that GT effects on oral epithelial miRNA expression in humans vary between individuals.

Pathogenesis and persistence of cryptoglandular anal fistula: a systematic review.

Anal fistulas continue to be a problem for patients and surgeons alike despite scientific advances. While patient and anatomical characteristics are important to surgeons who are evaluating patients with anal fistulas, their development and persistence likely involves a multifaceted interaction of histological, microbiological, and molecular factors. Histological studies have shown that anal fistulas are variably epithelialized and are surrounded by dense collagen tissue with pockets of inflammatory cells. Yet, it remains unknown if or how histological differences impact fistula healing. The presence of a perianal abscess that contains gut flora commonly leads to the development of anal fistula. This implies a microbiological component, but bacteria are infrequently found in chronic fistulas. Recent work has shown an increased expression of proinflammatory cytokines and epithelial to mesenchymal cell transition in both cryptoglandular and Crohn's perianal fistulas. This suggests that molecular mechanisms may also play a role in both fistula development and persistence. The aim of this study was to examine the histological, microbiological, molecular, and host factors that contribute to the development and persistence of anal fistulas.

microRNA from brush biopsy to characterize oral squamous cell carcinoma epithelium.

Few cancers are diagnosed based on RNA expression signatures. Oral squamous cell carcinoma (OSCC) is no exception; it is currently diagnosed by scalpel biopsy followed by histopathology. This study sought to identify oral tumor epithelial microRNA (miRNA) expression changes to determine if these changes could be used to diagnose the disease noninvasively. Analysis of miRNA profiles from surgically obtained OSCC tissue, collected under highly standardized conditions for The Cancer Genome Atlas, was done to determine the potential accuracy in differentiating tumor from normal mucosal tissue. Even when using small 20 subject datasets, classification based on miRNA was 90 to 100% accurate. To develop a noninvasive classifier for OSSC, analysis of brush biopsy miRNA was done and showed 87% accuracy in differentiating tumor from normal epithelium when using RT-qPCR or miRNAseq to measure miRNAs. An extensive overlap was seen in differentially expressed miRNAs in oral squamous cell carcinoma epithelium obtained using brush biopsy and those reported in saliva and serum of oral squamous cell carcinoma patients in several studies. This suggested that nonselective release of these miRNAs into body fluids from tumor epithelium was largely responsible for the changes in levels in these fluids seen with this disease. Using a variation in mirRPath we identified the KEGG pathway of neurotrophin signaling as a target of these miRNAs disregulated in tumor epithelium. This highlights the utility of brush biopsy of oral mucosa to allow simple acquisition of cancer relevant miRNA information from tumor epithelium.

A loss of profilin-1 in late-stage oral squamous cell carcinoma.

BACKGROUND: The genes for PFN1 and TMSB4 are both highly expressed in oral tissue and both encode actin monomer binding proteins thought to play a role in cell motility and possibly other crucial parts of tumor progression. METHODS: Oral brush cytology of epithelium from oral squamous cell carcinoma (OSCC) was used to measure PFN1 and TMSB4 mRNA in OSCC, while immunohistochemical analysis of tissue was used to check protein levels. RESULTS: High but variable expression of mRNAs encoding these two proteins was observed suggesting they may contribute to tumor characteristics in a subset of OSCCs. Both proteins were highly expressed in normal appearing basal epithelium, in the cytoplasm, and perinuclear area, while expression was minimal in upper epithelial layers. In OSCCs, expression of these proteins varied. In tumors classified as later stage, based on size and/or lymph node involvement, PFN1 levels were lower in tumor epithelium. A control gene, KRT13, showed expression in normal differentiated basal and suprabasal oral mucosa epithelial cells and as reported was lost in OSCC cells. CONCLUSION: Loss of PFN1 in tumor cells has been associated with lymph node invasion and metastasis in other tumor types, strengthening the argument that the protein has the potential to be a tumor suppressor in late-stage OSCC.

Similar Squamous Cell Carcinoma Epithelium microRNA Expression in Never Smokers and Ever Smokers.

