N-Alkoxy derivatization of indole-3-carbinol increases the efficacy of the G1 cell cycle arrest and of I3C-specific regulation of cell cycle gene transcription and activity in human breast cancer cells

Abstract

Indole-3-carbinol (I3C), 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 I3C that mediate this anti-proliferative response were investigated using synthetic and natural I3C 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 I3C derivatives that is significantly enhanced by the presence of increasing carbon lengths of the N-alkoxy substituents. Compared to I3C, the half maximal growth arrest responses occurred at 23-fold lower indole concentration for N-methoxy-I3C, 50-fold lower concentration for N-ethoxy-I3C, 217-fold lower concentration for N-propoxy-I3C, and 470-fold lower concentration for N-butoxy-I3C. At these lower concentrations, each of the N-alkoxy substituted compounds induced the characteristic I3C 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 I3C, whereas, both tryptophol and melatonine 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 I3C structure activity relationship for cytostatic activities, and implicates I3C-based N-alkoxy derivatives as a novel class of potentially more potent experimental therapeutics for breast cancer.

Indole-3-carbinol (I3C) increases apoptosis, represses growth of cancer cells, and enhances adenovirus-mediated oncolysis

“…When I3C was fed in the diet of A/J mice induced by tobacco smoke carcinogen, lung tumorigenesis was inhibited through inhibition of proliferation and induction of apoptosis in tumor cells. [9][10][11] Similar results were observed with mouse models of other cancers, such as breast cancer 12 and skin tumor. 13 Limited and inconclusive studies suggest that I3C may have anticarcinogenic properties in a wide range of cancers; thus it is used as a dietary supplement in the hope of cancer prevention.…”

Indole-3-carbinol and its N-alkoxy derivatives preferentially target ERα-positive breast cancer cells

“…However, ERa-positive breast cancer cell lines demonstrated greater sensitivity to I3C and its more potent N-alkoxy derivatives. 25 Contrary to previously published results, 17 I3C failed to inhibit NE activity or disrupt the generation of LMW-E. To identify pathways that accounted for sensitivity to I3C, we preformed proteomic and gene expression analyzes. We found that AhR, a direct molecular target of I3C, 11 mediated sensitivity to the antitumor effects of I3C in ERa-positive MCF-7 cells.…”

“…25 N-methoxy, N-ethoxy, N-propoxy, and N-butoxy derivatives of I3C, characterized by N-alkoxy substitution at the nitrogen position on the indole ring from one to 4 carbons in length, were synthesized according to the previously published methodology. 25 We first evaluated the expression of cyclin E (both full-length and LMW-E) in a panel of breast cancer cell lines (including luminal, TNBC, and HER2-positive subtypes) and several immortalized HMECs. Western blot analysis (Fig.…”

“…For this reason, several research groups have modified the chemical structure of I3C to enhance its antitumor properties. In cell culture models, N-alkoxy derivatives of I3C (N-methoxy I3C, N-ethoxy I3C, N-propoxy I3C, and Nbutoxy I3C) 25 have more potent anti-tumor properties compared to unmodified I3C (Fig. 1A) and also demonstrated a significant bias for ERa-positive breast cancer cell lines (Fig.…”

“…N-Methoxy-I3C, N-Ethoxy-I3C, N-Propoxy-I3C, and N-Butoxy-I3C were synthesized according to previously published methodology 25 by the Translational Chemistry Core Facility at the University of Texas MD Anderson Cancer Center (UT MDACC). N-Acetyl Cysteine (NAC) and tamoxifen were purchased from Sigma.…”