Oxidative stress accelerates intestinal tumorigenesis by enhancing 8-oxoguanine-mediated mutagenesis in MUTYH-deficient mice.

Oxidative stress-induced DNA damage and its repair systems are related to cancer etiology; however, the molecular basis triggering tumorigenesis is not well understood. Here, we aimed to explore the causal relationship between oxidative stress, somatic mutations in pre-tumor-initiated normal tissues, and tumor incidence in the small intestines of MUTYH-proficient and MUTYH-deficient mice. MUTYH is a base excision repair enzyme associated with human colorectal cancer. Mice were administered different concentrations of potassium bromate (KBrO3; an oxidizing agent)-containing water for 4 wk for mutagenesis studies or 16 wk for tumorigenesis studies. All Mutyh -/- mice treated with >0.1% KBrO3 developed multiple tumors, and the average tumor number increased dose dependently. Somatic mutation analysis of Mutyh -/-/rpsL transgenic mice revealed that G:C  > T:A transversion was the only mutation type correlated positively with KBrO3 dose and tumor incidence. These mutations preferentially occurred at 5'G in GG and GAA sequences in rpsL This characteristic mutation pattern was also observed in the genomic region of Mutyh -/- tumors using whole-exome sequencing. It closely corresponded to signature 18 and SBS36, typically caused by 8-oxo-guanine (8-oxoG). 8-oxoG-induced mutations were sequence context dependent, yielding a biased amino acid change leading to missense and stop-gain mutations. These mutations frequently occurred in critical amino acid codons of known cancer drivers, Apc or Ctnnb1, known for activating Wnt signal pathway. Our results indicate that oxidative stress contributes to increased tumor incidence by elevating the likelihood of gaining driver mutations by increasing 8-oxoG-mediated mutagenesis, particularly under MUTYH-deficient conditions.

Endogenous ROS production in early differentiation state suppresses endoderm differentiation via transient FOXC1 expression.

Oxidative stress plays a pivotal role in the differentiation and proliferation of cells and programmed cell death. However, studies on the role of oxidative stress in differentiation have mainly employed the detection of reactive oxygen species (ROS) during differentiation or generated by ROS inducers. Therefore, it is difficult to clarify the significance of endogenous ROS production in the differentiation of human cells. We developed a system to control the intracellular level of ROS in the initial stage of differentiation in human iPS cells. By introducing a specific substitution (I69E) into the SDHC protein, a component of the mitochondrial respiratory chain complex, the endogenous ROS level increased. This caused impaired endoderm differentiation of iPS cells, and this impairment was reversed by overproduction of mitochondrial-targeted catalase, an anti-oxidant enzyme. Expression of tumor-related FOXC1 transcription factor increased transiently as early as 4 h after ROS-overproduction in the initial stage of differentiation. Knockdown of FOXC1 markedly improved impaired endoderm differentiation, suggesting that endogenous ROS production in the early differentiation state suppresses endoderm differentiation via transient FOXC1 expression.

Characteristic mutations induced in the small intestine of Msh2-knockout gpt delta mice.

BACKGROUND: Base pair mismatches in genomic DNA can result in mutagenesis, and consequently in tumorigenesis. To investigate how mismatch repair deficiency increases mutagenicity under oxidative stress, we examined the type and frequency of mutations arising in the mucosa of the small intestine of mice carrying a reporter gene encoding guanine phosphoribosyltransferase (gpt) and in which the Msh2 gene, which encodes a component of the mismatch repair system, was either intact (Msh2+/+::gpt/0; Msh2-bearing) or homozygously knockout (KO) (Msh2-/-::gpt/0; Msh2-KO). RESULTS: Gpt mutant frequency in the small intestine of Msh2-KO mice was about 10 times that in Msh2-bearing mice. Mutant frequency in the Msh2-KO mice was not further enhanced by administration of potassium bromate, an oxidative stress inducer, in the drinking water at a dose of 1.5 g/L for 28 days. Mutation analysis showed that the characteristic mutation in the small intestine of the Msh2-KO mice was G-to-A transition, irrespective of whether potassium bromate was administered. Furthermore, administration of potassium bromate induced mutations at specific sites in gpt in the Msh2-KO mice: G-to-A transition was frequently induced at two known sites of spontaneous mutation (nucleotides 110 and 115, CpG sites) and at nucleotides 92 and 113 (3'-side of 5'-GpG-3'), and these sites were confirmed to be mutation hotspots in potassium bromate-administered Msh2-KO mice. Administration of potassium bromate also induced characteristic mutations, mainly single-base deletion and insertion of an adenine residue, in sequences of three to five adenine nucleotides (A-runs) in Msh2-KO mice, and elevated the overall proportion of single-base deletions plus insertions in Msh2-KO mice. CONCLUSIONS: Our previous study revealed that administration of potassium bromate enhanced tumorigenesis in the small intestine of Msh2-KO mice and induced G-to-A transition in the Ctnnb1 gene. Based on our present and previous observations, we propose that oxidative stress under conditions of mismatch repair deficiency accelerates the induction of single-adenine deletions at specific sites in oncogenes, which enhances tumorigenesis in a synergistic manner with G-to-A transition in other oncogenes (e.g., Ctnnb1).

