Correlation of the in vitro biotransformation of H3B-6527 in dog and human hepatocytes with the in vivo metabolic profile of 14C-H3B-6527 in a dog mass balance study.

1. H3B-6527 is an orally available covalent small molecule inhibitor of FGFR4 undergoing evaluation in adults with hepatocellular carcinoma. Absorption, metabolism, transport and elimination of H3B-6527 were investigated in vitro and in a 14C-H3B-6527 beagle dog mass balance study.2. Following intravenous dosing in dogs, unchanged 14C-H3B-6527 represents only 1.6% of the total dose in excreta. The low amount of radioactivity in the dog urine (4.9% of the administered dose), suggests that renal elimination is a minor pathway of clearance for H3B-6527. A majority of the radioactivity was observed in the feces up to 5 days after dose administration, suggesting that drug-related material was secreted in the bile, and that H3B-6527 clearance was mostly driven by metabolism.3. In vitro, H3B-6527 is a substrate of GSTs, CYP3A and P-glycoprotein.4. The major pathways of metabolism were similar in human and dog hepatocytes, and occurred via glutathione (GSH) conjugations and sequential hydrolysis, N-deethylation and hydroxylation.5. The metabolic profile of H3B-6527 was qualitatively similar in dog hepatocytes and plasma/excreta.

Tumoral CD105 is a novel independent prognostic marker for prognosis in clear-cell renal cell carcinoma.

BACKGROUND: Angiogenesis is essential for tumour growth and metastasis. There are conflicting reports as to whether microvessel density (MVD) using the endothelial marker CD105 (cluster of differentiation molecule 105) in clear-cell renal cell carcinomas (ccRCC) is associated with prognosis. Recently, CD105 has been described as a RCC cancer stem cell marker. METHODS: A total of 102 ccRCC were analysed. Representative tumour sections were stained for CD105. Vascularity (endothelial CD105) was quantified by MVD. The immunohistochemistry analysis detected positive (if present) or negative (if absent) CD105 tumoral staining. This retrospective population-based study was evaluated using Kaplan-Meier method, t-test and Cox proportional hazard model. RESULTS: We found that the expression of endothelial CD105 (MVD) negatively correlated with nuclear grade (P<0.001), tumour stage (P<0.001) and Leibovitch score (P<0.001), whereas the expression of tumoral CD105 positively correlated with these three clinicopathological factors (P<0.001). In multivariate analysis, tumoral CD105 was found to be an independent predictor of poor overall survival (P=0.002). CONCLUSIONS: We have shown for the first time that tumoral CD105 is an independent predictive marker for death risk and unfavourable prognosis in patients with ccRCC after curative resection.

[Pseudomalignant endoscopic appearance in hyperplastic polyp at esophagogastric junction: case report]. / Aspect endoscopique pseudocarcinomateux d'un polype hyperplasique au niveau de la jonction oesogastrique: à propos d'un cas.

Hyperplastic polyps are by far the most common benign tumors in the stomach larger than 5mm. Although the malignant potential of hyperplastic gastric polyps was originally denied, a low risk for carcinomatous conversion is now recognized. Its has been recommended that all gastric polyps of 5mm or more in diameter have to be removed. We present here the case of a large hyperplastic gastric polyp located at the esophagogastric junction with endoscopic malignant appearance. The description of its endoscopic and histological characteristics gives us the opportunity to discuss the difficulty of endoscopic diagnosis in the case of gastric polyps and to remind their prognosis.

[Renal vascular lesions and the occurrence of hypertension in patients with IgA nephropathy]. / Lésions vasculaires rénales et développement d'une HTA chez les patients atteints d'une glomérulonéphrite à dépôts mésangiaux d'IgA. Une histoire familiale d'hypertension artérielle.

