Dietary Indole Inhibition of Breast Cancer

Institution: University of California, Berkeley
Investigator(s): Gary Firestone, Ph.D. -
Award Cycle: 1997 (Cycle III) Grant #: 3IB-0110 Award: $150,000
Award Type: IDEA
Research Priorities
Etiology and Prevention>Prevention and Risk Reduction: ending the danger of breast cancer

Initial Award Abstract (1997)
There is a growing public and scientific interest in the influence of hormonally active substances in the diet on both the risk to develop breast cancer and the potential to control breast cancer with minimal side effects. The best drug treatment currently available for women with breast cancer is hormone therapy. The growth of approximately one-third of breast cancers is stimulated by estrogen, so treating patients with anti-estrogens (e.g., tamoxifen) will slow the growth of certain tumors. However, tamoxifen therapy is associated with many side effects. Our interest is focused on naturally occurring compounds in the diet that could either prevent or slow the growth of breast cancer. Of particular importance is the identification and characterization of dietary compounds that can act independently of estrogen and, for some patients, potentially be used in combination with anti-estrogen therapy to more effectively reduce breast cancer growth. A compound called indole-3-carbinol (I3C) is one such dietary substance present in cabbage, broccoli, and Brussels sprouts. I3C is currently undergoing phase 1 clinical trials as a therapy for human breast cancer patients, although relatively little is known about the molecular mechanism of its growth inhibitory effects.

We have determined that I3C can inhibit the growth of both estrogen-responsive and non-responsive human breast cancer cell lines. Our hypthothesis is that I3C can inhibit breast cancer cell growth through a cellular pathway that is independent of the cellular effects of estrogen. Recent experiments show that I3C dramatically reduces the level of an important protein, called CDK6, that helps control a growth regulatory process called the cell cycle. Our research goal is to determine the functional and proliferative consequences for the dietary indole regulation of the CDK6 cell cycle protein in cultured human breast cancer cells and in breast cancer cell-derived tumors. Molecular experimental strategies will be employed to alter the expression of CDK6 so that its level remains elevated in cultured human breast cancer cells even after treatment with I3C. In these laboratory generated breast cancer cells, we plan to assess the effects of up to one week treatments with I3C on the regulation of cell cycle component activities and the rate of DNA synthesis, which is a measure of cell growth. A second critical aim of the study will be to determine the effects of expressing large amounts of CDK6 on the ability of I3C to inhibit the growth of breast cancer cell derived tumors formed in mice. Determining the molecular mechanism by which I3C inhibits breast cancer cell growth will be important because dietary I3C potentially represents an easily obtainable anti-cancer agent to the general public for the potential use in the treatment and prevention of breast cancer at a low cost.

Final Report (1999)
The naturally occurring chemical indole-3-carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit growth of the MCF-7 estrogen--dependent human breast cancer cell line more effectively than either agent alone. This inhibition was accompanied by a decrease in adherent and anchorage independent growth and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also caused a more pronounced decrease in the cyclin dependent kinase 2 (CDK2) specific enzymatic activity date either compound alone, but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen decreased phosphorylation of the retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs. Several lines of evidence suggest that I3C works through a mechanism distinct from tamoxifen. I3C arrested the growth of estrogen receptor negative MDA-MB-231 cells, failed to compete with estrogen for estrogen receptor binding, and selectively inhibited the mRNA, and protein expression of CDK6. The rapid (15h) decrease of CDK6 mRNA is consistent win a transcriptional down-regulation of the CDK6 promoter. Using a PCR based strategy, we have cloned 2.5 kb and 1 kb fragments of the CDK6 promoter into luciferase expression vectors, and preliminary evidence indicates a 30% downregulation of these promoter fragments by I3C. Currently, we are investigating the regulation of this fragment by I3C, several I3C-related compounds, and tamoxifen. We have also deduced the genomic structure of the CDK6 gene, including the RNA start site, and determined that new protein synthesis is not necessary for downregulation of CDK6 transcripts.

This is the first study of the CDK6 gene promoter, and results of further study may establish a mechanistic link between I3C regulation of transcription factors and the control of breast cancer cell proliferation. Our results demonstrate that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.

Indole-3-carbinol Inhibits the Expresion of Cycli-dependent Kinase-6 and Induces a G1 Cell Cycle Arrest of Human Breast Cancer Cells Independent of Estrogen Receptor Signaling
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Cover CM, Hsieh SJ, Tran SH, Halladen G, Kim GS, Bjeldanes LF, and Firestone GL
Yr: 1998 Vol: 273 Nbr: 7 Abs: Pg:3838-3847

Indole-3-carbinol and Tamoxifen Cooperate to Arrest the Cell Cycle of MCF-7 Human Breast Cancer Cells
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Cover CM, Hsieh SJ, Cram EJ, Hong C, Riby JE, Beljanes LF, and Firestone GL
Yr: 1999 Vol: 59 Nbr: Abs: Pg:1244-1251