IJC International Journal of Cancer
Apc-driven colon carcinogenesis in Pirc rat is strongly reduced by polyethylene glycol Angelo Pietro Femia, Caterina Becherucci, Stefania Crucitta and Giovanna Caderni NEUROFARBA Department, Section of Pharmacology and Toxicology, University of Florence, Florence, Italy
Short Report
Polyethylene glycol (PEG) is one of the most powerful agents in reducing chemically induced carcinogenesis in rat colon. However, contrasting results in Min mice dampened the enthusiasm on this potentially strong and virtually safe, cancer chemopreventing agent. Pirc (F344/NTac-Apc am1137) rats carrying a germline heterozygous mutation in the Apc gene, spontaneously develop multiple tumours in the colon thus modelling both familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC). Given this similarity, we thought that these rats could be appropriate to test the efficacy of PEG 8000 in reducing carcinogenesis. Pirc male rats aged one month were treated with 5% PEG in drinking water for 2 or 6 months. Precancerous lesions were dramatically reduced after 2 months of PEG treatment (Mucin depleted foci (MDF)/colon were 99 617 and 126 8 in Controls and PEG-treated rats, respectively; p < 0.001; mean 6 SD). Similarly, colon tumors were significantly reduced after 6 months of treatment (tumors/rat were 8.1 6 2.3 and 3.6 6 2.2 in Controls and PEG-treated rats, respectively; p < 0.05; mean 6 SD). Colon proliferation, a parameter correlated to cancer risk, was also significantly lower in PEG-treated rats than in Controls, while apoptosis was not significantly affected. In conclusion, PEG markedly reduces colon carcinogenesis in Pirc rats mutated in Apc; we thus suggest that PEG may be used as chemopreventive agent to reduce cancer risk in FAP and CRC patients.
Polyethylene glycol (PEG), an osmotic laxative, markedly reduces colon carcinogenesis in azoxymethane or 1,2-dimethylhydrazine-induced rats, widely used models mimicking sporadic colorectal cancer (CRC).1–4 However, the enthusiasm on this potentially strong and virtually safe, chemopreventing agent was dampened by contrasting results obtained in Min mice.5 As a matter of fact, Naigamwalla et al. showed that PEG 8000 was not effective in reducing preneoplastic lesions in SWR mice and more worrying, it caused an overall increase in colon tumors in Apc-mutated Min Mice proficient or defective in DNA mismatch repair (MMR).5 Min mice, mutated in the Apc gene, replicate the key mutational event occurring in Familial Adenomatous Polyposis (FAP) and in sporadic CRC, but not the phenotypic manifestations since, at variance with human pathology, tumours in Min mice develop mostly in the small intestine and not in the colon.6–8 However, a higher dose of PEG 3350, another PEG formulation clinically used, has been shown effective in reducing
small intestinal tumors in Min Mice (MMR proficient) by other authors.9 Pirc rats (Polyposis In the Rat Colon) carry a germline heterozygous mutation of the Apc gene (F344/NTacApc am1137).6 Notably, at variance with Min mice, Pirc rats spontaneously develop a larger number of tumors in the colon, thus reproducing more faithfully the carcinogenesis process occurring in FAP and CRC. Importantly, Pirc has been shown to respond to known chemopreventive agents like celecoxib or sulindac pointing out the reliability of this model to find out effective chemopreventive treatments.6,10 Given these considerations we thought that Pirc rats may be the most appropriate model to settle the controversy on PEG effect on colon carcinogenesis. Accordingly, we treated Pirc rats with 5% PEG in the drinking water and we determined the number of mucin depleted foci (MDF), colon precancerous lesions in the colon, after two months or intestinal macroscopic tumors after six months of treatment.10
Key words: colon cancer prevention, MDF, polyethylene glycol, Pirc rat Grant sponsor: Istituto Toscano Tumori; Grant sponsor: University of Florence (Fondo ex-60%) DOI: 10.1002/ijc.29581 History: Received 10 Mar 2015; Accepted 16 Apr 2015; Online 24 Apr 2015 Correspondence to: Giovanna Caderni, NEUROFARBA Department, Section of Pharmacology and Toxicology, University of Florence, 6 Viale Pieraccini, 50139 Florence, Italy, E-mail: giovanna.caderni@unifi.it
Material and Methods
C 2015 UICC Int. J. Cancer: 137, 2270–2273 (2015) V
Animals and treatments
Pirc (F344/NTac-Apcam1137) rats, originally obtained by Taconic (Taconic Farms) were bred in CESAL (University of Florence, Italy) in accordance with the Commission for Animal Experimentation of the Italian Ministry of Health.10 The colony was maintained by mating heterozygous Pirc rats with wild type Fisher F344/NTac rats (Taconic Farms). Rats were maintained in polyethylene cages under an experimental protocol approved by the Commission for Animal Experimentation of the Italian Ministry of Health.
