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Inhibition of colon cancer cell proliferation in vitro and azoxymethane-induced aberrant crypt formation in vivo by balsalazide and metabolites
ASO0
AGA ABSTRACTS
• THE BENEFITS OF USING SUPPLEMENTAL L-ARGIN1NE IN A CHEMICAL INDUCED MODEL OF COLORECTAL CANCER QY Ma, M Hoper, BJ Rowlands. Department of Surgery , Institute of Clinical Science, Queen's University of Belfast, Northern Ireland, UK. Introduction: L-arginine inhibits mammary and transplantable solid turnouts in experimental models yet its effect on colorectal cancer has not been studied. The aim of this study was to investigate the effect of arginine on 1,2-dimethylhydrazine (DMH)-induced colonic cancer and its mechanism of action. Methodology: Male Wistar rats were treated with 20 weekly subcutaneous injections of DMH (20 mg/kg body weight). Arginine was given in a 1% solution of drinking water. Animals were divided into 3 groups. Group I (42 rats) was given DMH, group II (41 rats) DMI-I and whole period of arginine supplementation, Group HI (24 rats) DMH and first 10 weeks arginine only. Thymic lymphocyte profiferative response to the T-cell mitogen phytohaemaggiutinin (PHA) was determined using the uptake oftritiated thymidine. Stimulation index was calculated by the mean count per minute(cpm) stimulated cultures/mean epm unstimulated cultures. Result: Thirty eight out of 42 DMH animals developed colonic tumours compared to 28 out of 41 in Group II and 14 out of 24 in Group HI. Group I Group II ' Group llI Tumour incidence (%) 88 68* 58* No. oftumour/rat 2.9__+0.2 1.71+0.33" 1.04_+0.24" No. oftumour/tumour-beerin 5 rat 3_+0.25 2.15+0.36" 1.67_+0.28" Stimulation Index 15+6.1 36+8.1" 30.8+5.3* * P<0.05 compared to Group I (Mann-Whitney U test) Conclusion: Supplemental arginine significantly inhibited colonic tumorigenesis and stimulated thymie lymphocyte response. The reduction in tumorigenesis in the animals given supplemental arginine may be through a general stimulation of the host immune system.
• Inhibition of Colon Cancer Cell Proliferation In Vitro and AzoxymethaneInduced Aberrant Crypt Formation In Vivo by Balsalazide and Metabolites. DJ MacGregor, YS Kim, ME Sleisonger and LK Johnson*. Dept. of Med, DFMC and UCSF, San Francisco, CA; *Salix Pharmaceuticals, Inc., Pale Alto, CA USA. Introduction: Nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to reduce polyp recurrence in FAP patients, inhibit carcinogen-induced aberrant crypt loci number and multiplicity in rodents and inhibit proliferation of human colon cancer-derived cell lines in vitro. However, because of NSAIDinduced upper GI ulceration, their widespread chronic use for colon polyp or cancer chemoprevention therapy may be limited. Goals: The present goals were therefore to test, in similar assays, an alternative antiinflammatory agent which lacks such upper GI side effects. Balsalazide disodium (BSZ) is an aminosalicylate (5-ASA) linked to an inert carrier (4-aminobenzoyl-13alanine, 4ABA) currently undergoing Phase III studies for ulcerative colitis. BSZ lacks upper GI and renal side effects seen with standard NSAIDs and delivers high concentrations of 5-ASA to the colon. Methods: Cell proliferation was measured using HT-29 and LSI74T colon adenoma-derived cell lines in 96-wells microtiter plates. Cell densities were determined by the SRB-dye binding assay. Aberrant crypt studies used F344 rats (8 per group) treated with 2 weekly injections of AOM (15mg/kg). ACF were assessed 8 weeks after the first injection following methylene blue staining and fixation of the entire colon. Results: BSZ and its active metabolite, 5-ASA, inhibited proliferation of both cell lines in a dose dependent manner (BSZ ICs0---4mM,5-ASA ICs0=SmM). In contrast, 4-ABA was inactive. The 5-ASA inhibitory activity increased upon oxidation (ox5-ASA) to 90% with an ICs0=2mM. The drug effects were not cytotoxic and cell growth resumed upon drag withdrawal. Like the NSA1D indomethacin, growth inhibition by ox5-ASA caused accumulation of cells in the Go/G1 phase of the cell cycle. BSZ provided in the drinking water (125&250mg/kg) to AOM-treated rats resulted in a 49% (p<0.003) and 65% (p<0.001) reduction, respectively in the number of ACF. The number of ACF containing >_4crypts was reduced by 75% (p<0.001). Conclusions: BSZ significantly reduces the formation and size of early pre-adenoma lesions induced by the carcinogen, azoxymethane. This may be due to inhibition of aberrant crypt cell proliferation. The ICs0 for colon cancer cell growth inhibition seen for BSZ, 5-ASA and ox5-ASA are also within the range of the colonic concentrations achieved in patients treated with therapeutic levels of this drug. BSZ is therefore a promising candidate for further testing as a potential colon polyp/cancer chemoprevention agent. Funded in part by Salix Pharmaceuticals, Inc., Pale Alto, CA 94303 USA.
