Resistance to melanoma metastases in mice selected for high acute inflammatory response

The role of innate immunity in natural resistance to tumor progression was investigated in two mouse lines, AIRmax and AIRmin, selected by bi-directional selective breeding on the basis of high or low acute inflammatory response. Compared with AIRmin, AIRmax mice were shown to be resistant to 7,12-dimethylbenz[a]anthracene (DMBA)/12-O:-tetradecanoylphorbol-13-acetate-induced skin cancers and here we demonstrate that AIRmax are also able to restrain the development of metastases upon transfer of MHC compatible, incompatible or xenogeneic melanomas. An acute inflammatory response to melanoma cells was observed in AIRmax mice only, although both lines were found to mount similar specific immune responses to melanoma antigens. The genetically selected lines therefore represent a model system to analyze the positive correlation between multiple resistance to tumorigenesis and host inflammatory responsiveness.     

Azoxymethane-induced colon tumors and aberrant crypt foci in mice of different genetic susceptibility

Azoxymethane (AOM) is an organotropic colon carcinogen that is commonly used to induce colon tumors in rodents. Unlike its parent compound, 1,2-dimethylhydrazine (DMH), a tumor susceptibility phenotype in inbred mice with respect to AOM has not been established. Thus, this study was undertaken to determine whether genetic susceptibility extends to this carcinogen. SWR/J, A/J (both susceptible to DMH carcinogenesis) and AKR/J (resistant) mice were treated with 10 mg/kg AOM i.p. once a week for 8 weeks. Twenty-five weeks after the initial injection, tumor yield was determined. With a single exception, only SWR/J and A/J mice developed tumors, with a distribution that was limited to the distal colon (16.3±1.1 and 36.4±2.4, respectively). The formation of aberrant crypt foci (ACF), putative preneoplastic lesions, was also assessed in whole-mount colons using Methylene Blue staining. Consistent with tumor multiplicity, the total number of ACF was highest in A/J mice, followed by SWR/J mice. In addition, A/J mice had a significantly greater number of large ACF (five or more crypts per foci) than the other strains. Despite the absence of colon tumors, however, AKR/J mice did develop a significant number of ACF. This finding suggests that ACF in resistant mice are persistent but do not progress to tumors.     

DNA damage in liver,colon, stomach,lung and kidney of BALB/c mice treated with 1,2-dimethylhydrazine

DNA single-strand breaks induced in various organs of BALB/c mice by treatment with a single dose of 1,2-dimethylhydrazine (DMH) were studied by means of the alkaline elution method modified in order to allow the evaluation of DNA damage in vivo with no need of radioactive prelabelling. DNA damage was detected in liver, lung, kidney, stomach and colon mucosa, with the liver showing the greatest amount of damage. Its degree was dependent on the dose and route of administration. A differential effect was evident in colon mucosa from Swiss and C57BL/6 mice which are respectively susceptible and resistant to the induction of bowel tumors by DMH. The higher degree of DNA damage found in liver in comparison with colon mucosa is consistent with the previously reported higher degree of DNA methylation, but does not correlate with the specificity of this carcinogen in inducing tumors of the large intestine in mice given repeated subcutaneous injections.     

Sequential and morphological analyses of aberrant crypt foci formation in mice of differing susceptibility to azoxymethane-induced colon carcinogenesis