Worse prognosis of IgA nephropathy (IgAN) is associated to hypertension, high proteinuria, glomerular and vascular sclerosis. A family story of hypertension (FHT) in relatives could be a strong predictor of the occurrence of hypertension (HT) in children. Renal vascular lesions (RVL) are often observed in normotensive patients with IgAN. In order to evaluate a possible association between FHT and LVR in patients with IgAN, we investigated two groups of 73 IgAN patients, sex (56 males and 17 females) and age matched, according to the presence or not of FHT. FHT was diagnosed if relatives and/or at least one child under 60 years of age had treatment for HT or systolic and diastolic BP over 140/90 mmHg at the time of the survey. Patients entering into the study were followed during an average period of 5 to 8 years. At the end of the study, all patients were explored for HT and renal function. Creatinine clearance (CrCl) was evaluated by Cockcroft and Gault formula and renal failure was defined as CrCl<60mL/min. The results were as follow: at the time of renal biopsy, RVL were observed in 73% of males with FHT vs 16% of males without FHT (p<0.0001) and 70.6% of females with FHT vs 29.4% of females without FHT (p<0.001); at the end of the study period, HT was significantly associated to FHT in 89.6% of patients group with FHT vs 22.6% of HT patients in the group without FHT (p<.0001). Renal failure was present in 45.2% of patients with FHT vs 4.1% of patients without FHT (p<0.0001). These data suggest: VRL could be dependent of genetic factors; FHT should be an early predictor of VRL in patients with IgAN; FHT might be a risk factor for renal failure in patients with this renal disease.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone modulation of cytokine release in U937 human macrophages.

The nicotine-derived N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most abundant and potent carcinogens found in tobacco smoke. NNK induces lung tumors in rodents and is most likely involved in lung carcinogenesis in humans. Studies on the metabolism and carcinogenicity of NNK have been extensive. However, its effects on the immune system have not been investigated thoroughly. Considering that tobacco smoking partially suppresses the immune response in humans, and that immune surveillance plays a critical role in cancer development, we examined the effects of NNK on the production of selected cytokines. In a previous study, we observed an inhibition of NK cell activity and IgM secretory cell number in NNK-treated A/J mice [Rioux and Castonguay (1997) J Natl Cancer Inst 89: 874]. In this study, we demonstrate that U937 human macrophages activate NNK to alkylating intermediates by alpha-carbon hydroxylation and detoxify NNK by N-oxidation. We observed that NNK, following activation, induces the release of soluble tumor necrosis factor (TNF), but inhibits interleukin(IL)-10 synthesis. We also report that 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)- -butanone, and nitroso(acetoxymethyl)methylamine, which generate the same alkylating intermediates as NNK, have similar effects on TNF and IL-10. This suggests that pyridyloxobutylating and methylating intermediates generated from NNK are potent modulators of the immune response. The levels of IL-6, granulocyte/macrophage-colony-stimulating factor and macrophage chemotactic protein 1 were also decreased in supernatants of NNK-treated U937 macrophages. In contrast, IL-2 synthesis in Jurkat cells was inhibited by NNK treatment. This is the first study demonstrating that NNK, via its alkylating intermediates, alters the cytokine synthesis profile in human cells. Modulation of cytokine synthesis by NNK might partially explain the immunosuppresion observed in smokers. Inhibition of immune functions, resulting from NNK activation to alkylating agents, may facilitate lung tumor development.

The induction of cyclooxygenase-1 by a tobacco carcinogen in U937 human macrophages is correlated to the activation of NF-kappaB.

The nicotine-derived 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), present in tobacco smoke, is most likely involved in lung carcinogenesis in smokers. We demonstrated previously that non-steroidal anti-inflammatory drugs (NSAIDs) inhibit NNK-induced lung tumorigenesis, although the mechanism(s) is unknown. The present study demonstrates that, in U937 human macrophages, cyclooxygenase (COX)-1 and -2 are involved in the bioactivation of NNK to electrophilic mutagenic intermediates. We observed that acetylsalicylic acid and NS-398 decrease COX-dependent NNK activation in U937 cells by 66 and 37%, respectively. NSAIDs also decrease prostaglandin E(2) (PGE(2)) synthesis, which is induced in a dose-dependent manner, reaching a 7-fold increase, in NNK-treated human U937 cells. We observed that NNK induces COX-1 expression and activates the nuclear factor-kappaB (NF-kappaB), in U937 cells. N:-acetyl-L-cysteine and pyrrolidinedithiocarbamate, two inhibitors of reactive oxygen species (ROS), inhibit NNK-induced PGE2 synthesis by 41 and 44%, respectively. These data suggest that ROS, generated during pulmonary metabolism of NNK could act as signal transduction messengers and activate NF-kappaB, which will subsequently induce COX-1 activity and increase PGE(2) synthesis. These results reveal a novel aspect of tobacco carcinogenesis, and give us insight into the mechanisms of chemoprevention by NSAIDs. Accordingly, inhibition of NF-kappaB activation, leading to the inhibition of COX, offers a new approach in lung cancer prevention.

