Nur77 agonists induce proapoptotic genes and responses in colon cancer cells through nuclear receptor-dependent and nuclear receptor-independent pathways

Nerve growth factor-induced Balpha (NGFI-Balpha, Nur77) is an orphan nuclear receptor with no known endogenous ligands; however, recent studies on a series of methylene-substituted diindolylmethanes (C-DIM) have identified 1,1-bis(3'-indolyl)-1-(phenyl)methane (DIM-C-Ph) and 1,1-bis(3'-indolyl)-1-(p-anisyl)methane (DIM-C-pPhOCH3) as Nur77 agonists. Nur77 is expressed in several colon Cancer cell lines (RKO, SW480, HCT-116, HT-29, and HCT-15), and we also observed by immunostaining that Nur77 was overexpressed in colon tumors compared with normal colon tissue. DIM-C-Ph and DIM-C-pPhOCH3 decreased survival and induced 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 Nur77-active C-DIM compounds represent a new class of anti-colon Cancer drugs that act through receptor-dependent and receptor-independent pathways.