Thrombin cleaves recombinant soluble thrombomodulin into a lectin-like domain fragment and a fragment with protein C-activating cofactor activity.

Thrombomodulin (TM) is a transmembrane protein that plays an important role in regulating the coagulation system by acting as a cofactor for thrombin in protein C activation. Additionally, TM is involved in inflammation. Previous studies have shown that soluble fragments of TM of varying sizes, which are derived from membrane-bound TM, are present in plasma and urine. Soluble fragments of TM are speculated to exhibit biological activity. Among these, a lectin-like domain fragment (TMD1) is of particular importance. Recombinant TMD1 has previously been shown to attenuate lipopolysaccharide-induced inflammation. Here, we report that thrombin cleaves recombinant soluble TM, which is used for the treatment of disseminated intravascular coagulation associated with sepsis, into TMD1 and a fragment comprising the C-terminal portion of TM (TMD23), the latter of which retains the cofactor activity for activating protein C. Our findings suggest that thrombin not only activates protein C on membrane-bound TM but may also cleave TM to generate TMD1.

Production of anti-carbohydrate antibodies by phage display technologies: potential impairment of cell growth as a result of endogenous expression.

Anti-mannotriose (Man3) antibodies were previously isolated from a Keio phage library displaying human single chain variable fragments (scFvs) using a neoglycolipid, Man3- dipalmitoylphosphatidylethanolamine. Of three genes constructed, the 5A3 clone was expressed in mouse myeloma NS0 cells as a conjugate with human IgG(1) Fc (scFv-Fc) and characterized (Sakai, K., Shimizu, Y., Chiba, T., Matsumoto-Takasaki, A., Kusada, Y., Zhang, W., Nakata, M., Kojima, N., Toma, K., Takayanagi, A., Shimizu, N., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 253-262; Zhang, W., Matsumoto-Takasaki, A., Kusada, Y., Sakaue, H., Sakai, K., Nakata, M., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 263-270). Similarly, anti-Le(x) phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Le(x))-dipalmitoylphosphatidylethanolamine. Of five phage clones isolated, two scFv genes were constructed to express scFv-Fc proteins in NS0 cells. As was experienced with anti-Man3 scFv-Fc clones, only one anti-LNFPIII clone, 1F12, was successfully produced and purified as an scFv-Fc protein. Although anti-LNFPIII 1F12 and anti-Man3 5A3 scFv-Fc proteins were secreted into media, a decline in scFv-Fc production was observed with both stable clones during early passages. Transient expression of anti-LNFPIII and anti-Man3 scFv-Fc genes in COS-7 cells and subsequent analyses of scFv-Fc protein expression revealed accumulation of translated proteins in the endoplasmic reticulum for scFv-Fc proteins derived from clones that did not survive as stable clones. This report describes the following: (i) isolation of anti-LNFPIII scFv genes; (ii) purification of anti-LNFPIII scFv-Fc proteins from stably and transiently expressed cells; and (iii) extracellular or intracellular localization of two anti-LNFPIII and three anti-Man3 scFv-Fc proteins. The results suggest that expression of anti-Man3 and other anti-carbohydrate antibodies in mammalian cells is disadvantageous for cell growth.

Factor G utilizes a carbohydrate-binding cleft that is conserved between horseshoe crab and bacteria for the recognition of beta-1,3-D-glucans.

In the horseshoe crab, the recognition of beta-1,3-D-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes beta-1,3-D-glucans. To gain an insight into the recognition of beta-1,3-D-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than approximately 30 and 3 microM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble beta-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble beta-1,3-D-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a beta-sheet in a predicted beta-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for beta-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.

Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and IgG2b Fc-associated effector functions.

Replacement of aspartic acid by alanine at position 265 (D265A) in mouse IgG1 results in a complete loss of interaction between this isotype and low-affinity IgG Fc receptors (Fc gammaRIIB and Fc gammaRIII). However, it has not yet been defined whether the D265A substitution could exhibit similar effects on the interaction with two other Fc gammaR (Fc gammaRI and Fc gammaRIV) and on the activation of complement. To address this question, 34-3C anti-RBC IgG2a and IgG2b switch variants bearing the D265A mutation were generated, and their effector functions and in vivo pathogenicity were compared with those of the respective wild-type Abs. The introduction of the D265A mutation almost completely abolished the binding of 34-3C IgG2a and IgG2b to all four classes of Fc gammaR and the activation of complement. Consequently, these mutants were hardly pathogenic. Although oligosaccharide side chains of these mutants were found to contain higher levels of sialic acids than those of wild-type Abs, the analysis of enzymatically desialylated D265A variants ruled out the possibility that very poor Fc-associated effector functions of the D265A mutants were due to an increased level of the mutant Fc sialylation. Thus, our results demonstrate that aspartic acid at position 265 is a residue critically implicated in triggering the Fc-associated effector functions of IgG, probably by defining a crucial three-dimensional structure of the Fc region.

