- Email: [email protected]
Expression of RCAS1 is implicated in all stages of colorectal cancer development
T973'
Results: Both BITC and PE1TC caused a dose-dependent inhibition of prolite:ration as measured by 3H-thymidine incorporation, with 1Ca04 6~M and 2.4~M respectively The doubling times of cell populations exposed to IO}*MB1TC or PEITC were increased from 32h (control) to 220h and 120h respectively. Furthermore, this antiproliferative effect was associated with ceU-cycle arrest in the G2/M phase. The percentage of cells in G2/M increased steadily over 48h from 16.9%-+ 1.4% in controls m 48.3% +_4.1% (p<0.001) and 36.0%-+ 25% (p=0.001) for cells treated with BITC or PEITC respectively. This cell-cycle arrest was found to occur after mitotic spindle formation as cells ~yuchmnised at metaphase with nocodazole did not re-enter G0/G1 when subsequently treated with 10btM BITC or PEITC Conclusions: BITC and PEITC were found to be potent inhibitors of prolifel~ation of Coco2 ceils in vitro, and to cause celbcycle arrest during mitosis by an as yet undetermined mechanism. These results suggest that inhibition of proliferation by BITC and PEITC may represent an additional mechanism of cancer chemoprevention independent of their known effects on carcinogen metabotisra.
Hyperosmolarity Shifts Fas Signaling Pathway From Apoptosis To Proliferation by- Activation of p38 in Gastric Mucosal Cell Hanchen LL Calin Stoicov, Jear~marie Houghton Background&Aim. A high salt diet is associated with an increase risk of gastric cancer, hmvever, the molecular mechanism has not been elucidated. Fas acnvation classically leads to apoptosis, however, insufficient Fas signaling or an imbalance between downstream signaling components can initiate prolikrative signaling. We propose that an imbalance between Fas mediated apoptotic proliferative signaling is altered by' consumption of a high salt diet. Methods. Rat gastric mucosal ceils (RGM-1) were stably transfected with the pMSCV-puro vector containing mFas Ag cDNA, and low and high expressing clones isolated. The apoptotic and profit;erative response to FasL was determined in control medium or hyperosmotic medium (40mM or 80 mM sodium chloride) via a combination of FACS, DNA fragmentation assay, BrdU incorporation, NF-KB activation an_alysis, and WST-1 assay. Western blot. with phosphor-specific antibody was used to quami~ate the phosphorylation status of p38 MAPK as an index of p38 activation. Results. Exposure to hypemsmotic medium for 10 rain resulted in a marked increase in p38 MAPK phosphorylation which was inhibited by pretreannent witb SB203580, a specific inhibitor of p38 phosphoryiation. NF-KB activation increased 1.5 and 4 4 fold in cells exposed to 40 or 80mM NaCI respectively, an effect that was cnmpletdy aboliahed by the addition of SB203580. Low abundance clones increased proliferation (BrdU incorporation) in response to Fas L (12.5 ng/ml for 24 hours) from 10.09% to 14.42% which was dramatically augmented by NaCt 40ram in the presence of Fas L (2611%) but not in its absence (11.03%), and was dependent on p38 activation (NaC140raM, Fas L 12.5 ng/ml, SB203580 = I4.63%) To test if NaCI exposure could also protect against Fas mediated apoptosis, we employed a high Fas abundance clone which is highly sensitive to Fas mediated apoptosis After 24 hours exposure to 50ng/ml FasL, 76 -+ 0.03% of cells were apoptotic. This level decreased dramatically to 10.2 _+0.02% with the addition of 40 mM NaCI. This protective effect of NaCI was completely reversed by tire addition at SB203580 (79 -+ 0.04% apoptosis) WST-I assay confirmed increased cell survival in the NaCI treated group Conclusions. Hyperosnmlarity in the physiological range can active p38 MAPK signal, which may enhance Fas proliteeative signaling, and inhibit Fas apoptotic signaling. This growth signaling imbalance may contribute to the association between high salt diets and gasu'ic cancer,
T976
Expression of RCAS1 is implicated in all stages of Colorectal cancer development Kawm Leelawat, Takeshi Watanabe, Manabu Nakashima, Supatip Tujinda, Cheepsumm Suthipintawong, Vijittra Leardkamotkam Aims: The colorectal cancer is well established as an excellent model for the study of a sequence of genetic events associated with multistep carcinogenesis. The gradual accumulation of genetic alterations is necessary for the progression of benign polyps to cardnoma and eventually metastases. Each step of carcinogenesis produces many kinds of tumor antigens, which can be recognized and eliminated by the host immune system. To survive in the body, cancer cells must have ability to evade immune elimination. RCAS1 (Receptorbinding