Sulforaphane: Its Potential for Treatment of Breast Cancer

Institution: University of California, Santa Barbara
Investigator(s): Olga Azarenko, M.A. -
Award Cycle: 2006 (Cycle 12) Grant #: 12GB-0137 Award: $65,415
Award Type: Dissertation Award
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2006)

It has been argued that an increase in intake of fruits and vegetables to just five servings per day could lead to a decline of cancer rates, including breast cancer, by as much as 20%. Epidemiologic data has indicated that a diet rich in cruciferous vegetables such as broccoli, cabbage, and cauliflower provides better protection against breast cancer than overall ingestion of fruits and vegetables because crucifers contain unique plant compounds that play an important role in cancer protection. This project focuses on the anticancer activity of sulforaphane, one of the major relatively non-toxic organic isothiocyanates, which are considered to be the anti-carcinogenic chemicals responsible for the cancer-preventative activities in cruciferous vegetables. Sulforaphane, the most abundant isothiocyanate in the majority of crucifers, is a promising breast cancer protective agent. It has already been shown to reduce the formation of chemically induced mammary tumors in rats. Moreover, it also inhibits the growth of cultured human breast cancer cells.

Recent studies indicate that one of the targets of sulforaphane may be microtubules, the dynamic tube-like protein fibers in living cells that are responsible for proper cell division and mitosis. We have discovered that sulforaphane blocks cell cycle progression at mitosis in MCF-7 breast cancer cells in a manner similar to that of more powerful anti-mitotic anticancer drugs, such as vinblastine and taxol. Also, we found that the biochemical mechanism of action on the microtubules in mitotic spindles is similar to that of vinblastine and taxol. Importantly, these actions occur at levels close to those achieved in the human diet.

The goal of this project is to further test the activity of sulforaphane on breast cancer cells. Our thinking is that by combining sulforaphane with more powerful anticancer chemotherapeutic agents, it may be possible to simultaneously obtain good drug efficacy and reduced toxicity. Our project goals can be summarized in the following questions to be addressed:
(1) How does sulforaphane work against microtubules mechanistically? Does sulforaphane affect the structure and reduce dynamics of microtubules made of pure tubulin outside living cells?
(2) Does sulforaphane inhibit the growth and progression through the cell cycle of normal human mammary epithelial cells and/or cause cell death at concentrations effective in breast cancer cells?
(3) Does sulforaphane have any potential to be used with other more powerful and more toxic anticancer drugs in combination therapy to decrease harmful side effects?

To determine whether sulforaphane affects normal cells we will use human mammary epithelial cells that are commonly used in our laboratory. Cultured breast cancer cells will also be used for the experiments with drug combinations (sulforaphane and docetaxel) to assess the level of cancer cell growth inhibition and cell death.

Final Report (2008)
Epidemiological data have shown that a diet rich in cruciferous vegetables, such as broccoli, cabbage, and cauliflower provides better protection against breast cancer than overall ingestion of fruits and vegetables. Cabbages contain organic isothiocyanates, or mustard oils, which are believed to be responsible for the cancer-preventive and anti-carcinogenic activities in these vegetables.

The goal of the project was to analyze the anticancer activities of sulforaphane (SFN), the most abundant, relatively nontoxic organic isothiocyanate that has already been shown to reduce the incidence and rate of development of chemically induced mammary tumors in animals. Moreover, it inhibits the growth of cultured human breast cancer cells, leading to cell death.

We have discovered that SFN suppressed microtubule dynamic instability (the switching between growth and shortening at microtubule ends) and decreased the turnover of microtubules in MCF7 breast cancer cells at concentrations similar to those that inhibited cell growth in mitosis, causing cell death. A protein called tubulin is the building block subunit which assembles into microtubules, dynamic tube-like protein fibers that are responsible for the proper cell shape, motility, division and mitosis. Importantly, SFN stabilized microtubules in a manner qualitatively similar to that of clinically used powerful anti-mitotic anticancer drugs, such as taxanes (e.g., paclitaxel) and the vinca alkaloids (e.g., vinblastine).