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Femia et al.
What’s new? Polyethylene glycol (PEG) reduces colon cancer in rats treated with carcinogens. In this study, the authors found that PEG inhibits colon carcinogenesis in Pirc rats (for “polyposis in the rat colon”). Pirc rats carry a mutation in the Apc gene that mimics human familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC) more closely than other Apcmutated strains. Given the similarities between Pirc rats and human pathology, these results suggest that PEG trials in humans at risk for CRC may be worthwhile.
fed ad libitum with standard pellet diet (Teklad Global diet 2018, Harlan, Italy). PEG (8000) was purchased from ICNMP (Eschwege, Germany). Male Pirc rats aged one month were randomly allocated to Controls or PEG group, which were treated with 5% PEG in drinking water, as described.4 For the short-term experiment, rats (5 rats/group) were sacrificed after two months of treatment, while for the longterm carcinogenesis experiment (7 rats per group) animals were sacrificed after 6 months of treatment. Animals were monitored for body weight and possible rectal bleeding. Animals were sacrificed by CO2 asphyxia. Sample collection, determination of precancerous lesions and macroscopic tumors. At sacrifice the entire intestine (from
pylorus to anus) was dissected and flushed with cold saline. Small intestine was longitudinally cut and spread on filter paper with the mucosa surface upward to assess tumors as described.10 Similarly, the entire colon was longitudinally opened and macroscopic tumors assessed as described.10 To determine the presence of the precancerous MDF, the colon was pinned flat on a polystyrene board, fixed in buffered formalin and processed with the high-iron diamine Alcian blue staining (HID-AB) to enumerate MDF as described.10 Determination of proliferation and apoptosis in the intestinal mucosa
Proliferative activity in the apparently morphological normal mucosa (NM) was evaluated with immunohistochemistry determining proliferating cell nuclear antigen (PCNA) immunoreactivity in longitudinal sections of NM using a mouse monoclonal antibody (PC-10, Santa Cruz, CA).10 Proliferative activity was evaluated in at least 15 full longitudinal crypt sections of the NM and expressed as PCNA labeled cells (LC)/crypt or labeling index (LI): number of cells positive to PCNA/cells scored 3 100. To determine the distribution of the proliferative activity along the crypt, this was divided into three equal parts (lower, mid and upper compartment facing the lumen) and the percentage of labeling in each compartment over the total labeling present in the crypt was calculated. Apoptosis was evaluated in histological sections (4 mm thick) of the NM stained with hematoxylin-eosin, determining the number of apoptotic cells in at least 15 full entire longitudinal sectioned crypts, as reported.10 Apoptotic index C 2015 UICC Int. J. Cancer: 137, 2270–2273 (2015) V
(AI) is the number of apoptosis/cells in the crypt sections 3100. Statistical evaluation of the data
Data obtained from individual rats in the different experimental groups were summarized for quantitative continuous responses by calculating group means and standard deviations. Comparisons between PEG-treated group and Controls were analyzed with t test for unpaired samples performed with Graph-pad (p-level fixed at 0.05, two-sided).
