Our experiments have shown that flavonoids from citrus fruits inhibit proliferation of both estrogen receptor-negative (ER-) MDA-MB-435 and estrogen receptor-positive (ER+) MCF-7 human breast cancer cells in vitro. Subsequent studies in animal cancer models showed that substituting orange juice or grapefruit juice for drinking water inhibited mammary cancer development, although the animals grew normally. This indicated that the juices were acting specifically on the cancer cells. Flavonoids from orange juice and grapefruit juice appeared to be less effective inhibitors of mammary cancer in vivo than the juices themselves. It, therefore, seems possible that citrus limonoids may be largely responsible for the anti-cancer effects of the juices in vivo, since these triterpenoid compounds inhibited human breast cancer cells more effectively than the flavonoids in vitro.

The aim of this proposal is to investigate in detail the ability of citrus limonoids to inhibit growth and metastasis of ER- and ER+ human breast cancer cells following their injection into the mammary fat pads of immunodeficient nude mice. After toxicity studies have been done to determine the maximum tolerated dose, the compounds will be given by mouth to animals in which tumors formed from the human breast cancer cells have had some time to develop, in order to see whether they can arrest growth or cause regression of the primary tumors and inhibit metastases. In other studies, the limonoids will be investigated in an adjuvant setting where the tumors will be allowed more time to develop, then resected, and treatment begun to see whether they can inhibit regrowth of the tumors and metastases under these conditions. Limonoids for these studies will be provided by Drs. Hasagawa and Manners of the Western Regional Research Center, United States Department of Agriculture, Albany, CA. Dr. Hasagawa, in particular, has extensive experience in the isolation and characterization of these compounds.

As part of the above studies, experiments will be carried out to see whether the limonoids are inducing apoptosis as well as inhibiting proliferation of the cancer cells. More detailed investigation of the mechanisms involved in the anti-cancer activity of citrus limonoids will be carried out in collaboration with Drs. Marc Lippman and Robert Clarke at the Vincent T. Lombardi Cancer Center, Georgetown University, Washington, DC, whose expertise in this area of investigation is widely recognized.

The concentrations of limonoids and their metabolic products will also be measured in the mammary fat pad tumors of mice to which they have been given by mouth, to see whether the levels that can be achieved are comparable to those that have been shown to inhibit the human breast cancer cells effectively in vitro. This should provide information on the nature of the compounds responsible for any observed in vivo anti-cancer activity.

Research on the anti-cancer properties of these compounds in animals is an essential prerequisite to clinical trials designed to assess their usefulness in the treatment of human breast cancer.