The incidence of oral tumors in patients who never used mutagenic agents such as tobacco is increasing. In an effort to better understand these tumors we studied microRNA (miRNA) expression in tumor epithelium of never tobacco users, tumor epithelium of ever tobacco users, and nonpathological control oral epithelium. A comparison of levels among 372 miRNAs in 12 never tobacco users with oral squamous cell carcinoma (OSCC) versus 10 healthy controls was made using the reverse transcription quantitative polymerase chain reaction. A similar analysis was done with 8 ever tobacco users with OSCC. These comparisons revealed miR-10b-5p, miR-196a-5p, and miR-31-5p as enriched in the tumor epithelium in OSCC of both never and ever tobacco users. Examination of The Cancer Genome Atlas (TCGA) project miRNA data on 305 OSCCs and 30 controls revealed 100% of those miRNAs enriched in never smoker OSCCs in this patient group were also enriched in ever smoker OSCCs. Nonsupervised clustering of TCGA OSCCs was suggestive of two or four subgroups of tumors based on miRNA levels with limited evidence for differences in tobacco exposure among the groups. Results from both patient groups together stress the importance of miR196a-5p in OSCC malignancy in both never and ever smokers, and emphasize the overall similarity of miRNA expression in OSCCs in these two risk groups. It implies that there may be great similarity in etiology of OSCC in never and ever smokers and that classifying OSCC based on tobacco exposure may not be helpful in the clinic.

Gene expression based evidence of innate immune response activation in the epithelium with oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is a disease of the oral mucosa of unknown cause producing lesions with an intense band-like inflammatory infiltrate of T cells to the subepithelium and keratinocyte cell death. We performed gene expression analysis of the oral epithelium of lesions in subjects with OLP and its sister disease, oral lichenoid reaction (OLR), in order to better understand the role of the keratinocytes in these diseases. DESIGN: Fourteen patients with OLP or OLR were included in the study, along with a control group of 23 subjects with a variety of oral diseases and a normal group of 17 subjects with no clinically visible mucosal abnormalities. Various proteins have been associated with OLP, based on detection of secreted proteins or changes in RNA levels in tissue samples consisting of epithelium, stroma, and immune cells. The mRNA level of twelve of these genes expressed in the epithelium was tested in the three groups. RESULTS: Four genes showed increased expression in the epithelium of OLP patients: CD14, CXCL1, IL8, and TLR1, and at least two of these proteins, TLR1 and CXCL1, were expressed at substantial levels in oral keratinocytes. CONCLUSIONS: Because of the large accumulation of T cells in lesions of OLP it has long been thought to be an adaptive immunity malfunction. We provide evidence that there is increased expression of innate immune genes in the epithelium with this illness, suggesting a role for this process in the disease and a possible target for treatment.

A prototype tobacco-associated oral squamous cell carcinoma classifier using RNA from brush cytology.

BACKGROUND: Oral cancer in the form of squamous cell carcinoma (OSCC) is typically detected in advanced stages when treatment is complex and may not be curative. The need for surgical biopsy may contribute to delays in diagnosis and impede early detection. Multiple studies of RNA from surgically obtained tumor samples have revealed many genes differentially expressed with this disease. We sought to determine whether the identified mRNAs could be used as markers by a non-invasive detection system for OSCC using RNA from brush cytology. METHODS: Levels of mRNAs from 21 genes known to be differentially expressed in head and neck squamous cell carcinoma surgical samples, compared with controls, were shown to be quantifiable in oral brush cytology samples. These mRNAs were quantified in a training set of 14 tumor and 20 non-malignant brush cytology samples from tobacco/betel nut users. With the measurement of two additional mRNAs and analysis using support vector machines algorithm for class prediction of these cancers was produced. RESULTS: This OSCC classifier based on the levels of 5 mRNAs in RNA from brush cytology initially showed 0.93 sensitivity and 0.91 specificity in differentiating OSCC from benign oral mucosal lesions based on leave-one-out cross-validation. When used on a test set of 19 samples from 6 OSCCs and 13 non-malignant oral lesions, we found misclassification of only one OSCC and one benign lesion. CONCLUSIONS: This shows the promise of using RNA from brush cytology for early OSCC detection and the potential for clinical usage of this non-invasive classifier.

Analysis of RNA from brush cytology detects changes in B2M, CYP1B1 and KRT17 levels with OSCC in tobacco users.