KL-6 mucin in metastatic liver cancer tissues from primary colorectal carcinoma.

BACKGROUND/AIMS: KL-6 mucin is MUC1 bearing sialylated carbohydrate epitopes recognized by KL-6 antibody. This study aimed to assess subcellular localization of KL-6 mucin in liver metastatic tissues from colorectal carcinoma and hepatocellular carcinoma tissues. METHODOLOGY: Tissue samples were collected from 56 patients with liver metastasis of colorectal carcinoma and 92 patients with hepatocellular carcinoma who underwent a hepatectomy. Tissues were subjected to immunohistochemical analysis using KL-6 antibody. RESULTS: All 56 cases with metastatic liver cancer showed positive staining for KL-6 mucin in cancer tissues but not in non-cancerous tissues. All staining was observed in the circumferential membrane and/or cytoplasm of the cancer cells. In contrast, no staining for KL-6 mucin was observed in either cancerous or non-cancerous tissues of the 92 patients with hepatocellular carcinoma. CONCLUSIONS: KL-6 mucin may be an indicator for liver metastasis of colorectal carcinoma. Additionally, detection of KL-6 mucin may be helpful in distinguishing hepatocellular carcinoma from metastatic liver cancer.

Structural analyses of a hemolytic compound found in an extract of Hypsizygus marmoreus fruiting bodies at a low pH.

The current study determined the structure of a hemolytic compound found in an extract from the fruiting bodies of the edible mushroom Hypsizygus marmoreus when its pH was lowered. The hemolytic compound was purified using the modified Bligh and Dyer method followed by chromatography using reversed phase and silica gel columns. Structural analyses of the purified hemolytic compound were performed using NMR and ESI-MS. The deduced structure indicated a trans,trans-5,8-docosadienoic acid calcium salt. Although numerous proteinous hemolysins from various mushrooms have been described, the current study is the first to report on a low-molecular-weight hemolytic compound derived from an H. marmoreus extract.

Positive KL-6 mucin expression combined with decreased membranous beta-catenin expression indicates worse prognosis in colorectal carcinoma.

Interaction of MUC1 with beta-catenin plays a significant role in tumor progression and invasion. However, the clinical significance of coexpression of MUC1 and subcellular beta-catenin expression in colorectal carcinoma remains unclear. The present study evaluated the clinicopathological significance of their combined expression for predicting prognosis. Seventy-seven colorectal carcinomas were subjected to immunohistochemical staining with anti-MUC1 KL-6 mucin and anti-beta-catenin monoclonal antibody. Positive KL-6 mucin expression was correlated with decreased membranous beta-catenin expression (P=0.022), while no correlation was found between positive KL-6 expression and nuclear beta-catenin expression (P=0.142). Preservation of membranous beta-catenin expression was detected in 35 cases (45.5%) and decreased membranous beta-catenin expression was found in 42 cases (54.5%). Negative KL-6 expression was detected in 31 cases (41.3%) and positive expression was seen in 46 cases (59.7%). Combined positive KL-6 expression and decreased membranous beta-catenin expression was found in 30 patients (39.0%), whose survival was significantly worse than that of patients with other expression patterns for these two molecules (53.3 vs. 84.4%, P=0.005). Multivariate analysis showed that this combination was as an independent predictor of survival. We concluded that the combined pattern of positive KL-6 expression and decreased membranous beta-catenin expression by colorectal carcinoma is a useful biomarker for distinguishing a subgroup of patients with a worse prognosis.

Measurement of serum and tissue des-gamma-carboxyprothrombin in resectable hepatocellular carcinoma.

