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Increased oxidative stress and iron content in colonic neoplasia
A528
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 108, No. 4
G R O W T H I N H I B I T O R Y E F F E C T OF L I T H I U M SALT O F G A M M A L I N O L E N I C A C I D ON H U M A N P A N C R E A T I C C A N C E R CELLS IN-VITRO
D.Ravichandran, AC Cooper, CD Johnson, SJ Karran. University Surgical Unit, Southampton General Southampton, United Kingdom.
Hospital,
Lithium salt of Gamma Linolenic Acid (LiGLA) may prove useful in the palliative treatment of inoperable pancreatic cancer. It can be administered IV and has no significant side effects. We tested the susceptibility to LiGLA of 2 pancreatic ductal carcinoma cell fines (Pane 1 and MIA PaCa2, ECACC, Salisbury, UK). A human skin fibroblast cell line was used as the control. Cells were grown as monolayer on plastic under standard conditions in culture medium containing 10% FBS, harvested and seeded at 2500 cells in 100ul of medium per well in 96 well cell culture plates. LiGLA (Seotia,Guildford,UK) in 100ul of medium was added 24 hours later to achieve final concentrations varying from 5 to 490umol/l. Control experiments were n m with medium alone, Lithium Chloride(LiC1)(0.06 to 2mmol/1) to exclude a Lithium to exclude a nonspecific fat overload effect. Plateseffect, and Palmitic Acid (PA) (4 to 490umol/l) were kept at standard conditions, inspected daily and removed on the 7th day. Cell growth was assessed by a microculture Tetrazolium (MTT) assay. LiGLA showed a significant dose and time dependant growth inhibitory effect on both cancer cell lines. 50% growth inhibition was seen at 5 umul/l. In contrast the benign fibroblasts were not affected up to a concentration of 100 umol/l. PA and LiC1 had no significant effects on the growth of any cells. LiGLA has a selective growth inh!bitory effect on human pancreatic duetal carcinoma ceils at eoncentrations which may be achievable in-vivo, and may provide a useful therapeutie adjunct in patients with pancreatic cancer.
MULTIPLE PRIMARY CANCERS OF THE UPPER AERODIGESTIVE TRACT (UAT) MANIFEST DISTINCT ONCOGENE EXPRESSION AND GROWTH CHARACTERISTICS. U. Ribeiro Jr, A.V. Safage-Ribeiro,M.R.Clarke,M.C. Posner, V.A. Hawrylak, S.D. Finkelstein;J.C. Reynolds. Depts. of Surgery, Pathology & Medicine, Universityof Pittsburgh,Pittsburgh,PA UAT tumors occur in pa~ents with predisposing dsk factors. Such patients often develop multiple synchronousor metachronoustumors. However,the genetic basis for this predisposi~on is ill defined. The evaluation of shared patterns of gene~c alterations between individual pdmary cancers might elucidate etiologic factors or clinical course and possibility predict the appearance of a second pdmary.Aim: To evaluate the expression of oncogene,c-erbB-2 (c-neu); apoptosis gene, bcl-2; growth factor, TGF-alpha; and proliferatingcell nuclear antigen, PCNA in 17 patients each with 2-5 primary UAT malignancies. Methods: 41 separate primary tumors from 17 patients involving the esophagus(15), larynx (14), pharynx (8), lung (2), mouth (2)=~ndtongue (2) were examined using immunohistochemis~ for c-erbB-2, bc/-2,TGF-alpha, and PCNA. 40 were squamouscell carcinomas and 1 adenocarcinoma.Each tumor was sampled at several areas including pdmary and metastatic sites. Results: All patients had history of cigarette smoking and 13 had heavy alcohol usage. Eleven tumors were metachronous(6mo.-llyr.} and 11 were synchronous, bc/-2 expression was detected in 18 tumors (39%) from 12 patients. TGF-alpha was expressed in 33 tumors (80.5%). No correle~on was observed for either grade of intensity or distributionof bc/-2 and TGF-alpha among tumors from the same patient (p=0.21and p=0.32, respectively). PCNA labeling index (% positive stained cells divided by the total number of cells) in tumors ranged from 29.42% to 87.52% (mean=57.17%), PCNA expression showed complete discordance between tumors odgin~ng from the same patient (r=0.017, p=0.62). Membranousc-erbB-2 staining was seen in 2 patients (11.8%), one with synchronous tumors, both positive for c-erbB,2; the other expressed c-erbB-2 in only t of 5 tumors over a 3 year period. Metastases displayed the identical expression of its primary in all cases. Conclusions: Discordant phenotypic expression of c-erbB-2, bd-2, TGF-alpha and PCNA in patients with multiple UAT malignanciessuggests that malignanttransformationin this setting is the result of a mul~focal polyclonal process. Thus, each tumor seems to grow on an individual basis in accordancewith the field defecttheory.