Inhibition of COX-2 and induction of apoptosis: two determinants of nonsteroidal anti-inflammatory drugs' chemopreventive efficacies in mouse lung tumorigenesis.

Recent studies suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit lung tumorigenesis under conditions that are immunosuppressive. We hypothesized that this inhibition of mouse lung tumorigenesis requires induction of apoptosis and inhibition of COX (cyclooxygenase)-1, COX-2, and the incidence of K-ras mutation. The NSAIDs used in this study include acetylsalicylic acid (ASA) that is anti-inflammatory with COX-1 and COX-2 inhibition and N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398) that is a specific COX-2 inhibitor. We have previously demonstrated that ASA (147 and 294 mg/kg diet) and NS398 (7 mg/kg diet) inhibited lung tumorigenesis by 31%, 44%, and 34%, respectively, in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-treated A/J mice. No difference in the incidence and types of K-ras mutations was found between the lung tumors treated with NNK and those treated with NNK/ASA and NNK/NS398. In NNK-treated mice, ASA (394 mg/kg diet) or NS398 significantly increased the apoptotic index, from 0.07 to 0.30 or to 0.33, respectively. ASA (294 mg/kg diet) and NS398 also inhibited the expression of COX-2. Finally, modulation of gene expression by NS398 and ASA (294 mg/kg diet) was determined using Atlas cDNA expression arrays. Expression of cyclin B2 was decreased and expression of Fas-L and BAD were increased in lung tissues treated with both NS398 and ASA. Treatment with NS398 also increased expression of p57kip2 and myosin. These genes modulated by NSAIDs may play a role in mediating the observed chemopreventive effects of the NSAIDs in the mouse lung. Our results demonstrate that lung tumor prevention with NSAIDs involve both the induction of apoptosis and the inhibition of COX-2 expression.

Prevention of NNK-induced lung tumorigenesis in A/J mice by acetylsalicylic acid and NS-398.

Acetylsalicylic acid (ASA) is known to prevent cancer development, but its mechanism of action remains unclear. In this study, we compared the efficacies of this nonspecific cyclooxygenase (COX) inhibitor with N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), a specific COX-2 inhibitor. COX-2-specific inhibitors are less toxic than ASA. Lung tumorigenesis was induced in A/J mice by the administration of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the drinking water for 7 weeks (weeks 0 to +7). Groups of 25 A/J mice were fed ASA (588, 294, 147, or 73 mg/kg diet) before and throughout the assay (weeks -2 to +23). ASA at a dose of 588 mg/kg diet was the most effective because it reduced lung tumor multiplicity by 53%. The preventive effect of ASA increased with the dose, being of 32, 30, and 44% for 73, 147, and 294 mg/kg diet, respectively. NNK increased plasma prostaglandin E2 (PGE2) basal levels by 413%, whereas ASA attenuated this elevation in a dose-response manner (r2 = 0.99). Plasma PGE2 levels in ASA + NNK-treated mice correlate with the logarithm of the number of tumors (r2 = 0.99). NS-398 inhibited lung tumor multiplicity by 34% and returned plasma PGE2 to basal levels observed in untreated mice. Among the NNK-exposed mice, ASA and NS-398 treatment decreased the mean of the lung tumor volumes. Incubation of 82-132 and LM2 murine lung tumor cells with ASA or NS-398 decreased cell proliferation by 50% at concentrations higher than 100 microM. Incubations of NNK with COX-1 and -2 produced both activation and detoxification products by alpha-carbon hydroxylation and N-oxydation pathways, respectively. Bioactivation of NNK was more extensive by COX-2 than COX-1. Anti-COX-1 and -2, arachidonic acid, ASA, and NS-398 inhibited NNK bioactivation by COX-1 and -2 from 22-49%. Our data suggest that NNK is bioactivated by COX-2 in lung tissues and that COX-2-specific inhibitors might be promising chemopreventive agents.

