Cancer Res Treat.  2007 Jun;39(2):82-87.

The Chemopreventive Effect of Retinoids on Cellular NF-kappa B Activity Induced by NMU and NEU in Human Malignant Keratinocytes

• Moon KY

Abstract

PURPOSE: Retinoids have been shown to be effective in suppressing tumor development when chemical carcinogens such as N-nitroso-N-methylurea (NMU) and N- nitroso-N-ethylurea (NEU) were used to induce mammary tumors in a variety of animal models. However, the molecular mechanisms associated with the retinoid- mediated chemopreventive process, as linked to transcription factor NF-kappa B activation, for chemoprevention have not been elucidated. The purpose of this study was to determine the implications of NF-kappa B activation on the chemopreventive role of retinoids and their effect on cellular NF-kappa B activity that's induced by known alkylating chemical carcinogens such as NMU and NEU in human transfectant squamous cell carcinoma (SCC-13) cells.
MATERIALS AND METHODS
The activity of NF-kappa B, as regulated by chemical carcinogens and retinoids, was determined in cultured human SCC-13 keratinocytes that were transfected with the pNF-kappa B-SEAP-NPT plasmid; this permitted the expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-kappa B activity, and the plasmid contained the neomycin phosphotransferase (NPT) gene, which confers resistance to geneticin. The reporter enzyme activity was measured using a fluorescence detection assay method.
RESULTS
All-trans retinoic acid and 13-cis retinoic acid induced a reduction of NF-kappa B activity up to 64% and 65%, respectively, compared to the control. For the treatment of the human transfectant cells with chemical carcinogens, all-trans retinoic acid (5 mM) and 13-cis retinoic acid (5 mM) downregulated the cellular NF-kappa B activation up to 83% and 85% compared to the NF-kappa B activity that was upregulated by NMU (5 micro M) and NEU (5 micro M), respectively.
CONCLUSION
These results suggest that the chemopreventive effect of retinoids may be mediated by the down- regulated activation of NF-kappa B and that retinoids are implicated in the activation of NF-kappa B in human skin cells.

Keyword

Retinoids; NMU; NEU; Chemoprevention; NF-kappa B; Human keratinocytes

MeSH Terms

Alkaline Phosphatase
Carcinogens
Carcinoma, Squamous Cell
Chemoprevention
Fluorescence
Genes, Reporter
Humans*
Kanamycin Kinase
Keratinocytes*
Models, Animal
NF-kappa B*
Plasmids
Retinoids*
Skin
Transcription Factors
Tretinoin
Alkaline Phosphatase
Carcinogens
Kanamycin Kinase
NF-kappa B
Retinoids
Transcription Factors
Tretinoin

Figure

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  Fig. 1 Diagram of the pNF-κB-SEAP-NPT plasmid. The plasmid permits expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-κB activity and it contains the neomycin phosphotransferase (NPT) gene for the geneticin resistance in host cells.

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  Fig. 2 Dose-dependent downregulation by retinoic acid on the cellular NF-κB activity in human transfectant SCC-13 cells. Panel (A), All-trans retinoic acid effect on the cellular NF-κB activity; Panel (B), 13-cis retinoic acid effect on the cellular NF-κB activity. Retinoids were added to the culture medium after 48 h of incubation. The SEAP activities were measured 24 h after exposure to chemicals. Each value represents the mean±SD of three determinations. RLU stands for relative light units. A significant difference in the NF-κB activities between retinoid treated cells and the control is indicated by *p<0.01.

• 

  Fig. 3 Dose-dependent downregulation by All-trans retinoic acid on the cellular NF-κB activity induced by NMU and NEU in human transfectant SCC-13 cells. Panel (A), All-trans retinoic acid effect on the NMU-upregulated cellular NF-κB activity; Panel (B), All-trans retinoic acid effect on the NEU-upregulated cellular NF-κB activity. A significant difference in the NF-κB activities between the control and alkylating carcinogen treated cells is indicated by *p<0.01. A significant difference in the NF-κB activities between alkylating carcinogen treated cells and the all-trans retinoic acid treated cells is indicated by †p<0.01.

• 

  Fig. 4 Dose-dependent downregulation by 13-cis retinoic acid on the cellular NF-κB activity induced by NMU and NEU in human transfectant SCC-13 cells. Panel (A), 13-cis retinoic acid effect on the NMU-upregulated cellular NF-κB activity; Panel B, 13-cis retinoic acid effect on the NEU-upregulated cellular NF-κB activity. A significant difference in the NF-κB activities between the control and alkylating carcinogen treated cells is indicated by *p<0.01. A significant difference in the NF-κB activities between the alkylating carcinogen treated cells and the 13-cis retinoic acid treated cells is indicated by †p<0.01. Alkylating chemicals and retinoids were added to the culture medium at 0 and 48 h of incubations, respectively. The SEAP activities were measured at 24, 48 and 72 h after exposure to the chemicals. Each value represents the mean±SD of three determinations. RLU stands for relative light units.

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