• INCREASED OXIDATIVE STRESS AND IRON CONTENT IN COLONIC NEOPLASIA. GM Reddy, R Cerehia, L Stiel, M McKinley and GE Mnllin. Departments of Medicine and Pediatrics, North Shore UniversityHospital-C0rnellUniversityMedical College, Manhasset, NY. BACKGROUND: Oxygen free radicals have been strongly implicated in neoplastic processes by causing oxidative damage to DNA giving rise to mutagenesis and the metabolic activation of procarcinogens. Iron is a transition metal that catalyzes the formation of damaging reactive oxygen metabolites (ROMs) and lipid peroxides. Epidemiologicstudies have linkedcolorectalcancer to increaseddietary saturatedfat, red meat and iron intake. The aim of our study was to determine the levels of reactive oxygen species and iron content in colonic neoplasia. METHODS: Colonoscopic biopsies were obtained from: normal Controls (Cntrl) (n=16), colonic adenomas (Aden) (n=8), colorectal malignancies (Canc) (n=2l) and histologically confirmeddisease-freecolonic mucosa5 cm distant to cancer (NL:Ca)and adenomas (NL:Aden) for analysis. ROM's in mucosal biopsies were determined by chemiluninenscenceand iron content by flame atomicabsorption spectroseopsy(AAS). Protein analysis was determinedby the Lowry's method. ROMs are expressedas peak chemiluminescence(Cp) in photons per Ixgof protein (Cp/p) and mucosal iron content in ~g iron per mg protein. DNA cell cycle index (DNAi) was performed using flowcytometry.The Kruskal-WallisANOVAwas used for comparingROMs, Iron and DNA indicies among groups. Tile Mann-Whitney and Wilcoxon rank order non-parametric tests were then utilized to analyzedie significanceof the data. RESULTS: The median and the interquartile range (IQR) which indicates the range of values in the 25-75th percentile, was used to summarizethe data in the table below. 1 2 3 4 5 6 7 Dx
Cntrl
C a n c NL:Ca Aden NL:Ade
median 17 IQR 62
1494 3669
32 121
87 460
72 382
Fe median 3.4 IQR 8.4
9.5 13.7
9.3 6.8
32.4 21.8
20.0 23.8
comparison#,#; p value
a) 312; p=0.0004 b) 3,4; p=0.001 c) 3,6; p=0.02 d) 3,5; p=0.03 e) 5,2; p=0.02
a) 3,2; p=0.03 b) 5,2; p--0.001 c) 4,2; p=0.04 d) 6,2; p=0.01 e) 5,3; p=0.002 f) 5,4; p=0.02 In neoplastic lesions, iron content was found to significantly correlate with peak chemiluninenscence (Spearman's rho=0.6, p<0.01). DNAi was higher in neoplastic lesions (1.8, +/- 0.5) (mean, SEM) comparedto Controls (1.0, +/- 0.01) (p=0.01). CONCLUSIONS: Increased reactive oxygen species are found in colorectal cancers and tubular adenomas. The pro-oxidant transition metal iron, is increased in both neoplastic lesions and distant uninvolvedcolonic mucosa. Farther study is warrantedto determine if mucosal iron may serve as a marker to identify patients at risk for colorectalneoplasia.
