Sulforaphane: a naturally occurring mammary carcinoma mitotic inhibitor, which disrupts tubulin polymerization

Abstract

Sulforaphane (SUL), an isothiocyanate found in broccoli and other cruciferous vegetables, has been shown to induce phase II detoxification enzymes, inhibit chemically induced mammary tumors in rats, and more recently to induce cell cycle arrest and apoptosis in cancer cells of the colon. Here, we provide evidence that SUL also acts as a breast cancer anti-proliferative agent. The BALB/c mouse mammary carcinoma cell line F3II was treated with SUL at concentrations up to 15 microM and examined for markers of cell cycle arrest and apoptosis. Treatment of asynchronous F3II cells with 15 microM SUL resulted in G2/M cell cycle arrest, elevated p34cdc2 (cdc2) kinase activity, Bcl-2 down-regulation, evidence of caspase activation, and aggregation of condensed nuclear chromatin. Subsequent exposure of synchronized cells to 15 microM SUL resulted in elevated numbers of prophase/prometaphase mitotic figures, indicating cell cycle progression beyond G2 and arrest early within mitosis. Moreover, cells treated with 15 microM SUL displayed aberrant mitotic spindles, and higher doses of SUL inhibited tubulin polymerization in vitro. In addition, BALB/c mice injected s.c. with F3II cells and subsequently injected daily i.v. with SUL (15 nmol/day for 13 days) developed significantly smaller tumors (approximately 60% less in mass) than vehicle-treated controls. Western blot analysis of tumor proteins demonstrated significantly (P<0.05) reduced PCNA and elevated PARP fragmentation in samples from animals dosed with SUL. Taken together, these results indicate that SUL has mammary cancer suppressive actions both in cell culture and in the whole animal. Inhibition of mammary carcinogenesis appears in part to involve perturbation of mitotic microtubules and early M-phase block associated with cdc2 kinase activation, indicating that cells arrest prior to metaphase exit.

Induction of p21 protein protects against sulforaphane-induced mitotic arrest in LNCaP human prostate cancer cell line

“…On the other hand, the SFN-mediated G 2 -M phase cell cycle arrest is rapid and evident as early as 4 h posttreatment. The G 2 -M phase cell cycle arrest by SFN treatment at concentrations within the range used in the present study has also been observed in other cellular systems including HT29 human colon cancer cell line (15), F3II mouse sarcomatoid mammary carcinoma cell line (17), PC-3 and DU145 human prostate cancer cells (19,20), and human benign prostate hyperplasia epithelial cell line BPH-1 (23). In contrast, previous studies by Chiao et al (46) have documented a G 1 phase cell cycle arrest in LNCaP cells following a 24-h exposure to 3 and 9 Amol/L SFN.…”

“…Indeed, the immunofluorescence microscopy of SFN-treated LNCaP cells after staining with 4 ¶,6-diamidino-2-phenylindole as well as analysis of Ser 10 phosphorylation of histone H3 confirms mitotic arrest in our model. The SFN-mediated mitotic arrest is not restricted to the LNCaP cell line because similar response has been described previously in F3II mouse mammary carcinoma cells (17). Exposure of synchronized F3II cells to 15 Amol/L SFN results in elevated numbers of prophase/prometaphase mitotic figures, indicating progression beyond G 2 and arrest early in mitosis (17).…”

“…The SFN-mediated mitotic arrest is not restricted to the LNCaP cell line because similar response has been described previously in F3II mouse mammary carcinoma cells (17). Exposure of synchronized F3II cells to 15 Amol/L SFN results in elevated numbers of prophase/prometaphase mitotic figures, indicating progression beyond G 2 and arrest early in mitosis (17). Increased Ser 10 phosphorylation of histone H3 coupled with induction of cyclin B1 level on treatment with SFN has also been observed in MIA PaCa-2 pancreatic cancer cells (50).…”

Covalent Binding to Tubulin by Isothiocyanates

“…A recent study showed that SFN treatments can disrupt tubulin polymerization and spindle assembly, which in turn caused mitotic arrest (22). We demonstrated here that ITCs inhibit tubulin polymerization and disrupt microtubule networks in the same relative order of potency, providing evidence for models in which ITC binding to tubulin disrupts microtubule formation, which may then provoke cell cycle arrest and apoptosis.…”

“…It is conceivable that the structure of individual ITC compounds may influence their binding preferences as it dictates hydrophobicity, size, shape, and electrophilicity. The selective interactions obtained with intracellular proteins were suggested by previous studies in which ITCs were shown to inhibit preferentially, presumably via binding to enzymes, specific cytochrome P450 isozymes, depending on their structures (22,(23)(24)(25)(26). A recent study showed that two SFN derivatives, SFN-N-acetylcysteine and SFN-cysteine, were effective histone deacetylase (HDAC) inhibitors, whereas the SFN parent compound had little effect (27).…”