GASTROENTEROLOGY, Vol. 108, No. 4
• G E N O M I C D E F E C T S IN HEPATOCELLULAR CARCINOMA: T H E ROLE OF DNA M I S M A T C H REPAIR GENES. G . A , Macdonald. J.K. Greenson, H.D. Appalman, C.R. Boland. Departments of Internal Medicine and Pathology, University of Michigan, Ann Arbor, MI. Although hepatocellular carcinoma (HCC) is a major cause of cancer related deaths, the genomic events in the evolution of HCC are incompletely understood. Aim: To survey HCCs for evidence of defective DNA repair and loss of beterozygosity (LOH) for DNA mismatch repair (MMR) genes hMSH2 and hMLH1, and tumor suppresser genes APC/MCC, DCC and p53. Methods: Domains up to 5mm in diameter within benign tissue or HCC were isolated from 5 p~m sections of paraffin embedded surgical specimens. Following DNA extraction, PCR was performed on all samples using p32 end-labeled primers for variable number tandem repeats (VNTRs) D 2 S l 1 9 and D2S123 flanking the MMR gene hMSH2, and D3S1029, D5S346, D17S261 and D18S35 linked with the genes f o r hMLH1, APC/MCC, p53 and DCC respectively. D5S107 on 5q and an intronic VNTR of p53 were also studied. Samples were run on a denaturing polyacrylamide gel, and PCR preducts detected using autoradiography. A VNTR was considered informative when 2 bands were detected in non-neoplastic tissue. LOH was defined as >50% decrease in the relative intensity of one allele. Band shift, a marker of defective DNA MMR, was defined as a change in position of PCR product relative to bands in the benign tissue. Results: 22 patients had 23 HCCs resected. 3 patients had chronic hepatitis B, 3 hepatitis C, and 4 had a history of significant ethanol consumption. 132 domains were analyzed (range 3-13/patient), and 5 HCCs had evidence of defective DNA MMR with ->5 VNTR band shifts. HCC 1 HCC 2 HCC 3 HCC 4 HCC 5 Band shifts 9 7 6 5 5 hMSH2 LOH LOH LOH hMLH 1 LOH LOH LOH APC./MCC LOH LOH p53 LOH DCC LOH LOH LOH Of the remaining 18 HCCs, 5 had LOH at D18S35 only, 2 at p53, 1 at D5S107, and 1 at D2Sl19 without associated band shifts. 5 HCCs had <3 band shifts with no LOH and 4 had no LOH or band shifts. Conclusions: Defective DNA MMR occurs in a significant proportion of HCCs and is associated with LOH at DNA MMR genes, and a variety of tumor suppresser genes. The observation of LOH at the MMR genes in association with VNTR band shifts is not commonly seen in gastrointestinal tumors and suggests unique pathogenetic mechanisms in HCC.
