Experimental oral carcinoma of the tongue and buccal mucosa: possible biologic markers linked to cancers at two anatomic sites.

The application of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) can initiate and promote the development of oral squamous cell carcinoma of the tongue and buccal mucosa. In this study the level of expression of various markers related to the development of programmed cell death (PCD) in the respective oral carcinomas was analyzed. Sixteen male and female Syrian hamsters (Mesocrietus auratus) were treated with 0.05% DMBA for 16 weeks. Immunohistochemistry was used to observe the expression of p53, proliferating cell nuclear antigen (PCNA), Bcl-2, and nucleosome formation. Single-strand conformational polymorphism (SSCP) for exons 2-9 and sequence analysis of exon 9 of the p53 gene from normal buccal or tongue mucosa as well as the squamous cell carcinomas from the buccal mucosa or the tongue were determined. p53 (wild type) expression was significantly reduced in the tongue dysplastic mucosa or squamous cell carcinoma. The SSCP disclosed banding shifts or new bands in exons 2/3, 4, 8, and 9 for the tongue or buccal oral carcinomas (five of each). In exon 9 the mutation in codon 307 (ala)GCC-GTC(val) was present in the tongue but not in the buccal carcinoma. Other markers included the level of PCNA. PCNA was initially lower in the premalignant tongue lesions but increased in oral squamous cell carcinoma at both sites. In contrast, the amount of nucleosome formation in the tongue carcinomas was less than the level noted for buccal cancers but premalignant dysplasias in the tongue mucosa exhibited higher levels. The inhibitor of PCD, Bcl-2 was lower for dysplasias and carcinomas of the tongue compared to similar lesions of the buccal mucosa. These results indicate that oral carcinomas of different anatomical sites can exhibit differences in growth, oncogene mutation expression, and the development of PCD. The differences in Bcl-2 and nucleosome formation may signify their influence on oncogene expression and growth potential for developing transformed clones and established oral carcinomas.

Eustachio and "Libellus de dentibus" the first book devoted to the structure and function of the teeth.

Bartolomeo Eustachio (1520-1574), a major anatomist and physician of the Italian Renaissance, made very significant contributions toward our knowledge of the anatomy and physiology of the dentition, including the first descriptions of the structure of the dental pulp and of the periodontal membrane. His treatise on the teeth, "Libellus de Dentibus", published in 1563, has just been translated into English, and the remarkable descriptions of the dental pulp, the periodontal membrane, the development of both sets of teeth from dental follicles, the trigeminal nerve, and other oral structures have not been fully appreciated. We offer extensive translated material from his treatise to establish the unique insight that Eustachio had into the structure and function of the human dentition, based upon extensive dissections of both human and animal material. Eustachio also had very modern ideas concerning the treatment of periodontitis, including the curettage of granulation tissue to promote reattachment of the gingival tissue.

Experimental basis for cancer prevention by vitamin E.

Recent clinical trials have demonstrated the significant cancer preventive potential of vitamin E in many different cancer sites, ranging from oral and pharyngeal cancer to prostate cancer. There is an extensive experimental basis for this clinical cancer inhibition. The experimental background includes animal studies (experimental pathology, immunology and molecular biology, synergism, selectivity and safety), in vitro biochemical studies, and human studies (epidemiology and biomarkers, prevention of many pathologic entities other than cancer).

Leeuwenhoek and Vermeer, an association of genius.

Antony van Leeuwenhoek, the inventor of the microscope and originator of the microscopic sciences, had an interesting association with the great Dutch artist, Vermeer, whose paintings were recently displayed in major exhibitions in Holland and the U.S.A. Leeuwenhoek is of particular interest to dental medicine for the first description of the oral bacteria and the first microscopic description of the stratified squamous epithelium of the oral mucosa, with its different layers from stratum germinativum to stratum corneum.

Mechanisms of cancer inhibition by anti-oxidant nutrients.