BACKGROUND: The total des-gamma-carboxyprothrombin (DCP) produced in hepatocellular carcinoma (HCC) and the surrounding non-cancer liver tissue was estimated and the correlation between tissue DCP and serum DCP levels was examined. PATIENTS AND METHODS: Forty patients with a single primary HCC nodule served as the subjects. The DCP was quantified by electrochemiluminescence immunoassay and the tissue volumes were estimated by computed tomography (CT). RESULTS: The mean tissue DCP in the cancer tissue, non-cancer tissue and the whole HCC specimen was 147,400, 20,200 and 190,000 mAU, respectively, which correlated positively with the serum DCP level (r2 = 0.657, 0.452, and 0.684, respectively). The DCP expression levels in the cancer and non-cancer liver tissue were also confirmed by immunohistochemical detection of DCP. CONCLUSION: The elevation of serum DCP levels in HCC patients is influenced by DCP production not only in the cancer tissue, but also in the surrounding non-cancer liver tissue.

Elevation of serum KL-6 mucin levels in patients with cholangiocarcinoma.

BACKGROUND/AIMS: KL-6 mucin is a type of MUC1 mucin. Previous immunohistochemical studies by the authors revealed that, in liver cancer tissues, KL-6 mucin expressed only in intrahepatic cholangiocarcinoma (CC) but not in hepatocellular carcinoma (HCC). The current study performed serological analysis to evaluate KL-6 mucin levels in sera from various liver cancer patients and healthy individuals. METHODOLOGY: Enzyme-linked immunosorbent assay was used to measure KL-6 mucin levels in sera from 8 CC patients, 27 HCC patients, 30 metastatic liver cancer (MC) patients, and 19 healthy individuals. RESULTS: Serum KL-6 mucin levels were significantly higher in CC patients than in healthy individuals (p<0.0001), HCC patients (p<0.0001), and MC patients (p<0.001). Receiver operating characteristic analysis determined the cut-off level of serum KL-6 mucin levels to be 240 U/mL (CC vs. healthy, p<0.001) or 248 U/mL (CC vs. HCC, p<0.001). Using 248 U/mL as a cut-off value, the positive rate for CC patients, healthy individuals, and HCC patients was 100.0%, 15.8%, and 18.5%, respectively. CONCLUSIONS: Elevation of serum KL-6 levels was detectable in CC patients. Thus, KL-6 might be a useful serological marker to distinguish CC patients from HCC patients.

pH-Dependent exhibition of hemolytic activity by an extract of Hypsizygus marmoreus fruiting bodies.

The current study found that an extract from the fruiting bodies of the edible mushroom Hypsizygus marmoreus exhibited hemolytic activity against sheep red blood cells when its pH was lowered. Although hemolytic activity was not detected when an extract had a neutral pH, an extract with a low pH exhibited potent hemolytic activity. The maximal hemolytic activity was exhibited by an extract with a pH of 5.5. A heat-treated extract did not exhibit hemolytic activity before its pH was lowered, and that activity was inhibited in the presence of PMSF and EDTA. The turbidity of the extract increased during lowering of its pH, and the precipitate fraction exhibited hemolytic activity. Fractionation by a modified Bligh and Dyer method and TLC analyses suggested that a hemolytic compound in the extract might be a type of lipid. These results suggest that a hemolytic lipid-like compound in an extract of H. marmoreus fruiting bodies may be released by a non-active precursor substance(s) through metalloenzyme(s) while the extract has a low pH.

Structural alterations in outer arms of IgG oligosaccharides in patients with Werner syndrome.

Werner syndrome (WS) is a heredofamilial disorder characterized by clinicopathological premature aging. In healthy individuals, structural alteration of serum IgG oligosaccharides is known to be an aging phenotype. In the present study, we determined and compared oligosaccharide structures of serum IgG among WS patients, healthy age-sex-matched individuals, and healthy elderly individuals from both sexes in order to reveal whether WS patients exhibit an aging phenotype in terms of IgG oligosaccharide structure. Sialylation and galactosylation levels of IgG oligosaccharides from WS patients were similar to those from healthy elderly individuals in which sialylation and galactosylation levels were significantly lower than those from the healthy age-sex-matched individuals. In contrast, the bisecting N-acetylglucosaminylation level of IgG oligosaccharides from WS patients was comparable to that from the healthy age-sex-matched controls and significantly lower than that of the healthy elderly controls. There was no significant sexual difference in these modifications of IgG oligosaccharides. These results suggest that WS patients exhibit an aging phenotype for structural alterations such as sialylation and galactosylation in the outer arms of IgG oligosaccharides.