Aberrant crypt foci (ACF), putative preneoplastic lesions, are early morphological changes induced by the colon carcinogen azoxymethane (AOM). Although inbred mice differ markedly in their susceptibility to AOM carcinogenesis, we have previously shown that ACF develop in both resistant and sensitive mouse strains after AOM treatment. The purpose of this study was to examine the sequential development and identify the morphological characteristics of ACF induced by AOM in the distal colon of sensitive and resistant mice. A/J (highly susceptible), SWR/J (relatively susceptible) and AKR/J (resistant) mice were treated with 10 mg/kg AOM or saline i.p. once a week for 6 weeks and were killed at 1, 2, 4, 6, 9 and 24 weeks after the last injection. The distal colons were stained with methylene blue and the numbers of ACF and tumors determined. Tumors were present as early as 4 weeks after AOM exposure in SWR/J and A/J mice and increased in frequency throughout the study in both strains. No tumors developed in the AKR/J mice. ACF, however, formed in all strains of mice. The greatest difference between susceptible and resistant strains was in the number of large ACF that developed at later time points. Furthermore, morphometric analysis revealed that A/J mice had the highest percentage of dysplastic ACF, followed by SWR/J mice. These data indicate that the difference in cancer risk from AOM may be due to the lack of progression of smaller ACF in the resistant mice and to the development of dysplasia in a higher percentage of ACF from susceptible strains.     

Mucin-depleted foci have beta-catenin gene mutations, altered expression of its protein, and are dose- and time-dependent in the colon of 1,2-dimethylhydrazine-treated rats

Mucin-depleted foci (MDF) are purported preneoplastic lesions that can be easily visualized in the unsectioned colon of carcinogen-treated rats stained with high-iron diamine alcian blue (HID-AB). In F344 rats treated twice with 150 mg/kg of 1,2-dimethylhydrazine (DMH) and sacrificed after 5, 9, 13 and 28 weeks, MDF increased over time from 5 to 13 weeks, whereas they decreased at 28 weeks, when tumors appear. MDF multiplicity (crypts/MDF) linearly increased with time. Increasing doses of DMH (100, 150 and 200 mg/kg x 2 times) caused a dose-related increase in MDF. Mutations in Ctnnb1 gene codifying for beta-catenin were identified with PCR amplification and direct sequencing in 6/15 tumors (40%), 7/28 MDF (25%) and 2/27 (7%) aberrant crypt foci (ACF) identified in HID-AB-stained colon. All mutations in tumors and MDF caused amino acid substitution, while one mutation in ACF was silent. Beta-catenin detected at membrane level by immunohistochemistry was markedly reduced in MDF and tumors and, to a lesser extent, in ACF identified with HID-AB. By contrast, nuclear localization of beta-catenin was significantly increased in MDF and tumors, while no variation was observed in ACF. Beta-catenin cytoplasmic expression was also significantly increased in MDF and tumors but to a lesser extent in ACF. In conclusion, MDF are induced dose-dependently by DMH, increase in size with time, have mutations in the beta-catenin gene and marked alterations in beta-catenin cellular localization. Since all these phenomena are considered specific steps for colon tumorigenesis, these results further support the hypothesis that MDF are cancer precursors and can be proposed as endpoints in short-term carcinogenesis experiments.     

Induction of tumors in the colon and liver of the immunodeficient (SCID) mouse by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ)-modulation by long-chain fatty acids

We have recently shown that immunodeficient (SCID) mice, which lack functional T and B cells, are highly susceptible to low dose site specific induction of colon aberrant crypt foci (ACF), surrogates for colon tumors, by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ). To test whether long-term exposure to a high dose in the diet might prove carcinogenic to the SCID mouse colon, in contrast to other mice strains tested to date, the compound was administered at 300 p.p.m. in the diet to female 6-7-week-old SCID mice for 32 weeks. IQ induced high numbers of ACF, hyperplastic polyps, dysplasia, and colon adenomas, as well as hepatocellular altered foci and liver adenomas. Induction of colon tumors did not correlate with the main sites where ACF developed, the proximal colon, however, being seen mainly in the mid and distal colon. Induction of colon tumors correlated significantly with the incidence of dysplasia, crypt height, the mitotic index, cell proliferation and numbers of 8-hydroxydeoxyguanosine (8-OHdG)-positive cells in the colon crypt, particularly in mid and distal colon. Administration of 20% omega-6 polyunsaturated fatty acids (corn oil), omega-3 polyunsaturated fatty acids (perilla oil), or monounsaturated fatty acids (olive oil) simultaneously with IQ in the diet resulted in: (i) inhibition of colon and liver tumor induction by corn and perilla oil, whereas olive oil showed no effects; (ii) no reduction in total numbers of ACF by corn oil or perilla oil but significant suppression in the olive oil treated group; (iii) inhibition of tumor development particularly by omega-3 polyunsaturated fatty acids in perilla oil, correlating significantly with decreased cell proliferation in both colon and liver and a marked decrease in crypt heights and mitotic indices. Selective reduction in the numbers of 8-OHdG-positive nuclei, mainly in the middle and distal colon crypts, was also found to correlate with tumor inhibition. Thus, the results indicate carcinogenicity of IQ in the colon of the SCID mouse and preventive effects of polyunsaturated fatty acids.     

