DIM and Uterine / Cervical Health

Oral diindolylmethane (DIM): pilot evaluation of a nonsurgical treatment for cervical dysplasia.

Del Priore G, Gudipudi DK, Montemarano N, Restivo AM, Malanowska-Stega J, Arslan AA.

Indiana University School of Medicine, Dept of Ob-Gyn, Div Gyn Oncology, Indianapolis, IN 46202-5149, USA.

Gynecol Oncol. 2010 Mar;116(3):464-7.

OBJECTIVE: Standard surgical treatment for CIN may impair fertility generating a need for alternative treatment options. We tested the efficacy and toxicity of oral DIM in the treatment of CIN 2 or 3 lesions. METHODS: Patients with biopsy-proven cervical intraepithelial neoplasia (CIN) 2 or 3 scheduled for loop electrosurgical excision procedure (LEEP) were randomized 2:1 to receive diindolylmethane (DIM) (BioResponse-DIM, BioResponse, Boulder, CO) orally at approximately 2 mg/kg/day for 12 weeks or placebo (defatted rice bran, BioResponse). Subjects were evaluated every 3-4 months for 1 year. Analysis of data up to 1 year was assessed including Pap smear, HPV, colposcopy, biopsy and physical examination were performed at follow-up. Central pathology review confirmed all histology diagnoses. RESULTS: To date, 64 subjects (mean age 28 years, range 18-61) have been enrolled (45 in the DIM arm, 19 in the placebo arm), with 60 available for analysis. Average follow-up was 6 months. At enrollment, 58% were diagnosed with CIN 2 and 42% with CIN 3, 57% of subjects were Caucasian, 15% African American, 12% Hispanic and 17% Asian. During treatment 2 subjects (3%) complained of nausea (grade 2) at the 3- to 4-month visit. No systemic toxicities were observed (normal CBC, LFTs, comprehensive metabolic). Forty-six subjects had biopsies at first follow-up (77%). Twenty-one subjects (47%) in the DIM group had improved CIN with a decrease by 1-2 grades or a normal result. Median time to improvement was 5 months. Improved Pap smear was seen in 49% (22/45) with either a less severe abnormality or normal result. Colposcopy improved in twenty-five subjects in the DIM group (56%). Of these 25 subjects, 21 (84%) had improved colposcopic impression, 13 (52%) had a decrease in involved quadrants and 18 (72%) had a decrease in lesion number. Complete colposcopic response was observed in 4 subjects (9%). Stratifying by level of dysplasia, age, race, HPV status, tobacco use, contraceptive used did not alter the results. At median follow-up of 6 months, 85% of subjects have not required LEEP based on routine clinical triage of improving global assessment. There was no statistically significant difference in any outcome between the DIM and placebo group. CONCLUSION: Oral DIM at 2 mg/kg/day is well tolerated with no significant toxicity. We observed a high rate of clinically significant improvement in confirmed CIN 2 or 3 lesions among both treatment groups in this randomized clinical trial.

PMID: 19939441 [PubMed - indexed for MEDLINE]

Diindolylmethane inhibits cervical dysplasia, alters estrogen metabolism, and enhances immune response in the K14-HPV16 transgenic mouse model.

Sepkovic DW, Stein J, Carlisle AD, Ksieski HB, Auborn K, Bradlow HL.

The David and Alice Jurist Institute for Research, Hackensack University Medical Center, Hackensack, New Jersey 07601, USA.

Cancer Epidemiol Biomarkers Prev. 2009 Nov;18(11):2957-64.

