Melinda Myzak

Chemoprevention of Colorectal Cancer

Melinda Myzak
George B. Whatley Graduate Fellow
Linus Pauling Institute

Colorectal cancer has the third highest mortality rate of all cancers, with approximately 150,000 new cases diagnosed in the U.S. each year. Prevention is becoming an increasingly important area of research. Numerous epidemiological studies have demonstrated the importance of consumption of fruits and vegetables in cancer prevention, particularly in the prevention of colorectal cancer. Several recent studies have investigated the chemopreventive efficacy of broccoli consumption in humans and animals. Broccoli belongs to the Brassica genus, which also includes cauliflower, cabbage, and brussels sprouts. These vegetables contain compounds called glucosinolates that are enzymatically converted to isothiocyanates by a plant enzyme called myrosinase, which is released after chewing. Enzymes present in the intestinal tract of mammals can also catalyze this conversion. Broccoli contains high amounts of a glucosinolate called glucoraphanin, which is converted to the isothiocyanate, sulforaphane.

Sulforaphane has been shown to prevent chemically-induced intestinal and mammary tumor formation in animals. In cultured human cancer cells, sulforaphane can induce apoptosis (programmed cell death) and arrests the cell cycle, both of which are critically important in preventing tumor growth. Sulforaphane induces so-called "Phase 2" enzymes, which are part of the "detoxification" pathway that cells use to turn potentially harmful chemical carcinogens into harmless forms that can be excreted. Thus, sulforaphane may help eliminate potential carcinogens, preventing the initiation stage of tumor formation. However, this mechanism does not explain some of the other effects of sulforaphane, such as triggering cancer cells to die. We decided to address this question.

In cells, DNA is wrapped around proteins called histones. This allows the cell to efficiently "package" DNA and to control the expression of specific genes. Histones have "tails" that can determine how accessible the DNA is to transcription factors, which are proteins that bind to specific regions of DNA called promoters. In a DNA sequence, promoters are positioned before the actual gene and contain specific sequences recognized by transcription factors. Depending on the sequences and transcription factors, the gene is turned on or off. When a gene is turned on, the protein product of the gene is made.

Histone tails can be modified by addition or removal of acetyl groups. The addition of these chemical groups results in the tail moving away from the DNA, allowing transcription factors to interact with DNA. When acetyl groups are removed from the histone tail, it wraps around the DNA more tightly, so the DNA cannot interact with transcription factors. An acetylated histone is associated with DNA that has genes that are active, or turned on. Conversely, a deacetylated histone is associated with DNA that has genes that are inactive, or turned off. This switch between active and inactive genes is controlled by two groups of proteins called histone acetyltransferases, which add acetyl groups to histones, and histone deacetylases (HDACs), which remove acetyl groups from histones.

Cells contain tumor suppressor genes, whose role is to keep cells from becoming cancerous. In cancerous and precancerous cells these genes are associated with deacetylated histones, which results in their inactivity. Theoretically, if HDACs can be inhibited, these tumor suppressor genes can be turned back on. When the tumor suppressor genes are reactivated, cancer progression in cells may be halted and cancer cells may die. Several HDAC inhibitors have already been discovered. When inserted into the HDAC enzyme, these agents inhibit its activity. HDAC inhibitors have been shown to be effective in cultured cells from several types of cancer, and some are currently in clinical trials.

Based on the molecular structure of known HDAC inhibitors, we searched for possible dietary HDAC inhibitors. Since the structural characteristics of sulforaphane are similar to those of known HDAC inhibitors, sulforaphane may exert its chemopreventive activity through the inhibition of HDACs. We used a gene assay to investigate this. Specifically, the luciferase gene, which codes for a bioluminescent enzyme, was linked to a promoter that responds to HDAC inhibitors. When an HDAC inhibitor is added to cells in culture with these genes, more luciferase is made, and the resulting bioluminescence can be measured. Sulforaphane dose-dependently increased bioluminescence, indicating that it was acting as a potent HDAC inhibitor.

We also measured HDAC activity directly by incubating extracts of cellular nuclei (containing HDACs) with sulforaphane. In this experiment, sulforaphane did not have any effect on HDAC activity. However, extracts from live cells treated with sulforaphane showed a decrease in HDAC activity. Interestingly, when media from live cells treated with sulforaphane was incubated directly with the nuclear extracts, there was also a decrease in HDAC activity. These experiments suggested that a metabolite of sulforaphane—not sulforaphane itself—may be the actual HDAC inhibitor.

To confirm that a sulforaphane metabolite is the HDAC inhibitor, we inhibited the first step of sulforaphane metabolism. When cells were treated with this inhibitor before the addition of sulforaphane in our gene assay experiment, less luciferase was made, resulting in decreased bioluminescence. Additionally, sulforaphane did not inhibit HDAC activity when the inhibitor of sulforaphane metabolism was present. These results imply that metabolism of sulforaphane results in the formation of metabolite(s) that act as HDAC inhibitors. Further studies are under way to identify and characterize the active metabolite(s) of sulforaphane in HDAC inhibition.

In further support of the idea of sulforaphane as a novel HDAC inhibitor, overall histone acetylation was increased in cells treated with sulforaphane. In addition, these results were reproducible in cultured human colon cancer cells, including an increase in levels of a tumor suppressor protein. Future studies will continue to investigate HDAC inhibition by sulforaphane in other human cancer cells and animals. Thus far, my research has demonstrated a novel mechanism for colorectal cancer chemoprevention by sulforaphane and suggests that sulforaphane and its metabolites may have value in cancer therapy as well, through inhibition of HDAC activity.

I would like to thank Dr. George B. Whatley for his generous donation to the Linus Pauling Institute that has enabled me to conduct this research.

Last updated May, 2004

Micronutrient Research for Optimum Health

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