Genetic heterogeneity of inflammatory response and skin tumorigenesis in phenotypically selected mouse lines

Non-inbred AIR (AIRmax, AIRmin) and Car (Car-S, Car-R) mouse lines were generated from the same eight inbred mice through bidirectional selective breeding for acute inflammatory response and for susceptibility to two-stage skin tumorigenesis, respectively. Because AIR lines also showed a differential predisposition to skin tumorigenesis and Car lines differed in the extent of inflammatory response, we carried out genome-wide association studies using SNP arrays to identify the genetic elements affecting skin tumor susceptibility and inflammatory response in AIR and Car lines. We found that the phenotypic outcome reflects a specific genetic profile in each mouse line, suggesting that distinct genetic elements, selected by differential genetic drifts, and exerting pleiotropic effects in each mouse population, control the skin tumor susceptibility and inflammatory response phenotypes. These findings point to the complex link between skin tumor susceptibility and inflammatory response in mice.     

Effect of disulfiram on 1,2-dimethlhydrazine- and azoxymethane- induced aberrant crypt foci

We have previously reported a method for visualizing the mucosalsurface of fixed unsectioned rodent colons at the crypt leveland have identified lesions, termed aberrant crypt foci (ACF),in the colons of carcinogen-treated rodents. We hypothesizedthat ACF represent the precursor lesions (PL) of colon cancer.In the present study, the effect of feeding disulfiram (DSF)added to a semi-synthetic diet (0.5% or 1% by wt) on 1,2-dimethylhydrazine(DMH) and azoxymethane (AOM) induced ACF was investigated. DSFhas been shown to inhibit DMH and AOM-induced colon cancer.Therefore, it was reasoned that if ACF represent PL then theirinduction and growth should also be inhibited by DSF. CF1 femalemice were randomly divided into three groups of 30 each. Group1 was fed a diet containing 1% DSF for 9 days prior to and 1day after receiving a single i.p. injection of either DMH, AOMor saline. Group 2 was fed a diet containing 1% DSF for 9 daysprior to and 14 days after receiving a single i.p. injectionof DMH, AOM or saline, whereas group 3 received control dietthroughout the experimental duration. All animals were killed5 weeks after receiving the injections. It was observed thatfeeding DSF, for 9 days prior to and for either 1 day or 14days after the administration of a single injection of DMH,resulted in a complete inhibition of ACF. DSF feeding for 9days prior to and 1 day after AOM injection resulted in a significantlygreater number of ACF compared to the control group (12 ? 2.3vs 7.2 ? 1.2); whereas DSF feeding for a longer duration (i.e.9 days prior to and 14 days after AOM treatment) was associatedwith a significantly lower number of ACF compared to those fedDSF for only one day after AOM treatment (4.1 ? 0.6 vs 12.4? 2.3) and a lower number compared to the control group (4.1? 0.6 vs 7.2 ? 1.2).     

