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Nutrition and Cancer

Diallyl Disulfide Induces ERK Phosphorylation and Alters Gene Expression Profiles in Human Colon Tumor Cells^1,2

Graduate Program in Nutrition and the Nutrition Department, The Pennsylvania State University, University Park, PA 16802

³To whom correspondence should be addressed. E-mail: milnerj{at}mail.nih.gov.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Diallyl disulfide (DADS), a compound found in processed garlic, has been shown to arrest unsynchronized human colon tumor cells (HCT-15) in the G₂/M phase of the cell cycle. The present studies were designed to examine whether this cell cycle block related to alterations in protein kinase C (PKC), Ca²⁺/calmodulin-dependent protein kinase II (CAMK II) or extracellular signal-regulated kinase (ERK) activity. Exposing double thymidine synchronized HCT-15 cells to DADS (25, 50 and 100 µmol/L) for 4 h increased the G₂/M population by 30, 31 and 63%, respectively, compared with controls (P < 0.05). PKC and CAM KII activities were not influenced by increasing DADS exposure and thus did not correlate with the block of cells in the G₂/M phase. Although ERK activity increased by 44 and 60% after treatment with 100 and 500 µmol/L DADS (P < 0.05), it was not influenced by exposure to 25 or 50 µmol/L DADS. Western blot analysis revealed that although DADS (25, 50, 100 and 500 µmol/L) did not influence the quantity of ERK protein expressed, it did increase its phosphorylation by 39, 52, 73 and 61%, respectively, compared with controls (P < 0.05). These studies provide evidence that early alterations in ERK pathway signaling may contribute to the G₂/M arrest observed after DADS exposure. Preliminary data generated using the Clonetech Atlas Human Cancer cDNA Expression Array suggest that alterations in cell cycle, DNA repair and cellular adhesion factors accompany DADS exposure and may also be involved in mediating the block in G₂/M progression.

KEY WORDS: • diallyl disulfide • tumor proliferation • G₂/M phase • protein kinase • cDNA array

Colon cancer is the third leading cause of cancer death and the third most common type of malignancy in the United States (1). Despite recent advances in surgical and chemotherapeutic procedures, the 5-y survival rate for colon cancer is only 61% (1). Identifying alternative factors that may reduce the initiation and promotion of colon cancer is therefore important for minimizing the incidence and severity of this disease.

Garlic, sometimes referred to as a spice, herb or a vegetable, is recognized for its savory characteristics, which are associated with its high content of sulfur-containing compounds (2). Support for the ability of garlic (Allium sativum) to retard colon carcinogenesis in humans comes from recent meta-analyses that found that consumption of raw and cooked garlic (3.5g/wk) was associated with a 0.69 random-effects relative risk (3). Similarly, considerable preclinical evidence with model carcinogens and transplantable tumors supports a protective role of garlic and some of its allyl sulfur components. Specifically, animal studies demonstrate that allyl sulfides effectively inhibit 1,2-dimethylhydrazine and azoxymethane chemically induced colon tumor formation (4–8). Laboratory studies reveal that intraperitoneal and intragastric administration of allyl sulfide reduces the growth of transplanted HCT-15 colon tumor cells in nude mice (9). A similar depression in the proliferation of breast tumors and sarcoma cells was also noted (10,11). Consequently, allyl sulfides appear to effectively modify several phases of the in vivo cancer process, including tumor behavior.

Tumor growth is characterized by abnormalities in the mechanisms governing cell division and apoptosis (12,13). Antitumorigenic agents control tumor behavior by disrupting cell division and/or enhancing the apoptotic process (13,14). The antitumorigenic effects of water-soluble S-allylmercaptocysteine, and lipid-soluble diallyl sulfide and diallyl disulfide (DADS)³ relate both to a suppression in cell division and to an induction of apoptosis (10,15–19). Recently, studies found that the ability of DADS to disrupt cell division corresponds to suppressions in p34^cdc2 kinase activity and a block in the progression of cells from the G₂ into the M phase of the cell cycle (20). This suppression in p34^cdc2 activity was associated with a decrease in p34^cdc2/cyclin B₁ complex formation and its subsequent conversion to an active hypophosphorylated enzyme (21). Because p34^cdc2 formation and activation are critical for cells to undergo mitosis (22), this suppression in p34^cdc2 kinase is likely one of the final events leading to DADS’s blocking of G₂/M progression. It remains to be determined what early cellular events mediate this suppression in p34^cdc2 kinase activity and G₂/M progression.

