Protumoral roles of melanoma inhibitory activity 2 in oral squamous cell carcinoma.

BACKGROUND: The role of melanoma inhibitory activity 2 (MIA2) was examined in human oral squamous cell carcinoma (OSCC). METHODS: MIA2 role was examined by immunohistochemistry of human OSCCs and knockdown studies using human 3 OSCC cell lines with MIA2 expression. RESULTS: MIA2 expression was observed in 62 (66.7%) of 93 OSCCs and was associated with tumour expansion and nodal metastasis. Melanoma inhibitory activity 2 expression was inversely correlated with intratumoral infiltration of lymphocytes. Invasion and anti-apoptotic survival were reduced by MIA2 knockdown in HSC3 cells. MOLT-3 lymphocytes infiltrating the HSC3 cell layer was enhanced by MIA2 knockdown or MIA2 depletion with the antibody. In HSC3 cells, MIA2 knockdown decreased the expressions of vascular endothelial growth factor (VEGF), VEGF-C, and VEGF-D. The downregulation of VEGF-C and -D was caused by inhibition of p38 and extracellular signal-regulated kinase (ERK)1/2, respectively. Melanoma inhibitory activity 2 was co-precipitated with integrin α4 andα5 in HSC3 cells. Integrin α4 knockdown decreased p38 phosphorylation and increased apoptosis, whereas integrin α5 knockdown decreased c-Jun N-terminal kinase (JNK) phosphorylation and apoptosis. Inhibition of JNK decreased apoptosis in the HSC3 cells. CONCLUSION: These findings suggest that the roles of MIA2 might be based on the variety of the integrins and the subtypes of mitogen-activated protein kinase.

Downregulation of miR-126 induces angiogenesis and lymphangiogenesis by activation of VEGF-A in oral cancer.

BACKGROUND: MicroRNA (miRNA)-126 (miR-126) is an endothelial-specific miRNA located within intron 7 of epidermal growth factor-like domain 7 (EGFL7). However, the role of miR-126 in cancer is controversial. METHODS: We examined the function of miR-126 in oral squamous cell carcinoma (OSCC) cells. Furthermore, a series of 118 cases with OSCC were evaluated for the expression levels of miR-126. RESULTS: MicroRNA-126 (miR-126) was associated with cell growth and regulation of vascular endothelial growth factor-A activity, and demethylation treatment increased expression levels of miR-126 and EGFL7 in OSCC cells. A significant association was found between miR-126 expression and tumour progression, nodal metastasis, vessel density, or poor prognosis in OSCC cases. In the multivariate analysis, decreased miR-126 expression was strongly correlated with disease-free survival. CONCLUSION: The present results suggest that miR-126 might be a useful diagnostic and therapeutic target in OSCC.

CD10 enhances metastasis of colorectal cancer by abrogating the anti-tumoural effect of methionine-enkephalin in the liver.

OBJECTIVE: To examine the role of CD10, a characteristic marker of liver metastasis of colorectal cancers (CRCs). DESIGN: The effect of CD10 and Met-enkephalin (MENK) in CD10-positive and -negative human CRC cells was investigated under in vitro and in vivo conditions. Human CRC samples were examined. MAIN OUTCOME MEASURE: CD10-positive and CD10-knockdown HT29 cells and CD10-negative and CD10-transfected Colo320 cells in nude mice were treated with MENK and/or the CD10 inhibitor (thiorphan). Intracellular signalling of MENK and delta-opioid receptor (DOR) was examined by immunoblotting. RESULTS: MENK inhibited the growth, invasion and survival of CRC cells following thiorphan-induced CD10 inactivation. Thiorphan suppressed liver metastasis of CD10-positive CRC cells. Inoculation of mice with CRC cells induced MENK expression in the liver. Inhibition of hepatic MENK expression by cholesterol-conjugated antisense S-oligodeoxynucleotide increased liver metastasis of CRC cells even when the cells did not express CD10. DOR activation by MENK decreased the phosphorylation of epidermal growth factor receptor and extracellular signal-regulated kinase and increased p38-dependent apoptosis. Nitric oxide was found to induce DOR expression in CRC cells. Co-treatment with thiorphan and a nitric oxide donor had a marked anti-tumour effect on liver metastasis of HT29 cells. Of 68 CRC patients, 19 (28%) showed CD10 expression, which was dependent on the extent of liver metastasis. MENK concentration in metastasis-positive human liver was higher than that in the normal liver. CONCLUSION: CD10 expression in CRC cells abrogates the anti-tumour effect of hepatic MENK by degrading it, which enhances liver metastasis of CD10-positive CRC cells.