Results Short-term carcinogenesis experiment
Male Pirc rats, aged one month, were treated with 5% PEG in drinking water for two months. At sacrifice, a dramatic decrease in the number of precancerous MDF in the rats treated with PEG was observed (Fig. 1, left panel). This result was the basis for the following long-term carcinogenesis study. Long-term carcinogenesis experiment
Male Pirc rats, aged one month, were allocated to Control group or PEG 5% and treated for 6 months. The weight of the rats at the beginning of the experiment was 52 g 6 3 (7) and 56 g 6 7 (7) in Controls and PEG-treated, respectively, [mean 6 SD (animals/group)]. At the end of the experiment, when rats were seven month old, the body weight was 376 g 6 28 and 392 g 6 41 in Controls and PEG-treated rats, respectively. As previously reported, feces of PEG-treated rats contained more water [water content (as % of weight) was 52 6 2 and 74 6 2, in Controls and PEG-treated rats, respectively, p < 0.01].3 Not withstanding the higher water content the feces were well formed and no diarrhea was evident. Regarding carcinogenesis, we observed a significant lower number of colon tumors in the rats treated with PEG (Fig. 1, right panel). Moreover, colon tumors in the PEG group were significantly smaller than those in Control group (63.2 mm36 27 and 33.6 mm3 6 17 in Controls and PEGtreated rats, respectively, means 6 SD, p < 0.05). The number of small intestinal tumours was not affected by PEG (number of small intestinal tumours/rat was 5.4 6 4.2 and 4.7 6 2.4 in Controls and PEG-treated rats, respectively, means 6 SD). As it has been suggested that PEG may vary proliferation and apoptosis, we measured these two parameters in colon mucosa.9,11–13 The determination of proliferative activity in
Short Report
Diet composition and treatments of the animals. Rats were
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Apc-driven carcinogenesis in Pirc rat is reduced by PEG
Figure 1. Number of MDF/colon (left panel) and tumours (right panel) in Control and PEG treated rats. Bars represent mean 1 SE. For each experimental group there were 5 and 7 rats, for MDF and tumour determination, respectively. *, **: significantly different (p < 0.05 and 0.001, respectively) when compared with the control group with t test.
the apparently NM showed that the crypts were significantly shorter than those in Controls (Table 1); similarly, the number of PCNA labeled cells per crypt as well as the LI were significantly lower in the PEG-treated rats than in Controls. The distribution of the proliferative activity along the crypts was not affected by PEG. Apoptosis was slightly, but not significantly higher in PEG-treated rats than in Controls (AI: 0.37 6 0.15 and 0.27 6 0.11 in PEG treated and Controls, respectively). Since it has also been suggested that PEG may affect inflammation in the colon mucosa, we determined the expression of three genes connected with this process (i.e., Cox-2, Il-1b and TNF-a); this determination, carried out with RT-PCR, showed no differences between the two groups (data not shown).14
Short Report
Discussion The main result of this study is that PEG significantly reduces colon carcinogenesis in Pirc rats, a faithful model of FAP and CRC. In fact, we showed that PEG reduces the precancerous lesions MDF as well as colon tumors when animals were treated for longer time. Moreover, tumours developed in the PEG-treated rats were smaller than those in Controls. Small intestinal tumors were not significantly affected by PEG. Our results, are thus in line with the first findings by Corpet group reporting a strong inhibition of colon carcinogenesis by PEG.1–3 To our knowledge, there are no papers in Pubmed database reporting the effect of PEG in Pirc rats.
