In vitro activity: DIM-C-pPhOCH3 is an agonist Nur77 (Nerve growth factor-induced Bα, abbreviated as NGFI-Bα or Nur77). Nur77 is an orphan nuclear receptor with no known endogenous ligands and was reported to be overexpressed in colon tumors compared with normal colon tissue. DIM-C-pPhOCH3 was able to decrease the survival and induce apoptosis in RKO colon cancer cells, and this was accompanied by induction of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. The induction of apoptosis and TRAIL by DIM-C-pPhOCH3 was significantly inhibited by a small inhibitory RNA for Nur77 (iNur77); however, it was evident from RNA interference studies that DIM-C-pPhOCH3 also induced Nur77-independent apoptosis. Analysis of DIM-C-pPhOCH3-induced gene expression using microarrays identified several proapoptotic genes, and analysis by reverse transcription-PCR in the presence or absence of iNur77 showed that induction of programmed cell death gene 1 was Nur77 dependent, whereas induction of cystathionase and activating transcription factor 3 was Nur77 independent. DIM-C-pPhOCH3 (25 mg/kg/d) also inhibited tumor growth in athymic nude mice bearing RKO cell xenografts. These results show that DIM-C-pPhOCH3 represent a new class of anti-colon cancer drugs that act through receptor-dependent and receptor-independent pathways.

Kinase Assay: Microarray studies focused on early-induced genes, and RKO cells were treated with DMSO or 12.5 μmol/L DIM-C-pPhOCH3 for 2 and 6 h. RNA was isolated as described for the reverse transcription-PCR (RT-PCR) experiment and analyzed for gene expression using the Codelink Whole Genome Bioarrays (300026), and three replicates were determined for each time point and the DMSO control. The microarray data were analyzed using GeneSpring software version 7.2 (Agilent, Palo Alto, CA). The data were normalized in two steps. First, for each array, the expression value of each gene was divided by the median of all the values in that array. Second, for each gene, the expression value in each array was divided by the median value of that gene across all arrays. Genes with low-quality signals were excluded for statistical analysis. One-way ANOVA (assume equal variances) was carried out to identify differentially expressed genes. A gene was said to be differentially expressed if the Benjamini and Hochberg adjusted Ps were<0.05.

Cell Assay: Human colon carcinoma cell lines RKO and SW480 were provided by the University of Texas M.D. Anderson Cancer Center, Houston, Texas. HT-29, HCT116, and HCT-15 were obtained from the American Type Culture Collection (Manassas, VA). RKO, SW480, and HT-29 cells were maintained in DMEM/Ham's F-12 (Sigma, St. Louis, MO) without phenol red supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, and 5% fetal bovine serum (FBS) and 10 ml/L of 100× antibiotics antimycotic solution (Sigma). HCT116 and HCT-15 cells were maintained in RPMI 1640 (Sigma) supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, 0.45% glucose, 0.24% HEPES, 10% FBS, and 10 ml/L of 100× antibiotics antimycotic solution. Cells were maintained at 37°C in the presence of 5% CO2.