Mechanisms of Reduced Metastasis by Conjugated Linoleic Acid

Institution: University of California, Davis
Investigator(s): Kent Erickson, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6PB-0113 Award: $314,988
Award Type: Request for Applications
Research Priorities
Etiology and Prevention>Prevention and Risk Reduction: ending the danger of breast cancer

Initial Award Abstract (2000)
The overall goal of this project is to determine the mechanisms of how conjugated linoleic acid (CLA) reduces and potentially prevent the spread of breast cancer to other parts of the body, the process of metastasis. Most breast cancer deaths are caused by the spread of the tumor. Since very few women have tumors that have completely spread to distant organs when their cancer is first detected, if agents were discovered to block metastasis, great advances could be made in prevention of the most devastating part of the disease.

Conjugated linoleic acid is a component found in some sources of dietary fat. It has been also found to be a potent protective agent against breast cancer in experimental animals. Although some dietary fats, such as those from plant oils, increased breast cancer in animals and others, such as those from fish oils, reduced breast cancer growth, large changes in the amounts of dietary fat were necessary to achieve those alterations. In contrast, a relatively low dietary concentration of CLA can significantly alter experimental breast cancer. Consequently, small alterations in dietary fat consumption, either through natural sources or as a supplement, has a great potential for reducing or preventing tumor growth and its spread. Indeed we have shown that dietary CLA can significantly reduce breast tumor metastasis in mice.

In this project, we propose to investigate how CLA reduces breast cancer. We have chosen to focus on two important steps in the spread of tumor cells, the production of blood vessels and enzymes necessary for metastasis. Those were selected because our preliminary studies indicate that those steps may be altered by dietary fat. We believe these changes may be due to CLA's ability to reduce prostaglandin, a fat-based mediator which has been shown to influence metastasis and can also be altered by dietary fat. Moreover, CLA may have the ability to alter how genes are expressed.

From this work, we hope to determine how dietary CLA functions to reduce or prevent the spread of breast cancer. Proving that CLA alters blood vessel production in tumors would be significant since the major limitation of therapy to block new blood vessel formation has been to provide continuous levels of a potentially expensive or toxic agent on a long term basis. In contrast, the ability to provide an inexpensive agent in food, CLA, would circumvent this issue. Short-term studies have shown CLA to be safe in women. Although CLA has not been tested for its ability to prevent breast cancer in humans, these experiments should provide a basis, which will be necessary for future human studies to reduce the mortality of women diagnosed with the disease.

Final Report (2005)
Conjugated linoleic acid (CLA) is a fatty acid found in some sources of dietary fat and can be a potent protective agent against mammary tumors in experimental animals. We have recently published proof that CLA significantly reduced breast tumor spread in mice. In this study, we used a mouse model that displays many of the characteristics of human tumors to investigate the mechanism of how CLA reduces the malignant process.

Our focus to date has been to study the effect of dietary CLA on the expression of proteins that enhance or reduce the ability of tumors to grow or spread. For that study, mice were divided into dietary groups where the food consumed was similar except for the type and amount of CLA. Our observation was that animals fed the diets that had no CLA expressed proteins that enhanced tumorigenesis while animals fed diets that contained CLA expressed lower levels of the proteins that are known to enhance tumorigenesis. We also observed that the time it took tumors to establish was longer in animals that were fed the different CLA diets. Moreover, when animals fed diets containing CLA were injected with tumor cells directly into their blood stream, fewer tumors were found growing in their lungs compared to animals that were similarly treated except fed a diet containing no CLA. While there are many possible reasons for CLA's effect on tumor formation, growth and spread, we provide evidence that one reason may be that CLA, when given directly to tumor cells in culture, kills them. We also found that CLA altered the ability of tumor cells to invade and migrate in a tissue culture assay.

Future studies with CLA and the mechanism of its action on tumorigenesis will depend on finding what genes may be altered. We found that several genes were expressed differently when tumors were from animals fed CLA as opposed to animals that were fed only corn oil as a source of fat. (CLA treatment decreased VEGF MRNA levels and decreased the message expression of both COX-1 and COX-2 as measured by microarray.) Those genes are involved in various events important to several aspects of tumor initiation, growth and spread. These data add to accumulating evidence that CLA can be an important chemo-protective agent.