P R E V A L E N C E OF H E R E D I T A R Y NON POLYPOSIS C O L O N C A N C E R (HNPCC) IN SOUTH ITALY: A PROSPECTIVE INVESTIGATION. G.Rieqler, A.Savastano, G.Gagliardi, R.Carratu' and Gruppo SIED Campania: P.Borgheresi, A.Piscitelli, G.B.Rossi, M.A.Bianco, F.Montanaro, D.Cattaneo, A. Pezzullo, E.Sessa, P.Di Giorgio, V.Baldi, S.De Stelane, G . D e Palma, F.Guardascione, P . S p i n e l l i , F . S e l vaggi, P.Pastere, O.Saffiotti, M . M a r t o r a n o , P . M o r e l la, E.Parente, M.A.Bozzi, R.Iorio. A low frequence of H N P C C in a r e t r o s p e c t i v e s e q u e n c e of 115 colon cancer patients (1.7%) was recently reported (i}. Preliminary data are presented o n 252 c o n s e c u t i v e colorectal colon c a n c e r p a t i e n t s r e f e r r e d by 22 Groups p a r t i c i p a t i n g to a p r o s p e c t i v e investigation w h i c h s t a r t e d in J a n u a r y 94. The p r e v a l e n c e of the criteria which, when associated, are considered suggestive of HNPCC, is r e p o r t e d in the table: * vertical transmission * a g g r e g a t i o n in sibling * early age at onset (<51 years) * l o c a t i o n in the right colon * m u l t i p l e p r i m a r y tumors *mucinous carcinoma
n°
13 28 25 71 6 32
(5.8%) (12.4%) (10.2%) (28.3%) (2.4%) (13.3%)
Four criteria were associated in o n e p a t i e n t (0.4%), three in 7 (2.8%), two in 34 (13.5%), one in 83 (32.9%), none in 127 (50.4%). In this p r e l i m i n a r y series, two patients (0.79%) showed the minimal requisites according the "Amsterdam d e f i n i t i o n " of HNPCC (2). i) R i e g l e r G . : G a s t r o e n t e r o l o g y , 1 9 8 4 , suppl.106:A433 2) Vasen H.: Dis.Col.Rectum, 1991,34:424-5 Supported
by a Grant of M U R S T
60%
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 108, No. 4
G R O W T H I N H I B I T O R Y E F F E C T OF L I T H I U M SALT O F G A M M A L I N O L E N I C A C I D ON H U M A N P A N C R E A T I C C A N C E R CELLS IN-VITRO
D.Ravichandran, AC Cooper, CD Johnson, SJ Karran. University Surgical Unit, Southampton General Southampton, United Kingdom.
Hospital,
Lithium salt of Gamma Linolenic Acid (LiGLA) may prove useful in the palliative treatment of inoperable pancreatic cancer. It can be administered IV and has no significant side effects. We tested the susceptibility to LiGLA of 2 pancreatic ductal carcinoma cell fines (Pane 1 and MIA PaCa2, ECACC, Salisbury, UK). A human skin fibroblast cell line was used as the control. Cells were grown as monolayer on plastic under standard conditions in culture medium containing 10% FBS, harvested and seeded at 2500 cells in 100ul of medium per well in 96 well cell culture plates. LiGLA (Seotia,Guildford,UK) in 100ul of medium was added 24 hours later to achieve final concentrations varying from 5 to 490umol/l. Control experiments were n m with medium alone, Lithium Chloride(LiC1)(0.06 to 2mmol/1) to exclude a Lithium to exclude a nonspecific fat overload effect. Plateseffect, and Palmitic Acid (PA) (4 to 490umol/l) were kept at standard conditions, inspected daily and removed on the 7th day. Cell growth was assessed by a microculture Tetrazolium (MTT) assay. LiGLA showed a significant dose and time dependant growth inhibitory effect on both cancer cell lines. 50% growth inhibition was seen at 5 umul/l. In contrast the benign fibroblasts were not affected up to a concentration of 100 umol/l. PA and LiC1 had no significant effects on the growth of any cells. LiGLA has a selective growth inh!bitory effect on human pancreatic duetal carcinoma ceils at eoncentrations which may be achievable in-vivo, and may provide a useful therapeutie adjunct in patients with pancreatic cancer.