This study was designed to establish whether 3,3'-diindolylmethane (DIM) can inhibit cervical lesions, alter estrogen metabolism in favor of C-2 hydroxylation, and enhance immune function in the K14-HPV16 transgenic mouse model. Mice were bred, genotyped, implanted with E(2) pellets (0.25 mg/90-day release) under anesthesia, and divided into groups. Wild-type and transgenic mice were given either AIN76A diet alone or with 2,000 ppm DIM for 12 weeks. Blood and reproductive tracts were obtained. Blood was analyzed for estrogen metabolites and IFN-gamma. The cervical transformation zone was sectioned and stained for histology. Estradiol C-2 hydroxylation and serum IFN-gamma levels were significantly increased over controls in wild-type and transgenic mice receiving DIM. In wild-type mice without DIM, hyperplasia of the squamous epithelium was observed. Wild-type mice fed DIM displayed a normal thin epithelium. In transgenic mice without DIM, epithelial cell projections into the stroma (papillae) were present. An additional degree of nuclear anaplasia in the stratum espinosum was observed. Dysplastic cells were present. Transgenic mice fed DIM displayed some mild hyperplasia of the squamous epithelium. DIM increases estrogen C-2 hydroxylation in this model. Serum INF-gamma was increased, indicating increased immune response in the DIM-fed animals. Histopathology showed a marked decrease in cervical dsyplasia in both wild-type and transgenic mice, indicating that DIM delays or inhibits the progression from cervical dysplasia to cervical cancer. Using the K14-HPV16 transgenic mouse model, we have shown that DIM inhibits the development of E6/E7 oncogene-induced cervical lesions.

PMID: 19861518 [PubMed - indexed for MEDLINE]

In vivo, uterine-protective activity of absorption-enhanced diindolylmethane: animal and preliminary human use in combination with Tamoxifen.

Zeligs MA, Fulfs JC, Peterson R, Wilson SM, McIntyre L, Sepkovic DW, Bradlow HL.

Absorbable Diindolylmethane (BioResponse-DIM® [BR-DIM]) is a patented, orally active formulation of 3,3'-diindolylmethane (DIM), available as a dietary supplement. DIM is found in cruciferous vegetables, but also under investigation for its chemopreventive and pro-apoptotic activities. Based on DIM's known growth-inhibition of endometrial(1) and cervical cancer cell lines(2), oral BR-DIM was tested in vivo in a model of estrogen-driven uterine growth(3) and during monitored use in humans.

Groups of immature female rats were implanted with placebo or androstenedione pellets and fed either control, or BR-DIM (250 mg/kg/day [62.5 mg/kg/day DIM]) containing diets. Androstenedione served as a source of aromatase-derived estrogen. When sacrificed after 72 hrs, the BR-DIM group revealed significantly lower uterine/body weight indexes (p=0.04) compared to control rats. Thus, BR-DIM reduced estrogen-related uterine growth in rats.

In monitored human use, BR-DIM, at the label-recommended dose of 300 mg/day (providing 75 mg/day of DIM), was taken as a dietary supplement by a 41 year old with treated, Stage I breast cancer on Tamoxifen (20 mg/day). Dysfunctional uterine bleeding and Tamoxifen-related endometrial hyperplasia were present before BR-DIM use, as confirmed by transvaginal ultrasound and endometrial biopsy. Following 1 month of BR-DIM use, resolution of bleeding was noted. Normalization of endometrial contour and thickness were documented during 3 months of BR-DIM use, and lasting 1 month following cessation of its use (from 16.0 mm before, to 6.6 mm during, and 5.0 mm after DIM). 2 months after discontinuing BR-DIM, endometrial thickness had returned to the pre-treatment level (17.2 mm).

In testing urinary estrone metabolites in women with treated breast cancer, estrogen 2-hydroxylation was shown to not be influenced by Tamoxifen alone. During combined Tamoxifen use with BR-DIM, a shift to greater estrogen 2-hydroxylation and less 16-hydroxylation was demonstrated. Therefore, Tamoxifen did not inhibit promotion of estrogen 2-hydroxylation by BR-DIM. Women with treated breast cancer responded to BR-DIM with the same increased 2-hydroxylation of estrogen as previously demonstrated in healthy women(4).

Supported by these observations, BR-DIM deserves further evaluation as a potential uterine protective agent during combined use with Tamoxifen(5) and during estrogen exposure from HRT(6).

