Consumption of cruciferous vegetables is associated with reduced risk of various types of cancer. Isothiocyanates including sulforaphane and erucin are believed to be responsible for this activity. Erucin [1-isothiocyanato-4-(methylthio)butane], which is metabolically and structurally related to sulforaphane, is present in large quantities in arugula (Eruca sativa, Mill.), kohlrabi and Chinese cabbage. However, its cancer preventive mechanisms remain poorly understood. We found that erucin inhibits proliferation of MCF7 breast cancer cells (IC 50 = 28 µM) in parallel with cell cycle arrest at mitosis (IC 50 = 13 µM) and apoptosis, by a mechanism consistent with impairment of microtubule dynamics. Concentrations of 5–15 µM erucin suppressed the dynamic instability of microtubules during interphase in the cells. Most dynamic instability parameters were inhibited, including the rates and extents of growing and shortening, the switching frequencies between growing and shortening, and the overall dynamicity. Much higher erucin concentrations were required to reduce the microtubule polymer mass. In addition, erucin suppressed dynamic instability of microtubules reassembled from purified tubulin in similar fashion. The effects of erucin on microtubule dynamics, like those of sulforaphane, are similar qualitatively to those of much more powerful clinically-used microtubule-targeting anticancer drugs, including taxanes and the vinca alkaloids. The results suggest that suppression of microtubule dynamics by erucin and the resulting impairment of critically important microtubule-dependent cell functions such as mitosis, cell migration and microtubule-based transport may be important in its cancer preventive activities.

Copyright: © 2014 Azarenko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Introduction

Epidemiological data have demonstrated that dietary vegetables such as cabbage, cauliflower, kale, arugula, wild rocket and broccoli contain cancer preventive and anti-cancer activities. Isothiocyanates such as sulforaphane and erucin may be responsible for these actions [1], [2]. Several mechanisms are thought to play a role in the cancer preventive activities of isothiocyanates, including inhibition of Phase I carcinogen-activating enzymes [3], [4], induction of Phase II carcinogen detoxification enzymes [5], [6], inhibition of cancer cell proliferation by cell cycle arrest at G 2 /M, and removal of premalignant and malignant cells through the induction of apoptosis [7]–[10].

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane] is the most extensively studied isothiocyanate found in cruciferous vegetables. It is especially prominent in broccoli and broccoli sprouts [11]. However, much less is known about the anticancer activity of erucin [1-isothiocyanato-4-(methylthio)butane], a structurally-related sulfide analog of sulforaphane. (Fig. 1, inset). Cruciferous salad crops such as arugula and wild rocket ((Eruca sativa (Mill.) and Diplotaxis tenuifolia species), several varieties of Chinese cabbage, and kohlrabi, all commonly ingested, accumulate large quantities of glucoerucin [12]–[14], and erucin is produced from this precursor by the endogenous enzyme myrosinase when the vegetables are chopped or chewed. Erucin is also an in vivo metabolite of sulforaphane formed through reduction of sulforaphane’s thiomethyl group when various cabbages, especially broccoli and broccoli sprouts, are consumed [15]. Also, the inter-conversion of sulforaphane and erucin is a favored metabolic reaction in animal models and in human subjects who consume either fresh broccoli sprouts or a broccoli supplement [16]–[19]. Dietary isothiocyanates are well absorbed and attain good bioavailability [20]–[22].

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larger image TIFF original image Download: Figure 1. Effects of erucin on MCF7 cell proliferation, mitotic index, cell cycle progression and apoptosis. (A) Erucin (inset) inhibits proliferation along with mitotic arrest. Cells were incubated with a range of erucin concentrations for 72 hours and SRB cell proliferation assays were performed to assess cell proliferation (IC 50 = 28 µM, -•-). To determine the mitotic index (IC 50 = 13 µM, -▪-), cells were incubated with erucin for 24 hours, fixed, and stained with DAPI to visualize DNA (Materials and Methods). Data are the mean of three to four independent experiments; bars, ± SEM. (B) Erucin arrests cells at G2/M. Non-synchronized cells were treated with a range of erucin concentrations for 24 hours and analyzed by flow cytometry (Materials and Methods). White bars represent G1 phase, gray bars, S phase, and black bars, G2/M phase of the cell cycle. (C) Erucin induces time-, and concentration-dependent apoptosis. Cells were incubated with a range of erucin concentrations for 24 hours (-○-) and 48 hours (-□-) and the total number of apoptotic cells (early and late apoptotic) for each condition was determined by flow cytometry (Materials and Methods). Results are the mean ± SEM of at least three independent experiments performed in duplicate. https://doi.org/10.1371/journal.pone.0100599.g001

