Diallyl Disulfide Treatment Research Articles

The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells

Diallyl disulfide (DADS), an oil soluble constituent of garlic ( Allium sativum), has been reported to cause antimutagentic and anticarcinogenic effects in vitro and in vivo by modulating phases I and II enzyme activities. In recent years, several studies suggested that the chemopreventive effects of DADS can also be attributed to induction of cell cycle arrest and apoptosis in cancer cells. In the present study, we reported that DADS-induced cell cycle arrest at G2/M and apoptosis in human A549 lung cancer cells in a time- and dose-dependent manner. Additionally, a significant increase of intracellular reactive oxygen species (ROS) was induced in A549 cells less than 0.5 h after DADS treatment, indicating that ROS may be an early event in DADS-modulated apoptosis. Treatment of A549 cells with N-acetyl cysteine (NAC) completely abrogated DADS-induced cell cycle arrest and apoptosis. The result indicated that oxidative stress modulates cell proliferation and cell death induced by DADS.

Repetitive Treatments of Colon HT‐29 Cells with Diallyl Disulfide Induce a Prolonged Hyperacetylation of Histone H3 K14

Diallyl disulfide (DADS) is a sulfur compound derived from garlic. Several studies carried out in rodents have revealed protective effects of DADS against colon carcinogenesis. The antipromoting effects of DADS may be partly related to its ability to inhibit tumoral cell proliferation. In a previous study, we have shown that in two human colon tumor cell lines (HT-29 and Caco-2) seeded at a low density (0.2 x 10(6) cells/100-mm petri dish), DADS antiproliferative effects were associated with a transient increase of histone H3 K14 acetylation. Moreover, DADS could inhibit nuclear histone deacetylase activity. Therefore, in the present study, we examined the possible effects of different experimental conditions (HT-29 cells at high density, repetitive treatments with DADS) on the pattern of DADS-induced histone hyperacetylation. Using HT-29 cells seeded at a higher density (5 x 10(6) cells/100-mm petri dish), we found that DADS induced histone H3 K14 hyperacetylation rapidly (3 h). When administrated as single treatments, the DADS effect on histone H3 K14 remained transient. In contrast, repetitive treatment with DADS resulted in a prolonged hyperacetylation of histone H3 K14. Whatever the cell culture conditions were, DADS had no effect on histone H4 acetylation. Thus, in vitro, the cell density and pattern of DADS treatment influenced the HT-29 nuclear response to DADS. DADS belongs to food-borne molecules that may play a role in chromatin remodeling and contribute to the nutritional modulation of gene expression.

Enhancement of diallyl disulfide-induced apoptosis by inhibitors of MAPKs in human HepG2 hepatoma cells.

We examined the effects of diallyl disulfide (DADS), an oil-soluble organosulfur compound found in garlic, on human HepG2 hepatoma cells to better understand its effect on apoptosis and apoptosis-related genes. Our study has demonstrated that DADS affects cell proliferation activity and viability and elicits typical apoptotic morphologic changes (chromatic condensation and nuclear fragmentation) in human HepG2 hepatoma cells. Also, treatment with DADS induces a temporary increase in phosphorylated p38 MAPK (phospho-p38) and phosphorylated p42/44 MAPK (phospho-p42/p44) in a time- and concentration-dependent manner. Inhibition of activated/phosphorylated mitogen-activated protein kinase (MAPK) with phospho-p38 or phospho-p42/44 specific inhibitors, SB203580 or U0126, induces apoptosis without DADS treatment, indicating that at least the endogenous activated forms of p38 MAPK and p42/p44 MAPK markedly exert cytoprotective roles from cell apoptosis in the HepG2 hepatoma cells. Combined treatment with these inhibitors followed by DADS further enhances the DADS-induced apoptosis. Taken together, these results show that both DADS and the specific inhibitors of MAPKs could induce apoptosis in HepG2 hepatoma cells and that the MAPKs inhibitors further enhance the apoptotic effect in DADS-treated HepG2 hepatoma cells.

