Molecular imaging of aberrant crypt foci in the human colon targeting glutathione S-transferase P1-1.

Aberrant crypt foci (ACF), the earliest precursor lesion of colorectal cancers (CRCs), are a good surrogate marker for CRC risk stratification and chemoprevention. However, the conventional ACF detection method with dye-spraying by magnifying colonoscopy is labor- and skill-intensive. We sought to identify rat and human ACF using a fluorescent imaging technique that targets a molecule specific for ACF. We found that glutathione S-transferase (GST) P1-1 was overexpressed in ACF tissues in a screening experiment. We then synthesized the fluorogenic probe, DNAT-Me, which is fluorescently quenched but is activated by GSTP1-1. A CRC cell line incubated with DNAT-Me showed strong fluorescence in the cytosol. Fluorescence intensities correlated significantly with GST activities in cancer cell lines. When we sprayed DNAT-Me onto colorectal mucosa excised from azoxymethane-treated rats and surgically resected from CRC patients, ACF with strong fluorescent signals were clearly observed. The ACF number determined by postoperative DNAT-Me imaging was almost identical to that determined by preoperative methylene blue staining. The signal-to-noise ratio for ACF in DNAT-Me images was significantly higher than that in methylene blue staining. Thus, we sensitively visualized ACF on rat and human colorectal mucosa by using a GST-activated fluorogenic probe without dye-spraying and magnifying colonoscopy.

High antitumor activity of pladienolide B and its derivative in gastric cancer.

The antitumor activity of pladienolide B, a novel splicing inhibitor, against gastric cancer is totally unknown and no predictive biomarker of pladienolide B efficacy has been reported. We investigated the antitumor activity of pladienolide B and its derivative on gastric cancer cell lines and primary cultured cancer cells from carcinomatous ascites of gastric cancer patients. The effect of pladienolide B and its derivative on six gastric cancer cell lines was investigated using a MTT assay and the mean IC50 values determined to be 1.6 ± 1.2 (range, 0.6-4.0) and 1.2 ± 1.1 (range, 0.4-3.4) nM, respectively, suggesting strong antitumor activity against gastric cancer. The mean IC50 value of pladienolide B derivative against primary cultured cells from 12 gastric cancer patients was 4.9 ± 4.7 nM, indicative of high antitumor activity. When 18 SCID mice xenografted with primary cultured cells from three patients were administered the pladienolide B derivative intraperitoneally, all tumors completely disappeared within 2 weeks after treatment. Histological examination revealed a pathological complete response for all tumors. In the xenograft tumors after treatment with pladienolide B derivative, immature mRNA were detected and apoptotic cells were observed. When the expressions of cell-cycle proteins p16 and cyclin E in biopsied gastric cancer specimens were examined using immunohisctochemistry, positivities for p16 and cyclin E were significantly and marginally higher, respectively, in the low-IC50 group compared with the high-IC50 group, suggesting the possibility that they might be useful as predictive biomarkers for pladienolide B. In conclusion, pladienolide B was very active against gastric cancer via a mechanism involving splicing impairment and apoptosis induction.

Suppressive effect of RAS inhibitor manumycin A on aberrant crypt foci formation in the azoxymethane-induced rat colorectal carcinogenesis model.

BACKGROUND AND AIM: The chemopreventive effect of RAS inhibitors on colorectal cancer is unknown. Because aberrant crypt foci (ACF), earliest preneoplastic lesions, are highly positive for K-RAS mutation, RAS inhibitors are likely to be effective for chemoprevention. Therefore, in the present study, the suppressive effect of a RAS inhibitor, manumycin A, on ACF formation in an azoxymethane (AOM)-induced rat colorectal carcinogenesis model was investigated. METHODS: Rats injected with AOM were administered manumycin A (30 mg/kg) subcutaneously thrice weekly for 8 weeks or for 4 weeks (latter half), sacrificed at 8 weeks, and examined for ACF in the colorectum. Phosphorylated ERK and Ki-67 expression was evaluated by immunohistochemistry. Apoptosis was assessed by TUNEL staining. RESULTS: The mean number of ACF in the 8-week manumycin A group (72.9 ± 20.1) was significantly lower than in the vehicle group (155.6 ± 56.7, P < 0.01), and it was significantly lower even in the 4-week manumycin A group than in the vehicle group (92.2 ± 13.0 vs 222.3 ± 83.3, P < 0.01). The positive rate for phosphorylated ERK in the manumycin A group (13.5 ± 19.2%) was significantly lower than in the vehicle group (50.2 ± 19.8%, P < 0.01). The positive rate for Ki-67 in the manumycin A group (2.2 ± 3.4%) was significantly lower than in the vehicle group (14.7 ± 8.2%, P < 0.01). There were significantly more terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells in tissue samples from the manumycin A group versus the vehicle group (8.6 ± 9.7% vs 2.9 ± 2.0%, P < 0.05). CONCLUSION: Manumycin A suppressed ACF formation in the AOM-induced colorectal carcinogenesis model, demonstrating that RAS inhibitors may be very effective for chemoprevention of colorectal cancers.