RNA expression analysis of oral keratinocytes can be used to detect early oral cancer, but a limitation is the inability to obtain high quality RNA from oral tissue without using biopsies. While oral cytology cell samples can be obtained from patients in a minimally invasive manner, they have not been validated for quantitative analysis of RNA expression. Earlier we showed RNA from brush cytology of hamster Oral Squamous Cell Carcinoma (OSCC) demonstrated differential expression of B2M and CYP1B1 using real time RT-PCR in a dibenz[a,I]pyrene, tobacco carcinogen, induced model of this disease. Here we show reproducibility of this approach to measuring gene expression in humans. Cytology brush samples from 12 tobacco and betel related OSCC and 17 nonmalignant oral lesions revealed B2M mRNA was enriched in tumor samples while CYP1B1 mRNA was reduced, similar to what was seen in the model system. Additionally, we showed that KRT17 mRNA, a gene linked to OSCC in another brush cytology study, was also enriched in OSCC versus nonmalignant lesions, again supporting the promise of using RNA from brush oral cytology to reproducibly monitor oral gene expression.

Brand specific responses to smokeless tobacco in a rat lip canal model.

BACKGROUND: Different compositions of smokeless tobacco (ST) are widely thought to cause oral carcinoma at different rates but there is little direct evidence for this hypothesis. METHODS: We used a rat lip canal model to examine the mucosal changes induced by chronic daily exposure to four different brands of ST: Skoal, Copenhagen, Ettan Swedish Snus, and Stonewall, differing in measured levels of: tobacco specific nitrosamines (TSNAs), unprotonated nicotine, moisture, and pH. RESULTS: Exposure to the lip canal for 12 months produced changes in the mucosa marked by increases in S phase and M phase cells for the Skoal and Copenhagen exposed rats. This correlated with the high level of TSNAs and nicotine in these products. All the tobacco products, to different degrees, induced sites of moderate to severe dysplasia some with extensive rete peg outgrowth from the oral mucosa not seen in the controls. Many of these sites showed a loss of p16 expression. CONCLUSIONS: While all ST products caused dysplasia, the products with lower levels of TSNAs and unprotonated nicotine caused less, consistent with the model that tobacco with low levels of nitrosamines might potentially induce fewer carcinomas in human users.

Pediatric squamous cell carcinoma arising in an alpha-fetoprotein-producing mature cystic teratoma of the mandible.

Teratomas, most often diagnosed in younger patients, represent the most frequently identified subtype of pediatric germ cell tumors. It is very uncommon for teratomas to present in the head and neck region and demonstrate malignant transformation. We present a case of squamous cell carcinoma arising in an alpha-fetoprotein-producing cystic teratoma of the mandible in a 2-year-old female that is, to the best of our knowledge, the first such published report. The patient was treated with surgical excision along with chemotherapy and has remained disease-free 2 years after the conclusion of therapy.

Transcriptomic dissection of tongue squamous cell carcinoma.

BACKGROUND: The head and neck/oral squamous cell carcinoma (HNOSCC) is a diverse group of cancers, which develop from many different anatomic sites and are associated with different risk factors and genetic characteristics. The oral tongue squamous cell carcinoma (OTSCC) is one of the most common types of HNOSCC. It is significantly more aggressive than other forms of HNOSCC, in terms of local invasion and spread. In this study, we aim to identify specific transcriptomic signatures that associated with OTSCC. RESULTS: Genome-wide transcriptomic profiles were obtained for 53 primary OTSCCs and 22 matching normal tissues. Genes that exhibit statistically significant differences in expression between OTSCCs and normal were identified. These include up-regulated genes (MMP1, MMP10, MMP3, MMP12, PTHLH, INHBA, LAMC2, IL8, KRT17, COL1A2, IFI6, ISG15, PLAU, GREM1, MMP9, IFI44, CXCL1), and down-regulated genes (KRT4, MAL, CRNN, SCEL, CRISP3, SPINK5, CLCA4, ADH1B, P11, TGM3, RHCG, PPP1R3C, CEACAM7, HPGD, CFD, ABCA8, CLU, CYP3A5). The expressional difference of IL8 and MMP9 were further validated by real-time quantitative RT-PCR and immunohistochemistry. The Gene Ontology analysis suggested a number of altered biological processes in OTSCCs, including enhancements in phosphate transport, collagen catabolism, I-kappaB kinase/NF-kappaB signaling cascade, extracellular matrix organization and biogenesis, chemotaxis, as well as suppressions of superoxide release, hydrogen peroxide metabolism, cellular response to hydrogen peroxide, keratinization, and keratinocyte differentiation in OTSCCs. CONCLUSION: In summary, our study provided a transcriptomic signature for OTSCC that may lead to a diagnosis or screen tool and provide the foundation for further functional validation of these specific candidate genes for OTSCC.

