1,1-Bis(3'-indolyl)-1-(p-chlorophenyl)methane activates the orphan nuclear receptor Nurr1 and inhibits bladder cancer growth

Abstract

Nurr1 is an orphan nuclear receptor and a member of the nerve growth factor I-B subfamily of transcription factors with no known endogenous ligand or stimulator. We show, for the first time, evidence that Nurr1 is expressed in a panel of 11 human bladder cancer cell lines. A new class of methylene-substituted diindolylmethanes (C-DIM) were screened and 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl) activated the ligand-binding domain of Nurr1. Treatment of bladder cancer cells with Nurr1-active C-DIM resulted in decreased cell survival (MTT assay) and induction of cell death pathways, resulting in poly(ADP-ribose) polymerase cleavage and DNA fragmentation. The specificity of the Nurr1-active compound was shown using RNA interference in 253J B-V cells, whereby small interfering RNA against Nurr1 attenuated ligand-dependent activation of Nurr1 and poly(ADP-ribose) polymerase cleavage. Furthermore, activation of Nurr1 resulted in stimulation of tumor necrosis factor-related apoptosis-inducing ligand and small interfering RNA experiments attenuated tumor necrosis factor-related apoptosis-inducing ligand production. In an orthotopic model of human bladder tumors established in nude mice, administration of a Nurr1-active C-DIM suppressed bladder cancer growth. These results identify Nurr1 as a potential target for bladder cancer therapy and also identify a novel agent for activating Nurr1.

Figure 1

Expression of Nurr1 and Nur77 protein in urothelial carcinoma cells and structure-dependent Nurr1 activation by C-DIM. A, Nurr1 and Nur77 expression in bladder cancer cells. Whole-cell lysates from 253J B-V, 253J P, KU7, UM-UC3, UM-UC5, UM-UC6, UM-UC9, UM-UC10, UM-UC13, and UM-UC14 cells were analyzed by Western blot analysis as described in Materials and Methods. B to D, activation of GAL4-Nurr1/pGAL4, GAL4-Nor1/pGAL4, GAL4-Nur77/pGAL4, NuRE, PM-GAL4/pGAL4, or RXR-GAL4/pGAL4 by C-DIM compounds. KU7 bladder cancer cells were transfected with GAL4-Nurr1/pGAL4, GAL4-Nor1/pGAL4, GAL4-Nur77/pGAL4, NuRE, PM-GAL4/pGAL4, or RXR-GAL4/pGAL4 and treated with DMSO or different concentrations of the C-DIMs and luciferase. Columns, mean of at least three replicate experiments for each treatment group; bars, SE. *, P < 0.05.

Figure 2

Nurr1-active C-DIM-induced growth inhibition of urothelial carcinoma cells. A, time- and concentration-dependent effects of DIM-C-pPhCl on KU7 and 253J B-V cell proliferation. Cell were treated with DMSO or 2.5, 5, or 10 μmol/L DIM-C-pPhCl for 24, 48, and 72 h, and the percentage proliferation was determined where the number of cells in the solvent-treated (DMSO) group was set at 100%. Columns, mean of three replicate experiments for each concentration; bars, SE. *, P < 0.05. B, concentration-dependent induction of apoptosis by DIM-C-pPhCl on KU7 cells. KU7 cells were treated with the indicated concentrations of DIM-C-pPhCl for 48 h. Cells were then harvested, and DNA fragmentation characteristic of apoptosis was measured by propidium iodide staining and fluorescence-activated cell sorting analysis.

Figure 3

Effects of DIM-C-pPhCl on apoptosis. A, dose-dependent induction of apoptosis in KU7 cells by DIM-C-pPhCl. KU7 cells were treated with different concentrations of DIM-C-pPhCl for 48 h, and whole-cell lysates were analyzed by Western blot analysis. B, time-dependent induction of apoptosis by DIM-C-pPhCl in 253J B-V cells. 253J B-V cells were treated with 5 μmol/L C-DIM-pPhCl for 24 and 48 h, and whole-cell lysates were analyzed by Western blot analysis. C, effects of z-VAD-fmk in PARP cleavage. 253J B-V cells were treated with DIM-C-pPhCl alone or in combination with 10 μmol/L pan-caspase inhibitor z-VAD-fmk, which was administrated 1 h before the addition of 5 μmol/L DIM-C-pPhCl. After 48 h, whole-cell lysates were analyzed for cleaved PARP and TRAIL induction by Western blot analysis as described in Materials and Methods. D, induction of TRAIL mRNA by DIM-C-pPhCl in T24 cells. cDNAs were generated from 1 μg total RNAs from bladder cancer cells with or without 5 μmol/L DIM-C-pPhCl. Reverse transcriptase reaction was carried as described in Materials and Methods.