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung tumor formation by FGN-1 (sulindac sulfone).

The sulfone derivative of the non-steroidal anti-inflammatory drug (NSAID), sulindac, has been reported to inhibit mammary and colon tumor formation in rodent models of chemically-induced carcinogenesis. Unlike its parent compound, this metabolite lacks cyclo-oxygenase inhibitory activity. A tumor induction protocol, consisting of NNK administration in the drinking water over several weeks to model chronic human exposure, was used to test whether the sulfone (called FGN-1) could inhibit the formation of primary lung tumors in mice. A total of 150 female, AIN76A-fed, A/J mice received 9 mg of NNK each. Concentrations of FGN-1 that had been previously determined not to affect body weight gain were added to the food at levels of 0, 250, 500 and 750 mg/kg of diet (30 mice/group) starting 2 weeks before NNK administration and continuing for 22 weeks. At that time pleural surface tumors were counted. Tumor incidence decreased significantly from 96 % in the control diet and 93% in the 250 FGN-1 mg/kg diet to 63 and 67% in the 500 and 750 mg FGN-1/kg diet groups, respectively (P < 0.001 by chi-square analysis). Lung tumor multiplicity decreased from 18.1+/-3 tumors/ mouse (mean+/-SEM, control diet) to 12.3+/-3 (250), 5.3+/-1 (500) and 2.1+/-1 (750) (P < 0.0005 by post hoc ANOVA). In previous studies using this carcinogenesis protocol, the maximum tolerated dose of sulindac inhibited lung tumor multiplicity by no more than 50% with no effect on incidence. This dose-dependent reduction in tumorigenesis by a non-toxic dose of FGN-1 indicates a strong chemopreventive activity against experimental induction of lung carcinogenesis. The greater potency of the sulfone over sulindac and its lack of toxic side effects because of its inability to affect cyclo-oxygenase activity suggests that clinical testing in individuals at high risk for lung cancer should be considered.

Inhibitors of lipoxygenase: a new class of cancer chemopreventive agents.

5-Lipoxygenase is a key enzyme in the metabolism of arachidonic acid to leukotrienes. The preventive efficacy of 5-lipoxygenase inhibitors against lung tumorigenesis was determined in A/J mice given the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in drinking water from week 0 to week +7. Groups of 25 mice were fed either: acetylsalicylic acid (ASA), a cyclooxygenase inhibitor; A-79175, a 5-lipoxygenase inhibitor; MK-886, an inhibitor of the 5-lipoxygenase activating-protein; a combination of ASA and A-79175 from weeks -2 to +23. ASA, A-79175 and MK-886 reduced lung tumor multiplicity by 44, 75 and 52% respectively. Furthermore, A-79175 reduced tumor incidence by 20%. Administration of A-79175 and MK-886 decreased the mean tumor volume by 64 and 44% respectively. Lung tumor multiplicity was directly proportional to tumor volume. The combination of ASA and A-79715 was the most effective preventive intervention and reduced lung tumor multiplicity by 87% and lung tumor incidence by 24%, demonstrating that inhibition of both 5-lipoxygenase and cyclooxygenase is more effective than inhibition of either pathway alone. NNK treatment increased plasma prostaglandin E2 levels from 49 to 260 pg/ml and plasma LTB4 levels from 29 to 71 pg/ml. Incubation of 82-132 and LM2 murine lung tumor cells with MK-886 and A-79715 decreased cell proliferation in a concentration-dependent manner. Soybean lipoxygenases with or without murine lung microsomal proteins metabolized NNK by alpha-carbon hydroxylation (9.5% of the metabolites) and N-oxidation (3.9%). Activation of NNK by alpha-carbon hydroxylation was inhibited by addition of arachidonic acid and A-79715. Possible mechanisms of action of 5-lipoxygenase inhibitors include inhibition of tumor growth and lipoxygenase-mediated activation of NNK. These studies suggest that inhibitors of 5-lipoxygenase may have benefits as preventive agents of lung tumorigenesis.