Oxidative-stress-driven mutagenesis in the small intestine of the gpt delta mouse induced by oral administration of potassium bromate.

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5'-TGAA-3' sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.

Resveratrol and its Related Polyphenols Contribute to the Maintenance of Genome Stability.

Genomic destabilisation is associated with the induction of mutations, including those in cancer-driver genes, and subsequent clonal evolution of cells with abrogated defence systems. Such mutations are not induced when genome stability is maintained; however, the mechanisms involved in genome stability maintenance remain elusive. Here, resveratrol (and related polyphenols) is shown to enhance genome stability in mouse embryonic fibroblasts, ultimately protecting the cells against the induction of mutations in the ARF/p53 pathway. Replication stress-associated DNA double-strand breaks (DSBs) that accumulated with genomic destabilisation were effectively reduced by resveratrol treatment. In addition, resveratrol transiently stabilised the expression of histone H2AX, which is involved in DSB repair. Similar effects on the maintenance of genome stability were observed for related polyphenols. Accordingly, we propose that polyphenol consumption can contribute to the suppression of cancers that develop with genomic instability, as well as lifespan extension.

ROS control in human iPS cells reveals early events in spontaneous carcinogenesis.

Reactive oxygen species (ROS) generated during cellular respiration oxidize various cellular constituents, which cause carcinogenesis. Because most studies on the role of ROS in carcinogenesis have mainly been performed using tumor-derived cell lines, which harbor various types of mutation, it has been difficult to determine the molecular details that lead to cancer formation. To overcome this difficulty, we established human-induced pluripotent stem cell lines in which the intracellular ROS levels are controlled at various differentiation stages by manipulating the ROS-yielding mitochondria. By introducing a specific amino acid substitution (I69E) into the succinate dehydrogenase complex, subunit C protein, a component of mitochondrial respiratory chain complex II, the ROS level increased considerably. When ROS-overproducing cells at the early stage of endoderm differentiation were subcutaneously inoculated into the backs of nude mice, we observed tumor formation. These tumor-initiating cells were subjected to a comprehensive analysis by RNA sequencing. It was revealed that tumor-initiating cells showed 27 upregulated transcripts compared with control cells. The newly identified genes include those coding for PAX8 and FOSB (transcription factors) as well as FGF22, whose expressions are known to increase in developing embryos. These results suggest that these genes may play a pivotal role in cancer formation at the very early stages of cell differentiation.

Replication stress triggers microsatellite destabilization and hypermutation leading to clonal expansion in vitro.

Mismatch repair (MMR)-deficient cancers are characterized by microsatellite instability (MSI) and hypermutation. However, it remains unclear how MSI and hypermutation arise and contribute to cancer development. Here, we show that MSI and hypermutation are triggered by replication stress in an MMR-deficient background, enabling clonal expansion of cells harboring ARF/p53-module mutations and cells that are resistant to the anti-cancer drug camptothecin. While replication stress-associated DNA double-strand breaks (DSBs) caused chromosomal instability (CIN) in an MMR-proficient background, they induced MSI with concomitant suppression of CIN via a PARP-mediated repair pathway in an MMR-deficient background. This was associated with the induction of mutations, including cancer-driver mutations in the ARF/p53 module, via chromosomal deletions and base substitutions. Immortalization of MMR-deficient mouse embryonic fibroblasts (MEFs) in association with ARF/p53-module mutations was ~60-fold more efficient than that of wild-type MEFs. Thus, replication stress-triggered MSI and hypermutation efficiently lead to clonal expansion of cells with abrogated defense systems.

Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing.

Repeat destabilisation is variously associated with human disease. In neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. However, several discrepancies have been pointed out. Firstly, the MSI+ phenotype is not uniform in human neoplasms. Established classification utilises the frequency of microsatellite changes, i.e. MSI-H (high) and -L (low), the former regarded as an authentic MMR-defective phenotype. In addition, we have observed the qualitatively distinct modes of MSI, i.e. Type A and Type B. One discrepancy we previously pointed out is that tumours occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumours (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). In the present study, MSH2 mutations reported in Lynch syndrome (LS) kindred have been introduced into HeLa cells using the CRISPR/Cas9 system. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. Nevertheless, microsatellites were not markedly destabilised as in MSI-H tumours occurring in LS patients, and all the observed alterations were uniformly Type A, which confirms the results in mice. Our findings suggest added complexities to the molecular mechanisms underlying repeat destabilisation in human genome.

Mismatch repair dependence of replication stress-associated DSB recognition and repair.

Most cancers develop with one of two types of genomic instability, namely, chromosomal instability (CIN) or microsatellite instability (MSI). Both are induced by replication stress-associated DNA double-strand breaks (DSBs). The type of genomic instability that arises is dependent on the choice of DNA repair pathway. Specifically, MSI is induced via a PolQ-dependent repair pathway called microhomology-mediated end joining (MMEJ) in a mismatch repair (MMR)-deficient background. However, it is unclear how the MMR status determines the choice of DSB repair pathway. Here, we show that replication stress-associated DSBs initially targeted by the homologous recombination (HR) system were subsequently hijacked by PolQ-dependent MMEJ in MMR-deficient cells, but persisted as HR intermediates in MMR-proficient cells. PolQ interacting with MMR factors was effectively loaded onto damaged chromatin in an MMR-deficient background, in which merged MRE11/γH2AX foci also effectively formed. Thus, the choice of DNA repair pathway according to the MMR status determines whether CIN or MSI is induced.

Celecoxib and 2,5-dimethylcelecoxib inhibit intestinal cancer growth by suppressing the Wnt/ß-catenin signaling pathway.

We previously reported that celecoxib, a selective COX-2 inhibitor, strongly inhibited human colon cancer cell proliferation by suppressing the Wnt/ß-catenin signaling pathway. 2,5-Dimethylcelecoxib (DM-celecoxib), a celecoxib analog that does not inhibit COX-2, has also been reported to have an antitumor effect. In the present study, we elucidated whether DM-celecoxib inhibits intestinal cancer growth, and its underlying mechanism of action. First, we compared the effect of DM-celecoxib with that of celecoxib on the human colon cancer cell lines HCT-116 and DLD-1. 2,5-Dimethylcelecoxib suppressed cell proliferation and inhibited T-cell factor 7-like 2 expression with almost the same strength as celecoxib. 2,5-Dimethylcelecoxib also inhibited the T-cell factor-dependent transcription activity and suppressed the expression of Wnt/ß-catenin target gene products cyclin D1 and survivin. Subsequently, we compared the in vivo effects of celecoxib and DM-celecoxib using the Mutyh-/- mouse model, in which oxidative stress induces multiple intestinal carcinomas. Serum concentrations of orally administered celecoxib and DM-celecoxib elevated to the levels enough to suppress cancer cell proliferation. Repeated treatment with celecoxib and DM-celecoxib markedly reduced the number and size of the carcinomas without showing toxicity. These results suggest that the central mechanism for the anticancer effect of celecoxib derivatives is the suppression of the Wnt/ß-catenin signaling pathway but not the inhibition of COX-2, and that DM-celecoxib might be a better lead compound candidate than celecoxib for the development of novel anticancer drugs.

BUBR1 Insufficiency in Mice Increases their Sensitivity to Oxidative Stress.

