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Lovastatin augments apoptosis induced by COX-2 selective inhibitors in colon cancer cells
April 2000
3747 A 34 K OUTER MEMBRANE PROTEIN OF HELICOBACTER rrLORI (OIPA) APPROXIMATELY DOUBLES THE PRO-INFLAMMATORY POTENTIAL OF THE CAG PATHOGENICITY ISLAND. Yoshio Yamaoka, Dong H. Kwon, Fouad A. El-Zaatari, Michael S. Osato, David Y. Graham, VAMC and Baylor Coli of Medicine, Houston, TX. Background: According to the complete genome sequence, there are more than 32 outer membrane protein (OMP) families in H. pylori. The aim of this study was to examine the role of an omp HP0638 in the pathogenesis of H. pylori infection. Methods: Gene negative isogenic mutants of HP0638 and babA2 (which is responsible for adherence of H. pylori to Lewis-b [Leb ] blood group antigens on gastric epithelial cells) from 20 clinical isolates were created by inserting a chloramphenicol resistant cassette into the gene. The parental strains included 9 clinical isolates from Japan (8 cag pathogenicity island-positive and I cag-negative) and II isolates from the U.S. (8 cag-positive and 3 cag-negative). Mutant and wild H. pylori strains were co-cultured with gastric cancer cell lines (AGS and MKN45 cells) and IL-8 production in the supernate was assayed by ELISA. Results: babA2 gene knockouts had no effect on IL-8 production. Knockout of HP0638 in 75% of cag-positive strains reduced IL-8 production approximately 50% IL-8 compared to their parental strain. The 4 cag-positive strains in which IL-8 levels were unchanged by HP0638 knockout, all had CT dinucleotide repeats in the 5' region resulting a frame shift (i.e., slipped-strand repair) and truncation. All strains with naturally inactive HP0638 were from the US; Japanese strains were always on and thus, on average, may be more virulent. cag-negative isolates produce little IL-8 response, nevertheless, cag-negative strains that were HP0638 positive produced approximately twice the amount of IL-8 compared to cagnegative HP0638 negative strains. Conclusion: The HP0638 gene is proinflammatory and potentiates the pro-inflammatory action of the cag pathogenicity island. We hypothesis that this protein may be an important virulence factor in relation to the risk of clinically significant outcomes of H. pylori infection. We denote intact HP0638 gene Outer inflammatory protein (oipA). 3748 LOVASTATIN AUGMENTS APOPTOSIS INDUCED BY COX-2 SELECTIVE INHIBITORS IN COLON CANCER CELLS. Banke Agarwal, Petr Protiva, Prabhakar Swaroop, Ram Chuttani, William G. Ramey, Peter R. Holt, Beth Israel Deaconess Med Ctr, Boston, MA; St Luke's-Roosevelt Hosp Ctr, New York, NY; Brigham & Women's Hosp, Boston, MA. Background: Cox-2 selective inhibitors prevent colon cancer in animal models. We have earlier shown that lovastatin augments NSAID-induced apoptosis and potentiates the chemopreventive effect of sulindac on colon cancer in an animal model. Cox-2 selective inhibitors do not have the side effects that have limited the use of NSAIDs in chemoprevention of colon cancer. We asked whether combination of lovastatin with a Cox-2 selective inhibitor would enhance chemoprevention. Since NSAIDs and Cox-2 inhibitors are believed to inhibit colon cancer by inducing apoptosis in cancer cells, we studied the effect of lovastatin on apoptosis induced by the Cox-2 inhibitor (SC236- a structural analogue of Celecoxib) in colon cancer cells. Methods: Two colon cancer cell lines, SW480 and HCTll6 were used. Cells were incubated for 48 hours with SC236- 0, 25, 50 and 75 IAoM combined with lovastatin 0, 5, 10 or 20 IAoM. Apoptosis was measured by two separate assays -I) TUNEL positive cells and 2) sub-diploid cells and was quantified by flow cytometry. Results: SC236 caused a dose-dependent increase in apoptosis in both cell lines. Lovastatin also induced apoptosis in both cell lines in a dose-dependent manner. As shown below, lovastatin augmented apoptosis (measured as sub-diploid cells) induced by SC236 at all dose levels. Similar results were obtained when apoptosis was quantified as TUNEL positive cells. (figure) Conclusion: Lovastatin augments apoptosis induced by a selective Cox-2 inhibitor in two colon cancer cell lines. Since, NSAIDs and Cox-2 selective inhibitors prevent colon cancer by increasing apoptosis, our data would suggest that the combination of a
AGAA681
selective Cox-2 inhibitor with lovastatin may be more effective than Cox-2 inhibitor alone in the chemoprevention of colon cancer.