Inhibition of lung tumorigenesis by NSAIDS: a working hypothesis.

A 7-week treatment with the tobacco carcinogen NNK induced 8-10 lung adenomas per A/J mouse. NNK suppressed humoral and cellular immune responses and increased plasma PGE2 and LTB4 levels. This protocol is particularly suitable for testing NSAIDs and lipoxygenase inhibitors as cancer preventive agents. Sulindac and ASA inhibited lung tumorigenesis by 52 and 60%, respectively, attenuated the suppressive effect of NNK, and lowered the plasma PGE2 to basal levels. In contrast, naproxen neither inhibited lung tumorigenesis nor increased NNK-suppressed NK cell cytotoxicity. NSAIDs and lipoxygenase inhibitors had additive preventive efficacies against NNK-induced lung tumorigenesis. However, sulindac was not effective in preventing lung tumorigenesis induced by B[a]P, which lacks immunosuppressive activity. These results and those published by other investigators lead to the following hypothesis: Reactive intermediates derived from NNK interfere with the stimulation of the complex NF-kappa B/I kappa B. NF-kappa B is involved in the regulation of immune and inflammatory responses. The authors propose that NNK-derived intermediates induce the expression of COX-2 and lipoxygenase involved in NNK activation. This hypothesis provides a rationale for the lack of efficacy of naproxen to prevent tumorigenesis, to attenuate NNK-induced synthesis of PGE2, and to increase NK cell cytotoxicity. According to this hypothesis, PGE2 synthesis and induction of apoptosis contribute to varying degrees to the mechanism of cancer prevention.

Recovery from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced immunosuppression in A/J mice by treatment with nonsteroidal anti-inflammatory drugs.

BACKGROUND: We have previously reported that nonsteroidal anti-inflammatory drugs inhibit lung tumorigenesis induced by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. PURPOSE: The aims of this study were to determine if NNK suppresses humoral (i.e., antibody) and cellular immune responses in mice and if nonsteroidal anti-inflammatory drugs could attenuate these immune responses. METHODS: Female A/J mice (7-8 weeks old) were fed nonsteroidal anti-inflammatory drugs starting 2 weeks before the beginning of NNK treatment (9.1 mg per mouse in total) and continuing through the 7 weeks of NNK treatment. Eight groups (two control groups and six experimental groups) of 10 mice each were used per experiment. Animals in the two control groups received the same diet and water as animals in the six experimental groups; one control group received no nonsteroidal anti-inflammatory drugs or NNK and the other control group received only NNK. The primary humoral and cellular immune responses to the various treatments were assayed by the plaque-forming cell technique and by measurement of natural killer cell cytotoxic activity, respectively. At the end of each experiment, the animals were killed, blood was collected, plasma was prepared, and levels of the immune system modulator prostaglandin E2 were measured. RESULTS: NNK treatment inhibited the plaque-forming cell response by approximately 50%; this inhibition was attenuated by treatment with sulindac or acetylsalicylic acid (P = .0001 for both). In contrast, treatment with naproxen, which had no chemopreventive (i.e., tumor inhibitory) efficacy, further increased by 26% (P = .05) the immunosuppressive effect of NNK. The cytotoxic activity of splenic natural killer cells against YAC-1 cells was reduced by 60% (P = .002); treatment with acetylsalicylic acid (254 mg/kg of diet) reduced the NNK-induced natural killer cell cytotoxicity inhibition by 50% (P = .02), whereas the administration of the specific cyclooxygenase-2 inhibitor NS-398 (7 mg/kg of diet) resulted in an almost complete recovery (approximately 95%, P = .04) of natural killer cell activity. The prostaglandin E2 plasma concentration was approximately 100% greater in NNK-treated mice than in untreated mice. Treatment of the mice with nonsteroidal anti-inflammatory drugs attenuated this elevation (from approximately 25% to 100%), and NS-398 (7 mg/kg of diet) was the most effective (100%). CONCLUSIONS AND IMPLICATIONS: The ability of various nonsteroidal anti-inflammatory drugs to inhibit NNK-induced carcinogenesis appears to be directly related to the ability of these drugs to inhibit NNK-induced immunosuppression. Our results suggest that the chemopreventive effect of nonsteroidal anti-inflammatory drugs may be mediated through the modulation of prostaglandin E2 synthesis.

