N-Alkoxy derivatization of indole-3-carbinol increases the efficacy of the G1 cell cycle arrest and of 13C-specific regulation of cell cycle gene transcription and activity in human breast cancer cells

Indole-3-carbinol (13C), a naturally occurring component of Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts, induces a G1 cell cycle arrest of human breast cancer cells. Structure-activity relationships of 13C that mediate this anti-proliferative response were investigated using synthetic and natural 13C derivatives that contain substitutions at the indole nitrogen. Nitrogen substitutions included N-alkoxy substituents of one to four carbons in length, which inhibit dehydration and the formation of the reactive indolenine. Analysis of growth and cell cycle arrest of indole-treated human breast cancer cells revealed a striking increase in efficacy of the N-alkoxy 13C derivatives that is significantly enhanced by the presence of increasing carbon lengths of the N-alkoxy substituents. Compared to 13C, the half maximal growth arrest responses occurred at 23-fold lower indole concentration for N-methoxy 13C, 50-fold lower concentration for N-ethoxy 13C, 217-fold lower concentration for N-propoxy 13C, and 470-fold lower concentration for N-butoxy 13C. At these lower concentrations, each of the N-alkoxy substituted compounds induced the characteristic 13C response in that CDK6 gene expression, CDK6 promoter activity, and CDK2 specific enzymatic activity for its retinoblastoma protein substrate were strongly down regulated. 3-Methoxymethylindole and 3-ethoxymethylindole were approximately as bioactive as 13C, whereas both tryptophol and melatonin failed to induce the cell cycle arrest, showing the importance of the C-3 hydroxy methyl substituent on the indole ring. Taken together, our study establishes the first 13C structure-activity relationship for cytostatic activities, and implicates 13C-based N-alkoxy derivatives as a novel class of potentially more potent experimental therapeutics for breast cancer. (c) 2007 Elsevier Inc. All rights reserved.