BACKGROUND/AIM: Budding uninhibited by benzimidazole-related 1 (BUBR1) plays an important role in the spindle assembly checkpoint to prevent chromosome missegregation and aneuploidy during mitosis. We previously generated mutant mice that express BUBR1 at only 20% of the normal level (BubR1L/L mice). Here, we examined the effect of low BUBR1 expression on oxidative stress-induced carcinogenesis in mice. MATERIALS AND METHODS: We orally administered either a potassium bromate (KBrO3) solution (2 g/l) or tap water to BubR1L/L and wild-type (BubR1+/+)mice for 16 weeks and examined the subsequent incidence of tumours. RESULTS: KBrO3-treated BubR1L/L mice showed significantly higher mortality than the KBrO3-treated BubR1+/+ and control tap water-treated mice (p=0.0082). Histopathological and immunohistochemical analyses revealed that the spleens of surviving BubR1L/L mice were occupied by non-B-, non-T-cells with high proliferative potential. CONCLUSION: Our results indicate that low BUBR1 expression increases oxidative stress-induced mortality in mice, possibly caused by splenic neoplasms.

Nucleotide excision repair of oxidised genomic DNA is not a source of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a widely measured biomarker of oxidative stress. It has been commonly assumed to be a product of DNA repair, and therefore reflective of DNA oxidation. However, the source of urinary 8-oxodGuo is not understood, although potential confounding contributions from cell turnover and diet have been ruled out. Clearly it is critical to understand the precise biological origins of this important biomarker, so that the target molecule that is oxidised can be identified, and the significance of its excretion can be interpreted fully. In the present study we aimed to assess the contributions of nucleotide excision repair (NER), by both the global genome NER (GG-NER) and transcription-coupled NER (TC-NER) pathways, and sanitisation of the dGTP pool (e.g. via the activity of the MTH1 protein), on the production of 8-oxodGuo, using selected genetically-modified mice. In xeroderma pigmentosum A (XPA) mice, in which GG-NER and TC-NER are both defective, the urinary 8-oxodGuo data were unequivocal in ruling out a contribution from NER. In line with the XPA data, the production of urinary 8-oxodGuo was not affected in the xeroderma pigmentosum C mice, specifically excluding a role of the GG-NER pathway. The bulk of the literature supports the mechanism that the NER proteins are responsible for removing damage to the transcribed strand of DNA via TC-NER, and on this basis we also examined Cockayne Syndrome mice, which have a functional loss of TC-NER. These mice showed no difference in urinary 8-oxodGuo excretion, compared to wild type, demonstrating that TC-NER does not contribute to urinary 8-oxodGuo levels. These findings call into question whether genomic DNA is the primary source of urinary 8-oxodGuo, which would largely exclude it as a biomarker of DNA oxidation. The urinary 8-oxodGuo levels from the MTH1 mice (both knock-out and hMTH1-Tg) were not significantly different to the wild-type mice. We suggest that these findings are due to redundancy in the process, and that other enzymes substitute for the lack of MTH1, however the present study cannot determine whether or not the 2'-deoxyribonucleotide pool is the source of urinary 8-oxodGuo. On the basis of the above, urinary 8-oxodGuo is most accurately defined as a non-invasive biomarker of oxidative stress, derived from oxidatively generated damage to 2'-deoxyguanosine.

PSMC5, a 19S Proteasomal ATPase, Regulates Cocaine Action in the Nucleus Accumbens.

ΔFosB is a stable transcription factor which accumulates in the nucleus accumbens (NAc), a key part of the brain's reward circuitry, in response to chronic exposure to cocaine or other drugs of abuse. While ΔFosB is known to heterodimerize with a Jun family member to form an active transcription factor complex, there has not to date been an open-ended exploration of other possible binding partners for ΔFosB in the brain. Here, by use of yeast two-hybrid assays, we identify PSMC5-also known as SUG1, an ATPase-containing subunit of the 19S proteasomal complex-as a novel interacting protein with ΔFosB. We verify such interactions between endogenous ΔFosB and PSMC5 in the NAc and demonstrate that both proteins also form complexes with other chromatin regulatory proteins associated with gene activation. We go on to show that chronic cocaine increases nuclear, but not cytoplasmic, levels of PSMC5 in the NAc and that overexpression of PSMC5 in this brain region promotes the locomotor responses to cocaine. Together, these findings describe a novel mechanism that contributes to the actions of ΔFosB and, for the first time, implicates PSMC5 in cocaine-induced molecular and behavioral plasticity.

Differentiation-inducing factor-3 inhibits intestinal tumor growth in vitro and in vivo.

