Victoria Bentley and Shalini Rao: Sacred Heart School of Halifax

The Interactions of Red Clover with Tamoxifen

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Abstract

Introduction

Materials and Method

Results

Discussion

Conclusion

Bibliography

Contact Information

Acknowledgements

Appendix


Introduction:

Complementary and alternative modalities (CAMs), like red clover, are prevalent in our current lifestyles. The media typically endorses these alternative modalities. There is currently little done to screen natural medicines, and their potency is not government regulated. The interactions between natural and conventional medicines are not well known, and little research has been conducted on this topic. There is a problem substantiated in the fact that these interactions have the potential to be harm unaware patients. With this in mind, the purpose of this project is to investigate the interactions between red clover and Tamoxifen, when jointly administered to breast cancer cells.

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Breast Cancer

Cancer refers to a malignant growth of cells able to divide uncontrollably, and spread, destroying healthy cells. Breast cancer is a type of cancer most common in women, but also found in men, which originates in the cells of the breast. The primary function of the breast is to produce milk, and for this reason it is composed of milk glands, milk ducts, and fatty tissue. Most breast cancers begin in the glandular tissue, and are called adenocarcinomas, cancer may also start in the milk ducts (ductal carcinoma), or in groupings of glands called lobules (lobular carcinoma). The most common type of breast cancer is ductal carcinoma.

According to the National Cancer Institute of Canada, there will be an estimated 153 000 new cases of cancer, and 70 400 deaths due to cancer in 2006 in Canada. By examining the current incident and mortality rates in Canada, it can be estimated that 38% of Canadian women will have cancer at some point in their lives, and that 24% of Canadian women will die due to cancer (this is roughly one in every four women). In 2006, approximately 22 200 Canadian women will develop new cases of breast cancer, and 5 300 women will die from breast cancer. Over half of breast cancer cases occur in females between the ages of 50 and 69. For women, breast cancer is the cancer with the highest number of new cases in Canada, and is estimated to be the cancer with the second highest number of deaths in 2006, second to lung cancer. These statistics translate to an estimated 700 new cases of breast cancer, and 190 deaths due to breast cancer in Nova Scotia in 2006. The Atlantic provinces and Quebec have the highest incidence and mortality rates in Canada. The lifetime probability of developing breast cancer for women is grim, 1 in 8.9, and the likelihood of dying from the disease is 1 in 26.8.[1]

Over 70% of breast cancers in women are estrogen receptor (ER) positive, and their growth is dependent upon the hormone. This estrogenic characteristic of most breast cancers is what makes the use of anti-estrogens a viable solution when treating the cancer. There are two types of estrogen receptors, the first is ERa, and the second is ERb. Clinically, breast cancers are described as ERa+ (estrogen receptor a positive) or ERa- (estrogen a negative). Cells that are ERa+ grow in response to an estrogen signaling system, described as similar to the insulin factor system. MCF-7 cells are a human epithelial breast cancer cell line, commonly used in experimental models. MCF-7 cells are ERa+, and an adenocarcinoma breast cancer.

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Tamoxifen (Nolvadex)

Tamoxifen is the oldest of the selective estrogen receptor modulators. It is prescribed for women with hormone-receptor-positive breast cancer before and after menopause. Tamoxifen is taken as a preventive drug by women who have a family history of breast cancer, or are at high risk to otherwise develop breast cancer. It is the hormonal treatment of choice for pre-menopausal women. Tamoxifen may also be used for post-menopausal women, but it is not quite as effective for them as the aromatase inhibitors (a mechanism which deprives the tumor of estrogenic signals).

Tamoxifen is also administered to women who have non-invasive, hormone-receptor-positive breast cancer or have hormone-receptor-positive invasive breast cancer at any stage. Tamoxifen is commonly administer for up to five years, but women with a metastatic (advanced) disease can continue taking Tamoxifen for as long as the effects of it seem to be positive.

 Approximately two-thirds of cells are estrogen receptor positive (ER+). The MCF-7 cells that are discussed in this project were found through the experimental process to be ER+. Tamoxifen optimally targets ER+ cells. Essentially, Tamoxifen acts like estrogen in cells. It blocks the estrogenic effect at the estrogen receptor.

Tamoxifen mimics the shape of estrogen and binds tightly to the estrogen receptors in cells. When it binds with the estrogen receptor, it changes the shape of the signaling loop on the surface of the receptor. As Tamoxifen is larger than the hormone estrogen, it forces the activation loop out into an inactive conformation—blocking the signal to grow.

Tamoxifen-ER binds to the DNA to form a new complex, which is unable to function like the estrogen-ER complex. Therefore, the hormonal growth signal given off by the cell is turned off and the cell will stop proliferating. As Tamoxifen is such a weak estrogen, its estrogen signals don't stimulate cell growth of breast tissue. Also, because the Tamoxifen has stolen the place of the more powerful estrogen, it blocks estrogen-stimulated cancer cell growth. This mechanism of action is fundamental as it allows the Tamoxifen to act like an anti-estrogen.

Tamoxifen may also take the place of natural estrogen in the receptors of healthy breast cells. In doing so, it most likely discontinues abnormal growth and the development of a totally new breast cancer. By blocking natural estrogen from getting to the receptors, Tamoxifen is assisting in reducing the risk of breast cancer in women at high risk of breast cancer, and yet who have never had breast cancer before. Tamoxifen can also help women who have already had breast cancer in one breast, by decreasing the risk of a new breast cancer forming in the other breast. Of course, Tamoxifen is not a perfect SERM, and is not devoid of side effects. Of course, the risk of these side effects is sometimes trumped by the threat of breast cancer.