Inhibition of 1,2-dimethylhydrazine-induced colon tumorigenesis in Balb/c mice by dehydroepiandrosterone

Long-term administration of dehydroepiandrosterone (DHEA) toBalb/c mice significantly inhibits the rate of appearance of1,2-dimethylhydrazine (DMH)-induced macroscopic colon and analrumors and microscopic precursor and malignant lesions. Thesteroid, which has previously been shown to inhibit spontaneousbreast cancer and chemically induced lung tumors in mice, mayfind application as a chemopreventive in individuals at highrisk for developing colon cancer.     

Anti-proliferative and Apoptotic Effects of Basella rubra (L.) Against 1, 2-Dimethyl Hydrazine-induced Colon Carcinogenesis in Rats

Colorectal cancer is a very prevalent diagnosed cancer. The current study was performed in order to examine the role of BRAE (Basella rubra aqueous extract) in regulating aberrant crypt foci (ACF) formation, cell proliferation and inhibition of apoptosis in a colon carcinogenesis model in male Wistar rats. Rats were randomly allocated into six groups. Group I served as control, and group II acted as a drug control administered BRAE (250mg/kg b.w.) orally for 30 weeks. Rats in group III-VI were given subcutaneous injections of DMH (25mg/kg b.w. weekly) for 15 weeks to initiate colon carcinogenesis. Those in group IV and VI were administered BRAE along with DMH injections. Rats in group V were administered with BRAE after cessation of DMH injection. After 30 weeks of experimental period colons were obtained from experimental groups and analyzed for ACF incidence, argyrophilic nucleolar organizing region- associated proteins (AgNOR) count, histopathological and immunohistochemical changes. Only in DMH exposed groups were ACF and AgNOR numbers increased. Administration of BRAE appreciably decreased the numbers of ACF and AgNOR in BRAE treated groups. Histopathological findings revealed a high level of dysplastic changes with decreased number of goblet cells found only in only DMH injected rats. Administration of BRAE in treated group rats reversed these changes. Expression markers for cell proliferation (PCNA and Ki67) were elevated in DMH treated rats, but reduced with BRAE treatement. This expression was reversed with apoptosis markers (p53 and Caspase-3). Thus the results results of the present study were found to be significant and confirmed the potential efficacy of BRAE against colon carcinogenesis.     

Formation of Early Lesions in 1,2-dimethylhydrazine-induced Colon Carcinogenesis in Rats

Aberrant crypt foci (ACF) are recognized as preneoplastic lesions for colon cancer, and ACF in rodents arewidely used as an intermediate biomarker to predict tumorigenicity in the colon. However, a lack of correlationsbetween the formation of ACF and the development of colonic tumors has been reported in several studies. Forexample, 2-(carboxyphenyl) retinamide (2-CPR) and genistein were reported to inhibit the carcinogen-inducedformation of ACF, whereas both of them were later found to enhance colon tumorigenesis in rats treated withazoxymethane (AOM). Recently, we have identified β-catenin-accumulated crypts (BCAC) in the colon of ratsshortly after administration of AOM, and provided evidence that these are independent early lesions of classicalACF, and BCAC might be direct precursors for colon cancers. In the present study, we performed a comparativeanalysis of the modifying effects of 2-CPR and genistein on 1,2-dimethylhydrazine (DMH)-induced BCAC andACF in male F344 rats. Dietary administration of 2-CPR (315 ppm) significantly reduced the total number,multiplicity and size of ACF in DMH-exposed colonic mucosa, while genistein (250 ppm) had no significant effectson DMH-induced ACF formation. In contrast, both of 2-CPR and genistein significantly enhanced the multiplicityand size of DMH-induced BCAC when compared with DMH alone group. In addition, both 2-CPR and genisteinsignificantly increased the proliferating cell nuclear antigen (PCNA) index preferentially in BCAC. Togetherwith previous findings that 2-CPR and genistein are tumor promoters in the colon, our results support the conceptthat BCAC are precursors of colon tumors and suggest that these lesions are more reliable short-term biomarkersfor colon carcinogenesis in rodents than ACF.     