Cell division is governed by an intricate signaling system controlled at several stages by protein kinases (23). A number of these cellular kinases are implicated as potential modifiers of the G₂/M phase transition (24–27). For instance, extracellular signal-regulated kinase (ERK) promotes progression of cells through mitosis by facilitating p34^cdc2 kinase activation (24). Protein kinase C (PKC) appears to be involved in regulating p34^cdc2 kinase activity, lamin B phosphorylation and nuclear lamina disassembly (25,26). Last, calcium/calmodulin-dependent protein kinase II (CAM KII) is believed to be a key factor in targeting cyclin B₁ for ubiquitin-dependent proteolysis after the completion of mitosis (27). In the present studies, we examined whether alterations in PKC, CAM KII and/or ERK activities are events mediating the block in G₂/M progression by DADS.

To gain a more global understanding of DADS’s effects on cell division and overall tumor behavior, we also utilized cDNA array technology to screen HCT-15 cells for changes in gene expression. Data generated using a cancer-specific cDNA array offered insight into the cell cycle, DNA damage and extracellular matrix events that occur after exposure to DADS.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Chemicals.

The human colon tumor cell line (HCT-15) was purchased from the American Type Culture Collection (Rockville, MD). DADS was purchased from Fluka Chemika (Ronkonkoma, NY). SignaTECT protein kinase C and SignaTECT calcium/calmodulin-dependent protein kinase assay systems were obtained from Promega (Madison, WI). [-³²P]ATP (specific activity370 GBq/L) and [-³³P]dATP (specific activity370 GBq/L) were purchased from ICN Pharmaceuticals (Irvine, CA). The p44/42 mitogen-activated protein kinase (MAPK) assay kit and polyclonal anti-p44/42 MAPK antibody were purchased from Cell Signaling Technologies (Beverly, MA). Monoclonal anti-p-ERK antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase conjugated goat anti-mouse IgG was obtained from Pierce (Rockford, IL) and peroxidase labeled goat anti-rabbit IgG was purchased from Gibco BRL (Grand Island, NY). Atlas cDNA Expression Arrays were purchased from Clontech (Palo Alto, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).

Culture conditions.

HCT-15 cells were plated in tissue culture flasks and grown under a humidified, 5% CO₂ atmosphere at 37°C in RPMI 1640 medium (pH 7.2) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY), 1% penicillin/streptomycin (1 x 10⁷ units/L penicillin and 10 g/L streptomycin) and 1 mg/L insulin (28). Cells were plated at 4 x 10³/cm² for 24 h before synchronization by the double thymidine block method of Stein and Stein (29). Synchronized cells were treated with DADS 2 h after thymidine removal. DADS was dissolved in dimethyl sulfoxide (DMSO) before addition to cultures. Control cultures were treated with DMSO. The maximum quantity of DMSO added to the medium in these studies was 0.01%.

Cell proliferation.

HCT-15 cells were rinsed with PBS (pH 7.2) and harvested by trypsinization (2.5 g/L trypsin and 0.38 g/L EDTA) at the specified incubation times indicated below. Trypsin was deactivated by the addition of RPMI containing fetal bovine serum, and suspensions were centrifuged at 500 x g for 5 min. Cell pellets were resuspended in RPMI 1640 and counted using a hemocytometer to determine the number of viable cells.

Flow cytometry of cellular DNA.

