Discovery of antitumor effects of leczymes.

Sialic-acid binding lectin from bullfrog (Rana catesbeiana) eggs, cSBL, is a cytotoxic ribonuclease (RNase) belonging to the RNase A superfamily. cSBL is cytotoxic to tumor cells, such as malignant pleural mesothelioma by inducing apoptotic cell death caused by the degradation of RNA in tumor cells. In addition, we have reported some data that cSBL could be involved in the endoplasmic reticulum stress pathway, and it was also assumed to cause apoptotic cell death. The most significant property of cSBL is its specificity toward malignant cells. Furthermore, since the antitumor activity of cSBL was confirmed by in vivo experiments using mouse xenograft models, it is expected to be a candidate for clinical chemotherapy. Here, we summarize the history of cSBL, alternatively called "leczyme," with its present and future.

Sialic Acid-Binding Lectin from Bullfrog Eggs Exhibits an Anti-Tumor Effect Against Breast Cancer Cells Including Triple-Negative Phenotype Cells.

Sialic acid-binding lectin from Rana catesbeiana eggs (cSBL) is a multifunctional protein that has lectin and ribonuclease activity. In this study, the anti-tumor activities of cSBL were assessed using a panel of breast cancer cell lines. cSBL suppressed the cell growth of all cancer cell lines tested here at a concentration that is less toxic, or not toxic at all, to normal cells. The growth suppressive effect was attributed to the cancer-selective induction of apoptosis. We assessed the expressions of several key molecules associated with the breast cancer phenotype after cSBL treatment by western blotting. cSBL decreased the expression level of estrogen receptor (ER) α, while it increased the phosphorylation level of p38 mitogen-activated protein kinase (MAPK). cSBL also suppressed the expression of the progesterone receptor (PgR) and human epidermal growth factor receptor type 2 (HER2). Furthermore, it was revealed that cSBL decreases the expression of the epidermal growth factor receptor (EGFR/HER1) in triple-negative breast cancer cells. These results indicate that cSBL induces apoptosis with decreasing ErbB family proteins and may have great potential for breast cancer chemotherapy, particularly in triple-negative phenotype cells.

Catfish rhamnose-binding lectin induces G0/1 cell cycle arrest in Burkitt's lymphoma cells via membrane surface Gb3.

Silurus asotus egg lectin (SAL), an α-galactoside-binding protein isolated from the eggs of catfish, is a member of the rhamnose-binding lectin family that binds to Gb3 glycan (Galα1-4Galß1-4Glc). We have previously demonstrated that SAL reduces the proliferation of Gb3-expressing Burkitt's lymphoma Raji cells and confirm here that it does not reduce their viability, indicating that unlike other lectins, it is not cytotoxic. The aim of this study was to determine the signal transduction mechanism(s) underlying this novel SAL/Gb3 binding-mediated effect profile. SAL/Gb3 interaction arrested the cell cycle through increasing the G0/1 phase population of Raji cells. SAL suppressed the transcription of cell cycle-related factors such as c-MYC, cyclin D3, and cyclin-dependent protein kinase (CDK)-4. Conversely, the CDK inhibitors p21 and p27 were elevated by treatment with SAL. In particular, the production of p27 in response to SAL treatment increased steadily, whereas p21 production was maximal at 12 h and lower at 24 h. Activation of Ras-MEK-ERK pathway led to an increase in expression of p21. Notably, treatment of Raji cells with anti-Gb3 mAb alone did not produce the above effects. Taken together, our findings suggest that Gb3 on the Raji cell surface interacts with SAL to trigger sequential GDP-Ras phosphorylation, Ras-MEK-ERK pathway activation, p21 production, and cell cycle arrest at the G0/1 phase.

Sialidase NEU3 defines invasive potential of human glioblastoma cells by regulating calpain-mediated proteolysis of focal adhesion proteins.

