Kenichiro Doi

Lack of large intestinal carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine at low doses in rats initiated with azoxymethane

2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), an abundant food‐derived heterocyclic amine (HCA), has attracted particular attention as a human colon carcinogen. Humans are in fact exposed to continuous low doses of HCAs during lifetime. Therefore, we focused on rat large intestinal carcinogenicity of PhIP at levels that mimic practical human exposure. A total of 192 6‐week‐old male F344 rats were subcutaneously injected twice with 15 mg/kg body weight azoxymethane (AOM), then continuously fed various doses (0, 0.001, 0.01, 0.1, 1, 10, 50 and 200 ppm) of PhIP in the diet. At week 16, aberrant crypt foci (ACF) were quantitatively analyzed. At week 36, tumor occurrence was pathologically analyzed. Then immunohistochemical examinations were performed. PhIP was found to enhance strongly AOM‐initiated rat large intestinal tumorigenesis at high doses (50 and 200 ppm), while lower doses (0.001–10 ppm) had no apparent effects. High doses also caused variation in tumor histologic types and their distribution throughout the large intestinal segments. Frequencies of ACF/cm2 did not meaningfully vary between the groups. Cellular proliferation activity in normal‐appearing colonic mucosa was significantly increased at high doses. These novel findings may provide evidence of a low‐dose potential for PhIP, with a no‐observed effect level speculated to be 10 ppm in the present initiation‐promotion experimental model.

Induction of glutathione S-transferase placental form positive foci in liver and epithelial hyperplasia in urinary bladder, but no tumor development in male Fischer 344 rats treated with monomethylarsonic acid for 104 weeks.

The carcinogenicity of monomethylarsonic acid (MMA(V)), a major metabolite of inorganic arsenics in human and experimental animals, was investigated in male Fischer 344 rats. A total of 129 rats at 10 weeks of age were randomly divided into three groups and received drinking water containing MMA(V) at doses of 0 (Control), 50, and 200 ppm ad libitum for 104 weeks. No significant differences were found between the control and the MMA(V)-treated groups regarding clinical signs, mortality, hematological, and serum biochemistry findings. Quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci in liver revealed a significant increase of numbers and areas in the 200 ppm MMA(V)-treated group. In the urinary bladder MMA(V) induced simple hyperplasia and significantly elevated the proliferating cell nuclear antigen (PCNA)-positive index in the urothelium. A variety of tumors developed in rats of all groups, including the controls, but all were histologically similar to those known to occur spontaneously in F344 rats and there were no significant differences among the groups. Thus, it could be concluded that, under the present experimental conditions, MMA(V) induced lesions in the liver and urinary bladder, but did not cause tumor development in male F344 rats even after 2 years exposure.

Acid ceramidase is upregulated in AML and represents a novel therapeutic target

There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased overall survival of C57BL/6 mice engrafted with leukemic C1498 cells and significantly decreased leukemic burden in NSG mice engrafted with primary human AML cells. Collectively, these studies demonstrate that AC plays a critical role in AML survival through regulation of both sphingolipid levels and Mcl-1. We propose that AC warrants further exploration as a novel therapeutic target in AML.

Chemopreventive effect of JTE-522, a selective cyclooxygenase-2 inhibitor, on 1, 2-dimethylhydrazine-induced rat colon carcinogenesis

Selective COX-2 inhibitors have been suggested to be an effective strategy in the prevention of colon cancer without the adverse side effects of non-selective, nonsteroid anti-inflammatory drugs. The present experiment was designed to assess the potential chemopreventive properties of JTE-522, a new selective cyclooxygenase-2 inhibitor, on the induction of 1,2-dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF), a marker of rat colon carcinogenesis. A total of 80 male F344 rats were treated with 3 or 10 mg/kg of body weight JTE-522 or vehicle by oral gavage five times weekly from the start of the experiment. One week later, rats received s.c. injections of saline or 20 mg/kg of body weight DMH once weekly for four successive weeks. At the end of 12 weeks after the start of experiment, all rats were sacrificed and colons were evaluated for ACF. 10 mg/kg JTE522 significantly suppressed the total ACF/colon. No inhibitory effect was observed in the 3 mg/kg JTE-522 treatment group. This result suggests that JTE-522 possesses chemopreventive activity against colon carcinogenesis.