Synchronizes HCT-15 cells were harvested 4 h after DADS treatment, fixed in 70% ethanol and stored at -20°C until subsequent analysis. After centrifugation at 500 x g and the removal of the fixing agent, cellular DNA was stained by the addition of PBS containing 2 x 10⁵ units/L RNase and 18 mg/L propidium iodide and analyzed as previously described (20).

Cell lysis and protein quantification.

Synchronized HCT-15 cells were harvested 4 h after DADS addition and incubated in a lysis buffer composed of 25 mmol/L Tris-HCl (pH 7.4), 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 0.05% Triton X-100, 10 mmol/L ß-mercaptoethanol, 50 mg/L phenylmethylsulfonyl fluoride (PMSF), 2.2 mg/L aprotinin and 0.7 mg/L pepstatin A for analysis of PKC activity or in 20 mmol/L Tris-HCl (pH 8.0), 2 mmol/L EDTA, 2 mmol/L EGTA, 20 mg/L soybean trypsin inhibitor, 5 mg/L leupeptin, 25 mmol/L benzamidin, 50 mg/L PMSF, 2.2 mg/L aprotinin and 0.7 mg/L pepstatin A for examination of CAMK II activity. ERK extracts were obtained by incubating HCT-15 cells in 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerolphosphate, 1 mmol/L sodium vanadate, 1 mg/L leupeptin, 50 mg/L PMSF, 2.2 mg/L aprotinin and 0.7 mg/L pepstatin A. After brief sonification, extracts were incubated on ice for 10 min and centrifuged at 10,000 (CAM KII) or 14,000 x g (PKC and ERK) for 5–10 min at 4°C. The amount of protein in the resulting supernatant was determined using a standard kit (Pierce, Rockford, IL or Bio-Rad, Hercules, CA).

PKC and CAM KII activity.

Phospholipid stimulation of PKC activity and CAM KII stimulation by Ca²⁺ and calmodulin were measured using the methods described by Promega. Radioactivity was monitored using a Beckman model 3801 Scintillation Counter (Irvine, CA) to a final two-sigma error of 2%.

ERK activity.

ERK activity was assessed by a nonradioactive method according to the manufacturer’s directions (New England BioLabs, Beverly, MA). Bands were detected by enhanced chemiluminescence (Amersham Life Science, Bucks, UK). NIH Image 1.61 software (NIH, Bethesda, MD) was used to determine the relative density of each band. The amount of phosphorylated Elk-1 detected corresponded to ERK activity.

ERK Western blots.

Extracts from HCT-15 ERK lysates were subjected to electrophoresis on a 12% SDS acrylamide gel, transferred onto a nitrocellulose membrane and probed with anti-p44/42 MAP kinase or anti-P-ERK as the primary antibody and anti-mouse or anti-rabbit IgG conjugated peroxidase as a secondary antibody. Bands were detected and quantitated as described above.

RNA isolation.

Synchronized HCT-15 cells were exposed to DADS (0 or 50 µmol/L) for 12 h. Cells collected from 7 flasks (150 cm²) per treatment were pooled, and total RNA was isolated using Trizol reagent as directed by the manufacturer (Life Technologies, Grand Island, NY). Poly A⁺ RNA was obtained by passing total RNA over an oligo (dT) cellulose column known to preferentially bind mRNA. mRNA was eluted from the column using 3 mL of 1 mol/L Tris-HCl, 0.1 mol/L EDTA, 5 mol/L lithium chloride and 200 g/L SDS, and pelleted by the addition of 300 µL of 3 mol/L NaOAc and 2.5 mL of 100% ethanol overnight at -20°C. After centrifugation, mRNA pellets were washed with 80% ethanol, dried and stored in DEPC-treated water at -80°C.

cDNA expression arrays.

³³P-cDNA probes were prepared using the Atlas Human Cancer cDNA Expression Array kit developed by Clontech (Palo Alto, CA). Arrays were scanned by a Storm Phosphorimager and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Arrays were then stripped and reused in a second experiment involving control and DADS-treated samples. In this second experiment, arrays were switched so that the control array from the first experiment was used as the treatment array in the second experiment. This controlled for the nonspecific binding of the ³³P-cDNA probes to one array over the other.