The cancer inhibitory properties of anti-oxidant micronutrients have been well established in experimental animal models and cell culture studies. Human studies have also tended to indicate an inhibition of various forms of cancer and the regression of some precancerous lesions. The biological mechanisms for cancer inhibition and regression are now gradually becoming understood, and the anti-oxidant nutrients appear to act through a number of pathways common to most of the agents studied. These various micronutrients appear to act through a complex group of "common pathways" of anticancer activity based upon three major mechanisms: (1) tumour inhibition by immune cytokines; (2) stimulation of cancer suppressor genes, such as "wild type" p53, and diminished expression or dysregulation of oncogenes such as mutant p53 and H-ras; (3) inhibition of tumour angiogenesis through the inhibition of angiogenesis-stimulating factors such as TGF alpha. Retinoid action differs, in some respects, from other micronutrient anticancer mechanisms and appears to relate to its stimulation of cellular differentiation and resultant apoptosis of neoplastic cells. Combinations of anti-oxidant nutrients have been shown to be synergistic in their anticancer activity, probably due to their optimal anticancer activity at different oxygen potentials. Selectivity in the action on cancer cells, as opposed to normal cells, is a major feature of the anti-oxidant micronutrients.

Retinoid and carotenoid angiogenesis: a possible explanation for enhanced oral carcinogenesis.

The carotenoids beta-carotene and canthaxanthin and the retinoid 13-cis-retinoic acid (13-RA) have inhibited oral carcinogenesis in the hamster cheek pouch (16 wks, 3 times/wk at 1.4 mg/kg) induced by an 0.5% solution of 7, 12-dimethylbenz[a]anthracene (DMBA). However, 13-RA at a higher dose (> 2.0 mg/kg per treatment) increased squamous cell carcinoma growth (Eur J Cancer Clin Oncol 24, 839-850, 1988). 13-RA, beta-carotene, and canthaxanthin administered to 60 hamsters (16 wks, 3 times/wk, 10 mg/kg) altered neovascularization characterized by immunohistochemistry for transforming growth factor-alpha (TGF-alpha) and factor VIII. 13-RA + DMBA resulted in more smaller-sized tumors, with a reduced volume and tumor burden (tumor controls, 185.9; 13-RA + DMBA, 151.0). The carotenoids reduced the number and the sizes of the carcinomas formed (beta-carotene, 60 tumors, 142.3 x 10(3) mm3; canthaxanthin, 30 tumors, 116.1 x 10(3) mm3). Factor VIII and TGF-alpha were expressed in high intensity at cancer sites of the 13-RA + DMBA and DMBA groups with > 50 and > 10 cells, respectively, per x 400 field. In contrast, beta-carotene- and canthaxanthin + DMBA-treated pouches showed > 20 and 5 cells, respectively, per x 400 field for factor VIII and TGF-alpha. These results suggest that 13-RA treatment may increase vascular growth, but the carotenoids also produced enhanced levels of endothelial cell growth and TGF-alpha compared with the untreated mucosa. The carotenoids may enhance tumor growth under the appropriate carcinogenic environment.

Verification in syngeneic hamsters of in vitro transformation of hamster oral mucosa by 7,12-dimethylbenz(a)anthracene.

The carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) has been used to induce oral carcinogenesis of the hamster buccal mucosa in an experimental model that exhibits many of the genetic, biochemical and pathological features of human oral squamous cell carcinoma. To complement this in vivo process we have established an in vitro transformation procedure that involved the treatment of normal hamster oral mucosal keratinocytes (NHKs) with DMBA. Uptake of DMBA in NHKs was verified by observing autofluorescence of DMBA in the oral mucosal cells. Treatment doses ranged from 5, 50 and 200 ng and the NHKs were generally treated with DMBA for 1-14 days. The 200 ng dose proved to be toxic to these cells. The 5 and 50 ng treatments were found to maintain the viability of the NHKs and demonstrate anchorage-independent agarose growth, producing 18 and 40 colonies, respectively, after 14 days of treatment. Characterisation assays included determinations for cellular growth through plating efficiency, counting of cell colony number, and 3H-thymidine incorporation. Differentiation was ascertained by counting cornified cells, specification of either high or low molecular weight keratins, the percentage of cells expressing gamma glutamyl-transpeptidase (GGT), the level of p53 expression, and a determination of cell cycle. After 24 h the plating efficiency of the NHKs was found to be slightly increased following treatment with a 5 or 50 ng dose of DMBA compared to the untreated NHKs. After 14 days of incubation these doses also enhanced the number of colonies formed by the NHKs (e.g. plating efficiency). In contrast, the number of cornified cells was reduced in these colonies, while immunohistochemistry disclosed an increase in the number of NHKs expressing low molecular weight keratins, significantly lower levels of high molecular weight keratins and high levels for GGT. Flow cytometric analysis verified an increase in p53 expression (e.g. p53 wild type, 19% and p53 mutant, 66%). Cell cycle analysis of NHKs treated with DMBA (5 ng) demonstrated a shift in the number of cells in S phase (17.2%) and G2 + mitosis (11.0%). Cells from this DMBA treatment group were injected into syngeneic hamster recipient buccal pouches (10 x 10(6) cells/0.25 ml). Squamous carcinomas grew in four of six hamster buccal pouches as determined by histopathological analysis. The in vitro assay system will enhance our ability to define the genetic and molecular changes related to chemical carcinogenesis and oral malignant transformation.

Vitamin E inhibits experimental carcinogenesis and tumour angiogenesis.

In an experiment in which vitamin E inhibited carcinogenesis, it was found that tumour angiogenesis and tumour growth-factor alpha (TGF alpha) expression were also inhibited. Forty male golden hamsters were divided into four equal groups. Group 1 animals had the left buccal pouches painted three times weekly with 7,12-dimethylbenz(a)anthracene (DMBA) for 14 weeks. Group 2 animals had the same procedure of DMBA applications but also received alpha tocopherol. Groups 3 and 4 were vitamin E and untreated controls. Angiogenesis was studied with factor 8-related antigen (F8-RA) which identifies endothelial cells. TGF alpha was studied with the appropriate antibody. Staining was effected by the standard avidin-biotin horseradish peroxidase system. Mean tumour volume was significantly lower in the DMBA-vitamin E group compared to the tumour control group. Angiogenesis was significantly inhibited in the DMBA-vitamin E group and TGF alpha expression was also inhibited. It is suggested that inhibition of tumour angiogenesis by vitamin E may be an additional mechanism for the anticancer action of vitamin E.

Glutathione inhibits experimental oral carcinogenesis, p53 expression, and angiogenesis.

Previous studies have shown that reduced glutathione (GSH) inhibits experimental oral carcinogenesis in the hamster buccal pouch model. To gain further understanding of molecular mechanisms in the anticancer effect of GSH, these studies examined levels of p53 protein expression. 7,12-Dimethylbenz[a]anthracene (DMBA) was applied to the buccal pouches of 20 Syrian Golden hamsters (Mesocricetus auratus) in a 0.5% solution in mineral oil thrice weekly for 14 weeks. In 10 animals, 10 mg/kg reduced glutathione (GSH) in 0.5 ml of mineral oil was administered by mouth thrice weekly on days alternate to the DMBA painting. An additional 20 animals served as DMBA-untreated and GSH controls. At the termination of the experimental period, there were fewer tumors in the DMBA-GSH than in the DMBA tumor control group, and the tumors were smaller (tumor burden 315 vs. 3,040 mm3). Histologically, the DMBA-GSH group showed a marked reduction in dysplasia, carcinoma in situ, and invasive epidermoid carcinoma sites. Immunohistochemically, by use of monoclonal antibodies for wild-type p53 (PAb 246), changes were observed in protein expression levels at dysplastic sites and within the malignant tumors. Staining for p53 protein was slightly increased in dysplasia and squamous cell carcinoma in the tumor control animals (painted with DMBA) compared with the untreated controls that were free of tumors. In the GSH and DMBA treatment group, p53 protein expression levels were strongly increased in dysplastic and tumor sites. The significant inhibition of oral carcinogenesis associated with the administration of GSH was correlated with the increased levels of the wild-type p53 tumor suppressor gene, suggesting its possible use as a biomarker for GSH chemoprevention. The inhibition of oral carcinogenesis by reduced GSH was also related to a very significant inhibition of tumor angiogenesis, defined by factor VIII staining. Thus angiogenesis inhibition may be an additional mechanism for antioxidant chemoprevention, and this suggests another possible biomarker for antioxidant chemoprevention.