However, while the long-term carcinogenesis experiment was running, we have been acquainted with an abstract describing the beneficial effect of higher doses of PEG in Pirc rats, thus in line with our results.15 It is interesting to note that a French study on patients undergoing colonoscopy (1165 subjects) documented that the R ) was associuse of PEG-based laxative (PEG 4000, ForlaxV ated with a reduced risk of colon cancer.16 Similarly, a lower risk of CRC was reported by Charlton et al. in patients using Macrogol laxatives containing PEG.17 These reports, together with the previous and the present results, suggest indeed that PEG could be used to reduce cancer in subjects at risks such as those operated for sporadic CRC or FAP patients. As a matter of fact, in our study, although PEG caused an increase in fecal water content, no marked diarrhea was observed. As regard to the mechanisms of action of PEG effect, we found that PEG caused a significant shortening of colon crypts, as previously reported.13 We also found that PEG reduces colon proliferation, accordingly, Roy and coworkers reported that PEG suppressed epithelial proliferation downregulating EGFR in colonocytes.11,12,18 Our previous data on Pirc rats documented a higher proliferative activity in this strain compared with wt rats, a result in line with the increased proliferation in the NM of FAP patients and patients at risk (e.g., operated for adenomatous polyps) reported in several studies.10,19 Thus, the fact that PEG causes a decrease in this parameter bringing back it to lower values, is important. Variation in the distribution of the proliferative activity along the crypt, with a shift of the
Table 1. Colon proliferation parameters in control rats and in rats treated with 5% PEG in drinking water Groups
Distribution of proliferative activity (%)
Cells/crypt section
LC/crypt
LI
Bottom of the crypt
Mid of the crypt
Top of the crypt
Controls (6)1
96 6 4
44 6 6
47 6 5
50 6 3
49 6 5
261
PEG 5% (7)
84 6 42
31 6 32
37 6 52
50 6 5
48 6 4
261
Values are means 6 SD. 1 Number in parenthesis are the number of rats in which proliferation was determined. LC/crypt: PCNA labeled cells/crypt; LI: number of labeled cells/cells scored 3 100. The distribution of the proliferative activity along the crypt was calculated as described in details in the method section. 2 Significantly different (p < 0.01) when compared with the control group with t test.
C 2015 UICC Int. J. Cancer: 137, 2270–2273 (2015) V
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proliferative activity in the upper part, has been associated with increased risk of colon cancer.19 Accordingly, we measured this parameter in our rats, that was not affected by PEG. This result can be explained with the fact that although Pirc has high proliferation compared with wt, the distribution of colon proliferation in Pirc and wt rats is similar.10 Studies in vitro and in vivo reported that PEG also acts increasing apoptosis.9,13 On the contrary, we found only a
slight, not significant increase in this parameter. Similarly, we did not find any difference in the expression of genes related to inflammation process (i.e., Cox-2, Il-1b and TNF-a). In conclusion, PEG markedly reduces colon carcinogenesis in Pirc rats; given the similarities between this experimental model and human pathology we think that these results strongly support intervention trials with PEG in subjects at risk such as FAP or CRC patients.
References 1.
2.
3.
4.
5.
6.
8.
9.
10.
11.
12.
13.
progress report and recommendations. Gastroenterology 2013;144:705–17. Tomasetti C, Vogelstein B. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science 2015;347:78–81. Roy HK, Gulizia J, DiBaise JK, et al. Polyethylene glycol inhibits intestinal neoplasia and induces epithelial apoptosis in apc (min) mice. Cancer Lett 2004;15:35–42. Femia AP, Luceri C, Soares PV, et al. Multiple mucin depleted foci, high proliferation and low apoptotic response in the onset of colon carcinogenesis of the PIRC rat, mutated in apc. Int J Cancer 2015;136:E488–95. Roy HK, Koetsier JL, Tiwari AK, et al. Involvement of p21cip1/waf1 in the anti-proliferative effects of polyethylene glycol in colon carcinogenesis. Int J Oncol 2011;38:529–36. Roy HK, Kunte DP, Koetsier JL, et al. Chemoprevention of colon carcinogenesis by polyethylene glycol: suppression of epithelial proliferation via modulation of SNAIL/beta-catenin signaling. Mol Cancer Ther 2006;5:2060–9. Tache S, Parnaud G, Van Beek E, et al. Polyethylene glycol, unique among laxatives, suppresses aberrant crypt foci, by elimination of cells. Scand J Gastroenterol 2006;41:730–6.
14. Karlsson PC, Hughes R, Rafter JJ, et al. Polyethylene glycol reduces inflammation and aberrant crypt foci in carcinogen-initiated rats. Cancer Lett 2005;223:203–9. 15. Wali RK, DeLa Cruz M, Kunte D,et al. Polyethylene Glycol (PEG) suppresses adenomas and aberrant crypt foci (ACF) in the polyposis in Rat Colon Model (Pirc): implication for colorectal cancer prevention. American Gastroenterology Meeting 2012. Gastroenterology 2012;142, Supplement 1, Abstract #885. 16. Dorval E, Jankowski JM, Barbieux JP, et al. Polyethylene glycol and prevalence of colorectal adenomas. Gastroenterol Clin Biol 2006;30: 1196–9. 17. Charlton RA, Snowball JM, Bloomfield K, et al. Colorectal cancer risk reduction following macrogol exposure: a cohort and nested case control study in the UK. PLoS One 2013;8:e83203 18. Wali RK, Kunte DP, Koetsier JL, et al. Polyethylene glycol-mediated colorectal cancer chemoprevention: roles of epidermal growth factor receptor and snail. Mol Cancer Ther 2008;7: 3103–11. 19. Day DW, Jass JR, Price AB, et al. Morson &Dawson’s Gastrointestinal Pathology, 4th edn. Oxford: Blackwell Scientific Publications, 2003. 551–609.