The signals obtained from the two arrays were normalized to the nine housekeeping genes provided and compared with each other using the control array as a reference. The background intensity of each array was used as a threshold for filtering out signals that were too weak to be meaningfully interpreted.

Statistical analysis.

Data were analyzed using ANOVA and Fisher’s least significant difference test (StatView 4.51, Abacus Concepts, Berkeley, CA). Treatment differences with P < 0.05 were considered significantly different.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Cell growth and cell cycle progression.

Examination of cellular DNA profiles revealed that DADS increased the proportion of cells residing in the G₂/M phase of the cell cycle (Fig. 1). Treatment with 25, 50 or 100 µmol/L DADS for 4 h resulted in a 30, 31 and 63% increase in the G₂/M phase population, respectively (P < 0.05). Cell numbers increased by 1.9, 1.8, 1.6 and 1.7 x 10⁶ in response to 0, 25, 50 and 100 µmol/L DADS, respectively, and did not differ during this 4-h exposure.

View larger version (14K): [in this window] [in a new window]   FIGURE 1 Effect of increasing concentrations of diallyl disulfide (DADS) on the percentage of cells blocked in G2/M phase. Values are means ± SEM, n = 4. Values without a common letter differ, P < 0.05. The maximum SEM observed for all measurements was 2.6%.

The effect of DADS on PKC and CAM KII was examined to determine whether changes in activity accompanied the block in G₂/M progression. PKC and CAM KII activities were not influenced by DADS (25, 50 or 100 µmol/L) compared with controls (data not shown). The mean PKC activity for control lysates was 3.5 pmol ATP/(min · µg protein).

ERK activity.

Although significant elevations in ERK activity were not observed in response to 25 or 50 µmol/L DADS, exposure to 100 µmol/L increased activity by 44% compared with controls (P < 0.05; Fig. 2). Similar elevations in ERK activity occurred when cells were exposed to a higher concentration of DADS (500 µmol/L; Fig. 2).

View larger version (23K): [in this window] [in a new window]   FIGURE 2 Effect of increasing concentrations of diallyl disulfide (DADS) on extracellular signal-regulated kinase (ERK) activity in HCT-15 cells. Each bar represents the mean ± SEM Elk-1 phosphorylation by ERK, n = 4 determinants, except for 100 µmol/L DADS where n = 3. The upper panel shows a representative immunoblot. The mean ERK activity from control lysates was 285 arbitrary units and was designated as 100% in the graph. Values without a common letter differ, P < 0.05. The maximum SEM observed for all measurements was 20.0%.

Western blot analysis was used to determine whether changes in ERK protein expression and/or phosphorylation accounted for the ability of DADS to increase ERK activity. Differences in ERK protein expression were not detected in cultures grown in the presence or absence of DADS (0, 25, 50, 100 and 500 µmol/L; Fig. 3). However, elevations in ERK phosphorylation were found to accompany DADS exposure (Fig. 4). Adding 25, 50, 100 and 500 µmol/L DADS to cell cultures increased ERK phosphorylation by 39, 52, 73 and 61%, respectively (P < 0.05).

View larger version (25K): [in this window] [in a new window]   FIGURE 3 Effect of diallyl disulfide (DADS) (25, 50, 100 and 500 µmol/L) on extracellular signal-regulated kinase (ERK) protein expression in HCT-15 cells. Cell lysates were separated by SDS-PAGE and probed for ERK expression. The upper panel shows a representative immunoblot. The mean ERK expression from control lysates was 1063 arbitrary units and was designated as 100% in the graph. Each bar represents the mean ERK protein expression ± SEM, n = 4. The maximum SEM observed for all measurements was 9.2%.

