Chemopreventive Compounds Induce Apoptosis in Breast Cancer Cells

Davina Wu

Writer’s comment: As a student interested in health-related issues, particularly women’s health, I found that the scientific literature review assignment in Dr. Sondra Reid’s English 102B class was the perfect opportunity to explore developments at the forefront of breast cancer research. Due to the limited number of journal articles pertaining to the specific topic at that time, writing this literature review became a challenge. Thanks to Dr. Reid’s guidance and encouragement, I learned not only about current breast cancer research, but also how to be a better writer. Breast cancer and other types of cancer affect the lives of so many people. I am honored that my literature review is included in the UC Davis Prized Writing collection and hope that it will raise awareness with respect to active research in the ongoing battle against cancer.
—Davina Wu

Instructor’s comment: Davina Wu’s secure understanding of the science discussed in the articles she reviews is basic to the success of her paper. First, it helped her select a group of research articles similar enough to form a reasonable collective identity and, at the same time, different enough to establish an interesting range of approaches. Her grasp of content then helped her to see a disarmingly simple two-part structure within which to discuss the research—studies that examined the role of vitamins in inducing apoptosis, and those that examined the role of plant-derived compounds. Within that structure, finally, Davina clearly explains the essential findings from each study, creating a highly informative piece for her readers, who then benefit from her concise overview of recent developments in a specific approach to treating breast cancer. I am pleased that Davina’s good work in English 102B now reaches a broader public.
—Sondra Reid, English Department

Apoptosis, or programmed cell death, plays an integral part in maintaining tissue homeostasis. The purpose of apoptosis is to eliminate cells that are worn out, infected, or plagued by a malfunction in their internal machinery. Apoptosis employs an elegant set of mechanisms in which the morphology and internal organization of the cell changes. For example, chromatin condenses and the DNA fragments within the nucleus. The cell body eventually separates into small apoptotic vesicles that facilitate clearance by macrophages and phagocytic cells. Cell death by this method has advantages over necrosis; whereas necrosis can induce inflammation and other adverse effects, apoptosis does not. Because cancer can result if the delicate balance between cell proliferation and cell death is disrupted, cell death is an essential process.
         Scientists first acknowledged programmed cell death in the 1970s, but tremendous advancements in apoptosis research have occurred only in this past decade due to improvements in molecular and biochemical techniques. One area of active apoptosis research is focusing on breast cancer treatments. According to the American Cancer Society (2000), breast cancer is the second most common cancer in women; 40,800 women were estimated to die from the disease in the year 2000. Because the number of breast cancer cases resistant to existing treatments is on the rise, scientists are compelled to seek alternative means of treatment, such as new compounds that induce apoptosis. By targeting critical points along the apoptotic pathway, scientists hope to eliminate cancerous mammary cells while avoiding the adverse side effects characteristic of some treatments used today. Scientists currently at the forefront of breast cancer apoptosis research are investigating alternative compounds and their potential to induce cell death. The following is a review of recent studies that assess the effectiveness of vitamin E, vitamin D, pycogenol, and curcumin as inducers of apoptosis.

Vitamins Induce Apoptosis

Vitamin E Compounds
         Palm oil is rich in tocopherol and tocotrienol, two subgroups of vitamin E. Early studies by Nesaretnam et al. (1995) demonstrated that tocotrienol-rich-fraction of palm oil (TRF) inhibited human breast cancer cell proliferation. A study by Yu et al. (1999) suggested that the inhibition of cell proliferation was associated with apoptosis induced mainly by tocotrienols. By using DAPI (4-6-diamidino-2-phenylindole) staining of DNA, researchers were able to visualize condensed chromatin and DNA fragments, common apoptotic morphology. DNA fragments from apoptotic cells were then isolated and electrophoresed on an agarose gel. The amount of DNA laddering on a gel correlated with the extent of apoptosis. From the data, Yu et al. (1999) concluded that tocotrienols were more potent than tocopherols.
         Based on the previous vitamin E studies by Nesaretnam et al. (1995) and Yu et al. (1999), McIntyre et al. (2000) compared and contrasted the effects of tocopherol and tocotrienol isoforms on cancerous and non-cancerous cultures of mouse mammary epithelial cells. The results suggested that the isoforms a-, d-, and g-tocotrienol had greater potency of inducing apoptosis compared to the tocopherol isoforms, thus agreeing with the study performed by Yu et al. (1999). The DNA fragments separated on agarose gels indicated a greater number of fragments present in the malignant cells treated with tocotrienol, suggesting the effectiveness of tocotrienols and the greater sensitivity of malignant cell lines. Nesaretnam et al. (1995) hypothesized that tocotrienol had higher potency due to the relative ease with which the compound is taken up by the cells. McIntyre et al. (2000) made a similar hypothesis, suggesting that the chemical side chain of tocotrienol facilitated its uptake by breast cancer cells. Yu et al. (1999) and McIntyre et al. (2000) independently concluded that vitamin E, tocotrienol in particular, has great potential as a therapeutic agent for breast cancer.