Subcellular localization of KL-6 mucin in colorectal carcinoma cell lines: association with metastatic potential and cell morphology.

KL-6 mucin, a type of MUC1 mucin, is expressed in many malignant tissues including colorectal adenocarcinoma. Previous studies on colorectal adenocarcinoma tissues showed that subcellular localization of KL-6 mucin was associated with the tumor metastatic potential and the patient prognosis. In the present study, to further investigate the significance of subcellular localization of KL-6 mucin, we examined KL-6 mucin expression in colorectal carcinoma cell lines, COLO 201, LoVo, WiDr, and DLD-1, by means of Western blot, flow cytometric and immunocytochemical analyses in conjunction with xylitol treatment. COLO 201 cells characterized by free cells in culture and high metastatic potential revealed extremely high level expression of KL-6 mucin in the cytoplasm and cell surface. The cell surface mucin of COLO 201 cells could not be removed by xylitol treatment, suggesting that the mucin may tightly bind to the cell membrane. LoVo cells characterized by adhesive cells in culture and high metastatic potential express a low level of KL-6 mucin in their cytoplasm and cell surface. In contrast, WiDr and DLD-1 cells, both of which are characterized by low metastatic potential, express KL-6 mucin around the cell surface. The mucin of WiDr and DLD-1 cells could be removed by xylitol treatment, suggesting that KL-6 mucin of these two cell lines may be weakly attached around the cell surface. These results suggest that expression level and subcellular localization of KL-6 mucin may be related to the cell morphology in culture and metastatic potential in colorectal carcinoma cell lines. In addition, xylitol is an effective tool to remove KL-6 mucin weakly attached around the cell surface and to investigate the role of subcellular localization of KL-6 mucin.

KL-6 mucin is a useful immunohistochemical marker for cholangiocarcinoma.

This study aimed to clarify the clinical significance of the expression of KL-6 mucin, a type of MUC1, in primary liver cancer. Tissue specimens were collected from 21 patients with cholangiocarcinoma (CC), 78 with hepatocellular carcinoma (HCC), and 12 with combined hepatocellular and cholangiocarcinoma (cHCC-CC). Immunohistochemical analysis was done using a monoclonal antibody for KL-6 mucin as well as antibodies for Hep1 or CK7. KL-6 staining was positive in all the CC tissues examined, while it was not positive in any of the HCC tissues. Similar selectivity of KL-6 staining was also observed in the cHCC-CC specimens, and the cholangiocellular tissue could be clearly delineated by KL-6 staining. In contrast, 79.5% of HCC specimens and 25.0% of cHCC-CC specimens were positive for Hep1 in the hepatocellular tissues, while none of the CC or cHCC-CC specimens were positive in the cholangiocellular tissues. Staining for CK7 was positive in 95.2% of CC and 35.9% of HCC specimens, while 58.3 and 25.0% of cHCC-CC specimens displayed positivity for CK7 in the cholangiocellular and hepatocellular tissues, respectively. These results suggest that KL-6 may be a useful tumor marker for distinguishing CC from HCC. In addition, the high selectivity of KL-6 for cholangiocallular tissue may help to provide information for deciding the clinical strategy in cHCC-CC patients.

Induction of apoptosis in human hepatocellular carcinoma cells by synthetic antineoplaston A10.

Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedion) is a naturally occurring substance and was the first antineoplaston in the human body to be chemically identified. The effect of antineoplaston A10 on human hepatocellular carcinoma cell lines HepG2 and HLE has been examined. Antineoplaston A10 displayed anti-proliferative action inhibiting cell growth in a dose- and time-dependent manner in vitro as measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Incubation with antineoplaston A10 for 48 h induced apoptotic events such as a typical apoptotic morphology, formation of a characteristic ladder pattern of DNA migration and accumulation of sub-G1 phase cells. Next, hepatoma xenografts in nude mice were employed to study the antitumor effects of antineoplaston A10 in vivo. Oral administration of antineoplaston A10 delayed the growth of HepG2 and HLE cells in the mice without a reduction in body weight. A higher proportion of apoptotic cells in xenografts was observed by means of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. In addition, the level of expression of apoptotic marker p53 increased while that of anti-apoptotic protein bcl-2 decreased, as evaluated with immunohistochemical staining in the xenografts. These results suggested that antineoplaston A10 may inhibit the growth of human hepatoma cells through the induction of apoptosis.