Cancer Antigen expressed on SiSo cells) is a tumor-associated antigen, implicated in immune evasion of tumor cells by acting as a ligand that binds to its receptor on the immune cells such as NK ceils aud T call and subseqnently induces apoptosis of these cells The high expression of RCAS1 has been demonstrated by immunohistochen'fical staining in certain tumors such as cervix, breast, tung and stomach. However; the expression of RCAS1 in colorectal cancer has never been quantified. This study aims to investigate the expression of RCAS1 in colorectal cancer and to clarify the stages of coloreatal carcinogenesis, which express this antigen. Materials and methods: 50 colorectal cancers and 12 adenomatous polyps specimens obtained from Rajavithi Hospital were included in this study. Detection of RCAS1 expression was carried out on 4 ~m thick sections of fom'talin-fixed, paraffie embedded specimens by immunohistochemical-staining technique using monoclonal amiRCAS1 antibody and the freshly isolated tissues were processed for expressions at RCAS1 mRNA by RT-PCR. Tumor-infiltrating lymphocyies (TIL) in the specimens were detected in serial sectious by immunohistochemical-stainmg technique and the apoptosis of these cells were identified in situ by using Calorimetric TUNEL assay. Result: The immunosignal tbr RCAS1 protein was highly intense in all specimens of the adenomatous polyps and cotorectal cancers whereas it was weakly detected in the normal. Tbe results of RT-PCR from tumor tissues were also correlated with the result of immunohistochemical staining. The apoptosis of TIL was identified in sitn adjacent to the area of RCASlexpressed tumor celts. Conclusions: The result suggested that there was up regulation of RCAS1 in the early stage of colorectai carcinogenesis and persisted in all stages of cancer development. The expression of RCAS1 in the colorectal cancer might be implicated in evasion of immune surveillance by inducing apoptosia of TIL.
T974
ERRP, a Negative Regulator of EGFR, S~n~tlr Nat~lan Colon Cancer Cells to the Apoptotic Effect of Curcumi~ 5atish Maryala, Jasbir Rangi, ~ l i l ' R l k d l ~ . a u e r , Kiran. K. Nagothu, Richard Jaszewski, Arun K. Rishi, A d h i p . g , i ~ l ~ # ' r Accumulatingl'll~ic~ti~gesta t~iat curcumin (dileruloyhnethane), the active ingredient of turmeric, may' bel~L~mpmventive against several types of malignancios, including colorectal cancer. The antitumorigenic properties of curcumin have been attributed in part to the induction of apoptosis. Although ihe regulatory' mechanisms by which cureumin exerts its chcmopreventive effect are not fully understood, we have observed that curcumin inhibits the expression and activation of EGFR in colon cancer cells. Recently, we isolated a uegative regrdator of EGFR, termed ERRP (EGF-R Related Protein), ovemxpression of which inhibits proliferation and EGFR activation in colon cancer ceils. Purified recombinant ERRP was utilized to examine whether ERRP would sensitize colon cancer cells to the chemopreventive effect of curcumin. Exposure of HCT-116 cells to recombinant ERRP (l-I0p~g/ml) or curcumin (5-30~M) for 48 h inhibited proliteration in a dose-dependent mamler, mveahng a 60-70% inhibition with the highest dose. Cnscumin at doses of 5-30btM also stimulated apoptosis in HCT-116 cells, as detennined by the TUNEL assay. This was accompanied by a concomitant rise in caspase 8 and 3 activity. On the other hand, ERRP at a dose of l~g/ mt caused no significant increase in apoptosis in HCT-I16 cells To determine whether ERRP would sensitize colon cancer cells to curcumimmduced apoptosis, HCT-1t6 ceils were preincubated in the absence (control) or presence o~ a low dose of ERRP (1 ~ m l ) for 48 h, and subsequently incubated for anotber 48 h with or without 5btM c~urcnmin, a dose that causes only 15-20% apoprosis Preincubation of HCT-I16 cells with ERRP caused approximately 50% higher apoptosis than curcumiu alone. Curcumin-induced inhibit/on of proliferation of HCT-116 cells was also equally affected fnllowing preincubation with ERRP We conclude that ERRP exacerbates the curcumin-iaduced changes in proliferation and apoptosis of colon cancer cells
T977