Vitamin D Compounds
         Vitamin D is another vitamin under consideration for its ability to induce cell death in breast cancer cells. Simboli-Campbell et al. (1997) compared metabolic vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH2)D3), with the synthetic vitamin D analog EB1089. Cells from the human mammary adenocarcinoma cell culture line designated as MCF-7 were treated for 72 hours with either 100 nM 1,25(OH2)D3 or 100 nM EB1089 and were evaluated for signs of apoptosis. Researchers detected DNA fragments using Hoechst dye and in situ labeling with terminal transferase. Simboli-Campbell et al. (1997) concluded that EB1089 was more effective at inducing apoptosis than (1,25(OH2)D3). Another method for detecting apoptosis was flow cytometry, used to assess the proportion of cells in the Go/G1 phases of their life cycle. The increased number of detected cells arrested in their Go/G1 phases is important because cells are frequently observed to undergo apoptosis at this stage. Flow cytometry also indicated the greater potency of EB1089; a 48-hour treatment with 1 nM EB1089 induced the same percent of Go/G1 cells as the 100 nM 1,25(OH2)D3 treatment for the same length of time (Simboli-Campbell et al., 1997).
         While early studies indicated that EB1089 was more potent than 1,25(OH2)D3, researchers discovered another synthetic vitamin D analog, CB1093, with even greater apoptosis-inducing capability. Danielsson et al. (1997) used antibodies in ELISA assays and in situ labeling to detect DNA fragments resulting from apoptosis in cells treated with 1,25(OH2)D3 and the two analogs. Results demonstrated that a 10-fold lower concentration of CB1093 induced apoptosis compared to the required concentration of EB1089. Experiments conducted by Mathiasen et al. (1999) provided supporting evidence of the effectiveness of CB1093 as a potential chemopreventive agent. Variations in treatment concentrations indicated that CB1093 could be highly potent even at low concentrations and could induce cell death at a significantly faster rate.
         Research has also suggested that vitamin D and its two analogs are critical in reducing the expression of bcl-2, a gene associated with the inhibition of apoptosis. Simboli-Campbell et al. (1997) used Western blot analysis to detect the decrease of normal bcl-2 expression in the presence of vitamin D compounds. Western blot analysis specifically identified CB1093 as being more potent in reducing bcl-2 expression than EB1089 (Danielsson et al., 1997). Mathiasen et al. (1999) studied the effect of the vitamin D compounds on MCF-Fas-Bcl2-2, a cell line that over-expresses bcl-2. The excessive levels of bcl-2 inhibited the effects on apoptosis of vitamin D and its analogs on apoptosis, thus supporting the findings by Simboli-Campbell et al. (1997) and Danielsson et al. (1997) that interactions between a vitamin D analog and bcl-2 can determine whether or not a cell undergoes apoptosis.