Symposium Abstract (2003)
We have previously reported significantly decreased metastasis in mice fed high fat diets containing conjugated linoleic acid (CLA). Our overall goal has been to assess which steps or components in the metastatic cascade are altered by that fatty acid. While previous studies have shown that in vitro CLA treatment can directly affect the proliferation of mammary tumor cells, which is an important com- ponent of metastasis formation, it is unknown whether CLA is responsible for altering other impor- tant steps of the metastatic cascade, such as invasion and migration. Mouse mammary tumor cells with a high capacity for metastasis in vivo were treated with various concentrations of several different fatty acids and assessed for invasion and migration. Treatment of cells with c9, t11 and t10, c12 CLA isomers significantly decreased the number of cells that invaded and migrated through a base- ment membrane. By contrast, arachidonic acid and linoleic acid significantly stimulated increased invasion and migration. That increased invasion was abrogated when the eicosanoid inhibitor, indometha- cin, was added to the cultures. These same mam- mary tumor cells exhibited decreased eicosanoid production when cultured with either CLA isomer. Thus, decreased invasion and migration induced by CLA may be correlated with alteration of eicosanoid synthesis.

These in vitro results support our previously re- ported in vivo observations whereby CLA, in- domethacin and fish fatty acids were shown to decrease the lodgment, survival and proliferation stages of the metastatic cascade. The effects of CLA on invasion and migration may be due to decreased activity of the pro-invasion and migration metalloproteinases, MMP-2 and MMP-9. Expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were also altered by CLA. Those MMPs and their inhibitors can be regulated by eicosanoids. These data provide evidence that CLA affects tumor spread by altering more than one component or stage in the metastatic cascade. Moreover, those effects may be mediated through a common path- way, such as alteration of eicosanoid synthesis, which can effect the expression of genes involved in the specific metastatic stages.

Funded by CBCRP Grant 6PB-0113.

Symposium Abstract (2003)
Conjugated linoleic acid (CLA) is a dietary component that has been shown to reduce tumorigenesis at several sites. To date, the mechanism of action of CLA has not been clarified. We as well as other investigators have shown that CLA significantly decreased the growth and metastasis of breast tumor cell.

The purpose of this study was to determine whether CLA altered tumor cell growth and proliferation by altering the eicosanoid metabolism pathway. Mouse mammary tumor cell line 4526 was treated with various fatty acids including CLA isomers, c9, t11 and t10, c12, and linoleic acid (LA) for positive or ethanol for a vehicle control. The t10, c12 isomer significantly reduced cell proliferation in a dose dependent manner; c9, t11-CLA reduced cell proliferation by 17%, while LA increased proliferation by 10%. After treatment with the lipoxygenase inhibitor, NDGA, or the cycloxygenase inhibitor, in-domethacin, cell viability was measured. NDGA but not indomethacin treatment reduced cell viability in dose dependent manner.

To determine how t10, c12-CLA or NDGA inhibited tumor cell growth rate, apoptosis, cell proliferation, and cell cycle kinetics were measured. None of the fatty acids altered apoptosis but NDGA induced apoptosis. After treatment with 50 mM fatty acid or NDGA for 24hr, t10, c12-CLA and NDGA decreased, but 5 mM and 50 mM LA increased tumor cell proliferation. To determine whether cell proliferation was reduced as a result of cell cycle arrest, mammary tumor cells were treated with the CLA isomers, LA, or NDGA and cell cycle kinetics measured by flow cytometry. The t10, c12-CLA but not c9, t11-CLA and NDGA compared to control arrested the cell in the G1/G0 phase. Thus, the observed decrease in growth of mammary tumor cells in vitro especially by t10, c12-CLA may be due to decreased cell division and not increased cell death which was independent of eicosanoid pathway.

(Supported by the California Breast Cancer Research Program, 6PB-0113)

Symposium Abstract (2005)
In this study, we wanted to show that the antitumor effectiveness of the dietary fatty acid, conjugated linoleic acid (CLA), can change based on the type of dietary fat consumed. In the past, studies with animals have consistently shown that dietary CLA reduces mammary tumor incidence and spread or metastasis. Relatively low concentrations of CLA are actually required for those effects making it a very potent dietary component. To test our hypothesis, we fed mice diets that differed in fatty acid composition but not amount then assessed the effect of CLA concentration on mammary tumorigenesis. Mammary tumor metastasis was significantly decreased when beef tallow (BT) replaced half of a defined vegetable fat blend (VFB). In addition, that same BT/VFB diet lowered the level of CLA required to significantly decrease mammary tumor metastasis from 0.1% of the diet to 0.05%. Studies with cultured tumor cells showed that fatty acids normally found in abundant quantities in beef tallow, oleic, stearic and palmitic acids, enhanced the killing effects of CLA. An abundant vegetable oil fatty acid, linoleic acid, reduced the effectiveness of CLA toxicity on tumor cells in culture. Those data provide evidence that dietary BT, though low itself in CLA, may make dietary CLA more potent at reducing mammary tumorigenesis. Finding dietary components that have the ability to alter breast cancer outcome is absolutely essential. However, the relationship those factors may have with other dietary components needs to be assessed before they can be used as possible preventive or therapeutic agents. We believe that CLA can be a viable and cost effective chemopreventive agent as well as an adjuvant therapy for women with breast cancer. Supported by America's Beef Consumers.

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