MULTIPLE PRIMARY CANCERS OF THE UPPER AERODIGESTIVE TRACT (UAT) MANIFEST DISTINCT ONCOGENE EXPRESSION AND GROWTH CHARACTERISTICS. U. Ribeiro Jr, A.V. Safage-Ribeiro,M.R.Clarke,M.C. Posner, V.A. Hawrylak, S.D. Finkelstein;J.C. Reynolds. Depts. of Surgery, Pathology & Medicine, Universityof Pittsburgh,Pittsburgh,PA UAT tumors occur in pa~ents with predisposing dsk factors. Such patients often develop multiple synchronousor metachronoustumors. However,the genetic basis for this predisposi~on is ill defined. The evaluation of shared patterns of gene~c alterations between individual pdmary cancers might elucidate etiologic factors or clinical course and possibility predict the appearance of a second pdmary.Aim: To evaluate the expression of oncogene,c-erbB-2 (c-neu); apoptosis gene, bcl-2; growth factor, TGF-alpha; and proliferatingcell nuclear antigen, PCNA in 17 patients each with 2-5 primary UAT malignancies. Methods: 41 separate primary tumors from 17 patients involving the esophagus(15), larynx (14), pharynx (8), lung (2), mouth (2)=~ndtongue (2) were examined using immunohistochemis~ for c-erbB-2, bc/-2,TGF-alpha, and PCNA. 40 were squamouscell carcinomas and 1 adenocarcinoma.Each tumor was sampled at several areas including pdmary and metastatic sites. Results: All patients had history of cigarette smoking and 13 had heavy alcohol usage. Eleven tumors were metachronous(6mo.-llyr.} and 11 were synchronous, bc/-2 expression was detected in 18 tumors (39%) from 12 patients. TGF-alpha was expressed in 33 tumors (80.5%). No correle~on was observed for either grade of intensity or distributionof bc/-2 and TGF-alpha among tumors from the same patient (p=0.21and p=0.32, respectively). PCNA labeling index (% positive stained cells divided by the total number of cells) in tumors ranged from 29.42% to 87.52% (mean=57.17%), PCNA expression showed complete discordance between tumors odgin~ng from the same patient (r=0.017, p=0.62). Membranousc-erbB-2 staining was seen in 2 patients (11.8%), one with synchronous tumors, both positive for c-erbB,2; the other expressed c-erbB-2 in only t of 5 tumors over a 3 year period. Metastases displayed the identical expression of its primary in all cases. Conclusions: Discordant phenotypic expression of c-erbB-2, bd-2, TGF-alpha and PCNA in patients with multiple UAT malignanciessuggests that malignanttransformationin this setting is the result of a mul~focal polyclonal process. Thus, each tumor seems to grow on an individual basis in accordancewith the field defecttheory.
• INCREASED OXIDATIVE STRESS AND IRON CONTENT IN COLONIC NEOPLASIA. GM Reddy, R Cerehia, L Stiel, M McKinley and GE Mnllin. Departments of Medicine and Pediatrics, North Shore UniversityHospital-C0rnellUniversityMedical College, Manhasset, NY. BACKGROUND: Oxygen free radicals have been strongly implicated in neoplastic processes by causing oxidative damage to DNA giving rise to mutagenesis and the metabolic activation of procarcinogens. Iron is a transition metal that catalyzes the formation of damaging reactive oxygen metabolites (ROMs) and lipid peroxides. Epidemiologicstudies have linkedcolorectalcancer to increaseddietary saturatedfat, red meat and iron intake. The aim of our study was to determine the levels of reactive oxygen species and iron content in colonic neoplasia. METHODS: Colonoscopic biopsies were obtained from: normal Controls (Cntrl) (n=16), colonic adenomas (Aden) (n=8), colorectal malignancies (Canc) (n=2l) and histologically