DISTRIBUTION OF LABELLED CELLS PROVIDES REPRODUCIBLE MEASURE OF CRYPT PROLIFERATION IN RECTAL BIOPSIES. F.A. Maerae. D. Kilias, N.R. Hughes, G.P. Young, K. Sharpe. Depts. of Gastroenterology and Anatomical Pathology, Royal Melbourne Hospital and Depts. of Medicine and Statistics, University of Melbourne, Australia. Errors in measurement of proliferation in colonic crypts are inherent in techniques requiring incorporation of nucleotides and subjective assessment of staining cell positivity. The proliferating cell nuclear antigen (PCNA) technique involves direct staining of proliferating cells without incubation, but variable staining intensity leads to inter- and intra-observer and interlaboratory variability in labelling index calculations. The distribution of positive cells should be a more stable measure as it is not sensitive to positive threshold variations in counting. Rectal biopsies from 8 subjects were stained for PCNA and assessed by two readers using the same and different criteria for poaltivity, using initially unselected and then identical crypts for counting. The two criteria for positivity used were I. uniform dark brown staining of the nucleus and II. uniform dark brown staining of the nucleus and speckled nuclei. Distribution of positivity was compared in assessments made of (a) observer A using criterion I, (b) observer B using criterion II, (c) observer A using criterion 1I and (d) observer B counting identical crypts as used in (a) using criterion II. The comparisons were made using the likelihood ratio statistic (D) for testing for differences between the underlying distribution of positive cells obtained under (a) to (d). The expected value of D for the same distribution is 9; values greater than 16.92 indic,ate significant disagreement at the 5% level. LikelihQod ratio statistic for comDarin- readings (ak (bk (e3 and td~-(a3. Subject 1 2 3 4 5 6 7 8 D value 37.6* 11.4 11.0 12,4 4.7 22.4* 3.0 10.3 * p < 0.05. The disagreement in the distributions in the 2 subjects with significant results was due to (d)-(a) which is the difference in distribution of the extra positive cells counted by criterion II compared with criterion I using the same crypt set. The other six subjects' data showed good agreement. In our experience, reproducibility of measures of proliferation is best achieved by total distribution of labelled cells and may provide the best approach for multi-centre/observer studies. The effect of dietary interventions on this approach needs further evaluation. (Funded in part by Kellogg (Australia) Pry. Ltd.)
AGA ABSTRACTS
• THE BENEFITS OF USING SUPPLEMENTAL L-ARGIN1NE IN A CHEMICAL INDUCED MODEL OF COLORECTAL CANCER QY Ma, M Hoper, BJ Rowlands. Department of Surgery , Institute of Clinical Science, Queen's University of Belfast, Northern Ireland, UK. Introduction: L-arginine inhibits mammary and transplantable solid turnouts in experimental models yet its effect on colorectal cancer has not been studied. The aim of this study was to investigate the effect of arginine on 1,2-dimethylhydrazine (DMH)-induced colonic cancer and its mechanism of action. Methodology: Male Wistar rats were treated with 20 weekly subcutaneous injections of DMH (20 mg/kg body weight). Arginine was given in a 1% solution of drinking water. Animals were divided into 3 groups. Group I (42 rats) was given DMH, group II (41 rats) DMI-I and whole period of arginine supplementation, Group HI (24 rats) DMH and first 10 weeks arginine only. Thymic lymphocyte profiferative response to the T-cell mitogen phytohaemaggiutinin (PHA) was determined using the uptake oftritiated thymidine. Stimulation index was calculated by the mean count per minute(cpm) stimulated cultures/mean epm unstimulated cultures. Result: Thirty eight out of 42 DMH animals developed colonic tumours compared to 28 out of 41 in Group II and 14 out of 24 in Group HI. Group I Group II ' Group llI Tumour incidence (%) 88 68* 58* No. oftumour/rat 2.9__+0.2 1.71+0.33" 1.04_+0.24" No. oftumour/tumour-beerin 5 rat 3_+0.25 2.15+0.36" 1.67_+0.28" Stimulation Index 15+6.1 36+8.1" 30.8+5.3* * P<0.05 compared to Group I (Mann-Whitney U test) Conclusion: Supplemental arginine significantly inhibited colonic tumorigenesis and stimulated thymie lymphocyte response. The reduction in tumorigenesis in the animals given supplemental arginine may be through a general stimulation of the host immune system.