8W480
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3749 PPAR...1 IS A PUTATIVE DOWNSTREAM RECEPTOR OF COX-2 DERIVED PROSTACYCLIN DURING COLORECTAL CARCINOGENESIS. Rajnish A. Gupta, Aramandla Radhika, Jinyi Shao, Hongmiao Sheng, Sudhansu K. Dey, Raymond N. DuBois, Vanderbilt Univ Sch of Medicine, Nashville, TN; Univ of Kansas Med Ctr, Kansas City, KS. Previous studies using both genetic and pharmacological approaches have established that COX-2 plays an important role in colon cancer. However, there has been a poor understanding of potential eicosanoid receptors that may be activated by COX-2 derived products. The Peroxisome Proliferator Activated Receptors (PPARs) are lipid activated transcription factors that are members of the nuclear hormone receptor superfamily. Given the recent finding that the PPARSisoform is a transcriptional target of the B-CateninffCF complex (He et al. Cell 1999;99:335-345) the aims of this study were to I) Determine the expression pattern of PPARSin human and rodent colorectal tumors and 2) Determine whether selected COX metabolites could serve as activating ligands for PPARS. Methods: Northern Blotting was used to determine levels of PPARSmRNA in paired tumor and normal tissue from both human colon tumors and those derived from rats treated with the carcinogen azoxymethane (AOM). In situ hybridization was utilized to determine tissue localization of PPARS. Transient transfections using a PPAR responsive luciferase reporter were done to measure PPARStranscriptional activity in response to selected prostaglandins (PGs) in the HCA-7, HCT 116, and SW480 colon cancer cell lines (all three of which express PPARSRNA and protein). Results: PPARllis upregulated in colorectal tumors as compared to normal adjacent tissue in the rat AOM model (5 out of 5 matched pairs examined). In situ hybridization of human colorectal tumors suggests that PPARSis predominantly localized to the epithelium (5 matched samples analyzed). Addition of the stable P0I 2 analog cPOI, but not various other PG subtypes, resulted in a dose-dependent increase in PPARStranscriptional activity in all colon cell lines examined. Conclusions: We conclude that PPARS, similar to COX-2, is significantly elevated in colorectal neoplasias relative to normal mucosa in both human and AOM rat derived colorectal tumors. We also provide evidence that a COX-derived metabolite, PGI 2 , can serve as an activating ligand for PPARllin colon cancer cells. Previously published reports have established that wt APC negatively regulates the expression of both PPARlland COX-2. This observation, combined with our findings, leads us to hypothesize that in colorectal tumors arising in an APC mutant genotype, COX-2 may be promoting tumor formation at least in part via activation of PPARIl. Functional studies to further explore this hypothesis are currently underway.
3747 A 34 K OUTER MEMBRANE PROTEIN OF HELICOBACTER rrLORI (OIPA) APPROXIMATELY DOUBLES THE PRO-INFLAMMATORY POTENTIAL OF THE CAG PATHOGENICITY ISLAND. Yoshio Yamaoka, Dong H. Kwon, Fouad A. El-Zaatari, Michael S. Osato, David Y. Graham, VAMC and Baylor Coli of Medicine, Houston, TX. Background: According to the complete genome sequence, there are more than 32 outer membrane protein (OMP) families in H. pylori. The aim of this study was to examine the role of an omp HP0638 in the pathogenesis of H. pylori infection. Methods: Gene negative isogenic mutants of HP0638 and babA2 (which is responsible for adherence of H. pylori to Lewis-b [Leb ] blood group antigens on gastric epithelial cells) from 20 clinical isolates were created by inserting a chloramphenicol resistant cassette into the gene. The parental strains included 9 clinical isolates from Japan (8 cag pathogenicity island-positive and I cag-negative) and II isolates from the U.S. (8 cag-positive and 3 cag-negative). Mutant and wild H. pylori strains were co-cultured with gastric cancer cell lines (AGS and MKN45 cells) and IL-8 production in the supernate was assayed by ELISA. Results: babA2 gene knockouts had no effect on IL-8 production. Knockout of HP0638 in 75% of cag-positive strains reduced IL-8 production approximately 50% IL-8 compared to their parental strain. The 4 cag-positive strains in which IL-8 levels were unchanged by HP0638 knockout, all had CT dinucleotide repeats in the 5' region resulting a frame shift (i.e., slipped-strand repair) and truncation. All strains with naturally inactive HP0638 were from the US; Japanese strains were always on and thus, on average, may be more virulent. cag-negative isolates produce little IL-8 response, nevertheless, cag-negative strains that were HP0638 positive produced approximately twice the amount of IL-8 compared to cagnegative HP0638 negative strains. Conclusion: The HP0638 gene is proinflammatory and potentiates the pro-inflammatory action of the cag pathogenicity island. We hypothesis that this protein may be an important virulence factor in relation to the risk of clinically significant outcomes of H. pylori infection. We denote intact HP0638 gene Outer inflammatory protein (oipA). 