Resveratrol Selectively Engages Cell Death Signals Jason M. Erickson, Bashar M Attar, MaiT Jo Atten, Oksana Hohan Survival and replication of cancer cells is maintained by overriding apoptosis. Therefore, chemoprevention requires either stimulation of pmapoptotiv reactions or inhibition of antiapoptotic signals. Prm'iously, we showed that resveratrol inhibits proliferation of gas!tic adenocarcinoma cells and stimulates apoptosis by interacting with ceil signaling through PKC and oxidative stimuli. Here, we investigate the action of resvemtrol on components of death signaling pathways in gastric adenocarcinoma cells, aiming to define the contribution of tumor suppressor p53 to their expression and activity. METHODS: Gastric adenocarcinoma cell lines AGS, SNU-1, and KATO-III were c~dtured in RPMI-1640 media supplemented with 10 % FBS. After cell treatment with resveratml for specified times, p53 expression was detected by immunostaining and activities of caspases 1,3,6,8/10 arid 9 were datemuned by quantitation of calorimetric products from caspase-specific substrates. Protein levds of survivm, Bcl-2 and Bax were determined by immunoblotting Action of specific caspase inhibitors on cellular pmlderation was assessed by incorporation of )H-tfiyroidine into DNA. RESULTS: Resveratsol selectively downmgulated survivin protein expression in cells wah p53 (AGS and SNU-1) but was ineftective in KATO-III ceils with deleted p53. In twrn, resveratrol activated caspase 3 in cells with deleted p53, but had minimal efte'ct on other caspases. Increased mitochondriaI expression of proapoptotic Bax was seen in all cells upon exposure to resveratml. Antiapoptotic Bcl-2 remained unaffected by resveratrol treatment. Resveratrol altered survivin and Box expressim~ within 24 hours, but acm~ation of caspase 3 required longer treatment. Neither specihc nor broad range caspase mhibitors reversed resveratrol-induced inhibition of cellular proliferation. CONCLUSIONS: Resveratrol induces apoptosis in gastric adenocarcinoma cells by regulating both pro and antiapoptotic signaling molecules. However, engagement of specific apoptotic signals by resveratrol is selective and depends, in part, on p53 expression. Time dependent response to resveratml suggests that activation of caspase 3 is a late and irreversible event possibly elicited by changes in survivin and Box. These results indicate that resveratml interacts with rapid response dements of cdlular signaling, suppresses specific antiapoptotio signals, activates proapoptotic sig~als, and stimulates release of activated caspase 3 to induce apoptosis in gastric cancer cells. /
T975
Inhibition of Proliferation and Induction of Cell Cycle Arrest in Caco-2 Cells by Two Dietary Phytochemicals from Cruciferous Vegetables, Benzyl lsothiocyanate and Phenethyl Isothiocyanate James Visanii David Thompson, Philip Padfield Background: Benzyl isothiocyanate (BITC) and phenetbyl isothiocyanate (PEITC) are phenolic phytochemicals p~r as glucosinolate precursors in crucif~:rous vegetables, the consumption of which is reversely associated with risk of colon cancer. Both are putative cancer chemopreventives, and have been shown to attenuate tbe genotoxic efl~ectsof some carcinogens by modulating the actMties o[ phase I and 15base lI detox~fying enzymes. However, there are tva data available regarding the etfects of these phytocbemicals on cefi proliferation and cell cycle regulation. Aims: To characterize the effects of BITC and PEITC on prolderation and cell cycle regulation in Caco-2 cells i~ vitro. Methods: Coco-2 cells were treated with a range of concentrations of B1TC and PEITC. Rate of DNA sTntbesis was measured by ~H-thytuidine incorporation assay, proliferation was mea~sured by counting viable cdts. Cell cycle phase was measured by flow cytometry of propidium iodide stained cells with or without G2A~ block enforced by nocodazole All experiments were performed at least 3 times
AGA Abstracts
A-460
Results: Both BITC and PE1TC caused a dose-dependent inhibition of prolite:ration as measured by 3H-thymidine incorporation, with 1Ca04 6~M and 2.4~M respectively The doubling times of cell populations exposed to IO}*MB1TC or PEITC were increased from 32h (control) to 220h and 120h respectively. Furthermore, this antiproliferative effect was associated with ceU-cycle arrest in the G2/M phase. The percentage of cells in G2/M increased steadily over 48h from 16.9%-+ 1.4% in controls m 48.3% +_4.1% (p<0.001) and 36.0%-+ 25% (p=0.001) for cells treated with BITC or PEITC respectively. This cell-cycle arrest was found to occur after mitotic spindle formation as cells ~yuchmnised at metaphase with nocodazole did not re-enter G0/G1 when subsequently treated with 10btM BITC or PEITC Conclusions: BITC and PEITC were found to be potent inhibitors of prolifel~ation of Coco2 ceils in vitro, and to cause celbcycle arrest during mitosis by an as yet undetermined mechanism. These results suggest that inhibition of proliferation by BITC and PEITC may represent an additional mechanism of cancer chemoprevention independent of their known effects on carcinogen metabotisra.