Pre-neoplastic lesion, mucin-depleted foci, reveals de novo high-grade dysplasia in rat colon carcinogenesis

Aberrant crypt foci (ACF) and mucin-depleted foci (MDF) have recently been recognized as pre-neoplastic lesions in the colon of carcinogen-treated rodents. In the present study, we analyzed the sequential development of ACF and MDF histopathologically in the colon of rats from 5 to 40 weeks after DMH treatment. The numbers of ACF per colon increased over time during the experiment, and were much higher than the number in tumors, while the number of MDF per colon remained unchanged from the early stage (the 5th week after carcinogen exposure), and approximate to those in tumors. The incidence of ACF, which was much higher than that of tumors, also increased gradually in a time-dependent manner. The incidence of MDF, however, was similar to that of tumors and did not change significantly during the whole experiment. No lesion as dysplasia with high-grade (DHG) or adenocarcinoma (AC) were found in any large ACF from the 5th to 40th week histopathologically, whereas all of the large MDF showed DHG or AC features. Even at 5 weeks, MDF showed features of DHG. We classified these into two forms of MDF: flat and protruded MDF. At 40 weeks, the number of flat MDF per colon decreased significantly compared with that at 20 weeks (p<0.05), however, the number of protruded MDF per colon increased (p<0.01), and the percentage of DHG in a protruded MDF lesion decreased but that of AC increased remarkably. In conclusion, MDF may develop into cancer through the so-called 'de?novo cancer' pathway.     

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine increases the numbers of tumors, cystic crypts and aberrant crypt foci in multiple intestinal neoplasia mice

The multiple intestinal neoplasia (Min) mice have a mutation in the murine adenomatous polyposis coli (Apc) gene rendering them highly susceptible to spontaneous intestinal adenoma formation, similar to the familial adenomatous polyposis (FAP) syndrome in humans. We studied whether the most abundant mutagenic heterocyclic amine isolated from cooked food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), could influence early intestinal neoplasia in C57BL/6J-Min/+ and C57BL/6J- +/+ (wild-type) mice of both sexes. PhIP was given in 4 weekly i.p. injections of 50 mg/kg. Ten weeks after the start of the experiment, PhIP had significantly increased the numbers of small tumors and cystic crypts in the proximal section of the small intestine in male Min/+ mice, and the numbers of aberrant crypt foci (ACF) in the large intestines of both males and females. The effects of PhIP were more pronounced in male than in female Min/+ mice. In +/+ mice, no tumors or cystic crypts in the small intestine, and no tumors and only a very few ACF in the large intestine, were induced by PhIP. These results show that a substance frequently present in the human diet is able to enhance the neoplastic process induced by a genetic lesion, which is also commonly found both in inherited and sporadic colon carcinomas in humans.      

Protective effect of beta-carotene against colon tumors in mice

The effect of dietary beta-carotene on colon carcinogenesis induced by 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8] was studied in female inbred Swiss Webster (ICR) mice. At age 10 weeks and continuing throughout the experiment, mice received diets consisting mainly of natural foods (laboratory chow) and containing 2 or 22 mg beta-carotene/kg. At age 15 weeks they received 7 weekly sc injections of DMH (total dose: 196 mg DMH X diHCI/kg body wt). When autopsied 31 weeks after the first DMH injection, the incidence (percent of mice with tumors) and multiplicity (number of tumors/tumor-bearing mouse) of colon tumors were reduced by half in the mice supplemented with beta-carotene. There was a much greater decrease in adenocarcinomas than in adenomas. Mice observed for 13 additional weeks revealed that the mortality rate, due largely or wholly to colon cancer, was only about half in supplemented mice. Mice sacrificed 12 weeks after the first dose of DMH (i.e., well before tumors appeared) showed mild colon mucosal hyperplasia. beta-Carotene supplementation, however, did not alter this, indicating that the protective effect against colon cancer may have occurred at a late stage of carcinogenesis.     