3748 LOVASTATIN AUGMENTS APOPTOSIS INDUCED BY COX-2 SELECTIVE INHIBITORS IN COLON CANCER CELLS. Banke Agarwal, Petr Protiva, Prabhakar Swaroop, Ram Chuttani, William G. Ramey, Peter R. Holt, Beth Israel Deaconess Med Ctr, Boston, MA; St Luke's-Roosevelt Hosp Ctr, New York, NY; Brigham & Women's Hosp, Boston, MA. Background: Cox-2 selective inhibitors prevent colon cancer in animal models. We have earlier shown that lovastatin augments NSAID-induced apoptosis and potentiates the chemopreventive effect of sulindac on colon cancer in an animal model. Cox-2 selective inhibitors do not have the side effects that have limited the use of NSAIDs in chemoprevention of colon cancer. We asked whether combination of lovastatin with a Cox-2 selective inhibitor would enhance chemoprevention. Since NSAIDs and Cox-2 inhibitors are believed to inhibit colon cancer by inducing apoptosis in cancer cells, we studied the effect of lovastatin on apoptosis induced by the Cox-2 inhibitor (SC236- a structural analogue of Celecoxib) in colon cancer cells. Methods: Two colon cancer cell lines, SW480 and HCTll6 were used. Cells were incubated for 48 hours with SC236- 0, 25, 50 and 75 IAoM combined with lovastatin 0, 5, 10 or 20 IAoM. Apoptosis was measured by two separate assays -I) TUNEL positive cells and 2) sub-diploid cells and was quantified by flow cytometry. Results: SC236 caused a dose-dependent increase in apoptosis in both cell lines. Lovastatin also induced apoptosis in both cell lines in a dose-dependent manner. As shown below, lovastatin augmented apoptosis (measured as sub-diploid cells) induced by SC236 at all dose levels. Similar results were obtained when apoptosis was quantified as TUNEL positive cells. (figure) Conclusion: Lovastatin augments apoptosis induced by a selective Cox-2 inhibitor in two colon cancer cell lines. Since, NSAIDs and Cox-2 selective inhibitors prevent colon cancer by increasing apoptosis, our data would suggest that the combination of a
AGAA681
selective Cox-2 inhibitor with lovastatin may be more effective than Cox-2 inhibitor alone in the chemoprevention of colon cancer.
8W480
50 -
45 ~ . Lova 40
a
~' .
Lova 5 tLQLova 10
35
45
1-
40
.Lova 0 . Lova 10 Lova 2 0
35 !!!. 0; 30 o :>1 25
~ 30 e
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o
~
.g
20
.0 :l 15
'" 10 "#.
VI
;f!!. 10
HCT116
50
t
5
5 '
o -t
o o
25
50
SC 236 (fti\l)
75
o
25
50
75
SC 236 (~li\1 )
3749 PPAR...1 IS A PUTATIVE DOWNSTREAM RECEPTOR OF COX-2 DERIVED PROSTACYCLIN DURING COLORECTAL CARCINOGENESIS. Rajnish A. Gupta, Aramandla Radhika, Jinyi Shao, Hongmiao Sheng, Sudhansu K. Dey, Raymond N. DuBois, Vanderbilt Univ Sch of Medicine, Nashville, TN; Univ of Kansas Med Ctr, Kansas City, KS. Previous studies using both genetic and pharmacological approaches have established that COX-2 plays an important role in colon cancer. However, there has been a poor understanding of potential eicosanoid receptors that may be activated by COX-2 derived products. The Peroxisome Proliferator Activated Receptors (PPARs) are lipid activated transcription factors that are members of the nuclear hormone receptor superfamily. Given the recent finding that the PPARSisoform is a transcriptional target of the B-CateninffCF complex (He et al. Cell 1999;99:335-345) the aims of this study were to I) Determine the expression pattern of PPARSin human and rodent colorectal tumors and 2) Determine whether selected COX metabolites could serve as activating ligands for PPARS. Methods: Northern Blotting was used to determine levels of PPARSmRNA in paired tumor and normal tissue from both human colon tumors and those derived from rats treated with the carcinogen azoxymethane (AOM). In situ hybridization was utilized to determine tissue localization of PPARS. Transient transfections using a PPAR responsive luciferase reporter were done to measure PPARStranscriptional activity in response to selected prostaglandins (PGs) in the HCA-7, HCT 116, and SW480 colon cancer cell lines (all three of which express PPARSRNA and protein). Results: PPARllis upregulated in colorectal tumors as compared to normal adjacent tissue in the rat AOM model (5 out of 5 matched pairs examined). In situ hybridization of human colorectal tumors suggests that PPARSis predominantly localized to the epithelium (5 matched samples analyzed). Addition of the stable P0I 2 analog cPOI, but not various other PG subtypes, resulted in a dose-dependent increase in PPARStranscriptional activity in all colon cell lines examined. Conclusions: We conclude that PPARS, similar to COX-2, is significantly elevated in colorectal neoplasias relative to normal mucosa in both human and AOM rat derived colorectal tumors. We also provide evidence that a COX-derived metabolite, PGI 2 , can serve as an activating ligand for PPARllin colon cancer cells. Previously published reports have established that wt APC negatively regulates the expression of both PPARlland COX-2. This observation, combined with our findings, leads us to hypothesize that in colorectal tumors arising in an APC mutant genotype, COX-2 may be promoting tumor formation at least in part via activation of PPARIl. Functional studies to further explore this hypothesis are currently underway.