Potassium Bromate Enhances N-Ethyl-N-Hydroxyethylnitrosamine-Induced Kidney Carcinogenesis Only at High Doses in Wistar Rats: Indication of the Existence of an Enhancement Threshold

As susceptibility to carcinogens varies considerably among different strains of experimental animals, evaluation of dose-response relationships for genotoxic carcinogen in different strains is indispensable for risk assessment. Potassium bromate (KBrO3) is a genotoxic carcinogen inducing kidney cancers at high doses in male F344 rats, but little is known about its carcinogenic effects in other strains of rats. The purpose of the present study was to determine dose-response relationships for carcinogenic effects of KBrO3 on N-ethyl-N-hydroxyethylnitrosamine (EHEN)–induced kidney carcinogenesis in male Wistar rats. We found that KBrO3 showed significant enhancement effects on EHEN-induced kidney carcinogenesis at above 250 ppm but not at doses of 125 ppm and below when evaluated in terms of induction of either preneoplastic lesions or tumors in male Wistar rats. Furthermore, KBrO3 significantly increased the formation of oxidative DNA damage at doses of 125 and above but not at doses of 30 ppm and below in kidneys. These results demonstrated that low doses of KBrO3 exert no effects on development of EHEN-initiated kidney lesions and induction of oxidative DNA damage. Taking account of previous similar findings in male F344 rats, it is strongly suggested that a threshold dose exists for enhancement effects of KBrO3 on kidney carcinogenesis in rats.

Enhanced Urinary Bladder, Liver and Colon Carcinogenesis in Zucker Diabetic Fatty Rats in a Multiorgan Carcinogenesis Bioassay: Evidence for Mechanisms Involving Activation of PI3K Signaling and Impairment of p53 on Urinary Bladder Carcinogenesis

In the present study, modifying effects of diabetes on carcinogenesis induced in type 2 diabetes mellitus model Zucker diabetic fatty (ZDF) rats were investigated using a multiorgan carcinogenesis bioassay. Our re sults demonstrated enhancement of urinary bladder, colon and liver carcinogenesis in ZDF rats treated with five types of carcinogens (DMBDD). Elevated insulin and leptin and decreased adiponectin levels in the serum may be responsible for the high susceptibility of type 2 diabetes mellitus model rats to carcinogenesis in these organs. Possible mechanisms of increased susceptibility of diabetic rats to bladder carcinogenesis could be activation of the PI3K pathway and suppression of p53 in the urothelium in consequence of the above serum protein alterations.

Targeted Delivery of Ubiquitin-Conjugated BH3 Peptide-Based Mcl-1 Inhibitors into Cancer Cells

BH3 peptides are key mediators of apoptosis and have served as the lead structures for the development of anticancer therapeutics. Previously, we reported the application of a simple cysteine-based side chain cross-linking chemistry to NoxaBH3 peptides that led to the generation of the cross-linked NoxaBH3 peptides with increased cell permeability and higher inhibitory activity against Mcl-1 (Muppidi, A., Doi, K., Edwardraja, S., Drake, E. J., Gulick, A. M., Wang, H.-G., Lin, Q. (2012) J. Am. Chem. Soc.134, 1473422920569). To deliver cross-linked NoxaBH3 peptides selectively into cancer cells for enhanced efficacy and reduced systemic toxicity, here we report the conjugation of the NoxaBH3 peptides with the extracellular ubiquitin, a recently identified endogenous ligand for CXCR4, a chemokine receptor overexpressed in cancer cells. The resulting ubiquitin-NoxaBH3 peptide conjugates showed increased inhibitory activity against Mcl-1 and selective killing of the CXCR4-expressing cancer cells. The successful delivery of the NoxaBH3 peptides by ubiquitin into cancer cells suggests that the ubiquitin/CXCR4 axis may serve as a general route for the targeted delivery of anticancer agents.