Altered cytokeratin expression in carcinogenesis inhibition by antioxidant nutrients.

Epidermoid carcinomas were induced in hamster buccal pouches with use of 7.12 dimethylbenz[a]anthracene (DMBA). In five animals that served as tumor controls (Group 1), right buccal pouches were painted with DMBA (0.5% solution in mineral oil) thrice weekly for 14 weeks. In five animals (Group 2), right buccal pouches were painted with DMBA and reduced glutathione (GSH) was administered systemically by mouth. Five animals (Group 3) received vitamin E instead of glutathione. An additional 20 animals (Groups 4, 5, 6, and 7) were untreated, vehicle, glutathione, and vitamin E controls, respectively. Glutathione and vitamin E were given in doses of 10 mg/kg in 0.5 ml of mineral oil thrice weekly on days alternate to DMBA painting. Treatment by GSH and vitamin E reduced the number and size of tumors that were formed. Histopathologically, there were also fewer sites of dysplasia, carcinoma in situ, and early invasive epidermoid carcinoma than in the tumor control animals. The formalin-fixed and paraffin-embedded buccal pouch sections were stained immunohistochemically with use of monoclonal antibodies for cytokeratins. These included high-molecular-weight keratins (50,000-68,000 mol wt) 10, 13, and 8 (k10, k13, and k8, respectively). Oral carcinomas and dysplastic sites exhibited basal and suprabasal (spinous layer) high levels of k10, k13, and k8 staining. Treatment with GSH or vitamin E increased the suprabasal staining for high-molecular-weight keratins and reduced the protein expression for k10, k13, or k8. This pattern of staining was observed in dysplastic as well as in carcinoma sites. These results indicate that cytokeratin protein expression could contribute to a common biomarker analysis for chemoprevention.

A common pathway for the destruction of cancer-cells - experimental-evidence and clinical implications.

Chemopreventatives derived from different classes of chemical inhibit the growth of various cancers. This review concentrates on the chemopreventative activities of carotenoids, retinoids, and tocopherols. In this review we outline a common pathway for the antitumor action of chemopreventative agents. To establish this pathway we reviewed previous in vivo and in vitro studies indicating that the underlying modes of actions for chemopreventative activities were oxidative-reduction changes that developed in the tumor cell. Some of these changes were selective and did not appear in normal cells. The oxidative redox characteristics of the agents interacted with the oxidative redox systems of the tumor cell. Many chemopreventative agents are highly hydrophobic and interact with thiol peptides, and/or metalloproteins, such as glutathione reductase, superoxide dismutases, or flavoproteins. These could then become linked to prenylated membrane associated protein complexes or oxidized proteins. Interactions of the chemopreventative agents with these proteins can result in changes in expression of transcription factors nuclear binding proteins, such as, heat shock proteins (hsps) and p53. Dramatic reductions in mutant p53 and increased expression of wild type (antioncogene) p53 protein product has been associated with some chemopreventative agent inhibition of oral carcinogenesis. Enhanced hsp 70 and 90 expression has also been observed. These changes in protein expression occurred early during the process of oral carcinogenesis. The tumor cells accumulated in GO/G1 of the cell cycle and the induction of some apoptotic like changes (programmed cell death) were noted. Cell to cell communication and differentiation has also been increased with carotenoid, retinoid, and tocopherol treatment of transformed cells. Enhanced cytotoxic responses by macrophages and T lymphocytes at developing tumor sites were found to produce the oxygen reactive cytokine, tumor necrosis factor. The experimental evidence and hypothesized common pathway of antitumor action for chemoprevention may point to possible biomarkers that could be utilized in clinical intervention trials.