confirmeddisease-freecolonic mucosa5 cm distant to cancer (NL:Ca)and adenomas (NL:Aden) for analysis. ROM's in mucosal biopsies were determined by chemiluninenscenceand iron content by flame atomicabsorption spectroseopsy(AAS). Protein analysis was determinedby the Lowry's method. ROMs are expressedas peak chemiluminescence(Cp) in photons per Ixgof protein (Cp/p) and mucosal iron content in ~g iron per mg protein. DNA cell cycle index (DNAi) was performed using flowcytometry.The Kruskal-WallisANOVAwas used for comparingROMs, Iron and DNA indicies among groups. Tile Mann-Whitney and Wilcoxon rank order non-parametric tests were then utilized to analyzedie significanceof the data. RESULTS: The median and the interquartile range (IQR) which indicates the range of values in the 25-75th percentile, was used to summarizethe data in the table below. 1 2 3 4 5 6 7 Dx
Cntrl
C a n c NL:Ca Aden NL:Ade
median 17 IQR 62
1494 3669
32 121
87 460
72 382
Fe median 3.4 IQR 8.4
9.5 13.7
9.3 6.8
32.4 21.8
20.0 23.8
comparison#,#; p value
a) 312; p=0.0004 b) 3,4; p=0.001 c) 3,6; p=0.02 d) 3,5; p=0.03 e) 5,2; p=0.02
a) 3,2; p=0.03 b) 5,2; p--0.001 c) 4,2; p=0.04 d) 6,2; p=0.01 e) 5,3; p=0.002 f) 5,4; p=0.02 In neoplastic lesions, iron content was found to significantly correlate with peak chemiluninenscence (Spearman's rho=0.6, p<0.01). DNAi was higher in neoplastic lesions (1.8, +/- 0.5) (mean, SEM) comparedto Controls (1.0, +/- 0.01) (p=0.01). CONCLUSIONS: Increased reactive oxygen species are found in colorectal cancers and tubular adenomas. The pro-oxidant transition metal iron, is increased in both neoplastic lesions and distant uninvolvedcolonic mucosa. Farther study is warrantedto determine if mucosal iron may serve as a marker to identify patients at risk for colorectalneoplasia.
P R E V A L E N C E OF H E R E D I T A R Y NON POLYPOSIS C O L O N C A N C E R (HNPCC) IN SOUTH ITALY: A PROSPECTIVE INVESTIGATION. G.Rieqler, A.Savastano, G.Gagliardi, R.Carratu' and Gruppo SIED Campania: P.Borgheresi, A.Piscitelli, G.B.Rossi, M.A.Bianco, F.Montanaro, D.Cattaneo, A. Pezzullo, E.Sessa, P.Di Giorgio, V.Baldi, S.De Stelane, G . D e Palma, F.Guardascione, P . S p i n e l l i , F . S e l vaggi, P.Pastere, O.Saffiotti, M . M a r t o r a n o , P . M o r e l la, E.Parente, M.A.Bozzi, R.Iorio. A low frequence of H N P C C in a r e t r o s p e c t i v e s e q u e n c e of 115 colon cancer patients (1.7%) was recently reported (i}. Preliminary data are presented o n 252 c o n s e c u t i v e colorectal colon c a n c e r p a t i e n t s r e f e r r e d by 22 Groups p a r t i c i p a t i n g to a p r o s p e c t i v e investigation w h i c h s t a r t e d in J a n u a r y 94. The p r e v a l e n c e of the criteria which, when associated, are considered suggestive of HNPCC, is r e p o r t e d in the table: * vertical transmission * a g g r e g a t i o n in sibling * early age at onset (<51 years) * l o c a t i o n in the right colon * m u l t i p l e p r i m a r y tumors *mucinous carcinoma
n°
13 28 25 71 6 32
(5.8%) (12.4%) (10.2%) (28.3%) (2.4%) (13.3%)
Four criteria were associated in o n e p a t i e n t (0.4%), three in 7 (2.8%), two in 34 (13.5%), one in 83 (32.9%), none in 127 (50.4%). In this p r e l i m i n a r y series, two patients (0.79%) showed the minimal requisites according the "Amsterdam d e f i n i t i o n " of HNPCC (2). i) R i e g l e r G . : G a s t r o e n t e r o l o g y , 1 9 8 4 , suppl.106:A433 2) Vasen H.: Dis.Col.Rectum, 1991,34:424-5 Supported
by a Grant of M U R S T
60%