Reg IV expression is associated with cell growth and prognosis of adenoid cystic carcinoma in the salivary gland.

AIMS: Regenerating islet-derived family, member 4 (Reg IV) is associated with the progression of various cancers. The aim was to examine Reg IV expression in adenoid cystic carcinomas (ACCs) in salivary glands. METHODS AND RESULTS: Reg IV expression was detected by immunohistochemistry and compared with clinicopathological parameters. Expression of phosphorylated epidermal growth factor receptor (pEGFR), phosphorylated AKT (pAKT) and MUC2 was examined by immunohistochemistry. Reg IV function was assessed with Reg IV antisense S-oligodeoxynucleotides (AS) in ACC3 human ACC cells. Reg IV was expressed by salivary duct epithelia and acinus myoepithelia, but not in squamous epithelia. Reg IV expression was found in 41% (17/41) of ACCs, but in none of 40 oral squamous cell carcinomas (OSCCs) and was associated with nodal metastasis (P = 0.047) and poor prognosis (P = 0.012) in ACCs. Reg IV expression was associated with pEGFR (14/17, 82%) in Reg IV+ ACCs, but had no relationship with pAKT or MUC2 expression in ACCs. Cell growth was inhibited by AS treatment in Reg IV+ ACC3 cells, but not in HSC-4 OSCC cells, whereas in vitro invasion of neither cell types was affected by AS treatment. CONCLUSIONS: These results suggest that Reg IV might accelerate cell growth and disease progression of ACCs.

Interleukin-15 and transforming growth factor alpha are associated with depletion of tumor-associated macrophages in colon cancer.

We examined the effects of IL-15 and TGF-alpha on the inhibition of macrophage infiltration into colon cancer tissue, and secretion of amphoterin from colon cancer cells. Production of IL-15 and/or TGF-alpha was associated with depletion of tumor-associated macrophages (TAMs) in both Dukes' B and C tumors (P = 0.0324 and 0.0051, respectively). Production of IL-15 and/or TGF-alpha was also associated with amphoterin mRNA expression in colon cancer tissues with TAM depletion in both Dukes' B and C tumors (P = 0.0167 and P = 0.0062, respectively). WiDr human colon cancer cells treated with IL-15 and/or TGF-alpha induced reduction of nucleus-localized amphoterin and an increase in cytosolic and membranous amphoterin. Moreover, IL-15 and/or TGF-alpha treatment increased amphoterin secretion by WiDr cells. Most notably, IL-15 and TGF-alpha treatment induced the increase of cytosol/membrane localization and secretion of amphoterin and the most pronounced effect among the treatments carried out. These results suggested that IL-15/TGF-alpha promotes depletion of TAMs and secretion of amphoterin in colon cancer.

High concentration of deoxycholic acid abrogates in vitro transformation of IEC6 intestinal cells by azoxymethane.

In this study, we designed an in vitro azoxymethane (AOM)-induced carcinogenesis model and analyzed the effect of deoxycholic acid (DCA) on growth, apoptosis, genotoxicity, and transformation of IEC6 intestinal cells. CYP2E1 production was confirmed in IEC6 cells. The growth of IEC6 cells was enhanced by DCA (100 microg/ml). However, IEC6 cells treated with DCA (200 microg/ml) were inhibited and disappeared at 48 hrs after treatment. Apoptotic cells increased 11.2 times by treatment with DCA (200 microg/ml) as compared to cells with no treatment. DNA injury detected by comet assay was found in cells treated with AOM, but not in cells treated with DCA (100 microg/ml) and AOM. The number of colony formation in soft agar increased by AOM treatment. However, the number of foci treated with DCA (100 microg/ml) plus AOM was 69% that of cells treated with AOM alone. Two out of the 6 mice subcutaneously injected with AOM-treated IEC6 cells showed tumorigenesis, whereas IEC6 cells treated with DCA (100 microg/ml) plus AOM or DCA (100 microg/ml) alone did not form any tumor. Reduced protein expression of MLH1, Bcl-2 was detected in IEC6 cells treated with DCA (100 microg/ml). Production of Bax, pJNK, TGF-beta, TGFBRI, TGFBRII, and beta-catenin were higher in IEC6 cells treated with DCA (100 microg/ml) than that in cells with no treatment. These results suggest that high-dose DCA induced apoptosis and inhibited AOM-induced in vitro transformation of IEC6 cells.

BBM-928, a new antitumor antibiotic complex. II. Taxonomic studies on the producing organism.