Plant-derived Compounds Induce Apoptosis

         Scientists are also studying naturally occurring compounds from plants as potential therapeutic agents for treating breast cancer. One plant product currently under investigation is pycnogenol, which consists of a mixture of flavonoid compound from the bark of pine trees. Huynh and Teel (2000) evaluated the effectiveness of pycnogenol as an inducer of apoptosis in breast cancer cells based on their 1999 study that demonstrated the effect of pycnogenol on lung cancer cells. Normal human mammary cells from the MCF-10 line and breast cancer cells from the MCF-7 line were cultured in the presence of 0, 40, or 80 mg/ml pycnogenol (Huynh & Teel, 2000). Researchers used the DAPI staining method similar to the one used by Yu et al. (1999) and fluorescent microscopy to count cells with significant amounts of condensed chromatin and DNA fragments, major indices of apoptosis. Noncancerous MCF-10 cells showed no significant decrease in the number of healthy cells, whereas a considerable number of the cancerous MCF-7 cells were induced to die in a dose-dependent manner (Huynh & Teel, 2000). This study suggests that pycnogenol may one day be used to treat breast cancer.

         Another potential chemopreventive plant-derived compound is curcumin (diferuloyl methane), a major pigment of the rhizomes of turmeric, a major ingredient in curry. Ramachandran and You (1999) also used the MCF-10 and MCF-7 mammary cell lines when studying curcumin. Results indicated that a significantly higher percentage of the MCF-7 cells underwent apoptosis in comparison to the MCF-10 cells. Similar to findings by Huynh and Teel (2000) and McIntyre et al. (2000), the experiment by Ramachandran and You (1999) suggested that the cancerous cells were more sensitive to anti-apoptotic compounds than to the noncancerous cells. Thus, curcumin may be another potential agent to target breast cancer cells.

         The vitamins and plant-derived compounds currently under study have been highly effective at inducing apoptosis in breast cancer cells, thus demonstrating their immense promise as possible forms of treatment. However, the exact mechanism by which each compound signals a cell to die has not been determined because the intricate details of apoptosis are not yet fully understood. Programmed cell death involves multiple pathways; the exact pathway leading to apoptosis depends on the initiation signal and the particular genes and proteins that are activated. With a better understanding of apoptosis, scientists can apply that knowledge to design innovative breast cancer treatments, possibly involving vitamins and plant-derived compounds.

Literature Cited

American Cancer Society, Inc. Fast facts. American Cancer Society. 2000. (Nov. 2000).

Danielsson, C., Mathiasen, I.S., James, S.Y., Nayeri, S., Bretting, C., Hansen, C.M., Colston, K.W., Carlberg, C. (1997). Sensitive induction of apoptosis in breast cancer cells by a novel 1,25-Dihydroxyvitamin D3 analogue shows relation to promoter selectivity. Journal of Cellular Biochemistry 66: 552-562.

Huynh, H.T., Teel, R.W. (1999). Effects of intragastrically administered pycnogenol on NNK metabolism in F344 rats. Anticancer Research 19: 2095-2100.

Huynh, H.T., Teel, R.W. (2000). Selective induction of apoptosis in human mammary cancer cells (MCF-7) by pycnogenol. Anticancer Research 20: 2417-2420.

Mathiasen, I.S., Lademann, U., Jaattela, M. (1999). Apoptosis induced by vitamin D compounds in breast cancer cells is inhibited by bcl-2 but does not involve known caspases or p53. Cancer Research 59: 4848-4856.

McIntyre, B.S., Briski, K.P., Gapor, A., Sylvester, P.W. (2000). Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells. Proceedings of the Society for Experimental Biology and Medicine 224: 292-301.

Nesaretnam, K. Guthrie, N., Chambers, A.F., Carroll, K.K. (1995). Effect of tocotrienols on the growth of a human breast cancer cell line in culture. Lipids 30: 1139-1143.

Ramachandran, C., You, W. (1999). Differential sensitivity of human mammary epithelia and breast carcinoma cell lines to curcumin. Breast Cancer Research and Treatment 54: 269-278.

Simboli-Campbell M., Narvaez, C.J., VanWeelden, K., Tenniswood, M., Welsh, J. (1997). Comparative effects of 1,25(OH)2D3 and EB1089 on cell cycle kinetics and apoptosis in MCF-7 breast cancer cells. Breast Cancer Research and Treatment 42: 31-41.

Yu, W., Simmons-Menchaca, M., Gapor, A., Sanders, B.G., Kline, K. (1999). Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols. Nutrition and Cancer 33: 26-32.