Hyperosmolarity Shifts Fas Signaling Pathway From Apoptosis To Proliferation by- Activation of p38 in Gastric Mucosal Cell Hanchen LL Calin Stoicov, Jear~marie Houghton Background&Aim. A high salt diet is associated with an increase risk of gastric cancer, hmvever, the molecular mechanism has not been elucidated. Fas acnvation classically leads to apoptosis, however, insufficient Fas signaling or an imbalance between downstream signaling components can initiate prolikrative signaling. We propose that an imbalance between Fas mediated apoptotic proliferative signaling is altered by' consumption of a high salt diet. Methods. Rat gastric mucosal ceils (RGM-1) were stably transfected with the pMSCV-puro vector containing mFas Ag cDNA, and low and high expressing clones isolated. The apoptotic and profit;erative response to FasL was determined in control medium or hyperosmotic medium (40mM or 80 mM sodium chloride) via a combination of FACS, DNA fragmentation assay, BrdU incorporation, NF-KB activation an_alysis, and WST-1 assay. Western blot. with phosphor-specific antibody was used to quami~ate the phosphorylation status of p38 MAPK as an index of p38 activation. Results. Exposure to hypemsmotic medium for 10 rain resulted in a marked increase in p38 MAPK phosphorylation which was inhibited by pretreannent witb SB203580, a specific inhibitor of p38 phosphoryiation. NF-KB activation increased 1.5 and 4 4 fold in cells exposed to 40 or 80mM NaCI respectively, an effect that was cnmpletdy aboliahed by the addition of SB203580. Low abundance clones increased proliferation (BrdU incorporation) in response to Fas L (12.5 ng/ml for 24 hours) from 10.09% to 14.42% which was dramatically augmented by NaCt 40ram in the presence of Fas L (2611%) but not in its absence (11.03%), and was dependent on p38 activation (NaC140raM, Fas L 12.5 ng/ml, SB203580 = I4.63%) To test if NaCI exposure could also protect against Fas mediated apoptosis, we employed a high Fas abundance clone which is highly sensitive to Fas mediated apoptosis After 24 hours exposure to 50ng/ml FasL, 76 -+ 0.03% of cells were apoptotic. This level decreased dramatically to 10.2 _+0.02% with the addition of 40 mM NaCI. This protective effect of NaCI was completely reversed by tire addition at SB203580 (79 -+ 0.04% apoptosis) WST-I assay confirmed increased cell survival in the NaCI treated group Conclusions. Hyperosnmlarity in the physiological range can active p38 MAPK signal, which may enhance Fas proliteeative signaling, and inhibit Fas apoptotic signaling. This growth signaling imbalance may contribute to the association between high salt diets and gasu'ic cancer,
T976
Expression of RCAS1 is implicated in all stages of Colorectal cancer development Kawm Leelawat, Takeshi Watanabe, Manabu Nakashima, Supatip Tujinda, Cheepsumm Suthipintawong, Vijittra Leardkamotkam Aims: The colorectal cancer is well established as an excellent model for the study of a sequence of genetic events associated with multistep carcinogenesis. The gradual accumulation of genetic alterations is necessary for the progression of benign polyps to cardnoma and eventually metastases. Each step of carcinogenesis produces many kinds of tumor antigens, which can be recognized and eliminated by the host immune system. To survive in the body, cancer cells must have ability to evade immune elimination. RCAS1 (Receptorbinding Cancer Antigen expressed on SiSo cells) is a tumor-associated antigen, implicated in immune evasion of tumor cells by acting as a ligand that binds to its receptor on the immune cells such as NK ceils aud T call and subseqnently induces apoptosis of these cells The high expression of RCAS1 has been demonstrated by immunohistochen'fical staining in certain tumors such as cervix, breast, tung and stomach. However; the expression of RCAS1 in colorectal cancer