Vitamin C enhances the development of carcinomas in the hamster buccal pouch experimental model.

Forty young adult male Syrian hamsters (Mesocricetus auratus) were divided into four groups of 10 animals. The animals in group 1 (tumor control) had the right buccal pouches painted three times a week with a 0.5% solution of 7,12 dimethylbenz(a)anthracene in heavy mineral oil USP with the use of a number 4 sable brush. The animals in group 2 (experimental group) had the right buccal pouches painted with the same solution as group 1. In addition, they received 1 mg ascorbic acid in 0.5 ml mineral oil three times a week on days alternating with the other application. The ascorbic acid was administered by mouth with the use of a pipette. The animals in group 3 received 1 mg ascorbic acid in 0.5 ml mineral oil three times weekly, and the animals in group 4 were untreated controls. The animals were killed after 14 weeks. Tumors were counted and measured. Both right and left (control) pouches were photographed, excised, fixed in formalin, sectioned in paraffin, and studied histologically. The animals that received the ascorbic acid (vitamin C) had significantly larger tumors in the right buccal pouch, although actual numbers of gross tumors were only slightly increased. The figures for tumor burden in the animals in groups 1 and 2 were 364 versus 648 mm3. Histologic study revealed that the animals in group 2 had more anaplastic tumors and a significantly greater number of areas of dysplastic leukoplakia than the animals in group 1.

p53 in the anticancer mechanism of vitamin E.

Immunohistochemical techniques were used to study the expression of "wild type" p53 and "mutant" p53 in experimental cancer inhibition by vitamin E. The cancer model used was the squamous cell carcinoma of hamster buccal pouch induced by the carcinogen 7,12 dimethylbenz(a)anthracene (DMBA). Cancer development was studied sequentially for 8-14 weeks and specimens prepared for histological and immunohistochemical interpretation. Primary antibodies used were monoclonal antibodies for "wild type" and "mutant" p53. Specificity of antibodies was confirmed by flow cytometry. Peroxidase-antiperoxidase staining was used on the tissue specimens. In those animals receiving vitamin E the buccal pouch tumour development was significantly inhibited and there was a notable expression of "wild type" p53. There was also a relative absence of "mutant" p53 in the buccal pouch lesions of animals receiving vitamin E. These observations suggest that vitamin E may inhibit cancer formation by stimulating the expression of a cancer suppressor gene.

Inhibition of oral carcinogenesis by glutathione.

Forty young adult Syrian hamsters (Mesocricetus auratus) were divided into four groups of 10 animals each. In Group 1 (tumor control), the right buccal pouches were painted three times per week with a 0.5% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in heavy mineral oil (USP) with a no. 4 sable brush. In Group 2 (experimental group), the right buccal pouches were painted with DMBA, as in Group 1. In addition, Group 2 received 1 mg of reduced glutathione in 0.5 ml of mineral oil three times per week on days alternate to the DMBA application. The glutathione was administered systemically by mouth with a pipette. Group 3 received only glutathione, and Group 4 was untreated (control groups). Animals were sacrificed after 14 weeks, and tumors were counted and measured. Both right and left pouches were photographed, excised, fixed in formalin, sectioned in paraffin, and studied histologically. The animals receiving glutathione demonstrated significantly fewer and smaller tumors. The mean tumor burden was 315 mm3 in the glutathione-treated group and 3,040 mm3 in the untreated group. The statistical significance by Student's t test was < or = 0.0001. Histological study also revealed significantly fewer areas of dysplastic leukoplakia in the group treated with glutathione. This study represents the first demonstration of the anticancer effect of systemically administered reduced glutathione.