Short Report
7.
Corpet DE, Parnaud G. Polyethylene-glycol, a potent suppressor of azoxymethane-induced colonic aberrant crypt foci in rats. Carcinogenesis 1999;20:915–8. Parnaud G, Tache S, Peiffer G, et al. Polyethylene-glycol suppresses colon cancer and causes dose-dependent regression of azoxymethaneinduced aberrant crypt foci in rats. Cancer Res 1999;59:5143–7. Corpet DE, Parnaud G, Delverdier M, et al. Consistent and fast inhibition of colon carcinogenesis by polyethylene glycol in mice and rats given various carcinogens. Cancer Res 2000;60:3160–4. Femia AP, Caderni G, Bottini C, et al. Mucindepleted foci are modulated by dietary treatments and show deregulation of proliferative activity in carcinogen-treated rodents. Int J Cancer 2007; 120:2301–5. Naigamwalla D, Chia MC, Tran TT, et al. Polyethylene glycol 8000 and colon carcinogenesis: inhibition in the f344 rat, promotion in the min mouse. Cancer Res 2000;60:6856–8. Amos-Landgraf JM, Kwong LN, Kendziorski CM, et al. A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer. Proc Natl Acad Sci USA 2007;104:4036–41. Washington MK, Powell AE, Sullivan R, et al. Pathology of rodent models of intestinal cancer:
C 2015 UICC Int. J. Cancer: 137, 2270–2273 (2015) V
Apc-driven colon carcinogenesis in Pirc rat is strongly reduced by polyethylene glycol Angelo Pietro Femia, Caterina Becherucci, Stefania Crucitta and Giovanna Caderni NEUROFARBA Department, Section of Pharmacology and Toxicology, University of Florence, Florence, Italy
Short Report
Polyethylene glycol (PEG) is one of the most powerful agents in reducing chemically induced carcinogenesis in rat colon. However, contrasting results in Min mice dampened the enthusiasm on this potentially strong and virtually safe, cancer chemopreventing agent. Pirc (F344/NTac-Apc am1137) rats carrying a germline heterozygous mutation in the Apc gene, spontaneously develop multiple tumours in the colon thus modelling both familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC). Given this similarity, we thought that these rats could be appropriate to test the efficacy of PEG 8000 in reducing carcinogenesis. Pirc male rats aged one month were treated with 5% PEG in drinking water for 2 or 6 months. Precancerous lesions were dramatically reduced after 2 months of PEG treatment (Mucin depleted foci (MDF)/colon were 99 617 and 126 8 in Controls and PEG-treated rats, respectively; p < 0.001; mean 6 SD). Similarly, colon tumors were significantly reduced after 6 months of treatment (tumors/rat were 8.1 6 2.3 and 3.6 6 2.2 in Controls and PEG-treated rats, respectively; p < 0.05; mean 6 SD). Colon proliferation, a parameter correlated to cancer risk, was also significantly lower in PEG-treated rats than in Controls, while apoptosis was not significantly affected. In conclusion, PEG markedly reduces colon carcinogenesis in Pirc rats mutated in Apc; we thus suggest that PEG may be used as chemopreventive agent to reduce cancer risk in FAP and CRC patients.