View larger version (25K): [in this window] [in a new window]   FIGURE 4 Effect of diallyl disulfide (DADS) on extracellular signal-regulated kinase (ERK) phosphorylation status in HCT-15 cells. Cell lysates were separated by SDS-PAGE and probed for phosphorylated ERK using antibody specific for phosphorylated Tyr204 on p44 and p42. The upper panel shows a representative immunoblot. The mean ERK phosphorylation from control lysates was 242 arbitrary units and was designated as 100% in the graph. Each bar represents the mean expression of phosphorylated ERK ± SEM, n = 4. Values without a common letter differ, P < 0.05. The maximum SEM observed for all measurements was 19.0%.

To maximize our ability to detect changes in gene expression, we examined the effect of a 12-h DADS (50 µmol/L) exposure on gene expression profiles in synchronized HCT-15 cells. In the present study, 12% of the genes on the array were expressed above the detection limit. Tables 1, and 2 summarize the gene expression profile changes observed after DADS exposure. Overall, 36 genes were found to differ by at least 1.5-fold in DADS-treated cells compared with controls. mRNA expression levels revealed that 24 genes were up-regulated (Table 1), whereas 12 genes were down-regulated in response to DADS (Table 2).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Alterations in CAM KII activity did not accompany DADS (100 µmol/L) exposure in the present studies. Suppressions in PKC activity were also not observed, suggesting that modifications in the activity of these enzymes do not account for the ability of DADS to block G₂/M progression. Other studies found that depressions in platelet aggregation by ajoene, another sulfur compound in garlic, occurred independently of PKC and CAM KII regulation (34,35). Specifically, Rendu et al. (34) showed that ajoene (100 µmol/L) inhibited thrombin-induced aggregation without modifying PKC or CAM KII phosphorylation of protein P43 and the myosin light chain P20, respectively. Apitz-Castro et al. (35) found that direct activation of PKC by phorbol myristate acetate and its subsequent phosphorylation of P47 were also unaffected by ajoene (35). Collectively, these studies suggest that total PKC activity is not altered in response to DADS or ajoene. Whether differential regulation of PKC isozymes occurs in response to allyl sulfides in garlic remains to be determined.

The present studies reveal that elevations in ERK activity accompany exposure to high concentrations of DADS (100 and 500 µmol/L). Using Western blot analysis, we found that this increase in activity was associated with increased ERK protein phosphorylation. Increased ERK phosphorylation occurred in response to both low (25 and 50 µmol/L) and high (100 and 500 µmol/L) concentrations of DADS. The reason for the increase in phosphorylation in response to 25 and 50 µmol/L DADS is unclear because significant elevations in ERK activity were not observed in response to these concentrations. Studies by Wang et al. (36) suggested that ERK phosphorylation prevents cells from exiting mitosis. They found that injection of metaphase Xenopus tadpole cells with ERK-specific MAPK kinase-1 inhibited mitosis, whereas ERK dephosphorylation by the dual-specific phosphatase XCL100 enhanced the mitotic process. In the present studies, high levels of ERK phosphorylation may have served as a compensatory mechanism, mediating the block in G₂/M progression by DADS.

ERK activity fluctuates during the G₂/M transition with activation occurring early followed by severe inactivation in mitosis (37). Sustained activation of ERK has been shown to block G₂/M progression (38,39). Elevations in ERK activity after exposure to 100 and 500 µmol/L DADS may account for the increased ability of these concentrations to block HCT-15 cells in the G₂/M phase. Because exposure to these high concentrations of DADS (100 and 500 µmol/L) is also associated with apoptosis (19), the elevations in ERK activity observed in the present study may also represent early apoptotic events.

