Absorption-enhanced 3,3’-Diindolylmethane: Human Use in HPV-related, Benign and Pre-cancerous Conditions.
Zeligs, M.A., Sepkovic, D.W., Manrique, C., Macsalka, M., Williams, D.E.,and Bradlow, H.L
Proc. Am. Assoc. Cancer Res. 2003, Apr; 44.
3,3-Diindolylmethane is a dietary indole from cruciferous vegetables that has demonstrated pre-clinical therapeutic efficacy in models of DMBA-induced mammary cancer, transplanted human breast cancer, and in models of human papilloma virus (HPV) related disease.
Animal and human use of crystalline diindolylmethane has revealed the need for absorption-enhancing technology to allow adequate gastro-intestinal uptake. BioResponse-DIM, a patented formulation of diindolylmethane categorized and sold as a dietary supplement, utilizes solubility-enhancing micro-encapsulation technology to allow absorption of effective amounts of diindolylmethane.
Human use of this formulation promotes a dose-responsive upward effect on the urinary ratio of 2-OH/16-OH estrone metabolites, demonstrated by ELISA testing of urine before-and-after use. In previous prospective studies, a greater 2-OH/16-OH estrone urinary ratio has been associated with a lowered risk of future breast cancer. We are able to monitor compliance by measurement of urinary diindolylmethane using gas chromatography-mass spectrometry.
Human use of this preparation at higher doses has demonstrated treatment-related resolution of moderate and severe cervical dysplasia in preliminary open-label testing. A still higher dose, about 10 times above that possible from dietary exposure to diindolylmethane from vegetable sources, has resulted in the control of laryngeal papillomas and resolution of cutaneous and plantar warts in preliminary human testing. The clearing of HPV-related lesions is consistent with diindolylmethane's previously described, apoptosis-promoting and chemopreventive activity.
Diindolylmethane alters gene expression in human keratinocytes in vitro.
Carter TH, Liu K, Ralph W Jr, Chen D, Qi M, Fan S, Yuan F, Rosen EM, Auborn KJ.
North Shore-Long Island Jewish Research Institute, Manhasset,
NY 11030, USA.
Indole-3-carbinol (I3C) and its dimer 3,3'-diindolylmethane (DIM), obtained from dietary consumption of cruciferous vegetables, have multiple biochemical activities. Both compounds have been effective clinically in treating precancerous lesions of the cervix and laryngeal papillomas, pathologies with a human papillomavirus (HPV) component. Using cDNA microarrays, we examined early changes in gene expression after treatment with 100 micro mol/L DIM in C33A and CaSki cervical cancer cells and in an immortalized human epithelial cell line (HaCat), as well as in normal human foreskin keratinocytes (HFK). Multiple analyses were done after treating C33A cells for 6 h; other analyses included 4- and 12-h treatments of C33A and 6-h treatments of CaSki, HaCat and HFK cells. DIM consistently altered the expression of >100 genes at least twofold. Many of the stimulated genes encode transcription factors and proteins involved in signaling, stress response and growth.
Results were comparable between transformed cells with and without integrated HPV sequences, and many of the same genes were induced in these cancer-derived cells and in noncancer cells. Eight genes encoding bZip proteins were among the most consistently and robustly induced, including the stress-associated immediate early gene GADD153 (>50 fold in C33A) and nuclear factor-interleukin 6 (NF-IL6), also known as c/EBPbeta, (>5 fold in C33A), which has been shown to reduce expression of HPV oncogenes. Induction of GADD153, NF-IL6 and ATF3 was confirmed by Western analysis.
In functional analyses, DIM not only suppressed transcription of a luciferase gene driven by the HPV11 upstream regulatory region (URR) in C33A, CaSki, HaCat and HFK cells from >2-fold to 37-fold depending on the type of cells, but also reduced endogenous transcription of HPV16 oncogenes to undetectable levels in CaSki cells as determined by an RNase protection assay. Ectopic expression of GADD153 or NF-IL6 suppressed transcription in a dose-dependent manner driven by the HPV11 URR in C33A, CaSki, HaCat and HFK cells. These results identify unexpected ways in which dietary I3C and DIM invoke cellular responses and are consistent with a potential antiviral effect of DIM on keratinocytes, but they do not explain the differential sensitivity
Indole-3-carbinol and diindolylmethane
induce apoptosis of human cervical cancer cells and in murine HPV16-transgenic
preneoplastic cervical epithelium.
Dietary indole-3-carbinol (I3C) has clinical benefits for both cervical cancer and laryngeal papillomatosis, and causes apoptosis of breast cancer cells in vitro. We asked whether I3C and its major acid-catalyzed condensation product diindolylmethane (DIM), which is produced in the stomach after consumption of cruciferous vegetables, could induce apoptosis of cervical cancer cell lines. We also asked whether this effect could be observed in vivo. In vitro, both I3C and DIM caused accumulation of DNA strand breaks in three cervical cancer cell lines.
Induction of apoptosis was confirmed by nuclear morphology, nucleosome leakage, altered cytoplasmic membrane permeability and caspase 3 activation. Neither I3C nor DIM caused apoptotic changes in normal human keratinocytes. In C33A cervical cancer cells, DIM was more potent than I3C [dose at which the number of viable cells was 50% of that in untreated cultures (LD(50)) = 50-60 micromol/L for DIM and 200 micromol/L for I3C in a mitochondrial function assay] and faster acting. Furthermore, I3C reduced Bcl-2 protein in a time- and dose-dependent manner.
In HPV16-transgenic mice, which develop cervical cancer after chronic estradiol exposure, apoptotic cells were detected in cervical epithelium by TdT-mediated dUTP nick-end labeling staining and by immunohistochemical staining of active caspase 3 only in mice exposed to 17beta-estradiol (E2) and fed I3C. Rare apoptotic cells were also observed by hematoxylin and eosin staining in the spinous layer of the cervical epithelium in both control and transgenic mice. Estradiol reduced the percentage of these late-stage apoptotic cells in the cervical epithelium of transgenic, E2-treated mice, but this reduction was prevented by I3C.
These data confirm the proapoptotic action of I3C on transformed
cells in vitro, extend the observations to cervical cancer cells and to
DIM and show for the first time that dietary I3C results in increased
apoptosis in target tissues in vivo.