Purification and Partial Characterization of an Indomethacin Hydrolyzing Enzyme from Pig Liver

Purpose. Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. Methods. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of -chlorobenzoic acid liberated from indomethacin by HPLC. Results. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in -naphthylacetate and -nitrophenylacetate, which are typical substrates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 µM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.     

Occurrence of paralytic shellfish poison (PSP) in the starfish Asterina pectinifera collected from the Kure Bay, Hiroshima Prefecture, Japan.

Assays were made for paralytic toxicity of marine invertebrates inhabiting at the coasts of Hiroshima Bay, where the infestation of bivalves such as cultured oysters with paralytic shellfish poison (PSP) has been occurred. The starfish Asterina pectinifera collected at the estuary of Nikoh River, Hiroshima Bay, was found to contain moderate levels of paralytic toxicity. Its highest toxicities as PSP found on July 30, 1999 were 12.5 MU/g for whole body, 11.0 MU/g for integument tissues and 3.9 MU/g for viscera, respectively. The toxicity of integument was changed from 3.6 to 11.0 MU/g in 1 year. Its paralytic toxin principles were identified as PSP toxins, composing mainly from saxitoxin (STX) group toxins such as carbamoyl-N-hydroxy neosaxitoxin (hyneoSTX), and STX, by HPLC and LC-MS, accounting for over 90 mol%. The PSP toxins contained in the starfish A. pectinifera considered to be transferred from bivalves or detritus living in the same area, which were contaminated with PSP. However, the involved pathway may be different from that of Asterias amurensis which was infested directly through food chain from its food bivalves, for its toxin pattern.     

Induction of Squamous Cell Carcinomas in the Salivary Glands of Rats by Potassium Iodide

In a 2-year carcinogenicity study of potassium iodide (KI) in F344/DuCrj rats, squamous cell carcinomas (SCCs) were observed in the salivary glands of 4/40 males and 3/40 females receiving 1000 ppm KI in the drinking water. Ductular proliferation with lobular atrophy was observed at high incidence in the submandibular glands of the high-dose animals, and squamous metaplasia was frequently evident within the proliferative ductules and the larger interlobular ducts. A transition from metaplasia to SCC was apparent. The results suggest that squamous metaplasia in proliferative ductules, occurring secondarily to lobular impairment induced by KI, may develop into SCCs via a non-genotoxic, proliferation-dependent mechanism.     

Somatic Mutations of the PTEN/MMAC1 Gene in Fifteen Japanese Endometrial Cancers: Evidence for Inactivation of Both Alleles

Loss of heterozygosity (LOH) of chromosome 10q is observed in approximately 40% of endometrial cancers. Mutations in PTEN/MMAC1 , a gene recently isolated from the 10q23 region, are responsible for two dominantly inherited neoplastic syndromes, Cowden disease and Bannayan-Zonana syndrome. Somatic mutations of this gene have also been detected in sporadic cancers of the brain, prostate and breast. To investigate the potential role of this putative tumor suppressor gene in endometrial carcinogenesis as well, we examined 46 primary endometrial cancers for LOH at the 10q23 region, and for mutations in the entire coding region and exon-intron boundaries of the PTEN/MMAC1 gene. LOH was identified in half of the 38 informative cases, and subtle somatic mutations were detected in 15 tumors (33%). Our results suggest that of the genes studied so far in endometrial carcinomas, PTEN/MMAC1 is the most commonly mutated one, and that inactivation of both copies by allelic loss and/or mutation, a pattern that defines genes as "tumor suppressors,'contributes to tumorigenesis in endometrial cancers.     

Green tea component, catechin, induces apoptosis of human malignant B cells via production of reactive oxygen species.