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Red Clover

Red clover is a plant that was named Trifolium pratense by Carolus Linnaeus in 1753. Trifolium means 'three leaved' while pratense is Latin for "found in meadows”. Red clover is a plant whose flowers have been used in some cultures to treat certain medical problems. It is being studied in the relief of menopausal symptoms and it is also believe that this complementary and/or alternative medicine (CAM) may have anticancer effects. Red clover is also frequently referred to as purple clover and wild clover and it is the most commonly grown forage clover.

Red clover contains a type of phytoestrogen called isoflavones. A phytoestrogen is a plant hormone, which resembles human estrogen in chemical structure, yet is weaker. There are four types of isoflavones: genistein, daidzein, formononetin, and biochanin. Red clover contains genistein, which belongs to the family of drugs called enzyme inhibitors. Scientists believe genistein may be a significant anticancer force, particularly with hormone-related cancers such as breast cancer. However, the anticancer force of red clover has yet to be validated, and it is not used in lieu of more efficient cancer treatments.

Red clover is believed to act as a selective estrogen receptor modulator (SERM) when administered to cells. This means that the pharmacological aspect in red clover is designed to deliver the benefits of estrogen without its negative side effects. Tamoxifen, the conventional cancer drug that is discussed in the project, is also a SERM.

We see that red cover could potentially increase MCF-7 cell growth through its estrogenic effects, and that it also has the potential to decrease MCF-7 cell growth via genistein. What is key to this project is the net outcome of red clover when used to treat the cancer cells in correlation with Tamoxifen. Our rationale behind our project is the competitive binding aspect of Tamoxifen to ERs, which could mean that the added competition of red clover’s estrogenic activity may interfere with the efficacy of Tamoxifen.

Red clover is an herbal medicine which is used both topically and orally to treat many malignancies. It is often applied topically as a cure for cancerous growths. It is administered orally for many medical maladies, some of which include:

  • menopausal symptoms, hot flashes
  • breast mastalgia (cyclic breast pain of tenderness)
  • breast cancer prevention
  • indigestion, whooping cough, cough, asthma, bronchitis
  • sexually transmitted infections (STIs)

Taking complementary and alternative medicines has become something which is very popular in today’s society. In essence, taking CAMs has become something of a fad. People have begun to buy more and more medicines which do not require prescriptions. Soy, which comes from the same legume family as red clover, is appearing on the labels of many foods and beverages. Red clover is also used as a flavoring mechanism in foods and beverages. The growing use and existence of herbal therapies, for treatment of virtually any illness, necessitates study of the safety of these natural preparations.

Our red clover tincture was purchased through St. Francis Herb Farm. The tincture contained 250 mg red clover blossom/ mL of tincture. For more information please visit the above link.

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Estrogen

Estrogen is the primary hormone produced in the ovaries before menopause, and by other cells, such as fat cells, after menopause. The hormone is also secreted in lesser amounts from other locations in the female body. Estrogen is responsible for the development and presence of secondary female sexual characteristics, regulating the menstrual cycle, and plays a role in reproduction.

There are three estrogens produced by females, these being Estrone (E1), Estradiol (E2), and Estriol (E3). Estradiol is the predominant estrogen hormone produced by follicular cells in the ovary. It is also the most potent naturally occurring estrogen in the human body.

Phytoestrogens are plant compounds that have an estrogenic effect. Phytoestrogens have a similar chemical structure as that of estrogens, however the biological effect that they exert is somewhat weaker than the effect of phytoestrogens.  A common subclass of phytoestrogens is isoflavones, which are found in soy and red clover.

Within an estrogen-receptor-positive cell there will be an estrogen receptor. The receptor is how the estrogen produces an effect on a cell. The receptor allows the cell to recognize the estrogen, and once the estrogen has bound to the receptor will trigger a specific genetic sequence.

There are two types of estrogen receptors, estrogen receptor alpha (ERa) and estrogen receptor beta (ERb). The ERa has been researched more thoroughly, and more is known about its functions within the cell, as opposed to the ERb, which was only discovered in 1996. Due to the increased knowledge pertaining to ERa, clinically breast cancers are described as ERa+, or ERa-.  The two receptors have different affinities to selective estrogen receptor modulators (SERMs), and therefore respond differently to various SERMs. The functions of ERb are still fairly elusive, and much remains to be discovered about it. The action of an estrogen is determined based on its structure, the ER that it binds to (ERa, or ERb) and the interaction of the ligand with the ER. ERa and ERb differ in their distribution within cell tissue, and in their affinity to SERMs. It should be noted that a cell that is at one point mainly ERa could evolve to be ERb, adding a level of complexity. It is thought that certain phytoestrogens have a competitive preference for ERb. The way in which the DNA binding functions of ERa and ERb function are almost identical, although they have the ability to activate different genes that are responsive to estrogen.

We hypothesize that phytoestrogens in red clover will decrease the efficiency of Tamoxifen as a breast cancer treatment, therefore blocking the effects of the drug.

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[1] National Cancer Institute of Canada, Canadian Cancer Statistics 2006. (National Cancer Institute of Canada, 2006)

MCF-7 Cells
Tamoxifen Tablets 
Red Clover Blossom
Red Clover Capsules