• Inhibition of Colon Cancer Cell Proliferation In Vitro and AzoxymethaneInduced Aberrant Crypt Formation In Vivo by Balsalazide and Metabolites. DJ MacGregor, YS Kim, ME Sleisonger and LK Johnson*. Dept. of Med, DFMC and UCSF, San Francisco, CA; *Salix Pharmaceuticals, Inc., Pale Alto, CA USA. Introduction: Nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to reduce polyp recurrence in FAP patients, inhibit carcinogen-induced aberrant crypt loci number and multiplicity in rodents and inhibit proliferation of human colon cancer-derived cell lines in vitro. However, because of NSAIDinduced upper GI ulceration, their widespread chronic use for colon polyp or cancer chemoprevention therapy may be limited. Goals: The present goals were therefore to test, in similar assays, an alternative antiinflammatory agent which lacks such upper GI side effects. Balsalazide disodium (BSZ) is an aminosalicylate (5-ASA) linked to an inert carrier (4-aminobenzoyl-13alanine, 4ABA) currently undergoing Phase III studies for ulcerative colitis. BSZ lacks upper GI and renal side effects seen with standard NSAIDs and delivers high concentrations of 5-ASA to the colon. Methods: Cell proliferation was measured using HT-29 and LSI74T colon adenoma-derived cell lines in 96-wells microtiter plates. Cell densities were determined by the SRB-dye binding assay. Aberrant crypt studies used F344 rats (8 per group) treated with 2 weekly injections of AOM (15mg/kg). ACF were assessed 8 weeks after the first injection following methylene blue staining and fixation of the entire colon. Results: BSZ and its active metabolite, 5-ASA, inhibited proliferation of both cell lines in a dose dependent manner (BSZ ICs0---4mM,5-ASA ICs0=SmM). In contrast, 4-ABA was inactive. The 5-ASA inhibitory activity increased upon oxidation (ox5-ASA) to 90% with an ICs0=2mM. The drug effects were not cytotoxic and cell growth resumed upon drag withdrawal. Like the NSA1D indomethacin, growth inhibition by ox5-ASA caused accumulation of cells in the Go/G1 phase of the cell cycle. BSZ provided in the drinking water (125&250mg/kg) to AOM-treated rats resulted in a 49% (p<0.003) and 65% (p<0.001) reduction, respectively in the number of ACF. The number of ACF containing >_4crypts was reduced by 75% (p<0.001). Conclusions: BSZ significantly reduces the formation and size of early pre-adenoma lesions induced by the carcinogen, azoxymethane. This may be due to inhibition of aberrant crypt cell proliferation. The ICs0 for colon cancer cell growth inhibition seen for BSZ, 5-ASA and ox5-ASA are also within the range of the colonic concentrations achieved in patients treated with therapeutic levels of this drug. BSZ is therefore a promising candidate for further testing as a potential colon polyp/cancer chemoprevention agent. Funded in part by Salix Pharmaceuticals, Inc., Pale Alto, CA 94303 USA.
