Ring-substituted analogs of 3,3′-diindolylmethane (DIM) induce apoptosis and necrosis in androgen-dependent and –independent prostate cancer cells
- 450 Downloads
We recently reported that novel ring-substituted analogs of 3,3′-diindolylmethane (ring-DIMs) have anti-androgenic and growth inhibitory effects in androgen-dependent prostate cancer cells. The objectives of this study were to confirm the ability of 4,4′- and 7,7′-dibromo- and dichloro-substituted ring-DIMs to inhibit androgen-stimulated proliferation of androgen-dependent LNCaP human prostate cancer cells using a non-invasive, real-time monitoring technique. In addition, their ability to induce apoptotic and necrotic cell death in androgen-dependent as well as -independent (PC-3) prostate cancer cells was studied. Prostate cancer cells were treated with increasing concentrations of DIM and ring-DIMs (0.3–30 μM) and effects on cell proliferation were measured in real-time using an xCELLigence cellular analysis system. Chromatin condensation and loss of membrane integrity were determined by Hoechst and propidium iodide staining, respectively. Apoptotic protein markers were measured by immunoblotting and activation of caspases determined using selective fluorogenic substrates. Intra- and extracellular concentrations of DIM and ring-DIMs were assessed by electrospray ionization tandem mass spectrometry. Ring-DIMs inhibited androgen-stimulated LNCaP cell proliferation and induced apoptosis and necrosis in LNCaP and PC-3 cells with 2–4 fold greater potencies than DIM. DIM and the ring-DIMs increased caspases −3, −8 and −9 activity, elevated expression of Fas, FasL, DR4 and DR5 protein, and induced PARP cleavage in both cell lines. The cytotoxicity of the most potent ring-DIM, 4,4′-dibromoDIM, but not the other compounds was decreased by an inhibitor of caspase −3. The 4,4′-dibromoDIM was primarily found in the extracellular medium, whereas all other compounds were present to a much larger extent in the cell. In conclusion, ring-DIMs inhibited prostate cancer cell growth and induced cell death in LNCaP and PC-3 cells with greater potencies than DIM; they also structure-dependently activated different cell death pathways suggesting that these compounds have clinical potential as chemopreventive and chemotherapeutic agents in prostate cancer, regardless of hormone-dependency.
KeywordsProstate cancer Androgen-dependent Androgen-independent Diindolylmethane derivatives LNCaP PC-3 Apoptosis Necrosis Caspase activity Intracellular Concentrations
This work was made possible by an operating grant from the Canadian Institutes of Health Research (CIHR grant no. MOP-115019). The authors declare to have no conflict of interest.
- 6.Bjeldanes LF, Kim J-Y, Grose KR, Bartholomew JC, Bradfield CA (1991) Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in vivo: comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc Natl Acad Sci USA 88:9543–9547PubMedCentralCrossRefPubMedGoogle Scholar
- 10.Heath EI, Heilbrun LK, Li J, Vaishampayan U, Harper F, Pemberton P, Sarkar FH (2010) A phase I dose-escalation study of oral BR-DIM (BioResponse 3,3′- Diindolylmethane) in castrate-resistant, non-metastatic prostate cancer. American journal of translational research 2:402–411PubMedCentralPubMedGoogle Scholar
- 13.Wang TT, Schoene NW, Milner JA, Kim YS (2012) Broccoli-derived phytochemicals indole-3-carbinol and 3,3′-diindolylmethane exerts concentration-dependent pleiotropic effects on prostate cancer cells: comparison with other cancer preventive phytochemicals. Mol Carcinog 51:244–256CrossRefPubMedGoogle Scholar
- 14.Bhuiyan MM, Li Y, Banerjee S, Ahmed F, Wang Z, Ali S, Sarkar FH (2006) Down-regulation of androgen receptor by 3,3′-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in both hormone-sensitive LNCaP and insensitive C4-2B prostate cancer cells. Cancer Res 66:10064–10072CrossRefPubMedGoogle Scholar
- 18.Li Y, Wang Z, Kong D, Murthy S, Dou QP, Sheng S, Reddy GP, Sarkar FH (2007) Regulation of FOXO3a/beta-catenin/GSK-3beta signaling by 3,3′-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in prostate cancer cells. J Biol Chem 282:21542–21550CrossRefPubMedGoogle Scholar
- 23.Nachshon-Kedmi M, Yannai S, Haj A, Fares FA (2003) Indole-3-carbinol and 3,3′-diindolylmethane induce apoptosis in human prostate cancer cells. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 41:745–752CrossRefGoogle Scholar
- 26.Cho SD, Yoon K, Chintharlapalli S, Abdelrahim M, Lei P, Hamilton S, Khan S, Ramaiah SK, Safe S (2007) Nur77 agonists induce proapoptotic genes and responses in colon cancer cells through nuclear receptor-dependent and nuclear receptor-independent pathways. Cancer Res 67:674–683CrossRefPubMedGoogle Scholar
- 29.Qin C, Morrow D, Stewart J, Spencer K, Porter W, Smith R 3rd, Phillips T, Abdelrahim M, Samudio I, Safe S (2004) A new class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists that inhibit growth of breast cancer cells: 1,1-Bis(3′-indolyl)-1-(p-substituted phenyl)methanes. Mol Cancer Ther 3:247–260CrossRefPubMedGoogle Scholar
- 32.Bourque SD, Titorenko VI (2009) A quantitative assessment of the yeast lipidome using electrospray ionization mass spectrometry. Journal of visualized experiments, JoVEGoogle Scholar
- 41.Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 144:281–292PubMedCentralCrossRefPubMedGoogle Scholar