Polyethylene glycol (PEG), an osmotic laxative, markedly reduces colon carcinogenesis in azoxymethane or 1,2-dimethylhydrazine-induced rats, widely used models mimicking sporadic colorectal cancer (CRC).1–4 However, the enthusiasm on this potentially strong and virtually safe, chemopreventing agent was dampened by contrasting results obtained in Min mice.5 As a matter of fact, Naigamwalla et al. showed that PEG 8000 was not effective in reducing preneoplastic lesions in SWR mice and more worrying, it caused an overall increase in colon tumors in Apc-mutated Min Mice proficient or defective in DNA mismatch repair (MMR).5 Min mice, mutated in the Apc gene, replicate the key mutational event occurring in Familial Adenomatous Polyposis (FAP) and in sporadic CRC, but not the phenotypic manifestations since, at variance with human pathology, tumours in Min mice develop mostly in the small intestine and not in the colon.6–8 However, a higher dose of PEG 3350, another PEG formulation clinically used, has been shown effective in reducing
small intestinal tumors in Min Mice (MMR proficient) by other authors.9 Pirc rats (Polyposis In the Rat Colon) carry a germline heterozygous mutation of the Apc gene (F344/NTacApc am1137).6 Notably, at variance with Min mice, Pirc rats spontaneously develop a larger number of tumors in the colon, thus reproducing more faithfully the carcinogenesis process occurring in FAP and CRC. Importantly, Pirc has been shown to respond to known chemopreventive agents like celecoxib or sulindac pointing out the reliability of this model to find out effective chemopreventive treatments.6,10 Given these considerations we thought that Pirc rats may be the most appropriate model to settle the controversy on PEG effect on colon carcinogenesis. Accordingly, we treated Pirc rats with 5% PEG in the drinking water and we determined the number of mucin depleted foci (MDF), colon precancerous lesions in the colon, after two months or intestinal macroscopic tumors after six months of treatment.10
Key words: colon cancer prevention, MDF, polyethylene glycol, Pirc rat Grant sponsor: Istituto Toscano Tumori; Grant sponsor: University of Florence (Fondo ex-60%) DOI: 10.1002/ijc.29581 History: Received 10 Mar 2015; Accepted 16 Apr 2015; Online 24 Apr 2015 Correspondence to: Giovanna Caderni, NEUROFARBA Department, Section of Pharmacology and Toxicology, University of Florence, 6 Viale Pieraccini, 50139 Florence, Italy, E-mail: giovanna.caderni@unifi.it
Material and Methods
C 2015 UICC Int. J. Cancer: 137, 2270–2273 (2015) V
Animals and treatments
Pirc (F344/NTac-Apcam1137) rats, originally obtained by Taconic (Taconic Farms) were bred in CESAL (University of Florence, Italy) in accordance with the Commission for Animal Experimentation of the Italian Ministry of Health.10 The colony was maintained by mating heterozygous Pirc rats with wild type Fisher F344/NTac rats (Taconic Farms). Rats were maintained in polyethylene cages under an experimental protocol approved by the Commission for Animal Experimentation of the Italian Ministry of Health.
2271
Femia et al.
What’s new? Polyethylene glycol (PEG) reduces colon cancer in rats treated with carcinogens. In this study, the authors found that PEG inhibits colon carcinogenesis in Pirc rats (for “polyposis in the rat colon”). Pirc rats carry a mutation in the Apc gene that mimics human familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC) more closely than other Apcmutated strains. Given the similarities between Pirc rats and human pathology, these results suggest that PEG trials in humans at risk for CRC may be worthwhile.