Novel des-γ-carboxy prothrombin in serum for the diagnosis of hepatocellular carcinoma.

BACKGROUND AND AIM: Serum des-γ-carboxy prothrombin (DCP) levels using a newly developed electrochemiluminescence immunoassay (ECLIA, novel DCP [NX-DCP]) were measured, and the utility of NX-DCP and DCP/NX-DCP ratio for the diagnosis of hepatocellular carcinoma (HCC) was investigated. Antigenic differences in DCP between HCC and non-HCC patients were elucidated. METHODS: The subjects included 170 patients with HCC, 61 with benign liver disease, 12 with obstructive jaundice, and 10 warfarin users. NX-DCP was quantitated by sandwich ECLIA employing novel anti-DCP monoclonal antibodies, P11 and P16. Conventional DCP was quantitated by standard ECLIA. DCP extracted from serum by affinity-chromatography was analyzed by Western blotting. RESULTS: Conventional serum DCP levels were high in patients with HCC and obstructive jaundice, and in warfarin users, consistent with previous reports. Serum NX-DCP levels were high only in warfarin users and obstructive jaundice patients (vitamin K-deficient patients) but not in HCC patients. The DCP/NX-DCP ratio was significantly higher in the HCC group than in the benign liver disease, obstructive jaundice, and warfarin groups (P < 0.001). Receiver operating characteristic analysis showed significant superiority of the DCP/NX-DCP ratio over conventional DCP as a marker for HCC diagnosis (P < 0.05). Western blot analysis showed that P11 and P16 reacted strongly with DCP from a warfarin user and an obstructive jaundice patient but very faintly with DCP from an HCC patient. Immunohistochemistry on HCC samples and autopsied normal liver tissues from warfarin users showed similar results. CONCLUSIONS: The DCP/NX-DCP ratio is very useful for diagnosing HCC. DCP in HCC patients is distinct from that in vitamin K-deficient patients.

Reducing effect of feeding powdered nacre of Pinctada maxima on the visceral fat of rats.

An abdominal fat accumulation complicated by high blood triglycerides is regarded as a risk factor of metabolic syndrome. Feeding powdered nacre, mother of pearl, from Pinctada maxima, resulted in reduced body weight, visceral fat amount, and blood triglyceride level without influencing the food intake, body length, or amount of muscular tissue, suggesting that nacre powder specifically could decrease visceral fat.

Effects of estradiol and progesterone on the proinflammatory cytokine production by mononuclear cells from patients with chronic hepatitis C.

AIM: To investigate the effects of estradiol (E2) and progesterone on the unstimulated and oxidative stress-stimulated production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-8, and macrophage chemotactic protein (MCP)-1 by peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis C and healthy controls. METHODS: The PBMCs were separated from age-matched 72 males and 71 females with and without chronic hepatitis C, who were divided into two groups based on a mean menopausal age of 50 years. Oxidative stress was induced by hydrogen peroxide in the cells incubated in serum-free media. Cytokines in the culture supernatant were measured by an enzyme-linked immunosorbent assay. RESULTS: The highest levels of the spontaneous production of TNF-alpha, IL-1beta, IL-8, and MCP-1 by the unstimulated PBMCs were in the older male patients with chronic hepatitis C and the lowest levels were in the pre-menopausal female healthy controls. E2 inhibited the cytokine production by the unstimulated PBMCs from the older male and post-menopausal female patients, which was further stimulated by progesterone. The exposure to hydrogen peroxide in the PBMCs from the younger male and pre-menopausal female healthy subjects induced the production of cytokines. The change rates of the hydrogen peroxide-stimulated cytokine production were suppressed by E2 and enhanced by progesterone. CONCLUSION: These findings suggest that E2 may play a favorable role in the course of persistent liver injury by preventing the accumulation of monocytes-macrophages and by inhibiting proinflammatory cytokine production, whereas progesterone may counteract the favorable E2 effects.

Opposing effects of estradiol and progesterone on the oxidative stress-induced production of chemokine and proinflammatory cytokines in murine peritoneal macrophages.

In inflammatory and oxidative liver injury, virus proteins and reactive oxygen species are involved in the regulation of proinflammatory cytokine production by macrophages. This study investigated the effects of estradiol (E2) and progesterone on the unstimulated and oxidative stress-stimulated production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, macrophage inflammatory protein (MIP)-2, and macrophage chemotactic protein (MCP)-1 by peritoneal macrophages isolated from male and female mice. E2 inhibited the cytokine production of TNF-alpha, IL-1beta, MIP-2, and MCP-1 by the unstimulated macrophages from males and females, which was then further stimulated by progesterone. The exposure to hydrogen peroxide in the macrophages from both sexes induced the production of cytokine. The hydrogen peroxide-stimulated cytokine production was suppressed by E2 and enhanced by progesterone. The sex hormone effects on the unstimulated and stimulated macrophages were blocked by their receptor antagonists and showed no significant difference between male and female subjects. These findings suggest that E2 may play a favorable role in the course of persistent liver injury, by inhibiting proinflammatory cytokine production, which, in addition, progesterone may counteract the favorable E2 effects through their receptors.

