Activation of nerve growth factor-induced B alpha by methylene-substituted diindolylmethanes in bladder cancer cells induces apoptosis and inhibits tumor growth

Abstract

Nerve growth factor-induced B (NGFI-B) genes are orphan nuclear receptors, and NGFI-B alpha (Nur77, TR3) is overexpressed in bladder tumors and bladder cancer cells compared with nontumorous bladder tissue. 1,1-Bis(3'-indolyl)-1-(p-methoxyphenyl)-methane (DIM-C-pPhOCH(3)) and 1,1-bis(3'-indolyl)-1-(p-phenyl)methane have previously been identified as activators of Nur77, and both compounds inhibited growth and induced apoptosis of UC-5 and KU7 bladder cancer cells. The proapoptotic effects of methylene-substituted diindolylmethanes (C-DIMs) were unaffected by cotreatment with leptomycin B and were dependent on nuclear Nur77, and RNA interference with a small inhibitory RNA for Nur77 (iNur77) demonstrated that C-DIM-induced activation of apoptosis was Nur77-dependent. Microarray analysis of DIM-C-pPhOCH(3)-induced genes in UC-5 bladder cancer cells showed that this compound induced multiple Nur77-dependent proapoptotic or growth inhibitory genes including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), cystathionase, p21, p8, and sestrin-2. DIM-C-pPhOCH(3) (25 mg/kg/d) also induced apoptosis and inhibited tumor growth in athymic nude mice bearing KU7 cells as xenografts, demonstrating that Nur77-active C-DIMs exhibit potential for bladder cancer chemotherapy by targeting Nur77, which is overexpressed in this tumor type.

Fig. 1.

Nur77 expression in human bladder tumors and bladder cancer cell lines. Expression of Nur77 in low-grade (A), high-grade stage 2 (B), and high-grade stage 3 (C) bladder tumors and in RT4 and UC-14 (D) bladder cancer cell lines were determined by immunostaining with Nur77 antibodies as outlined under Materials and Methods.

Fig. 2.

Nur77-active C-DIMs decrease cell proliferation in UC-5 and KU7 cells. UC-5 cells (A) and KU7 cells (B) were treated with DMSO or different concentrations of C-DIMs (5, 10 and 15 μM) for 24 and 48 h, and the percentages of cells in the treatment groups compared with the solvent control (DMSO; set at 100%) were determined. Columns, mean of three replicate experiments for each treatment group; error bars, S.D. ∗, p < 0.05 compared with DMSO treatment group.

Fig. 3.

Nur77-active C-DIMs induce apoptosis in UC-5 and KU7 cells and also act through nuclear Nur77. A, effects of leptomycin B on PARP cleavage. UC-5 cells were treated for 24 h with DIM-C-pPhOCH₃ alone or in the presence of leptomycin B (0.05 ng/ml), and Western blot analysis of nuclear and cytosolic cell lysates was used to detect Nur77 and PARP cleavage. Sp1, loading control for nuclear fraction; N.S., nonspecific band for loading control. B, immunostaining for Nur77. UC-5 cells were treated with DMSO or 15 μM DIM-C-pPhOCH₃ for 24 h and immunostained with IgG or Nur77 antibodies. C, dose-dependent induction of apoptosis by C-DIMs. UC-5 and KU7 cells were treated with DMSO or different concentrations of DIM-C-pPhOCH₃ or DIM-C-Ph for 48 h. D, inhibition of Nur77-active C-DIMs-induced apoptosis by Z-VAD-fmk. UC-5 cells were treated with Nur77-active C-DIMs alone or in combination with 10 μM Z-VAD-fmk, and whole-cell lysates were analyzed by Western blot analysis.

Fig. 4.

Nur77-dependent induction of TRAIL and apoptosis in UC-5 cells. A, induction of TRAIL by DIM-C-pPhOCH₃ and DIM-C-Ph. Cells were treated with Nur77-active C-DIMs (15 μM) for 24 h, and whole-cell lysates were analyzed for TRAIL or β-tubulin. B and C, RNA interference with iNur77. UC-5 cells were transfected with iScr (nonspecific) or iNur77 and treated with DMSO or 15 μM DIM-C-pPhOCH₃, and whole-cell lysates were analyzed by Western blot analysis for Nur77, PARP cleavage, TRAIL, and β-tubulin proteins. Columns, mean of three replicate experiments for each treatment group; error bar, S.D. Protein levels were normalized to β-tubulin. B, ∗, P < 0.05, significant inhibition of Nur77 expression. C, ∗, significant induction of TRAIL and PARP cleavage; ∗∗, decreased expression of these proteins by iNur77.

Fig. 5.

Induction of gene expression by DIM-C-pPhOCH₃. A and B, induction of NAG-1, p8, CSE, and sestrin-2 in UC-5 cells. Cells were treated with DMSO or 15 μM DIM-C-pPhOCH₃ for 6 h, and NAG-1, p8, CSE, sestrin-2, and GAPDH (reference mRNA) were determined by RT-PCR. Induction responses were determined in three separate experiments. ∗, P < 0.05, significantly induced mRNA expression. C, effects of iNur77 on induced gene expression. UC-5 cells were transfected with iScr or iNur77, treated with DMSO or 15 μM DIM-C-pPhOCH₃ for 6 h, and analyzed by real-time PCR. Columns, mean of three replicate determinations for each treatment group; error bars, S.D. ∗, P < 0.05, significant induction by DIM-C-pPhOCH₃; #, significant decrease after transfection with iNur77.

Fig. 6.

Inhibition of tumor growth by DIM-C-pPhOCH₃. Male athymic nude mice bearing KU7 cell xenografts were treated with DIM-C-pPhOCH₃, and tumor volumes (A) and weight (B) were determined. The compound was given daily (25 mg/kg/day) in corn oil by oral gavage, and corn oil served as a solvent control. C, immunostaining for apoptosis. Tumor tissue from animals treated with solvent control or DIM-C-pPhOCH₃ were stained, and a TUNEL assay was used to detect apoptosis. D, whole-cell lysates from tumor tissues was analyzed by Western blot analysis using PARP antibody to detect apoptosis. Columns, mean of five replicate determinations for each group; error bars, S.E. ∗, P < 0.05, significant induction by DIM-C-pPhOCH₃.