Sialoglycoconjugate expression in primary colorectal cancer and metastatic lymph node tissues.

BACKGROUND/AIMS: Aberrant cell surface glycosylation, and especially excessive sialylation, is thought to have great importance in tumor malignancy. To investigate the clinicopathological significance of sialylation in colorectal cancer, we performed histochemical analyses using sialic acid-binding lectins. METHODOLOGY: Primary colorectal cancer and lymph node tissues obtained from 80 cases were subjected to lectin-immunohistochemical staining using Maackia amurensis leukoagglutinin (MAL) and Sambucus nigra agglutinin (SNA). The relationship between the staining characteristics and the various clinicopathological parameters was statistically analyzed. RESULTS: In primary cancer tissues, a high level of SNA staining was significantly related to worse prognosis and some pathological characteristics such as lymph node metastasis, whereas a high level of MAL staining was related to worse prognosis. In metastatic lymph node tissues, positive staining was frequently found for MAL and SNA, which wasremarkable in cases categorized as N2 metastasis. Furthermore, cases with MAL-positive staining in metastatic lymph node tissues evidently indicated worse prognosis than those with MAL-negative staining. CONCLUSIONS: Aberrant expression of SNA-positive sialoglycoconjugates in primary colorectal cancer tissues is important in terms of unfavorable pathological characteristics of the cancer. In addition, aberrant expression of MAL-positive sialoglycoconjugates in metastatic lymph node tissues might be related to worse prognosis.

Endogenous reactive oxygen species cause astrocyte defects and neuronal dysfunctions in the hippocampus: a new model for aging brain.

The etiology of astrocyte dysfunction is not well understood even though neuronal defects have been extensively studied in a variety of neuronal degenerative diseases. Astrocyte defects could be triggered by the oxidative stress that occurs during physiological aging. Here, we provide evidence that intracellular or mitochondrial reactive oxygen species (ROS) at physiological levels can cause hippocampal (neuronal) dysfunctions. Specifically, we demonstrate that astrocyte defects occur in the hippocampal area of middle-aged Tet-mev-1 mice with the SDHCV69E mutation. These mice are characterized by chronic oxidative stress. Even though both young adult and middle-aged Tet-mev-1 mice overproduced MitoSOX Red-detectable mitochondrial ROS compared to age-matched wild-type C57BL/6J mice, only young adult Tet-mev-1 mice upregulated manganese and copper/zinc superoxide dismutase (Mn- and Cu/Zn-SODs) activities to eliminate the MitoSOX Red-detectable mitochondrial ROS. In contrast, middle-aged Tet-mev-1 mice accumulated both MitoSOX Red-detectable mitochondrial ROS and CM-H2 DCFDA-detectable intracellular ROS. These ROS levels appeared to be in the physiological range as shown by normal thiol and glutathione disulfide/glutathione concentrations in both young adult and middle-aged Tet-mev-1 mice relative to age-matched wild-type C57BL/6J mice. Furthermore, only middle-aged Tet-mev-1 mice showed JNK/SAPK activation and Ca2+ overload, particularly in astrocytes. This led to decreasing levels of glial fibrillary acidic protein and S100ß in the hippocampal area. Significantly, there were no pathological features such as apoptosis, amyloidosis, and lactic acidosis in neurons and astrocytes. Our findings suggest that the age-dependent physiologically relevant chronic oxidative stress caused astrocyte defects in mice with impaired mitochondrial electron transport chain functionality.

Curcumin inhibits telomerase activity in human cancer cell lines.

Curcumin, one of the major components of tumeric, the dried rhizome of Curcuma longa L, has been shown to have anti-proliferating and anti-carcinogenic properties. In this study, we examined the effects of curcumin on cell growth and telomerase activity in human cancer cell lines Bel7402, HL60 and SGC7901. Curcumin (1-32 microM) showed anti-proliferating effects on these cell lines in a dose-dependent manner in vitro, and anti-tumor effects when curcumin (50-200 mg/kg) was orally administered to nude mice transplanted with the cancer cells. When the cells were treated with 1 microM of curcumin for 120 h, apoptotic cells were observed by means of the adridine orange/ethidium bromide staining method, single cell microgel electrophoresis and flow cytometric analysis. On the other hand, suppression of telomerase activity in extracts of the cells treated with 1 microM of curcumin was observed by means of a telomeric repeat amplification protocol - silver staining assay. These results suggest that curcumin could suppress telomerase activity in the cancer cell lines and that the decrease of telomerase expression followed by induction of apoptosis might be involved in the anti-proliferating effect of curcumin.