Like sulforaphane, erucin induces Phase II detoxification enzymes [23], [24] and inhibits Phase I enzymes [15]. It also has antioxidant activity [25] and increases expression of multidrug resistance transporters in human carcinoma cell lines [26]. In addition, erucin arrests cell cycle progression and induces apoptosis in human lung carcinoma, hepatoma and leukemia cell lines [27]–[30]. These actions may all play a role in erucin’s anticancer actions. The structural similarity between erucin and sulforaphane and the prominence of erucin in several widely-consumed cruciferous vegetables led us to explore the anti-proliferative effects of erucin and its effects on the polymerization and dynamics of microtubules in breast cancer cells and on the dynamics of microtubules reassembled from purified tubulin.

Microtubules are dynamic tube shaped protein polymers (25 nm in diameter) that play important roles in determining cell shape, polarity, cellular migration, signaling, and mitosis [31]–[33]. Microtubules can undergo two unusual non-equilibrium dynamic behaviors, dynamic instability, the switching between growth and shortening at microtubule plus ends [34], [35], and treadmilling, the net plus end assembly and minus end disassembly (reviewed in [31]). Microtubule dynamics are rapid during mitosis and are critical for the accurate and time-sensitive attachment of chromosomes to the mitotic spindle, movement of the chromosomes to form the metaphase plate, and production of proper tension at the kinetochores, all of which are essential for the passage through the metaphase/anaphase spindle checkpoint [36], [37]. Microtubule dynamics are also important in cell polarity, cell migration and metastasis [33].

Many commonly used anticancer drugs act by modulating microtubule dynamics including the taxanes (paclitaxel, docetaxel), the vinca alkaloids (e.g., vinblastine, vinorelbine, vincristine), maytansinoids, and eribulin [38], [39]. At a mechanistic level, microtubule-targeting agents act in two ways in cells and in vitro. At relatively high concentrations, they either reduce or augment the mass of assembled microtubules. At their lowest effective concentrations, they modulate the growing and shortening dynamics of microtubules without affecting the microtubule polymer mass [38]. Most microtubule-targeting compounds used for treatment of cancer can exert both activities, but as a rule they modulate microtubule dynamics at concentrations significantly lower than those required to increase or decrease the microtubule polymer mass. For example, taxol inhibits proliferation and mitosis in cultured tumor cells in the low nanomolar concentration range by suppressing microtubule dynamics, while the taxol concentration required to significantly increase the microtubule polymer mass is approximately 10 fold higher [40]. Similarly, the vinca alkaloids, which inhibit microtubule polymerization at high concentrations, inhibit cell proliferation at their lowest effective concentrations by altering the dynamics of microtubules without depolymerizing the microtubules [41]. Thus, the most potent actions of these drugs that lead to cell cycle arrest at mitosis and apoptotic cell death in cultured cells are on the dynamics of microtubules, not the quantity of assembled polymer [31], [38], [42].

Recently, we reported that sulforaphane inhibits proliferation and mitosis in MCF7 cells in the micromolar concentration range by suppressing microtubule dynamics and stabilizing microtubules in a manner similar to, but significantly weaker than that of clinically used microtubule targeting drugs [45]. Here, to understand and compare the antiproliferative and microtubule-targeting effects of erucin with those of sulforaphane, we analyzed the effects of erucin on proliferation, cell cycle progression, mitosis, apoptosis, and dynamic instability in MCF7 breast cancer cells stably transfected with enhanced green fluorescent protein EGFP-α-tubulin. We also analyzed the effects of erucin on the polymerization and dynamics of microtubules assembled from purified tubulin in vitro. We found that erucin inhibits proliferation of MCF7 cells in parallel with inhibition of cell cycle progression at G2/M and mitosis in a manner consistent with impairment of spindle microtubule dynamics. In addition, erucin strongly suppressed microtubule growing and shortening dynamics during interphase without affecting the microtubule polymer mass and did so in similar fashion with microtubules made from purified tubulin in vitro. Erucin also enhanced acetylation of microtubules in the cells, a marker for microtubule stabilization. These results strongly support the hypothesis that suppression of microtubule dynamics by erucin is important in its ability to prevent cancer.