In vitro growth changes of oral human keratinocytes after treatment with carotenoids, retinoid, and/or DMBA

Abstract

In vitro changes of normal human keratinocytes (NHKs) derived from the oral mucosa after treatment with the chemical carcinogen 7,12 dimethylbenz[a]anthracene (DMBA; 5, 50, 200 ng/10 ml) were evaluated. NHKs were also treated with chemopreventive nutrient agents that previously had enhanced growth of epidermal and oral keratinocytes or suppressed growth of oral squamous cell carcinoma. These agents included the carotenoids β‐carotene and canthaxanthin and the retinoid retinyl palmitate (60 μM). Plating efficiency, growth in agarose (independent growth), viability [tetra‐zolium salt (MTT) assay], and proliferation ([³H]thymidine labeling) defined the growth of NHKs. The number of corni‐fied cells and keratin expression (high‐molecular‐weight keratin) defined differentiation. γ‐Glutamyl transpeptidase, p53 expression, and tumorigenesis in mice defined oxidation and malignant transformation. Treatment with DMBA (50 ng/10 ml) was detected by autofluorescence; it produced an increase in pleomorphism and multinucleation and enhanced plating efficiency and the number of colonies grown in agarose. Chemopreventive treatment enhanced the number of colonies grown in agarose, but the MTT levels and [³H]thymidine incorporation‐proliferation (24 h) were reduced. Chemopre‐ventives also increased differentiation defined by the number of cornified cells and the expression of high‐molecular‐weight keratin‐positive cells. Malignant transformation potential was depressed by reducing γ‐glutamyl transpeptidase and mutant p53 expression, whereas tumor suppressor p53 was enhanced. NHKs treated with DMBA and injected into nude mice (nu/nu; 1 × 10⁶ cells/0.25 ml) produced tumor masses (3 of 3 animals), whereas the nutrient and DMBA groups produced smaller tumor masses, some with central ulcers (2 of 3 animals). Mock injection of untreated or nutrient‐treated NHKs without DMBA treatment did not produce a tumor mass (0 of 3 animals). β‐Carotene, retinyl palmitate, and canthaxanthin increased differentiation and reduced transformation induced by DMBA in oral NHKs.