Using caffeoyl pyrrolidine derivative LY52, a potential inhibitor of matrix metalloproteinase-2, to suppress tumor invasion and metastasis.

LY52 is a caffeoyl pyrrolidine derivative designed to fit the S'1 active pocket of gelatinases that act in tumor invasion and metastasis. Herein, we examined the effects of LY52 on expression of matrix metalloproteinase (MMP)-2 expression in human breast cancer MDA-MB-231 cells and on in vitro invasion and in vivo metastasis. LY52 significantly blocked MMP-2 activity as evidenced by a decrease in the degradation of succinylated gelatin. Gelatin zymography analysis showed that LY52 (0.1-200 microg/ml) inhibited expression of active MMP-2 in concanavalin A-stimulated MDA-MB-231 cells. Inhibition of MMP-2 expression was also observed in tissue of tumor xenografts in mice that were orally administered LY52 (25 or 100 mg/kg). Furthermore, LY52 displayed an inhibitory effect on in vitro invasion of MDA-MB-231 cells and pulmonary metastasis of B16F10 murine melanoma cells in mice without significant toxic effects. These results suggest that LY52 is a potential MMP-2 inhibitor that may effectively suppress tumor invasion and metastasis.

Caffeoyl pyrrolidine derivative LY52 inhibits tumor invasion and metastasis via suppression of matrix metalloproteinase activity.

BACKGROUND: LY52 is a caffeoyl pyrrolidine derivative designed to fit and extend into the active pocket of matrix metalloproteinase (MMP). In this study, the effects of LY52 on MMP-2 and MMP-9 activities and tumor invasion and metastasis were examined. MATERIALS AND METHODS: MMP expression in SKOV3 cells was analyzed by gelatin zymography. The anti-invasion and anti-metastasis abilities of LY52 were evaluated with penetration of SKOV3 cells through Matrigel-coated membrane in vitro and pulmonary metastasis of Lewis lung carcinoma in mice, respectively. RESULTS: LY52 significantly blocked the proteolytic activity of gelatinase. Gelatin zymography revealed that MMP-2 and MMP-9 expressions in SKOV3 cells were reduced in the presence of LY52. LY52 also suppressed SKOV3 cell invasion in vitro. Furthermore, a significant inhibition of pulmonary metastasis of Lewis lung carcinoma cells was observed in LY52-administrated mice. CONCLUSION: LY52 might suppress invasion and metastasis of carcinoma cells via inhibition of MMP-2 and MMP-9 proteolytic activities.

Clinical significance of subcellular localization of KL-6 mucin in primary colorectal adenocarcinoma and metastatic tissues.

AIM: To assess subcellular localization of KL-6 mucin and its clinicopathological significance in colorectal carcinoma as well as metastatic lymph node and liver tissues. METHODS: Colorectal carcinoma tissues as well as metastatic lymph node and liver tissues were collected from 82 patients who underwent colorectomy or hepatectomy. Tissues were subjected to immunohistochemical analysis using KL-6 antibody. RESULTS: Of the 82 colorectal carcinoma patients, 6 showed no staining, 29 showed positive staining only in the apical membrane, and 47 showed positive staining in the circumferential membrane and/or cytoplasm. Positive staining was not observed in non-cancerous colorectal epithelial cells surrounding the tumor tissues. The five-year survival rate was significantly lower in cases showing positive staining in the circumferential membrane and/or cytoplasm (63.0%) than those showing positive staining only in the apical membrane (85.7%) and those showing no staining (100%). Statistical analysis between clinicopathological factors and subcellular localization of KL-6 mucin showed that KL-6 localization in the circumferential membrane and/or cytoplasm was significantly associated with the presence of venous invasion (P = 0.0003), lymphatic invasion (P<0.0001), lymph node metastasis (P<0.0001), liver metastasis (P = 0.058), and advanced histological stage (P<0.0001). Positive staining was observed in all metastatic lesions tested as well as in the primary colorectal carcinoma tissues. CONCLUSION: The subcellular staining pattern of KL-6 in colorectal adenocarcinoma may be an important indicator for unfavorable behaviors such as lymph node and liver metastasis, as well as for the prognosis of patients.