Enhancement of diallyl disulfide-induced apoptosis by inhibitors of MAPKs in human HepG2 hepatoma cells

We examined the effects of diallyl disulfide (DADS), an oil-soluble organosulfur compound found in garlic, on human HepG2 hepatoma cells to better understand its effect on apoptosis and apoptosis-related genes. Our study has demonstrated that DADS affects cell proliferation activity and viability and elicits typical apoptotic morphologic changes (chromatic condensation and nuclear fragmentation) in human HepG2 hepatoma cells. Also, treatment with DADS induces a temporary increase in phosphorylated p38 MAPK (phospho-p38) and phosphorylated p42/44 MAPK (phospho-p42/p44) in a time- and concentration-dependent manner. Inhibition of activated/phosphorylated mitogen-activated protein kinase (MAPK) with phospho-p38 or phospho-p42/44 specific inhibitors, SB203580 or U0126, induces apoptosis without DADS treatment, indicating that at least the endogenous activated forms of p38 MAPK and p42/p44 MAPK markedly exert cytoprotective roles from cell apoptosis in the HepG2 hepatoma cells. Combined treatment with these inhibitors followed by DADS further enhances the DADS-induced apoptosis. Taken together, these results show that both DADS and the specific inhibitors of MAPKs could induce apoptosis in HepG2 hepatoma cells and that the MAPKs inhibitors further enhance the apoptotic effect in DADS-treated HepG2 hepatoma cells.

Critical role of allyl groups and disulfide chain in induction of Pi class glutathione transferase in mouse tissues in vivo by diallyl disulfide, a naturally occurring chemopreventive agent in garlic.

We have shown previously that the chemoprotective activity of diallyl disulfide (DADS), a naturally occurring anticancer agent in garlic, against benzo[a]pyrene (BP)-induced forestomach carcinogenesis in mice correlates strongly with its inductive effects on the expression of Pi class glutathione (GSH) transferase mGSTP1-1. The present structure-activity relationship studies were designed to define the role of allyl groups and the disulfide chain in mGSTP1-inducing activity of DADS. Hepatic mGSTP1 mRNA levels rose rapidly upon treatment of mice with DADS, reached a maximum between 12 and 24 h (< or =5.7-fold induction) and fell to control levels by 48 h after DADS treatment. Induction of mGSTP1 mRNA in the forestomach was maximal between 6 and 12 h after DADS treatment (< or =4.7-fold induction). The mGSTP1 mRNA expression was either unaltered (liver) or moderately increased (forestomach) upon treatment of mice with dipropyl disulfide (DPDS), which is a naturally occurring saturated analog of DADS. These results indicated that the allyl groups are critical for the mGSTP1-inducing activity of DADS. A statistically significant increase in the expression of mGSTP1 mRNA was also observed in the liver and forestomach of mice treated with diallyl monosulfide (DAMS), albeit to a much lesser extent compared with DADS. These results indicated that the oligosulfide chain length in garlic organosulfides (OSCs) is equally important for their mGSTP1-inducing activity. The role of the disulfide chain in DADS-mediated induction of mGSTP1 was further investigated by testing a pair of alkadienes (1,7-octadiene and 1,8-nonadiene) having structural similarity to DADS. Both DADS and the alkadienes carry allyl groups at both ends of a linear molecule and the distance between the allylic carbon atoms is similar in both compounds, but the central disulfide chain of DADS is replaced with an alkyl chain in the alkadienes. The alkadienes were either ineffective or moderately active in increasing mGSTP1 expression. In conclusion, the results of the present study clearly indicate that the presence of terminal allyl groups as well as the central disulfide chain is required for maximum induction of mGSTP1 in vivo by garlic-derived OSCs.

Mechanisms of protection against aflatoxin B(1) genotoxicity in rats treated by organosulfur compounds from garlic.