A histochemical and immunohistochemical investigation of guanase and nedasin in rat and human tissues.

Human guanase is known as a specific enzyme in the liver, kidney, and brain. However, its functional significance remains poorly understood. In addition, interestingly, a different organ distribution between humans and rats was suggested. Here, we performed immunohistochemical staining with anti-human nedasin (neuronal and endocrine discs large/SAP102 associated protein), whose sequence was identical to that of guanase, antibody and histochemical staining for guanase in normal tissues of rat and human liver, kidney, and small intestine, and compared the results. Guanase activity was observed uniformly in the rat hepatocytes, biliary epithelium and vascular endothelium cells, while it was localized to the hepatocytes and biliary epithelium in the human liver. When the histochemical staining for guanase and the immunohistochemical staining for nedasin were compared, the stained regions were different in the rat liver but were almost consistent in all human tissues. Totally consistent staining results were also obtained between rats and humans in the other organization except the liver. Based upon the research reports to date, the experiments on guanase and nedasin in rat organs performed in this study are considered to have important implications in the investigation of their physiological significance.

Histochemical and immunohistochemical investigation of guanase and nedasin in human tissues.

Guanase is known as an enzyme released from the liver. Recently, cloning and sequencing of the guanase gene were reported. In addition, almost simultaneously, it was reported that an unknown protein that binds to neuronal and endocrine lethal(1)-discs large (NE-dlg), one of the membrane-associated guanylate kinase homologues (MAGUK) family proteins involved in synaptic connection between neurons, was cloned and named nedasin (NE-dlg associated protein), whose sequence was almost identical to that of guanase. We immunostained fresh frozen sections of surgically removed human liver, kidney, and small intestine with anti-nedasin antibody, and simultaneously performed histochemical staining for guanase for comparison. Histochemically, guanase activity was observed in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the mucosal epithelium on the small intestine, and in the proximal tubule on the kidney. Immunohistochemically, a brown discoloration due to DAB oxidation was seen in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the proximal tubule but in the distal tubule a little on the kidney, in the mucosal epithelium on the small intestine. The stained region of the liver and the small intestine were different from that of the kidney. The different staining properties dependent on the organs were considered to be due to different isozymes. The physiological significance of guanase may vary with the isozymes, further studies are considered necessary.

Effects of estradiol and progesterone on tumor necrosis factor alpha-induced apoptosis in human hepatoma HuH-7 cells.

Oxidative stress, including the generation of reactive oxygen species (ROS), is known to be involved in apoptosis. Preventing apoptosis may thereby induce a malignant transformation of liver tumor cells. Estradiol (E2) is a potent endogenous antioxidant. We examined the proapoptotic role of progesterone as well as the antiapoptotic role of E2 in human hepatoma HuH-7 cells in a state of early apoptosis induced by tumor necrosis factor (TNF) alpha. The TNF alpha-induced ROS generation, lipid peroxidation, antioxidant enzyme consumption, a proapoptotic predominant expression of Bcl-2 family proteins, and a disruption of mitochondrial membrane potential were all inhibited by E2, and then they were further stimulated by progesterone in HuH-7 cells. The inhibitory effects of E2 were blocked by coincubation with progesterone. Treatment with the progesterone receptor antagonist RU486 led to the blockage of the progesterone-mediated responses to E2 pretreatment in TNF alpha-induced apoptosis. These findings demonstrate that E2 inhibits the TNF alpha-induced early apoptosis in hepatoma cells, by suppressing the oxidative stress processes, whereas progesterone acts in a manner opposite from the effects of E2, and the inhibitory effects of E2 were blocked by progesterone, thus leading to the apoptosis of hepatoma cells.

Clinical value of the determination of serum guanase activity in patients with chronic hepatitis type C.

The study examines the clinical significance of guanase (GU) measurement in patients with hepatitis C. 688 patients in whom either ALT was abnormal, or in whom HBsAg or HCVAb was detected in the serum, were enrolled into this study. The percentage of cases in which normal ALT while elevated GU was compared among the different disease groups. Then, the percentage of cases with normal ALT but elevated GU was compared between HBV and HCV groups. For the entire population, a significant correlation was observed between ALT and GU (r=0.872). The overall percentage of cases with normal ALT but elevated GU activity was 11.4%. In HCV group, 449 cases had normal ALT. Of these cases, 20.3% had elevated GU, while ALT was normal. Before 1989, no test to check donated blood for HCV antibody was available. However, screening of donated blood for high GU was associated with a reduced incidence of post-transfusion hepatitis. This is probably because following the screening, blood donated by patients with hepatitis C who had normal ALT but elevated GU was rejected. After the introduction of HCV antibody measurement, GU measurement is still useful to reveal the pathophysiological condition in-patients with chronic hepatitis type C.