An actinomycete strain No. G455-101 isolated from a soil sample collected in Luzon Island, Philippines produced a new antitumor antibiotic complex BBM-928. The organism was determined to be a new species of the genus Actinomadura and designated Actinomadura luzonensis nov. sp. The type strain, No. G455-101, has been deposited under the number ATCC 31491.

Taxonomy of the antibiotic Bu-2313-producing organism. Microtetraspora caesia sp. nov.

An aerobic actinomycete strain isolated from an Indian soil sample and designated No. E864-61 was found to produce in submerged fermentation a new antibiotic complex, Bu-2313 (components A and B). Strain E864-61 forms single, pairs or chains of three to eight spores on the aerial mycelium and its aerial mass color is grayish blue-green. The cell wall of strain E864-61 contains meso-diaminopimelic acid and galactose. Strain E864-61 has been classified as a new species of the genus Microtetraspora and designated as Microtetraspora caesia sp. nov.

Streptoalloteichus, a new genus of the family Actinoplanaceae.

A new genus Streptoalloteichus is proposed in the family Actinoplanaceae to distinguish species of actinomycetes which form short or long spore-chains on aerial mycelium, bears oval sporangia with motile spores and has a characteristic cell-wall composition of strain C677-91 type. Strain C677-91 (ATCC 31217, FERM-P No. 4070) was named Streptoalloteichus hindustanus gen. nov. and sp. nov. The actinomycete strain C677-91 produces spore-chain clusters and sclerotia in the aerial mycelium which are morphologically similar to those found in some species of Streptomyces. The cultural characteristics of the strain on agar media also resemble those of Streptomyces species and the colonies have no distinct color. Strain C677-91 produces sporangia or sporangia-like vesicles which contain one to several spores in the vegetative mycelium. The sporangiospores possess a single long polar flagellum and are motile. The cell wall of strain C677-91 contains meso-alpha,epsilon-diaminopimelic acid, alanine, glutamic acid, galactose, mannose, rhamnose and glucosamine. Strain C677-91 has several important characteristics in common with Streptomyces tenebrarius including the production of nebramycin factors but the latter strain does not produce sporangia.

Repression of MLH1 and MGMT genes in colon mucosa adjacent to implanted cancer in athymic mouse.

Hyperplastic mucosa adjacent to colon cancer, being a reactive change, accelerates cancer progression and its metastasis through expression of angiogenic factors. We investigated promoter methylation in hyperplastic mucosa adjacent to orthotopic KM12SM colon cancer in mice. In the hyperplastic mucosa adjacent to KM12SM tumors in the cecum of athymic mice, reductions in the levels of the mutL homologue 1 (MLH1) and O6-methylguanine-DNA methyltransferase (MGMT) proteins were detected by immunohistochemistry and immunoblotting. To examine the effects of growth factors and cytokines on promoter methylation and repressed expression of the MLH1 and MGMT genes, a rat intestinal epithelial cell line, IEC6, was treated with epidermal growth factor (EGF) and interleukin (IL)-15 for 35 days. Protein levels of MLH1 and MGMT were reduced in EGF- and IL-15-treated IEC6 cells. A methylation-sensitive restriction enzyme assay revealed that CpG methylation was present in the promoter regions of the MLH1 and MGMT genes in DNAs extracted from hyperplastic mucosa adjacent to KM12SM tumors. These findings suggest that promoter CpG methylation affects expression of MLH1 MGMT genes in hyperplastic mucosa adjacent to colon cancer.

[Oxidation of hydrogen sulfide with cell-free extract of Hyphomicrobium neptunium ATCC 15444].

Soluble (Fr. 1) and membrane (Fr. 2) fractions were prepared from the cell-free extract of Hyphomicrobium neptunium ATCC 15444, and their effects on the oxidization of hydrogen sulfide (H2S) were studied. When H2S gas was supplied to Fr. 1 and Fr. 2, sulfur in both fractions and the majority of thiosulfate ion in Fr. 1 were detected. The sulfide-oxidizing activity in Fr. 2 but not in Fr. 1 was inhibited by the addition of diethyldithiocarbamate, suggesting that Fr. 1 and Fr. 2 have different types of sulfide-oxidase, and that Fr. 2 would include cytochrome c dependent sulfide oxidase. It was also found that thiosulfate ion was formed from sulfur and sulfite ion by adding Fr. 1. However, the pronase-treatment of Fr. 1 did not influence on the formation of thiosulfate ion. These results suggest that H2S was oxidized to sulfur and sulfite ion by two types of sulfide oxidase and sulfur oxidase in H. neptunium ATCC 15444, and that the sulfite ion changed rapidly to thiosulfate through a non-enzymatic reaction with sulfur.