Diallyl sulfide (DAS) and diallyl disulfide (DADS), two garlic constituents, were found previously to inhibit aflatoxin B(1) (AFB(1))-initiated carcinogenesis in rat liver, DADS being the most effective. In order to study the mechanisms involved in this protection, we have examined the ability of liver microsomes and cytosols from DAS- and DADS-treated rats to modulate the mutagenicity and the metabolism of AFB(1). We also examined the effects of these compounds on the expression of cytochromes P450 (CYP) and phase II enzymes known to be involved in AFB(1) metabolism. Administration of DAS (1 mmol/kg for 4 days) to rats resulted in significant inhibition of microsome-mediated mutagenicity of AFB(1), whereas DADS treatment did not alter AFB(1) mutagenicity. DAS treatment increased the metabolism of AFB(1) mainly towards the formation of AFQ(1) and AFM(1), which might account for the reduction of AFB(1) microsomal-mediated mutagenicity. DADS treatment slightly affected the oxidative metabolism of AFB(1). DAS and DADS induced CYP3A2, CYP2B1 and CYP2B2, DAS being more potent. Cytosols from DAS- and DADS-treated rats produced a significant inhibition of AFB(1)-8,9-epoxide (AFBO)-induced mutagenicity and significantly increased the cytosolic formation of AFB(1)-glutathione conjugates, DADS treatment being more effective. Western blot analysis showed that DADS is a potent inducer of glutathione S-transferase A5 (rGSTA5) and AFB(1) aldehyde reductase 1 (rAFAR1), while DAS is a weak inducer of these enzymes. Finally, we demonstrated that antibodies raised against rGSTA5 strongly reduced the antimutagenic activity of cytosols from DAS- and DADS-treated rats against AFBO. All together, these results demonstrate that DAS prevents AFB(1) mutagenicity through a dual mechanism, i.e. by modulating both the phase I and II metabolism of AFB(1), whereas DADS acts mainly by increasing the phase II metabolism of AFB(1). The induction of rGSTA5 and rAFAR1 is probably the main mechanism by which allyl sulfides give protection against AFB(1)-induced carcinogenesis.

Effects of the garlic compound diallyl disulfide on the metabolism, adherence and cell cycle of HT-29 colon carcinoma cells: evidence of sensitive and resistant sub-populations.

Diallyl disulfide (DADS) is a major organosulphur compound present in garlic with an anti-mitotic potential against colon neoplastic lesions in vivo and colon tumour cell growth in vitro. Using the human colon adenocarcinoma HT-29 Glc(-/+) cell line we identified sub-populations of tumoural cells with markedly different characteristics in terms of metabolic capacities, adhesion properties and distribution in the cell cycle phases. After 1 and 2 days treatment with 100 microM DADS HT-29 cells were largely released into the culture medium. These floating cells accumulated in the G(2)/M phase and were characterized by a 5-fold reduction in cell capacity for de novo protein synthesis. Polyamine metabolism, which is necessary for intestinal epithelial cell attachment and growth, was also severely affected, since 3-fold reductions in polyamine biosynthesis and net accumulation of putrescine were measured after DADS treatment. However, oxidation of L-glutamine, the main precursor of the tricarboxylic acid cycle in these cells, and de novo synthesis of glutathione, a tripeptide involved in tumoural cell chemoresistance, were not affected by DADS treatment. In contrast, the adherent sub-population of HT-29 cells, although partially accumulated in G(2)/M phase, were characterized by unaffected metabolic capacities when compared with control cells except for putrescine accumulation, which was transiently decreased, and L-glutamine oxidation, which was increased 2-fold. DADS-resistant cells selected within 5 days were then able to proliferate at a similar rate to control untreated cells. The DADS-induced changes in HT-29 metabolic capacities, adhesion properties and the cell cycle are discussed from a causal perspective.

Effects of organosulfur compounds from garlic oil on the antioxidation system in rat liver and red blood cells

The modulation of garlic oil (GO) and three allyl compounds, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), on the antioxidation system in rat livers and red blood cells was examined. Rats were orally administered GO (200 mg/kg body weight), DAS (20, 80 mg/kg body weight), DADS (80 mg/kg body weight) or DATS (70 mg/kg body weight) three times a week for 6 weeks. Control rats received corn oil (2 ml/kg body weight) alone. GO, DADS and DATS treatment significantly increased the glutathione (GSH) content (48–84%) in red blood cells ( P<0.05). DATS displayed a greater enhancement than GO and DADS ( P<0.05). Hemolysis induced by tert-butyl hydroperoxide was not suppressed by GO or allyl compound treatment although higher GSH content was evident. Hepatic GSH was not influenced by garlic components. In rat livers, DADS and DATS significantly increased the activity of GSH reductase (46 and 54%, respectively) and of GSH S-transferase (GST) (63 and 103%, respectively), but decreased the GSH peroxidase activity (27 and 28%, respectively). In contrast, GSH reductase and GST activities in the DAS group, either 20 or 80 mg/kg body weight, were similar to the control group. A decrease of GSH peroxidase activity was observed in rats dosed with 80 mg/kg body weight ( P<0.05). An increase in GST activity and a decrease in GSH peroxidase activities were also noted in GO-treated rats ( P<0.05). In red blood cells, three GSH-related antioxidant enzyme activities were not affected by garlic oil and its organosulfur components. Immunoblot assay showed that, accompanying the increase in hepatic GST activity, GO, DADS, DAS (80 mg/kg body weight) and DATS increased the expression of GST Ya, Yb1 and Yc proteins. Results indicate that GO and three allyl compounds play a differential role in modulation of the GSH-related antioxidant system in rat livers and red blood cells.