Existence of No Hepatocarcinogenic Effect Levels of 2-amino-3,8-dimethylimidazo(4,5-f )quinoxaline with or without Coadministration with Ethanol

There is increasing evidence of the existence of no effect levels for genotoxic carcinogens. However, only limited information is available regarding dose-response curves for combination effects of multiple carcinogens at low dose. In the present study, 280 male F344 rats were divided into 14 groups to determine the effects of co-administration of various doses of 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx) and 10% ethanol on the development of glutathione S-transferase placental form (GST-P)-positive foci in the liver. The results provided concrete evidence for the existence of no effect levels for hepatocarcinogenicity of MeIQx either in presence or absence of ethanol and, therefore, for a practical threshold for this genotoxic carcinogen.

Chemopreventive effects of 13α,14α-epoxy-3β-methoxyserratan-21β-ol (PJJ-34), a serratane-type triterpenoid, in a rat multi-organ carcinogenesis bioassay.

A novel serratane-type triterpenoid, 13alpha,14alpha-epoxy-3beta-methoxyserratan-21beta-ol (PJJ-34) derived from cuticles of Picea jezoensis Carr. var. jezoensis, has proved to be highly effective at suppressing carcinogenesis both in vitro and in vivo. To investigate possible anti-carcinogenic efficacy at the whole-body level, male Fischer 344 rats were subjected to an established rat multi-organ carcinogenesis bioassay (DMBDD model). After initiation with five carcinogens, groups 1-3 (20 in each) were intragastrically (i.g.) administered PJJ-34 dissolved in 1 ml of 0.5% CMC (5 times/week) at doses of 0, 5 and 10mg/kg body weight (b.w.), respectively, until the end of week 30. PJJ-34 did not show apparent toxicity. Incidences of adenomas (100-->75%) and carcinomas (63-->30%) in the lung were significantly decreased in the 5mg/kg b.w. group, and multiplicity of alveolar hyperplasias and total lung tumors (adenomas+carcinomas) were significantly reduced by both 5 and 10mg/kg. The incidence of colorectal tumors was also significantly decreased in the 10mg/kg group (63-->28%) along with the multiplicity. Rat liver pre-neoplastic lesions, glutathione S-transferase placental form (GST-P) foci, and tumor development in the other organs were not affected. Immunohistochemical indices for proliferating cell nuclear antigen (PCNA) and cyclin D1 in normal alveolar epithelium of the lung were significantly suppressed at both doses. In conclusion, PJJ-34 is chemopreventive against lung and colon carcinogenesis without exerting apparent toxicity, and suppression of cell proliferation could play a key role in the underlying mechanisms.

Ethanol-Extracted Brazilian Propolis Exerts Protective Effects on Tumorigenesis in Wistar Hannover Rats

The present study was conducted over a course of 104 weeks to estimate the carcinogenicity of ethanol-extracted Brazilian green propolis (EEP). Groups of 50 male and 50 female Wistar Hannover rats, 6-week-old at commencement were exposed to EEP at doses of 0, 0.5 or 2.5% in the diet. Survival rates of 0.5% and 2.5% EEP-treated male and female rats, respectively, were significantly higher than those of respective control groups. Overall histopathological evaluation of neoplasms in rat tissues after 2 years showed no significant increase of tumors or preneoplastic lesions in any organ of animals administered EEP. Significantly lower incidences of pituitary tumors in 0.5% EEP male and 2.5% EEP female groups, malignant lymphoma/leukemia in both 2.5% EEP-treated males and females and total thyroid tumors in 0.5% EEP male group were found. Administration of EEP caused significant decreases of lymphoid hyperplasia of the thymus and lymph nodes in 2.5% EEP-treated rats, tubular cell hyperplasia of kidneys in all EEP groups, and cortical hyperplasia of adrenals in EEP-treated females. In the blood, significant reduction of neutrophils in all EEP-treated males and band neutrophils in 2.5% EEP-treated females was found indicating lower levels of inflammation. Total cholesterol and triglicerides levels were significantly lower in the blood of 2.5% EEP-treated female rats. In conclusion, under the conditions of the 2-year feeding experiment, EEP was not carcinogenic, did not induce significant histopathological changes in any organ, and further exerted anti-inflammatory and antitumorigenic effects resulting in increase of survival of Wistar Hannover rats.