[An oxidation enzyme for hydrogen sulfide from Hyphomicrobium neptunium ATCC15444].

We described in our previous paper that the cell-free extract from Hyphomicrobium neptunium ATCC 15444 oxidized hydrogen sulfide to sulfur. We tried to purify the enzyme used for this oxidation to determine the molecular nature of this enzyme. The 40% ammonium sulfate precipitate from the supernatant of cell sonicate of H. neptunium was chromatographed on a column of Phenyl Sepharose CL-4B and its active fractions were collected. These fractions were further purified through Superose 12 and then TSK gel G3000SW column chromatographies. A protein of molecular weight about 54,000 and isoelectric point pI 6.8 was isolated. However, this final protein was found to reduce its activity to less than one-tenth of those of ammonium sulfate precipitate and of the fraction from Phenyl Sepharose CL-4B column chromatography. This might be caused either by the loss of accessory component or by its requirement of low molecular factors necessary for its activation at the stage of gel filtration. The neutral isoelectric point of this enzyme could be suitable as the function of H. neptunium because its final product was S0, and it grows at neutral pH. In contrast, the final oxidative product of hydrogen sulfide by Thiobascillus is sulfuric acid, and they grow at acidic pH.

Co-treatment with deoxycholic acid and azoxymethane accelerates secretion of HMGB1 in IEC6 intestinal epithelial cells.

OBJECTIVES: High-mobility group box 1 (HMGB1) is a nuclear protein that acts as a ligand of the receptor for advanced glycation end products (RAGE) and its expression enhances progression of cancer. However, the mechanism underlying HMGB1 secretion is still unclear. In this study, we examined the effect of deoxycholic acid (DCA), a promoter of colon carcinogenesis, on HMGB1 secretion. MATERIALS AND METHODS: We used an in vitro transformation model comprised of IEC6 intestinal epithelial cells treated with azoxymethane (AOM) and/or DCA. HMGB1 expression and secretion were examined by Western and Northern blot analyses, and ELISA. Intracellular translocation of HMGB1 was examined by protein fractionation. RESULTS: AOM + DCA-treated IEC6 cells showed upregulation of HMGB1 mRNA expression and increased level of HMGB1 protein in culture medium, but decreased level of HMGB1 protein in the nucleus. AOM + DCA treatment increased level of histone H4 acetylation, which induced translocation of HMGB1 from the nucleus to the cytoplasm and increased HMGB1 secretion. Leptomycin B inhibited extranuclear translocation and secretion of the HMGB1 protein. CONCLUSION: These findings suggest that DCA affects intracellular localization and secretion of HMGB1.

Reg IV enhances peritoneal metastasis in gastric carcinomas.

OBJECTIVES: The role of Regenerating (Reg) IV on peritoneal metastasis was examined in gastric cancer using. MATERIAL AND METHODS: Reg IV-transfected human gastric cancer cells (MKN28-R1, MKN28-R2, TMK1-R1), control transfectants (MKN28-R0, TMK1-R0), and REG4-knocked down MKN45 cells were examined in in vitro and in nude mice peritoneal metastasis models. RESULTS AND DISCUSSION: Increase of expression and secretion of Reg IV, and levels of BCL-2, BCL-XL,survivin, phosphorylated AKT, and phosphorylated EGFR, and decrease of nitric oxide-induced apoptosis were found in Reg IV-transfectants, whereas those were abrogated in the knockdown cells. In mice models, increased number and size of peritoneal tumors and decreased apoptosis were found in Reg IV-transfectants, whereas those were abrogated by the knockdown cells. Mice survivals were worsened in Reg IV-transfectants-inoculated mice, but were improved in Reg IV-knockdown cell-inoculated mice. Levels of Reg IV protein in peritoneal lavage fluids increased in Reg IV-transfectants inoculated mice, but decreased in Reg IV-knockdown cell inoculated mice. In metastasized human gastric cancers, Reg IV positivity in peritoneum-metastasis cases was higher than those in negative cases. Reg IV was detected in peritoneal lavage fluids from human gastric cancer patients, in whose lavages keratin mRNA was detected by reverse transcriptase-polymerase chain reaction. Collectively, Reg IV might accelerate peritoneal metastasis in gastric cancer. Reg IV in lavage fluids might be a good marker for peritoneal metastasis.

