The Effects of Sorbic Acid, All-trans-retinoic Acid StructuralAnalog, on Retinoids Metabolism in Squamous Cell CarcinomaAMC-HN-6 Cell Line

Choi, Jee Ho; Chung, Eui Chang; Sung, Kyung Jeh; Moon, Kee Chan; Koh, Jai Kyoung; Kim, Sang Yoon; Han, In Seok

Abstract

BACKGROUND: All-trans-retinoic acid metabolism by cytochrome P450 is one of the major mechanisms that can regulate the level of retinoids in cells. Therefore, enhanced metabolism of all-trans retinoic acid by all-trans retinoic acid induced cytochrome P450 would probably decrease the therapeutic effects of active retinoids. We previously reported that the tail of all-trans retinoic acid (the carboxyl-terminus) may bind to a binding site of cytochrome P450 in part by electrostatic forces, and the head of RA (the beta-cyclogeranylidene ring) may bind to an active site of cytochrome P450 in part by hydrophobic forces. It is very interesting to study the interactions between the RA binding site of cytochrome P450 induced by all-trans retinoic acid and the structural analogs of all-trans retinoic acid and its effects of RA metabolism.
OBJECTIVE
The purpose of this study is to examine the effects of sorbic acid, that has a similar structure with the tail of all-trans retinoic acid, on RA metabolism in head-neck squamous cell carcinoma cell line AMC-HN-6 which showed a much increased induction of cytochrome P450 by all-trans retinoic acid.
METHODS
We examined the effects of sorbic acid on RA metabolism in all-trans retinoic acid specific cytochrome P450-inducible AMC-HN-6 cell line using cytochrome P450 enzyme assay with total cell lysates and microsomal proteins. Radioisotope-labeled polar metabolites of all-trans retinoic acid were separated by thin layer chromatography and the radioactivity was measured by beta-counter. Metabolic activity was expressed as the percentage of total radioactivity of polar metabolites.
RESULTS
The results are summurized as follows: 1. RA metabolism of AMC-HN-6 cell line was inhibited by actinomycin D and cyclohexamide and was also inhibited by ketoconazole, the cytochrome P450 inhibitor, in a concentration-dependent manner. 2. Cytochrome P450-mediated oxidation was induced by all-trans retinoic acid, 13-cis-RA, 9-cis-RA, and retinal, but not by retinol in AMC-HN-6 cell line.3. Sorbic acid inhibited RA metabolism of AMC-HN-6 cell line in a concentration-dependent manner when the enzyme assay was performed on microsomal protein but could not inhibit RA metabolism in total cell lysate enzyme assay.
CONCLUSION
The conversion of all-trans retinoic acid to polar metabolites is inhibited by sorbic acid in microsomal enzyme assay of AMC-HN-6 cell line, but not in total cell assay. These results suggest that sorbic acid can bind to the active binding site of cytochrome P450 but binding affinity of sorbic acid to RA binding molecules such as CRABP-I,-II, RARs, RXRs may be stronger than that of sorbic acid to cytochrome P450.