Heparan sulfates (HS) bind a diversity of protein ligands on the cell surface and in the extracellular matrix and thus can modulate cell signaling. The state of sulfation in glucosamines and uronic acids within the chains strongly influences their binding. We have previously cloned and characterized two human extracellular endoglucosamine 6-sulfatases, HSulf-1 and HSulf-2, which selectively liberate the 6-O sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact HS and heparins. These enzymes serve important roles in development and are upregulated in a number of cancers. To determine whether the Sulfs act on the trisulfated disaccharides that exist on the cell surface, we expressed HSulfs in cultured cells and performed a flow cytometric analysis with the RB4CD12, an anti-HS antibody that recognizes N- and O-sulfated HS saccharides. The endogenously expressed level of the cell surface RB4CD12 epitope was greatly diminished in CHO, HEK293, and HeLa cells transfected with HSulf-1 or HSulf-2 cDNA. In correspondence with the RB4CD12 finding, the N, 6-O, and 2-O trisulfated disaccharides of the HS isolated from the cell surface/extracellular matrix were dramatically reduced in the Sulf-expressed HEK293 cells. We then developed an ELISA and confirmed that the RB4CD12 epitope in immobilized heparin was degraded by purified recombinant HSulf-1 and HSulf-2, and conditioned medium (CM) of MCF-7 breast carcinoma cells, which contain a native form of HSulf-2. Furthermore, HSulf-1 and HSulf-2 exerted activity against the epitope expressed on microvessels of mouse brains. Both HSulf activities were potently inhibited by PI-88, a sulfated heparin mimetic with anti-cancer activities. These findings provide new strategies for monitoring the extracellular remodeling of HS by Sulfs during normal and pathophysiological processes.

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 μM. 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 μM), 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.

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Alk(en)yl sulfides have been found to be responsible for the anticancer, antithrombotic, and antioxidant effects of garlic. We sought to identify the most potent structure of sulfides that exhibits a hepatoprotective effect against carbon tetrachloride (CCl(4))-induced acute liver injury in rats. Rats were pretreated with diallyl trisulfide (DATS) i.g. at a dose of 500 micromol/kg body weight for 5 d. On d 6, CCl(4) was administered i.g. at a dose of 2.5 mL/kg body weight. Twenty-four hours after CCl(4) administration, rats were killed and plasma and liver samples collected. DATS pretreatment significantly suppressed the CCl(4)-induced elevation of plasma aspartate aminotransferase and alanine aminotransferase activities (P < 0.05). Histological observations supported the hepatoprotective effects. Western blot and spectrophotometric analyses indicated that DATS suppressed cytochrome P450 2E1 activity and its protein level and elevated those of glutathione S-transferase. Dipropyl trisulfide (DPTS), which is a saturated alkyl chain analogue of DATS, did not affect CCl(4)-induced liver toxicity or drug-metabolizing enzymes. These results suggest that hepatoprotective activity of trisulfides is due to their regulation of drug-metabolizing enzymes. Furthermore, the effects of 6 kinds of alk(en)yl trisulfides, including DATS and DPTS, on phase II enzyme activity were examined in rats. Alk(en)yl trisulfides were administered i.g. (500 micromol/kg body weight) to rats for 5 d. Only the allyl group-containing DATS and allyl methyl trisulfide enhanced these activities.

We reported the regulation of protein function by oxidative modification of the specific cysteine residue(s) by diallyl trisulfide (DATS). In this study, we examined if DATS modifies the cysteine residue of thioredoxin (Trx) by urea-polyacryl amide gel electrophoresis. DATS modified two specific cysteine residues in Trx and this oxidative modification of cysteine residues would be sole causative of the apoptosis induced by DATS in leukemic cells.