The AAPS Journal

, Volume 15, Issue 3, pp 864–874 | Cite as

Epigenetic Modifications of Nrf2 by 3,3′-diindolylmethane In Vitro in TRAMP C1 Cell Line and In Vivo TRAMP Prostate Tumors

  • Tien-Yuan Wu
  • Tin Oo Khor
  • Zheng-Yuan Su
  • Constance Lay-Lay Saw
  • Limin Shu
  • Ka-Lung Cheung
  • Ying Huang
  • Siwang Yu
  • Ah-Ng Tony KongEmail author
Research Article Theme: Natural Products Drug Discovery in Cancer Prevention


3,3′-diindolylmethane (DIM) is currently being investigated in many clinical trials including prostate, breast, and cervical cancers and has been shown to possess anticancer effects in several in vivo and in vitro models. Previously, DIM has been reported to possess cancer chemopreventive effects in prostate carcinogenesis in TRAMP mice; however, the in vivo mechanism is unclear. The present study aims to investigate the in vitro and in vivo epigenetics modulation of DIM in TRAMP-C1 cells and in TRAMP mouse model. In vitro study utilizing TRAMP-C1 cells showed that DIM suppressed DNMT expression and reversed CpG methylation status of Nrf2 resulting in enhanced expression of Nrf2 and Nrf2-target gene NQO1. In vivo study, TRAMP mice fed with DIM-supplemented diet showed much lower incidence of tumorigenesis and metastasis than the untreated control group similar to what was reported previously. DIM increased apoptosis, decreased cell proliferation and enhanced Nrf2 and Nrf2-target gene NQO1 expression in prostate tissues. Importantly, immunohistochemical analysis showed that DIM reduced the global CpG 5-methylcytosine methylation. Focusing on one of the early cancer chemopreventive target gene Nrf2, bisulfite genomic sequencing showed that DIM decreased the methylation status of the first five CpGs of the Nrf2 promoter region, corroborating with the results of in vitro TRAMP-C1 cells. In summary, our current study shows that DIM is a potent cancer chemopreventive agent for prostate cancer and epigenetic modifications of the CpG including Nrf2 could be a potential mechanism by which DIM exerts its chemopreventive effects.


3,3′-diindolylmethane (DIM) epigenetic methylation Nrf2 prostate cancer 



We thank Dr. Chungxiou Wang as a histopathologist for her assistance in histology evaluation. We also thank Dr. Barbara Foster, Rowell Park Cancer Institute, Buffalo, NY, who generously provided the TRAMP-C1 cells. We thank all members in Dr. Kong’s group for their generous help in discussion and preparation of this manuscript.

Conflict of Interest Statement

None declared


This work was supported by Institutional Funds to Dr. Ah-Ng Tony Kong.

Supplementary material

12248_2013_9493_Fig7_ESM.jpg (44 kb)
Supplementary Fig. 1

Immunohistochemical analysis of the effects of cell proliferation, PCNA. Representative photomicrographs (×40 magnification) of PCNA-stained (dark brown color) TRAMP prostate tissue section and percentage levels of cell proliferation. The scale bar stands for 500 μm. p < 0.05, significantly different from the control and was based on Mann–Whitney U test. (JPEG 43 kb)

12248_2013_9493_MOESM1_ESM.tif (44.7 mb)
High-resolution image (TIFF 45,813 kb)
12248_2013_9493_Fig8_ESM.jpg (46 kb)
Supplementary Fig. 2

Immunohistochemical analysis of the effects of apoptosis, TUNEL. Representative photomicrographs (×40 magnification) of TUNEL stained (dark brown color) TRAMP prostate tissue section and percentage levels of apoptosis. The scale bar stands for 500 μm. *p < 0.05, significantly different from the control by Mann–Whitney U test. Number sign indicates significantly different between D-G1 and D-G2 (p = 0.043). (JPEG 46 kb)

12248_2013_9493_MOESM2_ESM.tif (44.6 mb)
High-resolution image (TIFF 45,718 kb)
12248_2013_9493_MOESM3_ESM.doc (32 kb)
Supplementary Table I Murine primers for qPCR (DOC 32 kb)
12248_2013_9493_MOESM4_ESM.doc (28 kb)
Supplementary Table II Confirmation of genotype of the TRAMP mice (DOC 28 kb)
12248_2013_9493_MOESM5_ESM.doc (28 kb)
Supplementary Table III DIM inhibit palpable tumor and metastasis in TRAMP males. aNumbers represent the presence of palpable tumor showed at the end of the experiment at 24 weeks of age. Fisher’s exact test was used to compare the incidence of palpable tumor between the control and the DIM-treated mice killed at 24 weeks of age. *p values < 0.05 were considered as significant. bNumbers represent the presence of lymph nodes metastasis showed at the end of experiment when the mice were killed. Fisher’s exact test was used to compare the incidence of lymph node metastasis between the control and the DIM treated mice sacrificed at 24 weeks of age. #p values < 0.05 were considered as significant. (DOC 27 kb)
12248_2013_9493_MOESM6_ESM.pdf (13 kb)
Supplementary Table IV TRAMP mice body weights, wet GU weights, and normalized wet GU weights by body weights measured weekly. (PDF 12 kb)


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Copyright information

© American Association of Pharmaceutical Scientists 2013

Authors and Affiliations

  • Tien-Yuan Wu
    • 1
  • Tin Oo Khor
    • 1
  • Zheng-Yuan Su
    • 1
  • Constance Lay-Lay Saw
    • 1
  • Limin Shu
    • 1
  • Ka-Lung Cheung
    • 1
  • Ying Huang
    • 1
  • Siwang Yu
    • 2
  • Ah-Ng Tony Kong
    • 1
    Email author
  1. 1.Center for Cancer Prevention Research, Department of Pharmaceutics, Ernest Mario School of PharmacyRutgers, The State University of New JerseyPiscatawayUSA
  2. 2.Department of Chemical Biology, School of Pharmaceutical SciencesPeking University Health Science CenterBeijingChina

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