3,3’-Diindolylmethane (DIM) and its ring-substituted halogenated analogs (ring-DIMs) induce differential mechanisms of survival and death in androgen-dependent and –independent prostate cancer cells

Alexander A Goldberg,Jesus Olivero-Verbél,Hossam Draz,Stephen H Safe,Diana Montes-Grajales,J Thomas Sanderson

doi:10.18632/genesandcancer.60

Abstract

We recently reported that novel ring-substituted analogs of 3,3'-diindolylmethane (ring-DIMs) induce apoptosis and necrosis in androgen-dependent and -independent prostate cancer cells. In this paper, we have focused on the mechanism(s) associated with ring-DIM-mediated cell death, and on identifying the specific intracellular target(s) of these compounds. The 4,4'- and 7,7'-dichloroDIMs and 4,4'- and 7,7'-dibromoDIMs induced the death of LNCaP, C42B and DU145 prostate cancer cells, but not that of immortalized normal human prostate epithelial (RWPE-1) cells. Ring-DIMs caused the early loss of mitochondrial membrane potential (MMP) and decreased mitochondrial ATP generation in prostate cancer cells. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore, inhibited ring-DIM-mediated cell death, and salubrinal, an inhibitor of ER stress, inhibited cell death mediated only by 4,4'-dihaloDIMs. We found that although salubrinal did not inhibit the onset of ER stress, it prevented 4,4'-dibromoDIM mediated loss of MMP. Salubrinal potentiated cell death in response to 7,7'-dihaloDIMs and DIM, and this effect concurred with increased loss of MMP. Using in silico 3-D docking affinity analysis, we identified Ca2+/calmodulin-dependent kinase II (CaMKII) as a potential direct target for the most toxic ring-DIM, 4,4'-dibromoDIM. An inhibitor of CaMKII, KN93, but not its inactive analog KN92, abrogated cell death mediated by 4,4'-dibromoDIM. The ring-DIMs induced ER stress and autophagy, but these processes were not necessary for ring-DIM-mediated cell death. Inhibition of autophagy with bafilomycin A1, 3-methyladenine or by LC3B gene silencing sensitized LNCaP and C42B, but not ATG5-deficient DU145 cells to ring-DIM- and DIM-mediated cell death. We propose that autophagy induced by the ring-DIMs and DIM has a cytoprotective function in prostate cancer cells.

Highlights

Prostate cancer accounts for almost one third of all cancer deaths in the United States and is the second highest cause of cancer-related death in males [1]
To further investigate the mechanism of ring-DIMinduced toxicity, we looked at membrane potential (MMP), mitochondrial ATP generation and endoplasmic reticulum (ER) stress in response to ringDIM exposure
We have previously reported that ring-DIMs and DIM induce apoptosis and necrosis in androgen receptor-positive (AR+) AD LNCaP and in androgen receptor-negative (AR-) AI PC-3 prostate cancer cells

Summary

Introduction

Prostate cancer accounts for almost one third of all cancer deaths in the United States and is the second highest cause of cancer-related death in males [1]. Most prostate tumours are initially androgen-dependent (AD). A large contingent will progress to an aggressive androgenindependent (AI) form, which are more drug-resistant and lead to increased morbidity and mortality among patients. Prostate cancer is treated with a combination of radiotherapy, chemical castration, androgen receptor (AR) antagonists (hydroxyflutamide, bicalutamide), or inhibitors of steroidogenesis (abiraterone). Patients treated with hydroxyflutamide or bicalutamide often suffer from severe side-effects as a result of the anti-androgenic therapy [2, 3], necessitating the search for novel chemotherapeutic agents with fewer deleterious effects. It is imperative to search for novel therapeutic targets which may aid the development of a new generation of drugs effective in the elimination of AI prostate tumours

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