Inhibition of lung tumourigenesis by sulindac: comparison of two experimental protocols.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen in mice and is most likely involved in the aetiology of tobacco-induced lung cancer. Two protocols using NNK and A/J mice have been developed. In the single-dose protocol, each mouse was injected once with 2 mg of NNK. In the 7-week protocol, each mouse received 9.1 mg of NNK in drinking water during 7 weeks. Mice were killed 16 weeks after NNK treatment. We observed a near-Gaussian distribution in the number of tumours per mouse in the single protocol, but not in the 7-week protocol. In the 7-week protocol, a significant number (8.6%) of mice had more than 20 tumours/mouse. In the single-dose protocol, no mouse had more than 20 tumours. Sulindac at a dose of 123 mg/kg of diet inhibits lung tumourigenesis in the 7-week protocol, but not in the single-dose protocol. We observed that the inhibition of tumourigenesis in the 7-week protocol was proportional to the logarithm of the dose of sulindac between 15 and 123 mg/kg of diet. Treatment of mice for 7 weeks inhibits the primary humoral response to sheep red blood cells by 70%. This observation is particularly significant considering that NNK is present in tobacco smoke and that tobacco smoking suppresses both the specific and non-specific humoral and cellular immunity. Single injections of 2.0, 3.5 or 5.0 mg of NNK had no effect on this response. Our results suggest that the immunosuppressive effects of NNK contribute to its high carcinogenic potency particularly in sustained or life-time exposure models. We hypothesize that sulindac promotes the recovery of immune system from the NNK-mediated suppression observed in the 7-week protocol. This study illustrates the importance of selecting the most appropriate protocol of carcinogen treatment in investigating the efficacies of cancer chemopreventive agents.

Carcinoma of the ampulla of Vater: prognostic factors after curative surgery: a series of 45 cases.

BACKGROUND: Some adjuvant or neoadjuvant therapy could be important for patients operated on for tumours of the ampulla of Vater, especially for those having a higher risk of recurrence. AIM: To evaluate prognostic factors after curative surgery based on a series of 45 cases of malignant tumours of the Oddi sphincter. PATIENTS: From 1970 to 1992, a curative resection was performed in 45 patients (age 62.8 (SD 10.1) years) with adenocarcinoma of the ampulla. Surgical procedures included pancreatoduodenectomy (n = 42) and resection of the ampulla (n = 3). Actuarial survival was 44 (SD 9)% at five years. METHODS: Various prognostic variables were studied: clinical manifestations, macroscopic aspect, differentiation, noninvasive adenomatous component, mucin histochemistry, immunohistochemistry (CEA, CA19.9, p53, Ki67), and accepted classifications (Blumgart and Kennedy, Martin, Yamaguchi and Enjoji, Talbot et al, pTNM). RESULTS: Variables with prognostic power, in order of importance were: Classification of Talbot et al; CA19.9; pTNM; sialomucins; classification of Yamaguchi and Ejoji; Martin classification; sulphomucins; non-invasive adenomatous component (positive > negative); jaundice; tumour localisation. CONCLUSIONS: This series confirmed the prognostic power of former classifications and showed the prognostic power of other variables (mucin, non-invasive adenomatous component, CA19.9).