RNA from brush oral cytology to measure squamous cell carcinoma gene expression.

BACKGROUND: RNA expression analysis of oral keratinocytes can be used to detect early stages of disease such as oral cancer or to monitor on-going treatment responses of the same or other oral diseases. A limitation is the inability to obtain high quality RNA from oral tissue without using biopsies. While oral cytology cell samples can be obtained from patients in a minimally invasive manner they have not been validated for quantitative analysis of RNA expression. METHODS: As a starting point in the analysis of tumor markers in oral squamous cell carcinoma (OSCC), we examined RNA in brush cytology samples from hamsters treated with dibenzo[a,l]pyrene to induce oral carcinoma. Three separate samples from each animal were assessed for expression of candidate marker genes and control genes measured with real-time RT-PCR. RESULTS: Brush oral cytology samples from normal mucosa were shown to consist almost exclusively of epithelial cells. Remarkably, ss-2 microglobulin and cytochrome p450, 1B1 (CYP1B1) RNA showed potential utility as markers of OSCC in samples obtained in this rapid and non-surgical manner. CONCLUSION: Brush oral cytology may prove useful as a source of RNA for gene expression analysis during the progression of diseases of the oral epithelium such as OSCC.

Cytotoxicity of liposomal alpha-tocopheryl succinate towards hamster cheek pouch carcinoma (HCPC-1) cells in culture.

There is compelling evidence for the cancer chemopreventive effects of vitamin E and related compounds. Of all the vitamin E derivatives that have been investigated to date, vitamin E acid succinate is the most effective anti-cancer agent. This report describes the preparation and testing of liposomal formulation of mono alpha-tocopheryl ester of succinic acid (alpha-TOS) for cytotoxicity against hamster cheek pouch carcinoma cell line (HCPC-1). Small unilamellar vesicles (SUV) of phosphatidylcholine incorporating 70 microM alpha-TOS were superior to alpha-TOS alone or SUV without incorporated alpha-TOS, as inducers of apoptosis in HCPC-1 cells. Liposomal alpha-TOS perturbed the lipid structure in cells, promoted apoptosis, and decreased cell viability. The mechanism of action of alpha-TOS appears to involve membrane damage and induction of ceramide mediated apoptosis.

Molecular and cellular effects of green tea on oral cells of smokers: a pilot study.

Studies in cell culture and laboratory animals have shown that green tea and its major component, epigallocatechin-3-gallate, inhibit cell growth and reduce tumor incidence. However, results of epidemiological studies have generated inconsistent, sometimes conflicting data regarding protection by green tea against human cancers. To clarify the findings of these laboratory studies in application to humans, we conducted a pilot intervention study with three heavy smokers (> 10 cigarettes/day) and three nonsmokers (never smokers) in order to evaluate the molecular and cellular effects of drinking green tea using human oral cells as an investigative tool. Green tea total extract (400-500 mg/cup, 5 cups/day) was administered in drinking water to the subjects for four weeks. Two oral cytology samples were taken weekly for measurements of tobacco carcinogen-induced DNA damage, including bulky adducts and oxidized bases, cell growth, DNA content, and apoptosis. The study showed that during the course of green tea administration smoking-induced DNA damage was decreased, cell growth was inhibited, and the percentage of cells in S phase was reduced, cells accumulated in G1 phase (cyclin D1 positive), DNA content became more diploid and less aneuploid, and p53, Caspase-3, and TUNEL, markers of apoptosis, were increased. The study, although preliminary, indicates that drinking green tea reduced the number of damaged cells in smokers by inducing cell growth arrest and apoptosis, a mechanism similar to that observed in cultured cells and animals. These results warrant a large-scale intervention trial to further verify the role of green tea in the prevention of oral cancer in smokers.