Effects of allyl sulfur compounds and garlic extract on the expression of Bcl-2, Bax, and p53 in non small cell lung cancer cell lines.

Allyl sulfur compounds play a major role in the chemoprevention against carcinogenesis. The present study compared the antiproliferative effects of diallyl sulfide (DAS), diallyl disulfide (DADS) and garlic extract on p53-wild type H460 and p53-null type H1299 non small cell lung cancer cells (NSCLC). The DAS and DADS treatment of both H460 and H1299 cells resulted in the highest numbers of cells in apoptotic state as measured by acridine orange staining, however, garlic extract treatment did not induce any significant apoptotic cells by MTT assay. DADS was found to be more effective in inducing apoptosis on NSCLC. The level of p53 protein in H460 cell was increased following DADS treatment. DAS and garlic extract treatment of H460 cells induced a rise in the level of Bax and a fall of Bcl-2 level. These results demonstrate that DAS, DADS and garlic extract are effective in reduction of anti-proliferative gene in NSCLC and suggest that modulation of apoptosis-associated cellular proteins by DAS, DADS and garlic extract may be the mechanism for apoptosis which merit further investigation as potential chemoprevention agents.

Diallyl disulfide inhibits p34 cdc2 kinase activity through changes in complex formation and phosphorylation

Previous studies from our laboratory demonstrated that diallyl disulfide (DADS), an oil-soluble allyl sulfur compound found in processed garlic, markedly suppressed p34(cdc2) kinase activity and induced a G(2)/M phase arrest in cultured human colon tumor (HCT-15) cells. The present studies reveal that suppression of p34(cdc2) kinase activity by DADS does not result from direct interactions with the protein, but through changes in factors influencing the formation and conversion of the enzyme to its active form. Flow cytometric analyses showed that the increased proportion of cells in the G(2)/M phase following DADS treatment was accompanied by an increase in cyclin B(1) protein expression. A temporal and dose-dependent response in cyclin B(1) expression was observed in cells treated with DADS. Western blot analysis revealed that 50 microM DADS did not influence the quantity of p34(cdc2) protein expressed, but did decrease the amount associated with cyclin B(1) by 26% (P < 0.05). Exposure of unsynchronized cells to 25 or 50 microM DADS caused a trend towards increased p34(cdc2) hyperphosphorylation (17 and 22%, respectively). Exposure of synchronized cells to 100 microM DADS increased p34(cdc2) hyperphosphorylation by 15% (P < 0.05). Consistent with its ability to slightly increase the quantity of hyperphosphorylated p34(cdc2), DADS, 25 or 50 microM, decreased cdc25C protein expression by 23 and 46%, respectively (P < 0.05). The present studies suggest that the ability of DADS to inhibit p34(cdc2) kinase activation occurs because of decreased p34(cdc2)/cyclin B(1) complex formation and modest p34(cdc2) hyperphosphorylation.