SFN also suppressed the growing and shortening dynamics of purified microtubules in a manner similar to that in living cancer cells, indicating that its effects on dynamics in cells were due to a direct action on the microtubules. Moreover, the effects of SFN on microtubules, both in cells and with purified polymers, occurred at SFN concentrations well below those required to inhibit polymerization of the microtubules (i.e., below those required to reduce the polymer mass). In addition, using biochemical techniques we determined that SFN’s effects on dynamics are more likely due to an action of SFN itself or SFN-tubulin complexes directly on tubulin in the microtubules. My findings confirmed the hypothesis that tubulin is one of the SFN’s important cellular targets.

The anti-proliferative effects of SFN were examined in combination with the microtubule-targeted chemotherapeutic agent paclitaxel in cultured breast cancer cells. Preliminary results showed that SFN does not interfere with the actions of paclitaxel. In fact, SFN enhances the effects produced by paclitaxel on cancer cell growth inhibition, mitotic arrest, and induction of apoptosis. Furthermore, I have found that their anti-proliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability. The combination of SFN and paclitaxel at their anti-proliferative IC50 (15 ?M for SFN and 7.5 nM for paclitaxel) altered most of the parameters of dynamic instability synergistically. For example, together the drugs inhibited overall microtubule dynamicity by 75%, but each drug individually inhibited dynamicity by 45 and 49%, respectively. These results suggest that, in combination with microtubule-targeted drugs, SFN may synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation with important consequences for combination clinical therapy.

The findings of this project propose a novel mechanism of anti-proliferative action for SFN and strongly support the hypothesis that inhibition of mitosis by microtubule stabilization is important for SFN’s chemopreventive activity.

Symposium Abstract (2007)
Isothiocyanates (ITCs) are important anticarcinogenic phytochemicals abundant in cruciferous vegetables, such as broccoli, broccoli sprouts, cabbage, and cauliflower. Recent studies have revealed that ITCs exert cytotoxic effects on various cancer cell lines through the inhibition of cell growth, induction of cell cycle arrest and activation of cell death, and that some ITCs might be selective exclusively towards cancer cells. In this study we compared the effects of major ITC, sulforaphane (SFN) on breast cancer (MCF7) and normal human mammary epithelial cells (HMEpC). We determined that SFN inhibited cell proliferation ~2-fold more potently in MCF7 than in HMEpC cells (IC50 SFN: 9 μM and 19 μM, respectively). We also found that ³15 μM SFN blocked MCF7 cells in prometaphase stage of mitosis by disrupting spindle formation and preventing proper chromosome segregation. Interestingly, SFN did not induce mitotic arrest in HMEpC cells at these concentrations; however, some aberrant mitotic spindles with abnormal chromosome segregation were observed. Cell cycle analysis by flow cytometry confirmed that SFN induced G2/M phase delay in MCF7 cells (15 μM SFN, 24 h: 46 ± 1.5 % in G2/M vs. 29 ± 1.3 % in controls), while the cell cycle of HMEpC cells was not affected. Furthermore, SFN induced microtubule depolymerization in interphase MCF7 and HMEpC cells in a concentration dependent manner. MCF7 cell growth inhibition was accompanied by a significant time- and dose-dependent induction of apoptosis. Surprisingly, high concentrations of SFN (>15 μM, 24h) significantly reduced the viability of HMEpC cells. Our results indicate that low concentrations of SFN may trigger cell cycle arrest in mitosis by perturbing normal microtubule polymerization and suppressing microtubule dynamic instability, leading to apoptosis in MCF7 cells. However, high concentrations of SFN also inhibited proliferation of HMEpC cells and disrupted cytoplasmic microtubule polymerization without blocking cells in mitosis. These results indicate that high concentrations of SFN are active in both normal and cancerous cells. Further experiments are under way to elucidate the mechanisms of action of SFN on human breast cancer cells vs. normal human mammary epithelial cells. Our data lead to important questions regarding proper implementation and suitability of isothiocyanates and SFN in particular in cancer chemoprevention.

Suppression of microtubule dynamic instability and turnover in MCF7 breast cancer cells by sulforaphane.
Index Medicus: Carcinogenesis
Authors: Azarenko O, Okouneva T, Singletary KW, Jordan MA, Wilson L
Yr: 2008 Vol: 29 Nbr: 12 Abs: Pg:2360-8