Utilization of cDNA array technology proved to be a powerful tool for examining the effects of DADS on the expression of genes involved in HCT-15 tumorigenesis. To our knowledge, the present studies are the first to provide a comprehensive overview of the genetic events accompanying the block in G₂/M progression by DADS. Moreover, consistent with its ability to block cell cycle progression, DADS exposure was found to modify the expression of several cell cycle proteins (Tables 1, and 2). cDNA expression data revealed that DADS suppressed the expression of cyclin-dependent protein kinase 6 (cdk6) and the p19INK4 cyclin-dependent kinase inhibitor, whereas it up-regulated cell division control protein 2 (cdc2), cdk2, cdk3, cdk4 and cyclin E. These up-regulations indicate that both G₁- and G₂-specific cyclin-dependent kinases are affected by DADS exposure. Whether these changes in transcription result in increased protein expression, however, is unclear. Our previous studies showed that DADS inhibited p34^cdc2 complex expression and phosphorylation without altering cdc2 protein levels (21). Clearly, genetic and post-translational modifications both appear to play important roles in mediating the antitumorigenic effects of DADS.

Data generated using cDNA array technology reveal that the antiproliferative effects of DADS also relate to alterations in cellular matrix gene expression. Specifically, DADS exposure down-regulated the expression of aggrecan 1, tenascin R, vitronectin and cadherin 5, whereas it up-regulated 40S ribosomal protein SA, platelet-derived growth factor-associated protein, and glia-derived neurite-promoting factor levels. These changes in matrix protein expression likely reflect depressions in cellular adhesion because decreased adhesion was observed in human colon tumor (HT-29) cells exposed to allyl sulfide (50 µmol/L) (40) and DADS (100 µmol/L) (41). Recently, Franz et al. (42) reported that the increase in HT-29 cell detachment by garlic relates to increased epidermal growth factor receptor and integrin- 6 mRNA expression. Suppressions in integrin-mediated adhesion have also been found to correspond to the ability of ajoene to inhibit viral adhesion and platelet aggregation (43,44). Whether suppressions in integrin-mediated adhesion also account for the antitumorigenic effects associated with DADS remains to be determined because integrin expressions in the present study were below detection levels. The ability to determine changes in integrin expression in these studies may have been precluded by the examination of a single time after exposure. Additional studies are required to characterize more fully the changes in gene expression patterns that are critical in explaining the block in G₂/M progression.

Unfortunately, ERK, PKC and CAM KII cDNAs were not present on the arrays used in the current studies. Nevertheless, cDNA expression analysis did find that DADS exposure induces a marked increase (12.87-fold) in macrophage migration inhibitory factor (MIF) expression (Table 1). Although the exact nature of MIF up-regulation by DADS is unclear, studies by Mitchell et al. (45) showed that the release of MIF by NIH/3T3 cells culminates in ERK phosphorylation and activation. Consequently, the induction of MIF expression by DADS may be associated with the compound’s effect on ERK activation. Additional studies are warranted to characterize the effects of DADS on MIF release and determine its effect on ERK activation and cell cycle arrest.

In summary, the present studies revealed that the block in G₂/M progression occurring in response to DADS precedes detectable changes in proliferation. Changes in PKC or CAM KII did not accompany DADS exposure; however, elevations in ERK activity and ERK phosphorylation were observed. The ability of DADS to modify HCT-15 tumor proliferation also related to changes in the expressions of several genes involved in tumorigenesis. Identifying these and other targets for DADS will assist in clarifying the mechanisms accounting for its antitumorigenic benefits.

	ACKNOWLEDGMENTS

 
The authors would like to thank Nan-Qian Li for her assistance with the cDNA array experiments.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 2001, April 2001, Orlando, FL [Knowles, L. M. & Milner, J. A. (2001) Diallyl disulfide modifies intracellular signaling in human colon tumor cells. FASEB J. 15: A993 (abs.)].

² Supported in part by grants 97-35200-4679 and 97-36215-5191 from the U.S. Department of Agriculture.

⁴ Abbreviations used: CAMK II, calcium/calmodulin-dependent protein kinase; cdc2, cell division control protein 2; cdk6, cyclin-dependent protein kinase 6; DADS, diallyl disulfide; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MIF, macrophage migration inhibitory factor; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride.

Manuscript received 30 April 2003. Initial review completed 30 May 2003. Revision accepted 20 June 2003.

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