Differentiation-inducing factor-1 (DIF-1) produced by Dictyostelium discoideum strongly inhibits the proliferation of various types of cancer cells by suppression of the Wnt/ß-catenin signal transduction pathway. In the present study, we examined the effect of differentiation-inducing factor-3 (DIF-3), a monochlorinated metabolite of DIF-1 that is also produced by D. discoideum, on human colon cancer cell lines HCT-116 and DLD-1. DIF-3 strongly inhibited cell proliferation by arresting the cell cycle at the G0/G1 phase. DIF-3 reduced the expression levels of cyclin D1 and c-Myc by facilitating their degradation via activation of GSK-3ß in a time and dose-dependent manner. In addition, DIF-3 suppressed the expression of T-cell factor 7-like 2, a key transcription factor in the Wnt/ß-catenin signaling pathway, thereby reducing the mRNA levels of cyclin D1 and c-Myc. Subsequently, we examined the in vivo effects of DIF-3 in Mutyh(-/-) mice with oxidative stress-induced intestinal cancers. Repeated oral administration of DIF-3 markedly reduced the number and size of cancers at a level comparable to that of DIF-1. These data suggest that DIF-3 inhibits intestinal cancer cell proliferation in vitro and in vivo, probably by mechanisms similar to those identified in DIF-1 actions, and that DIF-3 may be a potential novel anti-cancer agent.

UV-induced mutations in epidermal cells of mice defective in DNA polymerase η and/or ι.

Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to sunlight-induced skin cancer, which is caused by deficiency in DNA polymerase η (Polη). Polη catalyzes accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions. DNA polymerase ι (Polι) is a paralog of Polη that has been suggested to participate in TLS past UV-induced lesions, but its function in vivo remains uncertain. We have previously reported that Polη-deficient and Polη/Polι double-deficient mice showed increased susceptibility to UV-induced carcinogenesis. Here, we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Polη- and/or Polι-deficient mice. While Polη-deficient mice showed significantly higher UV-induced mutation frequencies than wild-type mice, Polι deficiency did not influence the frequencies in the presence of Polη. Interestingly, the frequencies in Polη/Polι double-deficient mice were statistically lower than those in Polη-deficient mice, although they were still higher than those of wild-type mice. Sequence analysis revealed that most of the UV-induced mutations in Polη-deficient and Polη/Polι double-deficient mice were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T pairs associated with Polη deficiency suggests that Polη contributes to accurate TLS past both thymine- and cytosine-containing dimers in vivo. A significant decrease in G:C to A:T transition in Polη/Polι double-deficient mice when compared with Polη-deficient mice suggests that Polι is involved in error-prone TLS past cytosine-containing dimers when Polη is inactivated.

BubR1 insufficiency inhibits neointimal hyperplasia through impaired vascular smooth muscle cell proliferation in mice.

OBJECTIVE: BubR1, a cell cycle-related protein, is an essential component of the spindle checkpoint that regulates cell division. Mice with BubR1 expression reduced to 10% of the normal level display a phenotype characterized by progeria; however, the involvement of BubR1 in vascular diseases is still unknown. We generated mice in which BubR1 expression was reduced to 20% (BubR1(L/L) mice) of that in wild-type mice (BubR1(+/+)) to investigate the effects of BubR1 on arterial intimal hyperplasia. APPROACH AND RESULTS: Ten-week-old male BubR1(L/L) and age-matched wild-type littermates (BubR1(+/+)) were used in this study. The left common carotid artery was ligated, and histopathologic examinations were conducted 4 weeks later. Bone marrow transplantation was also performed. Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aorta to examine cell proliferation, migration, and cell cycle progression. Severe neointimal hyperplasia was observed after artery ligation in BubR1(+/+) mice, whereas BubR1(L/L) mice displayed nearly complete inhibition of neointimal hyperplasia. Bone marrow transplantation from all donors did not affect the reconstitution of 3 hematopoietic lineages, and neointimal hyperplasia was still suppressed after bone marrow transplantation from BubR1(+/+) mice to BubR1(L/L) mice. VSMC proliferation was impaired in BubR1(L/L) mice because of delayed entry into the S phase. VSMC migration was unaffected in these BubR1(L/L) mice. p38 mitogen-activated protein kinase-inhibited VSMCs showed low expression of BubR1, and BubR1-inhibited VSMCs showed low expression of p38. CONCLUSIONS: BubR1 may represent a new target molecule for treating pathological states of vascular remodeling, such as restenosis after angioplasty.

Abnormality in Wnt signaling is causatively associated with oxidative stress-induced intestinal tumorigenesis in MUTYH-null mice.

