Alkenyl group is responsible for the disruption of microtubule network formation in human colon cancer cell line HT-29 cells

Abstract

Alk(en)yl trisulfides (R-SSS-R') are organosulfur compounds produced by crushed garlic and other Allium vegetables. We found that these compounds exhibit potent anticancer effects through the reaction with microtubules, causing cell cycle arrest. Nine alk(en)yl trisulfides including dimethyl trisulfide, diethyl trisulfide, dipropyl trisulfide (DPTS), dibutyl trisulfide, dipentyl trisulfide, diallyl trisulfide (DATS), dibutenyl trisulfide, dipentenyl trisulfide and allyl methyl trisulfide were synthesized and added to cultures of HT-29 human colon cancer cells at a concentration of 10 muM. The trisulfides with alkenyl groups such as DATS, but not those with alkyl groups, induced rapid microtubule disassembly at 30-60 min as well as cell cycle arrest during the mitotic phase approximately at 4 h after the treatment. Both DATS-induced microtubule disassembly and the cell cycle arrest were cancelled by the simultaneous treatment of the cancer cells with 2 mM L-cysteine, glutathione (GSH) or N-acetyl-L-cysteine. Reciprocally, L-buthionine-(S,R)-sulfoximine (500 muM), an inhibitor of GSH synthesis, enhanced the power of DATS in inducing the cell cycle arrest. These results indicate that alk(en)yl trisulfide react with sulfhydryl groups in cysteine residues of cellular proteins such as microtubule proteins. Thus, the present study provides evidence that trisulfides with alkenyl groups have potent anticancer activities, at least in part, directed toward microtubules. These findings suggest that alkenyl trisulfides and their structurally related compounds may provide novel and effective anticancer agents.

Fig. 1.

Chemical structures of alk(en)yl trisulfides used in this study. These compounds can be divided into three groups: (A) alkyl trisulfides, (B) alkenyl trisulfides and (C) mixed alk(en)yl trisulfides.

Fig. 2.

Effects of alk(en)yl trisulfides on microtubule formation. HT-29 cells were treated with alkyl trisulfides (panel B), alkenyl trisulfides (panel C) or mixed alk(en)yl trisulfides (panel D) for the times as indicated. Panel A shows untreated control cells. Morphology of the microtubules formed in the cells was observed by the indirect immunofluorescence method using anti-β-tubulin antibody, as described in the text. Scale bar 20 μm. Panel E shows assembly of phosphocellulose-purified tubulin measured by turbidity assay, in which tubulin (1.5 mg/ml) mixed with 1 μM DATS or DPTS at 4°C was warmed at 37°C to initiate the polymerization. The increase in absorbance was monitored at 340 nm.

Fig. 3.

Cell cycle progression of HT-29 cells cultured in the presence of alk(en)yl trisulfides having various structures. HT-29 cells were cultured in the presence or absence of alkyl trisulfides (dimethyl trisulfide, diethyl trisulfide, DPTS, dibutyl trisulfide and dipentyl trisulfide), alkenyl trisulfides (DATS, dibutenyl trisulfide and dipentenyl trisulfide) or mixed ank(en)yl trisulfide (allyl methyl trisulfide) at a concentration of 10 μM for 12 h. Then, the cell cycle distribution in G₂/M phase was analyzed by using a flow cytometer, as described in Materials and Methods (panel A). Values are the mean ± SE of three independent experiments. ** (P < 0.01) represents the significant differences from value of control group (vehicle) determined by Dunnett’s multiple comparison test. Time-course estimations of the effects of alkyl trisulfides or alkenyl trisulfides (10 μM) on the cell cycle progression are shown in panels B and C, respectively. Panel D shows growth inhibition curves for HT-29 cells cultured with DATS or DPTS. Data represent the average ± SEM of three experiments.

Fig. 4.

Induction of mitotic arrest by DATS through the inhibition of mitotic spindle formation. Detection of Ser10-phosphorylated histone H3, a sensitive marker for cells at the M phase, was detected in HT-29 cells cultured with DATS (10 μM) for 12 h by using a flow cytometer (panel A). Spindle formation of HT-29 cells cultured with vehicle or DATS (10 μM) was assessed by the immunofluorescence method using anti-β-tubulin antibody, as described in Materials and Methods (green; panel B). The nucleus was counterstained with propidium iodide (magenta). Scale bar 20 μm.

Fig. 5.

DATS-induced microtubule disarrangement and cell cycle arrest are cancelled by the pretreatment of the cell with L-cysteine. Panel A: HT-29 cells were cultured with vehicle (a), 2 mM L-cysteine (b), 10 μM DATS (c) or both 10 μM DATS and 2 mM L-cysteine (d) for 12 h. The microtubule distribution of the cells was assessed by the immunofluorescence method using anti-β-tubulin antibody (green). The nucleus was counterstained with propidium iodide (magenta). Scale bar 20 μm. Panel B: HT-29 cells were preincubated with 2 mM L-cysteine for 2 h prior to the DATS treatment. Then, the cells were further incubated with or without 10 μM DATS for 12 h. The cell cycle distribution of the cells was analyzed by using a flow cytometer. Values are the mean ± SE of three independent experiments. The letters on the bars show the statistical relations and if different from each other, they differ with P < 0.01 in Tukey–Kramer’s multiple comparison test.

Fig. 6.

BSO enhanced and sustained the DATS-induced cell cycle arrest but had little influence on DPTS-treated cells. HT-29 cells were pretreated with 500 μM BSO for 24 h and then the cells were treated with 10 μM DATS (A and C) or DPTS (B and D) for the times as indicated. The cell cycle distribution of the cells was analyzed by using a flow cytometer.