References

1. Chen DZ, Qi M, Auborn KJ, Carter TH. Indole-3-carbinol and diindolylmethane induce apoptosis of human cervical cancer cells and in murine HPV16-transgenic preneoplastic cervical epithelium. J Nutr. 2001 Dec;131(12):3294-302.
2. Leong H, Firestone GL, Bjeldanes LF. Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells result from an estrogen receptor-mediated increase in transforming growth factor-alpha expression.Carcinogenesis. 2001 Nov;22(11):1809-17.)
3. Katzenellenbogen BS, Ferguson ER, Lan NC. Fundamental differences in the action of estrogens and antiestrogens on the uterus: comparison between compounds with similar duration of action. Endocrinology. 1977 May;100(5):1252-9.
4. Zeligs, M.A., Sepkovic, D.W., Manrique, C., Macsalka, M., Williams, D.E.,and Bradlow, H.L. Absorption-enhanced 3,3’-Diindolylmethane: Human Use in HPV-related, Benign and Pre-cancerous Conditions. Proc. Am. Assoc. Cancer Res. 2002 Apr; 43, 3198 (Abstract).
5. Kennedy MM, Baigrie CF, Manek S. Tamoxifen and the endometrium: review of 102 cases and comparison with HRT-related and non-HRT-related endometrial pathology. Int J Gynecol Pathol. 1999 Apr;18(2):130-7.
6. Gull B, Karlsson B, Milsom I, Granberg S. Factors associated with endometrial thickness and uterine size in a random sample of postmenopausal women. Am J Obstet Gynecol. 2001 Aug;185(2):386-91.
7. Crowell JA, Page JD, Zeligs MA, Guyton KZ. Drug Metabolism Enzyme Induction by Indole-3-Carbinol and Diindolylmethane in Rats, NIH Meeting Abstract, Amsterdam, September, 2002.

 


Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells result from an estrogen receptor-mediated increase in transforming growth factor-alpha expression.

Leong H, Firestone GL, Bjeldanes LF.

Carcinogenesis 2001 Nov; 22(11):1809-17.

3,3'-Diindolylmethane (DIM), a major in vivo product of indole-3-carbinol (I3C), is a promising anticancer agent derived from vegetables of the Brassica genus including broccoli, Brussels sprouts and cabbage. We report here that DIM has a potent cytostatic effect in cultured human Ishikawa endometrial cancer cells.

A combination of northern blot and quantitative PCR analyses revealed that DIM induced the level of TGF-alpha transcripts by approximately 4-fold within 24 h of indole treatment. DIM also induced a 4-fold increase in the activity of the estrogen response marker, alkaline phosphatase (AP). Co-treatment of cells with the estrogen receptor (ER) antagonist ICI, or with the inhibitor of PKA-mediated activation of the ER, H89, ablated the DIM induction of both TGF-alpha expression and AP activity.

Furthermore, DIM increased the maximum stimulatory effect of estrogen on TGF-alpha expression. Co-treatment with the protein synthesis inhibitor, cycloheximide, abolished the inductive effects of DIM, indicating differences in the mechanistic requirements of DIM and estrogen. DIM treatment also stimulated levels of secreted TGF-alpha protein by >10-fold. The ectopic addition of TGF-alpha inhibited the growth of Ishikawa cells, whereas incubation with a TGF-alpha antibody partially reversed the growth inhibitory effects of DIM.

Taken together, these results extend our previous findings of the ligand independent estrogen receptor agonist activity of DIM, and uncover an essential role for the stimulation in TGF-alpha expression and the TGF-alpha activated signal transduction pathway in the potent cytostatic effects of DIM in endometrial cancer cells.

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Indole-3-carbinol and diindolylmethane induce apoptosis of human cervical cancer cells and in murine HPV16-transgenic preneoplastic cervical epithelium.

Chen DZ, Qi M, Auborn KJ, Carter TH.

J Nutr 2001 Dec; 131(12):3294-302

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 pro-apoptotic 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.

 


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