PURPOSE: Green tea polyphenol, (-)-epigallocatechin-3-gallate, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of (-)-epigallocatechin-3-gallate as a novel therapeutic agent for the patients with B-cell malignancies including multiple myeloma. EXPERIMENTAL DESIGN: We investigated the effects of (-)-epigallocatechin-3-gallate on the induction of apoptosis in HS-sultan as well as myeloma cells in vitro and further examined the molecular mechanisms of (-)-epigallocatechin-3-gallate-induced apoptosis. RESULTS: (-)-Epigallocatechin-3-gallate rapidly induced apoptotic cell death in various malignant B-cell lines in a dose- and time-dependent manner. (-)-Epigallocatechin-3-gallate-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials (Deltapsim); the release of cytochrome c, Smac/DIABLO, and AIF from mitochondria into the cytosol; and the activation of caspase-3 and caspase-9. Elevation of intracellular reactive oxygen species (ROS) production was also shown during (-)-epigallocatechin-3-gallate-induced apoptosis of HS-sultan and RPMI8226 cells as well as fresh myeloma cells. Antioxidant, catalase, and Mn superoxide dismutase significantly reduced ROS production and (-)-epigallocatechin-3-gallate-induced apoptosis, suggesting that ROS plays a key role in (-)-epigallocatechin-3-gallate-induced apoptosis in B cells. Furthermore, a combination with arsenic trioxide (As2O3) and (-)-epigallocatechin-3-gallate significantly enhanced induction of apoptosis compared with As2O3 alone via decreased intracellular reduced glutathione levels and increased production of ROS. CONCLUSIONS: (-)-Epigallocatechin-3-gallate has potential as a novel therapeutic agent for patients with B-cell malignancies including multiple myeloma via induction of apoptosis mediated by modification of the redox system. In addition, (-)-epigallocatechin-3-gallate enhanced As2O3-induced apoptosis in human multiple myeloma cells.     

Regulation of biosynthesis and phosphorylation of P210 protein during differentiation induction of K 562 cells

The changes of P210^bcr/abl and other tyrosine phosphorylated proteins in K562 cells during growth and differentiation were studied by metabolic labeling with ³²PO₄ and immunoprecipitation with anti-phosphotyrosine sera. The anti-phosphotyrosine sera recognized P210^bcr/abl and other phosphoproteins with mol wt of 150, 115, 100, 70 and 64 kD. The 2-day incubation of K562 cells with inducers for differentiation, hemin, sodium butyrate and TPA, decreased the level of P210^bcr/abl protein and other phosphoproteins. Inhibitors of tyrosine protein kinase, amiloride and genistein, also reduced the phosphorylation of P210^bcr/abl protein and other substrates, but did not induce differentiation of the cells. Although most of these additives inhibited the cell growth, cytotoxic agents such as adriamycin, vincristine and Ara-C did not affect the level of P210^bcr/abl protein. The experiments using ³⁵S-methionine labeled cells and the immunoprecipitation with anti-abl sera suggested that reduced biosynthesis but not dephosphorylation of P210^bcr/abl protein mainly accounted for the reduction of P210^bcr/abl protein reacting to anti-phosphotyrosine sera in differentiation-induced cells. These results indicate that the reduction of P210^bcr/abl protein synthesis plays some roles in cellular differentiation of K562 cells.     

Frequent expression of CD30 in extranodal NK/T‐cell lymphoma: Potential therapeutic target for anti‐CD30 antibody‐based therapy