GASTROENTEROLOGY, Vol. 108, No. 4
• G E N O M I C D E F E C T S IN HEPATOCELLULAR CARCINOMA: T H E ROLE OF DNA M I S M A T C H REPAIR GENES. G . A , Macdonald. J.K. Greenson, H.D. Appalman, C.R. Boland. Departments of Internal Medicine and Pathology, University of Michigan, Ann Arbor, MI. Although hepatocellular carcinoma (HCC) is a major cause of cancer related deaths, the genomic events in the evolution of HCC are incompletely understood. Aim: To survey HCCs for evidence of defective DNA repair and loss of beterozygosity (LOH) for DNA mismatch repair (MMR) genes hMSH2 and hMLH1, and tumor suppresser genes APC/MCC, DCC and p53. Methods: Domains up to 5mm in diameter within benign tissue or HCC were isolated from 5 p~m sections of paraffin embedded surgical specimens. Following DNA extraction, PCR was performed on all samples using p32 end-labeled primers for variable number tandem repeats (VNTRs) D 2 S l 1 9 and D2S123 flanking the MMR gene hMSH2, and D3S1029, D5S346, D17S261 and D18S35 linked with the genes f o r hMLH1, APC/MCC, p53 and DCC respectively. D5S107 on 5q and an intronic VNTR of p53 were also studied. Samples were run on a denaturing polyacrylamide gel, and PCR preducts detected using autoradiography. A VNTR was considered informative when 2 bands were detected in non-neoplastic tissue. LOH was defined as >50% decrease in the relative intensity of one allele. Band shift, a marker of defective DNA MMR, was defined as a change in position of PCR product relative to bands in the benign tissue. Results: 22 patients had 23 HCCs resected. 3 patients had chronic hepatitis B, 3 hepatitis C, and 4 had a history of significant ethanol consumption. 132 domains were analyzed (range 3-13/patient), and 5 HCCs had evidence of defective DNA MMR with ->5 VNTR band shifts. HCC 1 HCC 2 HCC 3 HCC 4 HCC 5 Band shifts 9 7 6 5 5 hMSH2 LOH LOH LOH hMLH 1 LOH LOH LOH APC./MCC LOH LOH p53 LOH DCC LOH LOH LOH Of the remaining 18 HCCs, 5 had LOH at D18S35 only, 2 at p53, 1 at D5S107, and 1 at D2Sl19 without associated band shifts. 5 HCCs had <3 band shifts with no LOH and 4 had no LOH or band shifts. Conclusions: Defective DNA MMR occurs in a significant proportion of HCCs and is associated with LOH at DNA MMR genes, and a variety of tumor suppresser genes. The observation of LOH at the MMR genes in association with VNTR band shifts is not commonly seen in gastrointestinal tumors and suggests unique pathogenetic mechanisms in HCC.
DISTRIBUTION OF LABELLED CELLS PROVIDES REPRODUCIBLE MEASURE OF CRYPT PROLIFERATION IN RECTAL BIOPSIES. F.A. Maerae. D. Kilias, N.R. Hughes, G.P. Young, K. Sharpe. Depts. of Gastroenterology and Anatomical Pathology, Royal Melbourne Hospital and Depts. of Medicine and Statistics, University of Melbourne, Australia. Errors in measurement of proliferation in colonic crypts are inherent in techniques requiring incorporation of nucleotides and subjective assessment of staining cell positivity. The proliferating cell nuclear antigen (PCNA) technique involves direct staining of proliferating cells without incubation, but variable staining intensity leads to inter- and intra-observer and interlaboratory variability in labelling index calculations. The distribution of positive cells should be a more stable measure as it is not sensitive to positive threshold variations in counting. Rectal biopsies from 8 subjects were stained for PCNA and assessed by two readers using the same and different criteria for poaltivity, using initially unselected and then identical crypts for counting. The two criteria for positivity used were I. uniform dark brown staining of the nucleus and II. uniform dark brown staining of the nucleus and speckled nuclei. Distribution of positivity was compared in assessments made of (a) observer A using criterion I, (b) observer B using criterion II, (c) observer A using criterion 1I and (d) observer B counting identical crypts as used in (a) using criterion II. The comparisons were made using the likelihood ratio statistic (D) for testing for differences between the underlying distribution of positive cells obtained under (a) to (d). The expected value of D for the same distribution is 9; values greater than 16.92 indic,ate significant disagreement at the 5% level. LikelihQod ratio statistic for comDarin- readings (ak (bk (e3 and td~-(a3. Subject 1 2 3 4 5 6 7 8 D value 37.6* 11.4 11.0 12,4 4.7 22.4* 3.0 10.3 * p < 0.05. The disagreement in the distributions in the 2 subjects with significant results was due to (d)-(a) which is the difference in distribution of the extra positive cells counted by criterion II compared with criterion I using the same crypt set. The other six subjects' data showed good agreement. In our experience, reproducibility of measures of proliferation is best achieved by total distribution of labelled cells and may provide the best approach for multi-centre/observer studies. The effect of dietary interventions on this approach needs further evaluation. (Funded in part by Kellogg (Australia) Pry. Ltd.)