MUTYH is a DNA glycosylase that excises adenine paired with 8-oxoguanine to prevent mutagenesis in mammals. Biallelic germline mutations of MUTYH have been found in patients predisposed to a recessive form of familial adenomatous polyposis (MAP: MUTYH-associated polyposis). We previously reported that Mutyh-deficient mice showed a high susceptibility to spontaneous and oxidative stress-induced intestinal adenoma/carcinoma. Here, we performed mutation analysis of the tumor-associated genes including Apc, Ctnnb1, Kras and Trp53 in the intestinal tumors of Mutyh-deficient mice. In the 62 tumors, we identified 25 mutations in Apc of 18 tumors and 36 mutations in Ctnnb1 of 36 tumors. Altogether, 54 out of the 62 tumors (87.1%) had a mutation in either Apc or Ctnnb1; no tumor displayed mutations simultaneously in the both genes. Similar to MAP, 60 out of 61 mutations (98.3%) were identified as G:C to T:A transversions of which 85% occurred at either AGAA or TGAA sequences. Immunohistochemical analyses revealed the accumulation of ß-catenin in the nuclei of tumors. No mutation was found in either Kras or Trp53 in the tumors. These results indicate that the uncontrolled activation of Wnt signaling pathway is causatively associated with oxidative stress-induced intestinal tumorigenesis in the Mutyh-deficient mice.

DIF-1 inhibits tumor growth in vivo reducing phosphorylation of GSK-3ß and expressions of cyclin D1 and TCF7L2 in cancer model mice.

We reported that differentiation-inducing factor-1 (DIF-1), synthesized by Dictyostelium discoideum, inhibited proliferation of various tumor cell lines in vitro by suppressing the Wnt/ß-catenin signaling pathway. However, it remained unexplored whether DIF-1 also inhibits tumor growth in vivo. In the present study, therefore, we examined in-vivo effects of DIF-1 using three cancer models: Mutyh-deficient mice with oxidative stress-induced intestinal tumors and nude mice xenografted with the human colon cancer cell line HCT-116 and cervical cancer cell line HeLa. In exploration for an appropriate route of administration, we found that orally administered DIF-1 was absorbed through the digestive tract to elevate its blood concentration to levels enough to suppress tumor cell proliferation. Repeated oral administration of DIF-1 markedly reduced the number and size of intestinal tumors that developed in Mutyh-deficient mice, reducing the phosphorylation level of GSK-3ß Ser(9) and the expression levels of early growth response-1 (Egr-1), transcription factor 7-like 2 (TCF7L2) and cyclin D1. DIF-1 also inhibited the growth of HCT-116- and HeLa-xenograft tumors together with decreasing phosphorylation level of GSK-3ß Ser(9), although it was not statistically significant in HeLa-xenograft tumors. DIF-1 also suppressed the expressions of Egr-1, TCF7L2 and cyclin D1 in HCT-116-xenograft tumors and those of ß-catenin, TCF7L2 and cyclin D1 in HeLa-xenograft tumors. This is the first report to show that DIF-1 inhibits tumor growth in vivo, consistent with its in-vitro action, suggesting that this compound may have potential as a novel anti-tumor agent.

Mismatch repair deficient mice show susceptibility to oxidative stress-induced intestinal carcinogenesis.

We have previously established an experimental system for oxidative DNA damage-induced tumorigenesis in the small intestine of mice. To elucidate the roles of mismatch repair genes in the tumor suppression, we performed oxidative DNA damage-induced tumorigenesis experiments using Msh2-deficient mice. Oral administration of 0.2% Potassium Bromate, KBrO3, effectively induced epithelial tumors in the small intestines of Msh2-deficient mice. We observed a 22.5-fold increase in tumor formation in the small intestines of Msh2-deficient mice compared with the wild type mice. These results indicate that mismatch repair is involved in the suppression of oxidative stress-induced intestinal tumorigenesis in mice. A mutation analysis of the Ctnnb1 gene of the tumors revealed predominant occurrences of G:C to A:T transitions. The TUNEL analysis showed a decreased number of TUNEL-positive cells in the crypts of small intestines from the Msh2-deficient mice compared with the wild type mice after treatment of KBrO3. These results suggest that the mismatch repair system may simultaneously function in both avoiding mutagenesis and inducing cell death to suppress the tumorigenesis induced by oxidative stress in the small intestine of mice.