has never been quantified. This study aims to investigate the expression of RCAS1 in colorectal cancer and to clarify the stages of coloreatal carcinogenesis, which express this antigen. Materials and methods: 50 colorectal cancers and 12 adenomatous polyps specimens obtained from Rajavithi Hospital were included in this study. Detection of RCAS1 expression was carried out on 4 ~m thick sections of fom'talin-fixed, paraffie embedded specimens by immunohistochemical-staining technique using monoclonal amiRCAS1 antibody and the freshly isolated tissues were processed for expressions at RCAS1 mRNA by RT-PCR. Tumor-infiltrating lymphocyies (TIL) in the specimens were detected in serial sectious by immunohistochemical-stainmg technique and the apoptosis of these cells were identified in situ by using Calorimetric TUNEL assay. Result: The immunosignal tbr RCAS1 protein was highly intense in all specimens of the adenomatous polyps and cotorectal cancers whereas it was weakly detected in the normal. Tbe results of RT-PCR from tumor tissues were also correlated with the result of immunohistochemical staining. The apoptosis of TIL was identified in sitn adjacent to the area of RCASlexpressed tumor celts. Conclusions: The result suggested that there was up regulation of RCAS1 in the early stage of colorectai carcinogenesis and persisted in all stages of cancer development. The expression of RCAS1 in the colorectal cancer might be implicated in evasion of immune surveillance by inducing apoptosia of TIL.
T974
ERRP, a Negative Regulator of EGFR, S~n~tlr Nat~lan Colon Cancer Cells to the Apoptotic Effect of Curcumi~ 5atish Maryala, Jasbir Rangi, ~ l i l ' R l k d l ~ . a u e r , Kiran. K. Nagothu, Richard Jaszewski, Arun K. Rishi, A d h i p . g , i ~ l ~ # ' r Accumulatingl'll~ic~ti~gesta t~iat curcumin (dileruloyhnethane), the active ingredient of turmeric, may' bel~L~mpmventive against several types of malignancios, including colorectal cancer. The antitumorigenic properties of curcumin have been attributed in part to the induction of apoptosis. Although ihe regulatory' mechanisms by which cureumin exerts its chcmopreventive effect are not fully understood, we have observed that curcumin inhibits the expression and activation of EGFR in colon cancer cells. Recently, we isolated a uegative regrdator of EGFR, termed ERRP (EGF-R Related Protein), ovemxpression of which inhibits proliferation and EGFR activation in colon cancer ceils. Purified recombinant ERRP was utilized to examine whether ERRP would sensitize colon cancer cells to the chemopreventive effect of curcumin. Exposure of HCT-116 cells to recombinant ERRP (l-I0p~g/ml) or curcumin (5-30~M) for 48 h inhibited proliteration in a dose-dependent mamler, mveahng a 60-70% inhibition with the highest dose. Cnscumin at doses of 5-30btM also stimulated apoptosis in HCT-116 cells, as detennined by the TUNEL assay. This was accompanied by a concomitant rise in caspase 8 and 3 activity. On the other hand, ERRP at a dose of l~g/ mt caused no significant increase in apoptosis in HCT-I16 cells To determine whether ERRP would sensitize colon cancer cells to curcumimmduced apoptosis, HCT-1t6 ceils were preincubated in the absence (control) or presence o~ a low dose of ERRP (1 ~ m l ) for 48 h, and subsequently incubated for anotber 48 h with or without 5btM c~urcnmin, a dose that causes only 15-20% apoprosis Preincubation of HCT-I16 cells with ERRP caused approximately 50% higher apoptosis than curcumiu alone. Curcumin-induced inhibit/on of proliferation of HCT-116 cells was also equally affected fnllowing preincubation with ERRP We conclude that ERRP exacerbates the curcumin-iaduced changes in proliferation and apoptosis of colon cancer cells
T977