Relationship between dose, time, and tumor yield in mouse dimethylhydrazine-induced colon tumorigenesis

Colorectal tumor yield and volume data were obtained using 355 CF1 mice serially sacrificed up to 84 weeks following various dose levels of the carcinogen 1,2-dimethylhydrazine dichloride (DMH). Several conclusions were reached: (a) With increasing doses of DMH, there was an increased tumor yield and decreased latency period. (b) With repeated doses, there was a rapidly cumulative tumor yield. (c) New tumors continued to accumulate in the colon and rectum even at long intervals after the DMH treatments. This was substantiated by a positive correlation between the number of tumors per colon and the delay after DMH. In addition, when several tumors were present in the same mouse, their sizes were graded rather than uniform. These observations are consistent with a 2- or multi-step carcinogenesis mechanism. The latter implies that DMH induces a permanent transmissible alteration within some cells which thereafter will be at risk for further alterations capable of initiating cancer growth.     

Augmentation of 1,2-dimethylhydrazine-induced colon cancer by experimental colitis in mice: role of dietary vitamin E

Because ulcerative colitis predisposes to colonic cancer, for determination of the effect of colitis on experimental colon carcinogenesis, rectal instillations of peptides that attract and activate neutrophils were used to induce colitis in CD-1 (ICR) BR mice receiving 20 weekly injections of the carcinogen 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8]. From week 4 through week 15 of DMH injections, twice-weekly enemas of formyl-norleucyl-leucyl-phenylalanine were given to DMH-treated mice. The effect of the antioxidant vitamin E in the diet (1,750 IU/kg diet) was studied in another group of mice treated with DMH and having colitis. Four weeks after DMH was discontinued, cancer occurred in 9 of 28 (32%) animals with DMH plus control enemas, in 22 of 29 (76%) animals with DMH plus colitis (P = .001), and in 16 of 28 (57%) animals with DMH plus colitis plus supplemental vitamin E (P = .11 compared with the group with DMH and colitis). Colitis enhances DMH-induced colonic carcinogenesis.     

Predominant T helper type 2-inflammatory responses promote murine colon cancers

Colon cancer is one of the most serious complications of inflammatory bowel diseases, especially ulcerative colitis (UC). Previous studies have shown that characteristic immunological event during inflammation in UC is the expression of T helper-type 2 (Th2) cell-derived cytokines. In this study, we investigated the influence of a predominant Th2-type cytokine response in colitis on carcinogen-induced colon tumors. Wild type (WT), interferon gamma (IFN-gamma) gene deficient (-/-) [Th2 dominant] or interleukin (IL)-4(-/-) [Th1-dominant] mice of BALB/c background were used in this study. To compare tumor formation, mice were given the carcinogen azoxymethane (AOM) and intrarectal administration of trinitrobenzene sulfonic acid (TNBS), to induce colitis. Thirty-three weeks after initial treatment, the total colon was examined. When IFN-gamma(-/-) mice were treated with AOM and TNBS, significantly higher number of tumors were seen (8.4 +/- 1.7) than in WT (3.3 +/- 2.9) or IL-4(-/-) (3.1 +/- 3.4) mice, which received identical treatments. A separate set of experiment, using less doses of AOM and TNBS also showed the higher frequency of tumor formation in IFN-gamma(-/-) mice than in IL-4(-/-) mice. Histologically, the tumors were well- or moderately-differentiated adenocarcinomas. No invasion into the submucosal or serosal layers of the intestine was seen. In immunohistological staining, some tumors in IFN-gamma(-/-) mice showed distinct nuclear expression of beta-catenin, in contrast to the strong membrane staining seen in tumors of IL-4(-/-) mice. In conclusion, colonic inflammation associated with Th2-dominant cytokine responses enhanced the formation of malignant neoplasms.