Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells.

Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.

Selective cytostatic and cytotoxic effects of glucosinolates hydrolysis products on human colon cancer cells in vitro.

Glucosinolates hydrolysis products are attracting increasing attention since many studies have suggested that they may be involved in the anticarcinogenic property of cruciferous vegetables. In this study, we show that diindolylmethane (DIM) and sulforaphane, produced during the hydrolysis of glucobrassicin and glucoraphanin, respectively, exert a dose-dependent cytotoxicity on human colon adenocarcinoma HT29 cells. Moreover, these products are able to inhibit quiescent cells to re-enter the cell cycle. Interestingly, our results clearly show that low doses of DIM and sulforaphane, although very effective on undifferentiated intestinal HT29 cells, do not affect the viability of the differentiated CaCo2 cells. The reversibility of their effects has also been tested and is discussed.

Minimum antibiotic levels for selecting a resistance plasmid in a gnotobiotic animal model.

The minimum antibiotic concentrations for selecting an R plasmid in vivo were determined in germfree mice colonized by two isogenic strains of Escherichia coli, one of which carried an R plasmid. Seventy groups of three gnotobiotic mice were given low doses of ampicillin, colistin, flumequin, gentamicin, tetracycline, or streptomycin via drinking water for 2 weeks. The equilibrium between susceptible and resistant populations of bacteria was monitored daily in feces and compared with that of control mice given pure water. This model yielded reproducible data, and dose and response were strongly correlated. The minimum selecting doses ranged from 0.9 to 12.8 micrograms/ml of water, depending on the antibiotic and the R plasmid. The use of mathematical models and complementary in vitro experiments accounted for the effect of the low antibiotic levels.

Antibiotic residues and R-plasmid selection: are in vitro methods good models?

Three clones of E. coli, one of which was harbouring a tetracycline resistance plasmid were inoculated together into the stomach of axenic mice. Without antibiotic selective pressure, the R-Plasmid bearing strain became dominant in the faeces of mice, while the R-plasmid free strain was eliminated. When the R-plasmid bearing strain was given to mice 4 days after the inoculation with the R-plasmid free strain, it was repressed and remained at the stable level of 10(4.5) organisms per g of faeces. But a rapid spread of the R-plasmid was observed, tetracycline resistant bacteria become dominant within one day, and replace the tetracycline sensitive E. coli. The tetracycline resistance plasmid did not disadvantage the mediating strain in the gut, even in the absence of antibiotic pressure. In contrast Lebek and Egger (1983), studying the same strains in vitro, found that in a chemostat the plasmid bearing strain was overgrown by the plasmid free strain. These results strongly suggest that in vitro interactions between E. coli strains cannot be directly extrapolated to in vivo conditions. For the determination of the no-effect level of antibiotic residue on the selection of R-factor in the gut, studies should be made in vivo.