Resveratrol Selectively Engages Cell Death Signals Jason M. Erickson, Bashar M Attar, MaiT Jo Atten, Oksana Hohan Survival and replication of cancer cells is maintained by overriding apoptosis. Therefore, chemoprevention requires either stimulation of pmapoptotiv reactions or inhibition of antiapoptotic signals. Prm'iously, we showed that resveratrol inhibits proliferation of gas!tic adenocarcinoma cells and stimulates apoptosis by interacting with ceil signaling through PKC and oxidative stimuli. Here, we investigate the action of resvemtrol on components of death signaling pathways in gastric adenocarcinoma cells, aiming to define the contribution of tumor suppressor p53 to their expression and activity. METHODS: Gastric adenocarcinoma cell lines AGS, SNU-1, and KATO-III were c~dtured in RPMI-1640 media supplemented with 10 % FBS. After cell treatment with resveratml for specified times, p53 expression was detected by immunostaining and activities of caspases 1,3,6,8/10 arid 9 were datemuned by quantitation of calorimetric products from caspase-specific substrates. Protein levds of survivm, Bcl-2 and Bax were determined by immunoblotting Action of specific caspase inhibitors on cellular pmlderation was assessed by incorporation of )H-tfiyroidine into DNA. RESULTS: Resveratsol selectively downmgulated survivin protein expression in cells wah p53 (AGS and SNU-1) but was ineftective in KATO-III ceils with deleted p53. In twrn, resveratrol activated caspase 3 in cells with deleted p53, but had minimal efte'ct on other caspases. Increased mitochondriaI expression of proapoptotic Bax was seen in all cells upon exposure to resveratml. Antiapoptotic Bcl-2 remained unaffected by resveratrol treatment. Resveratrol altered survivin and Box expressim~ within 24 hours, but acm~ation of caspase 3 required longer treatment. Neither specihc nor broad range caspase mhibitors reversed resveratrol-induced inhibition of cellular proliferation. CONCLUSIONS: Resveratrol induces apoptosis in gastric adenocarcinoma cells by regulating both pro and antiapoptotic signaling molecules. However, engagement of specific apoptotic signals by resveratrol is selective and depends, in part, on p53 expression. Time dependent response to resveratml suggests that activation of caspase 3 is a late and irreversible event possibly elicited by changes in survivin and Box. These results indicate that resveratml interacts with rapid response dements of cdlular signaling, suppresses specific antiapoptotio signals, activates proapoptotic sig~als, and stimulates release of activated caspase 3 to induce apoptosis in gastric cancer cells. /
T975
Inhibition of Proliferation and Induction of Cell Cycle Arrest in Caco-2 Cells by Two Dietary Phytochemicals from Cruciferous Vegetables, Benzyl lsothiocyanate and Phenethyl Isothiocyanate James Visanii David Thompson, Philip Padfield Background: Benzyl isothiocyanate (BITC) and phenetbyl isothiocyanate (PEITC) are phenolic phytochemicals p~r as glucosinolate precursors in crucif~:rous vegetables, the consumption of which is reversely associated with risk of colon cancer. Both are putative cancer chemopreventives, and have been shown to attenuate tbe genotoxic efl~ectsof some carcinogens by modulating the actMties o[ phase I and 15base lI detox~fying enzymes. However, there are tva data available regarding the etfects of these phytocbemicals on cefi proliferation and cell cycle regulation. Aims: To characterize the effects of BITC and PEITC on prolderation and cell cycle regulation in Caco-2 cells i~ vitro. Methods: Coco-2 cells were treated with a range of concentrations of B1TC and PEITC. Rate of DNA sTntbesis was measured by ~H-thytuidine incorporation assay, proliferation was mea~sured by counting viable cdts. Cell cycle phase was measured by flow cytometry of propidium iodide stained cells with or without G2A~ block enforced by nocodazole All experiments were performed at least 3 times
AGA Abstracts
A-460