fed ad libitum with standard pellet diet (Teklad Global diet 2018, Harlan, Italy). PEG (8000) was purchased from ICNMP (Eschwege, Germany). Male Pirc rats aged one month were randomly allocated to Controls or PEG group, which were treated with 5% PEG in drinking water, as described.4 For the short-term experiment, rats (5 rats/group) were sacrificed after two months of treatment, while for the longterm carcinogenesis experiment (7 rats per group) animals were sacrificed after 6 months of treatment. Animals were monitored for body weight and possible rectal bleeding. Animals were sacrificed by CO2 asphyxia. Sample collection, determination of precancerous lesions and macroscopic tumors. At sacrifice the entire intestine (from
pylorus to anus) was dissected and flushed with cold saline. Small intestine was longitudinally cut and spread on filter paper with the mucosa surface upward to assess tumors as described.10 Similarly, the entire colon was longitudinally opened and macroscopic tumors assessed as described.10 To determine the presence of the precancerous MDF, the colon was pinned flat on a polystyrene board, fixed in buffered formalin and processed with the high-iron diamine Alcian blue staining (HID-AB) to enumerate MDF as described.10 Determination of proliferation and apoptosis in the intestinal mucosa
Proliferative activity in the apparently morphological normal mucosa (NM) was evaluated with immunohistochemistry determining proliferating cell nuclear antigen (PCNA) immunoreactivity in longitudinal sections of NM using a mouse monoclonal antibody (PC-10, Santa Cruz, CA).10 Proliferative activity was evaluated in at least 15 full longitudinal crypt sections of the NM and expressed as PCNA labeled cells (LC)/crypt or labeling index (LI): number of cells positive to PCNA/cells scored 3 100. To determine the distribution of the proliferative activity along the crypt, this was divided into three equal parts (lower, mid and upper compartment facing the lumen) and the percentage of labeling in each compartment over the total labeling present in the crypt was calculated. Apoptosis was evaluated in histological sections (4 mm thick) of the NM stained with hematoxylin-eosin, determining the number of apoptotic cells in at least 15 full entire longitudinal sectioned crypts, as reported.10 Apoptotic index C 2015 UICC Int. J. Cancer: 137, 2270–2273 (2015) V
(AI) is the number of apoptosis/cells in the crypt sections 3100. Statistical evaluation of the data
Data obtained from individual rats in the different experimental groups were summarized for quantitative continuous responses by calculating group means and standard deviations. Comparisons between PEG-treated group and Controls were analyzed with t test for unpaired samples performed with Graph-pad (p-level fixed at 0.05, two-sided).
Results Short-term carcinogenesis experiment
Male Pirc rats, aged one month, were treated with 5% PEG in drinking water for two months. At sacrifice, a dramatic decrease in the number of precancerous MDF in the rats treated with PEG was observed (Fig. 1, left panel). This result was the basis for the following long-term carcinogenesis study. Long-term carcinogenesis experiment
Male Pirc rats, aged one month, were allocated to Control group or PEG 5% and treated for 6 months. The weight of the rats at the beginning of the experiment was 52 g 6 3 (7) and 56 g 6 7 (7) in Controls and PEG-treated, respectively, [mean 6 SD (animals/group)]. At the end of the experiment, when rats were seven month old, the body weight was 376 g 6 28 and 392 g 6 41 in Controls and PEG-treated rats, respectively. As previously reported, feces of PEG-treated rats contained more water [water content (as % of weight) was 52 6 2 and 74 6 2, in Controls and PEG-treated rats, respectively, p < 0.01].3 Not withstanding the higher water content the feces were well formed and no diarrhea was evident. Regarding carcinogenesis, we observed a significant lower number of colon tumors in the rats treated with PEG (Fig. 1, right panel). Moreover, colon tumors in the PEG group were significantly smaller than those in Control group (63.2 mm36 27 and 33.6 mm3 6 17 in Controls and PEGtreated rats, respectively, means 6 SD, p < 0.05). The number of small intestinal tumours was not affected by PEG (number of small intestinal tumours/rat was 5.4 6 4.2 and 4.7 6 2.4 in Controls and PEG-treated rats, respectively, means 6 SD). As it has been suggested that PEG may vary proliferation and apoptosis, we measured these two parameters in colon mucosa.9,11–13 The determination of proliferative activity in
Short Report
Diet composition and treatments of the animals. Rats were
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Apc-driven carcinogenesis in Pirc rat is reduced by PEG
Figure 1. Number of MDF/colon (left panel) and tumours (right panel) in Control and PEG treated rats. Bars represent mean 1 SE. For each experimental group there were 5 and 7 rats, for MDF and tumour determination, respectively. *, **: significantly different (p < 0.05 and 0.001, respectively) when compared with the control group with t test.