Extranodal NK/T‐cell lymphoma, nasal type (ENKTL) is a subtype of non‐Hodgkin lymphoma with a poor prognosis. Although first‐line treatments for patients with localized ENKTL have been established, there is no gold standard treatment for patients with advanced ENKTL and refractory and/or relapsed disease. Anti‐CD30 antibody‐based therapy, including brentuximab vedotin (BV), has been shown to target malignant lymphomas with CD30 expression. In particular, this therapeutic agent has recently been suggested to be effective for Hodgkin lymphoma and mature T‐cell lymphoma. However, the efficacy of BV toward ENKTL has not yet been established. Therefore, we investigated the expression of CD30 in a large cohort to evaluate BV as a potential treatment for ENKTL. In this study, 97 Japanese patients with newly diagnosed ENKTL between January 2007 and December 2015 were enrolled. Flow cytometry and immunohistochemistry were performed for the evaluation of CD30 expression. If the cut‐off value of CD30 expression is 1% or more, there were 55 positive cases (56.5%). According to the localization of lesion, the frequency of CD30 expression was significantly higher in the non‐nasal type than in the nasal type (P = .0394). No differences were observed in almost all clinical characteristics between CD30‐positive cases and CD30‐negative cases. In addition, the expression of CD30 was not a prognostic factor for either overall survival or progression‐free survival. In conclusion, frequent expression of CD30 in ENKTL suggests anti‐CD30 antibody‐based therapy may be an effective treatment.     

Dietary glycemic and insulin scores and colorectal cancer survival by tumor molecular biomarkers

Accumulating evidence suggests that post‐diagnostic insulin levels may influence colorectal cancer (CRC) survival. Yet, no previous study has examined CRC survival in relation to a post‐diagnostic diet rich in foods that increase post‐prandial insulin levels. We hypothesized that glycemic and insulin scores (index or load; derived from food frequency questionnaire data) may be associated with survival from specific CRC subtypes sensitive to the insulin signaling pathway. We prospectively followed 1,160 CRC patients from the Nurses' Health Study (1980–2012) and Health Professionals Follow‐Up Study (1986–2012), resulting in 266 CRC deaths in 10,235 person‐years. CRC subtypes were defined by seven tumor biomarkers (KRAS, BRAF, PIK3CA mutations, and IRS1, IRS2, FASN and CTNNB1 expression) implicated in the insulin signaling pathway. For overall CRC and each subtype, hazard ratio (HR) and 95% confidence interval (95% CI) for an increase of one standard deviation in each of glycemic and insulin scores were estimated using time‐dependent Cox proportional hazards model. We found that insulin scores, but not glycemic scores, were positively associated with CRC mortality (HR = 1.19, 95% CI = 1.02–1.38 for index; HR = 1.23, 95% CI = 1.04–1.47 for load). The significant positive associations appeared more pronounced among PIK3CA wild‐type cases and FASN‐negative cases, with HR ranging from 1.36 to 1.60 across insulin scores. However, we did not observe statistically significant interactions of insulin scores with PIK3CA, FASN, or any other tumor marker (p interaction > 0.12). While additional studies are needed for definitive evidence, a high‐insulinogenic diet after CRC diagnosis may contribute to worse CRC survival.     

Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non‐Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B‐cell lymphoma cells harboring gain‐of‐function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL‐AF9, MLL‐AF4, and AML1‐ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications.     

CXCL12/CXCR4 activation by cancer‐associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer

Cancer‐associated fibroblasts (CAFs) are reportedly involved in invasion and metastasis in several types of cancer, including gastric cancer (GC), through the stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying these tumor‐promoting effects are not well understood, which limits the potential to develop therapeutic targets against CAF‐mediated CXCL12/CXCR4 signaling. CXCL12 expression was analyzed in resected GC tissues from 110 patients by immunohistochemistry (IHC). We established primary cultures of normal fibroblasts (NFs) and CAFs from the GC tissues and examined the functional differences between these primary fibroblasts using co‐culture assays with GC cell lines. We evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF‐573,228) on the invasive ability of GC cells. High CXCL12 expression levels were significantly associated with larger tumor size, increased tumor depth, lymphatic invasion and poor prognosis in GC. CXCL12/CXCR4 activation by CAFs mediated integrin β1 clustering at the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient than PF‐573,228 at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin β1 in GC cells, resulting in GC progression. Taken together, the inhibition of CXCL12/CXCR4 signaling in GC cells may be a promising therapeutic strategy against GC cell invasion.