Reversal of expression of 15-lipoxygenase-1 to cyclooxygenase-2 is associated with development of colonic cancer.

AIMS: Two different pathways of linoleic acid (LA) metabolism have opposite effects on the development of colonic cancer: a protumoral prostaglandin cascade metabolized by cyclooxygenase (COX)-2, and an antitumoral peroxisome proliferator-activated receptor (PPAR)-gamma ligands metabolized by 15-lipooxygenase (LOX)-1. The aim was to examine the switching of the two LA metabolic pathways in colonic adenomas and carcinomas. MATERIALS AND METHODS: The expression of 15LOX-1 mRNA and COX-2 protein was examined in 54 adenomas, 21 pTis carcinoma-in-adenoma lesions and 36 pT3/p Stage II carcinomas of the colon by in-situ hybridization and immunohistochemistry, respectively. RESULTS: 15LOX-1 expression was found in 89% (48 of 54) of adenomas, 43% (nine of 21) of adenomas and 10% (two of 21) of carcinomas in carcinoma-in-adenoma lesions, but not in pT3 carcinomas (P < 0.0001). In contrast, COX-2 production was found in 11% (six of 54) of adenomas, 52% (11 of 21) of adenomas and 71% (15 of 21) of carcinomas in carcinoma-in-adenoma lesions, and 92% (33 of 36) of pT3 carcinomas (P < 0.0001). Concurrence of 15LOX-1 down-regulation and COX-2 up-regulation was found in 6% (three of 54) of adenomas, 33% (seven of 21) of adenomas and 71% (15 of 21) of carcinomas in carcinoma-in-adenoma lesions, and 92% (33 of 36) of pT3 carcinomas (P < 0.0001). CONCLUSIONS: These results suggest that switching of LA metabolism by reversal of the expression of 15LOX-1 and COX-2 is associated with acquisition of malignant potential in colonic neoplasia.

Receptor for advanced glycation end products (RAGE) is important in the prediction of recurrence in human oral squamous cell carcinoma.

AIMS: Receptor for advanced glycation end products (RAGE) has recently been recognized as a cancer-associated protein responsible for cancer progression and metastasis in gastrointestinal cancers. The aim was to examine the role of RAGE in oral squamous cell carcinoma (OSCC). METHODS AND RESULTS: RAGE expression was examined by immunohistochemistry in 74 OSCC patients and evaluated with a grading based on Allred's score. RAGE expression was compared with clinicopathological parameters including clinical stage, invasive depth, nodal metastasis, disease recurrence and disease-free survival. High-grade expression of RAGE (RAGE-H) was observed in 30 (40.5%) of 74 OSCCs. RAGE-H was associated with depth of invasion (P < 0.0001) and local recurrence (P < 0.0001), but not with histological differentiation, clinical stage or nodal metastasis. Disease-free survival in patients with RAGE-H was significantly worse than in those with low-level RAGE expression. Multivariate analysis showed RAGE-H to be an independent prognostic factor for disease-free survival in OSCC patients (P = 0.0022). CONCLUSION: RAGE is a relevant factor in predicting disease recurrence and patients' prognosis in OSCC.

Determinants for prediction of malignant potential by metalloproteinase:E-cadherin ratio in prostate core needle biopsy.

According to a good correlation between in situ hybridization-based metalloproteinase-2/9:E-cadherin ratio (MER) and the pathological stage of prostate cancer, we set the cutoff line of MER at 6.0 (MER>6) to distinguish between organ-confined (pT2) and advanced diseases (pT3a-b/N1). In this study, we looked at the factors affecting MER and leading to a misprediction of the pathological stage. We examined MER in 39 paired specimens of prostate core needle biopsy and prostatectomy from the same patient and compared these MERs. In 34 (87%) of 39 cases, the MER of biopsy was correlated with the final pathological stage (pT2 vs. pT3a-b/N1). MER ranges in pT3a-b/N1 cancer were significantly wider than those in pT2 cancer (p < 0.01). The number of MER>6 fields in Gleason score 8-9 cancer was larger than that in Gleason score 7 cancer (p < 0.0001). In 5 cases where there was a failure to distinguish pT2 from pT3a-b/N1, the misdiagnosis was significantly associated with a small number of biopsies (4 or 6 specimens; p = 0.0469), a small amount of tumor tissue in biopsy specimens (less than 5 mm; p = 0.0492), and a wide MER range (more than 5.0; high intratumoral heterogeneity; p = 0.0202). Considering these factors increases the usefulness of preoperative prediction of the final pathological stage by MER in prostate cancer.