the apparently NM showed that the crypts were significantly shorter than those in Controls (Table 1); similarly, the number of PCNA labeled cells per crypt as well as the LI were significantly lower in the PEG-treated rats than in Controls. The distribution of the proliferative activity along the crypts was not affected by PEG. Apoptosis was slightly, but not significantly higher in PEG-treated rats than in Controls (AI: 0.37 6 0.15 and 0.27 6 0.11 in PEG treated and Controls, respectively). Since it has also been suggested that PEG may affect inflammation in the colon mucosa, we determined the expression of three genes connected with this process (i.e., Cox-2, Il-1b and TNF-a); this determination, carried out with RT-PCR, showed no differences between the two groups (data not shown).14
Short Report
Discussion The main result of this study is that PEG significantly reduces colon carcinogenesis in Pirc rats, a faithful model of FAP and CRC. In fact, we showed that PEG reduces the precancerous lesions MDF as well as colon tumors when animals were treated for longer time. Moreover, tumours developed in the PEG-treated rats were smaller than those in Controls. Small intestinal tumors were not significantly affected by PEG. Our results, are thus in line with the first findings by Corpet group reporting a strong inhibition of colon carcinogenesis by PEG.1–3 To our knowledge, there are no papers in Pubmed database reporting the effect of PEG in Pirc rats.
However, while the long-term carcinogenesis experiment was running, we have been acquainted with an abstract describing the beneficial effect of higher doses of PEG in Pirc rats, thus in line with our results.15 It is interesting to note that a French study on patients undergoing colonoscopy (1165 subjects) documented that the R ) was associuse of PEG-based laxative (PEG 4000, ForlaxV ated with a reduced risk of colon cancer.16 Similarly, a lower risk of CRC was reported by Charlton et al. in patients using Macrogol laxatives containing PEG.17 These reports, together with the previous and the present results, suggest indeed that PEG could be used to reduce cancer in subjects at risks such as those operated for sporadic CRC or FAP patients. As a matter of fact, in our study, although PEG caused an increase in fecal water content, no marked diarrhea was observed. As regard to the mechanisms of action of PEG effect, we found that PEG caused a significant shortening of colon crypts, as previously reported.13 We also found that PEG reduces colon proliferation, accordingly, Roy and coworkers reported that PEG suppressed epithelial proliferation downregulating EGFR in colonocytes.11,12,18 Our previous data on Pirc rats documented a higher proliferative activity in this strain compared with wt rats, a result in line with the increased proliferation in the NM of FAP patients and patients at risk (e.g., operated for adenomatous polyps) reported in several studies.10,19 Thus, the fact that PEG causes a decrease in this parameter bringing back it to lower values, is important. Variation in the distribution of the proliferative activity along the crypt, with a shift of the
Table 1. Colon proliferation parameters in control rats and in rats treated with 5% PEG in drinking water Groups
Distribution of proliferative activity (%)
Cells/crypt section
LC/crypt
LI
Bottom of the crypt
Mid of the crypt
Top of the crypt
Controls (6)1
96 6 4
44 6 6
47 6 5
50 6 3
49 6 5
261
PEG 5% (7)
84 6 42
31 6 32
37 6 52
50 6 5
48 6 4
261
Values are means 6 SD. 1 Number in parenthesis are the number of rats in which proliferation was determined. LC/crypt: PCNA labeled cells/crypt; LI: number of labeled cells/cells scored 3 100. The distribution of the proliferative activity along the crypt was calculated as described in details in the method section. 2 Significantly different (p < 0.01) when compared with the control group with t test.
C 2015 UICC Int. J. Cancer: 137, 2270–2273 (2015) V
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proliferative activity in the upper part, has been associated with increased risk of colon cancer.19 Accordingly, we measured this parameter in our rats, that was not affected by PEG. This result can be explained with the fact that although Pirc has high proliferation compared with wt, the distribution of colon proliferation in Pirc and wt rats is similar.10 Studies in vitro and in vivo reported that PEG also acts increasing apoptosis.9,13 On the contrary, we found only a
slight, not significant increase in this parameter. Similarly, we did not find any difference in the expression of genes related to inflammation process (i.e., Cox-2, Il-1b and TNF-a). In conclusion, PEG markedly reduces colon carcinogenesis in Pirc rats; given the similarities between this experimental model and human pathology we think that these results strongly support intervention trials with PEG in subjects at risk such as FAP or CRC patients.
References 1.
2.
3.
4.
5.
6.
8.
9.
10.
11.
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