Liver subcellular fractions from rats treated by organosulfur compounds from Allium modulate mutagen activation

The effects of in vivo administration of naturally occurring organosulfur compounds (OSCs) from Allium species were studied on the activation of several mutagens. Male SPF Wistar rats were given p.o. one of either diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) or dipropyl disulfide (DPDS) during 4 consecutive days and the ability of hepatic S9 and microsomes from treated rats to activate benzo[ a]pyrene (BaP), cyclophosphamide (CP), dimethylnitrosamine (DMN), N-nitrosopiperidine ( N-PiP) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) was determined in the Ames test. Administration of DAS, DPS and DPDS resulted in a significant increase of the activation of BaP, CP, N-PiP and PhIP mediated by S9 and microsomes while DADS treatment only increased the mutagenicity of PhIP. In contrast, S9 from DADS-treated rats significantly inhibited the mutagenicity of N-PiP and BaP. DAS, DADS and DPS strongly inhibited DMN mutagenicity while DPDS enhanced it. To understand the mechanisms underlying these effects, the modifications of the activities of specific isozymes of CYP involved in the activation of these mutagens were studied. DAS, DPS and DPDS strongly enhanced pentoxyresorufin O-dealkylase (PROD) activity related to CYP2B and slightly increased ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities related to CYP1A family. DADS exerted the same effects than other OSCs but to a lesser extent. p-Nitrophenol hydroxylase (PNPH) activity related to CYP2E1 was inhibited by DAS and DADS, whereas DPDS significantly increased this activity. Hence, the effects of OSCs on the mutagenicity of several genotoxic compounds are mediated by modification (enhancement or inhibition) of specific CYP involved in their activation.

Effects of diallyl sulfide and diallyl disulfide on cisplatin-induced changes in glutathione and glutathione-S-transferase activity.

The effects of diallyl sulfide (DAS) and diallyl disulfide (DADS) on cisplatin-induced changes in glutathione (GSH) and glutathione-S-transferase (GST) activity in rat liver and kidney was investigated. Cisplatin treatment significantly (p < 0.05) decreased GSH and GST activity in both liver and kidney. DADS treatment significantly (p < 0.05) enhanced GSH and GST activity in rat liver and kidney. Furthermore, DADS treatment reversed the effect of cisplatin on GSH and GST activity both in liver and kidney. Administration of DADS with cisplatin could enhance GSH and GST activity and lower cisplatin-induced nephrotoxicity.

Novel Anti-Carcinogenic Activity of an Organosulfide from Garlic: Inhibition of H-RASOncogene Transformed Tumor Growthin Vivoby Diallyl Disulfide Is Associated with Inhibition of p21H-rasProcessing

In this study, we report a novel anticarcinogenic activity of an organosulfur compound from garlic, diallyl disulfide (DADS). DADS treatment significantly inhibited the growth of H-rasoncogene transformed tumors in nude mice. As compared to controls, the appearance of tumors was also delayed markedly by oral administration of DADS. The inhibition of tumor growth by DADS treatment correlated with the inhibition of p21H-rasmembrane association in the tumor tissue. The levels of membrane associated p21H-raswere markedly lower in the tumor tissues of DADS treated mice as compared to controls. An opposite trend, however, was evident for cytosolic p21H-ras. Furthermore, DADS treatment resulted in a significant inhibition of hepatic as well as tumoral 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. These results indicate that DADS suppresses the growth of H-rasoncogene transformed tumors in nude mice by inhibiting the membrane association of tumoral p21H-ras.

Diallyl Disulfide Suppresses the Growth of Human Colon Tumor Cell Xenografts in Athymic Nude Mice

The present studies examined the antiproliferative effects of diallyl disulfide (DADS) on the growth of human colon tumor cell line, HCT-15, xenografts in 6-wk-old female NCr nu/nu mice with an initial body weight of 20–22 g. Intraperitoneal injection of 1 mg DADS thrice weekly reduced tumor volume by 69% (P < 0.05) without apparent ill consequences such as altered growth of the host. Providing this quantity of DADS intragastrically also inhibited growth of the HCT-15 tumor. At equivalent DADS dosages, intraperitoneal treatment was proportionately more effective (P < 0.05) in reducing tumor growth than gastric intubation. Tumor inhibition caused by DADS (0.5 mg thrice weekly) was similar to that occurring with 5-fluorouracil (5-FU) treatment (0.5 mg thrice weekly). Combining DADS and 5-FU was no more effective in inhibiting tumor growth than using either compound alone. However, concurrent DADS treatment significantly (P < 0.05) inhibited the depression in leukocyte counts and spleen weight and prevented the elevated plasma urea caused by 5-FU treatment. These data suggest that DADS, a constituent of garlic oil, reduces the toxicity of 5-FU and is an effective antitumorigenic agent against xenografts resulting from an established human colon tumor cell line.