Fig 2: Working Model for regulation of prostate cancer. Schematic diagram shows the proposed mechanism underlying the inhibitory effects of I3C and DIM on LPS induction of IL-1β mRNA and protein, mediated through AHR, and not the CD84-mediated pathway. I3C and DIM down-regulated CD84 mRNA, but not protein levels in an AHR-dependent manner. Activation of AHR is indicated by upregulation of CYP1B1, a well-documented AHR-responsive gene. The classic AHR ligand, b-NF, exerts similar effects as I3C/DIM on IL-1β, CD84, and CYP1B1 mRNA, but only b-NF inhibited LPS-induction of IL-6 mRNA levels. Solid line: Effects of I3C/DIM. Dotted line: Effects of b-NF. Bold X: Based on our data, the pathway does not occur. DIM is known to be an acid condensation product of I3C. The effectiveness of I3C and DIM on the pathway examined is best differentiated by dose dependence of the compounds. Specifically, DIM elicits an effect at a much lower concentration than I3C. However, our results indicate that both I3C and DIM can influence the AHR pathway. Hence, I3C and DIM may directly influence immune cells and also modulate cancer cell-stromal cell interaction to exert their cancer protective effects. The overall effect would reduce monocyte/macrophage infiltration, inflammation and potential for angiogenesis in the prostatic tumor. Further investigations are necessary to validate this hypothesis in vivo in prostate cancer.

Fig 3: Effect of AHR siRNA on I3C (25 µM), DIM (10 µM) and b-NF (10 µM) modulation of IL-1β, CD84 and CYP1B1 mRNA in u-THP-1 cells. Experiments using AHR siRNA were conducted in u-THP-1 cells as described in Materials and Methods. After treatments, total RNA was isolated and AHR, CD84, CYP1B1 and IL-1β mRNA were determined using RT-PCR. (A) Effects of AHR siRNA on AHR mRNA levels; (B) Effects of AHR siRNA on CYP1B1 mRNA levels; (C) Effects of AHR siRNA on CD84 mRNA levels; (D) Effects of AHR siRNA on LPS induction of IL-1β mRNA levels. Results are expressed as mean ± SD (n = 3). * indicates significant difference from vehicle control at p ≤ 0.05.

Fig 4: Effects of I3C, DIM and PMA on CD84. (A) I3C and DIM on CD84 protein in u-THP-1. THP-1 cells were treated with I3C (5 µM) or DIM (5 µM) for 96 h, cells were harvested for Western analysis and quantitation as described in Materials and Methods. Results expressed as mean ± SD (n = 3); (B) Effects of PMA on CD84 protein levels. THP-1 cells were differentiated using PMA as described in Materials and Methods. After treatment, cells were harvested for protein and CD84 protein levels were determined by Western blot analysis. Results expressed as mean ± SD (n = 3). * indicates significant difference from each other at p = 0.05; (C) Time-dependent effects of PMA on CD84 protein in THP-1 cells. U-THP-1 cells were treated with PMA for 0–96 h and cells harvested at indicated time points for western analysis and quantitation as described in Materials and Methods. Results expressed as mean ± SD (n = 3). * indicates significant difference from 48 h at p = 0.05.

Fig 5: Effects of I3C, DIM on CD84 mRNA levels in THP-1 cells. THP-1 cells were differentiated using PMA as described in Materials and Methods. U-THP-1 and d-THP-1 cells were treated with or without I3C or DIM as described in Materials and Methods. After treatment with the compounds, cells were exposed to LPS for 2 h, total RNA isolated and CD84 mRNA expression determined using RT-PCR. The treatment results were normalized to LPS-induced IL-1β mRNA level in the absence of compounds and expressed as % control (n = 3, mean ± SD). (A) Effect of I3C; (B) Effect of DIM. * indicates significant difference from vehicle control at p ≤ 0.05.