Figure 4

Nurr1-dependent induction of TRAIL and cleaved PARP in 253J B-V cells. Cells were transfected with nonspecific control (iNS) or iNurr1 and treated with DMSO or 10 μmol/L DIM-C-pPhCl, and whole-cell lysates were analyzed by Western blot analysis for Nurr1, PARP cleavage, TRAIL, and actin (loading control) proteins (A). Columns, mean of three replicate experiments for each treatment group; bars, SE. Protein levels were normalized to actin. Significant (P < 0.05) inhibition of Nurr1 expression (B), decreased expression induction of PARP cleavage (C), and TRAIL (D) by iNurr1 are quantitated and 100% protein expression was assigned to levels in cells treated with DIM-C-pPhCl and transfected with nonspecific control. Each experiment was reproduced three times independently.

Figure 5. In vivo

anticarcinogenic activity of DIM-C-pPhCl. A, effects of DIM-C-pPhCl on primary bladder tumors. Three different treatment groups consist of DMSO control (CTRL; n = 14), 12.5 mg/kg/d DIM-C-pPhCl (DIM1; n = 13), and 25 mg/kg/d DIM-C-pPhCl (DIM2; n = 13). Each group was treated thrice a week right after randomization. 253J B-V cells (2 × 10⁵) were implanted into bladder wall of male nude mice. Mice were injected with luciferin i.p. and were imaged on the charge-coupled device camera 10 min after injection. Bioluminescent imaging images were collected for 1 or 10 s for each group. Different image acquisition times were needed to avoid saturating the charge-coupled device camera. Bioluminescence is presented as a pseudocolor scale: red, highest photon flux; blue, lowest photon flux. Bioluminescence from primary tumors was quantified by region of interest analysis of images obtained on the indicated time points (X axis) after cell implantation. Background bioluminescence was subtracted from each tumor region of interest value. Mean ± SE photon flux in each group. Bottom, representative bioluminescent imaging images of primary 253J B-V bladder tumors 2 and 4 wk after initial treatment. B, tumor size measurements. Actual tumor sizes were measured 4 wk after implantation of cancer cells. Mean ± SE tumor volume. *, P < 0.05. C, effects of DIM-C-pPhCl on human bladder xenograft mice survival. Kaplan-Maier plots were generated, and survival time of animals was analyzed for significance by log-rank survival analysis. *, P < 0.05.

Figure 6

Histopathologic changes of mice tumor after DIM-C-pPhCl treatment. A, H&E staining of tumor from DMSO control [original magnification, ×40 (a) and ×200 (d)], 12.5 mg/kg/d DIM-C-pPhCl [original magnification, ×40 (b) and ×200 (e)], and 25 mg/kg/d DIM-C-pPhCl [original magnification, ×40 (c) and ×200 (f)]. Grouping was determined as described in Materials and Methods. B, PCNA staining of DMSO control (original magnification, ×200), 12.5 mg/kg/d DIM-C-pPhCl (original magnification, ×200), and 25 mg/kg/d DIM-C-pPhCl (original magnification, ×200). Grouping were determined as described in Materials and Methods. Proliferation was determined using PCNA index. CTRL, control; DIM1, 12.5 mg/kg/d DIM-C-pPhCl; DIM2, 25 mg/kg/d DIM-C-pPhCl. To calculate PCNA index, the tissue was photographed using Optotronics Tec 470 camera linked to a computer and digital printer. The intensity of the immunostaining was quantified in multiple points in five different areas of each sample by an image analyzer using Optimas image analysis software (Media Cybernetics) to obtain an average measurement. The density of proliferative cells was expressed as an average of the five highest densities identified within a single ×200 field. *, P < 0.05. C, TUNEL staining of DMSO control (original magnification, ×200), 12.5 mg/kg/d DIM-C-pPhCl (original magnification, ×200), and 25 mg/kg/d DIM-C-pPhCl (original magnification, ×200). Grouping were determined as described in Materials and Methods. Apoptosis of bladder tumors was quantified for TUNEL expression. *, P < 0.05.