BACKGROUND: Glioblastoma multiforme is one of the most malignant tumors of the human central nervous system characterized by high degree of invasiveness. Focusing on this invasive nature, we investigated whether ganglioside-specific sialidase NEU3 might be involved, because gangliosides are major components of brain tissues, and cell surface sialic acids, as target residues of sialidase catalysis, are thought to be closely related to cell invasion. METHODS: NEU3 mRNA levels of human glioblastoma specimens were evaluated by quantitative RT-PCR. Human glioblastoma cell lines, U251, A172, and T98G were used for cell invasion and migration by transwell and cell scratching assay. The molecules involved in the signaling cascade were investigated by western blot and immunofluorescent microscopy. RESULTS: NEU3 expression was down-regulated in human glioblastoma specimens. In the human glioblastoma cell lines, NEU3 overexpression reduced invasion and migration by promoting the assembly of focal adhesions through reduced calpain-dependent proteolysis, but NEU3 silencing resulted in accelerating cell invasion via disassembly of focal adhesions. In NEU3-silenced cells, elevation of calpain activity and GM3 accumulation were observed, as results of reduced sialidase hydrolysis, localization of calpain and GM3 at the cell lamellipodium being demonstrated by immunofluorescence microscopy. CONCLUSION: Sialidase NEU3 was found to exert a great influence on cell invasion in regulation of calpain activity and focal adhesion disassembly and consequent invasive potential of glioblastoma cells. GENERAL SIGNIFICANCE: This first demonstration of sialidase involvement in invasive potential of gliolastoma cells may point to NEU3 as an attractive treatment target of human gliomas.

Lissoclibadin 1, a Polysulfur Aromatic Alkaloid from the Indonesian Ascidian Lissoclinum cf. badium, Induces Caspase-Dependent Apoptosis in Human Colon Cancer Cells and Suppresses Tumor Growth in Nude Mice.

Lissoclibadins, polysulfur aromatic alkaloids, were isolated from the Indonesian ascidian Lissoclinum cf. badium. Lissoclibadins 1 (1), 3 (2), 4 (3), 7 (4), 8 (5), and 14 (6) inhibited the growth of four human solid cancer cell lines: HCT-15 (colon adenocarcinoma), HeLa-S3 (cervix adenocarcinoma), MCF-7 (breast adenocarcinoma), and NCI-H28 (mesothelioma). Lissoclibadin 1 (1) exerted the most potent cytotoxic effects in vitro and mainly promoted apoptosis through an intrinsic pathway with the activation of a caspase-dependent pathway in HCT-15 cells. In vivo studies demonstrated that 1 suppressed tumor growth in nude mice carrying HCT-15 cells without significant secondary adverse effects. In conclusion, the results obtained in the present study demonstrate that 1 has potential as a chemotherapeutic candidate for preclinical investigations.

Phosphatidic acid-mediated activation and translocation to the cell surface of sialidase NEU3, promoting signaling for cell migration.

The plasma membrane-associated sialidase NEU3 plays crucial roles in regulation of transmembrane signaling, and its aberrant up-regulation in various cancers contributes to malignancy. However, it remains uncertain how NEU3 is naturally activated and locates to plasma membranes, because of its Triton X-100 requirement for the sialidase activity in vitro and its often changing subcellular location. Among phospholipids examined, we demonstrate that phosphatidic acid (PA) elevates its sialidase activity 4 to 5 times at 50 µM in vitro at neutral pH and promotes translocation to the cell surface and cell migration through Ras-signaling in HeLa and COS-1 cells. NEU3 was found to interact selectively with PA as assessed by phospholipid array, liposome coprecipitation, and ELISA assays and to colocalize with phospholipase D (PLD) 1 in response to epidermal growth factor (EGF) or serum stimulation. Studies using tagged NEU3 fragments with point mutations identified PA- and calmodulin (CaM)-binding sites around the N terminus and confirmed its participation in translocation and catalytic activity. EGF induced PLD1 activation concomitantly with enhanced NEU3 translocation to the cell surface, as assessed by confocal microscopy. These results suggest that interactions of NEU3 with PA produced by PLD1 are important for regulation of transmembrane signaling, this aberrant acceleration probably promoting malignancy in cancers.

Sialidase NEU3 contributes neoplastic potential on colon cancer cells as a key modulator of gangliosides by regulating Wnt signaling.

The plasma membrane-associated sialidase NEU3 is a key enzyme for ganglioside degradation. We previously demonstrated remarkable up-regulation of NEU3 in various human cancers, with augmented malignant properties. Here, we provide evidence of a close link between NEU3 expression and Wnt/ß-catenin signaling in colon cancer cells by analyzing tumorigenic potential and cancer stem-like characteristics. NEU3 silencing in HT-29 and HCT116 colon cancer cells resulted in significant decrease in clonogenicity on soft agar and in vivo tumor growth, along with down-regulation of stemness and Wnt-related genes. Analyses further revealed that NEU3 enhanced phosphorylation of the Wnt receptor LRP6 and consequently ß-catenin activation by accelerating complex formation with LRP6 and recruitment of GSK3ß and Axin, whereas its silencing exerted the opposite effects. NEU3 activity-null mutants failed to demonstrate the activation, indicating the requirement of ganglioside modulation by the sialidase for the effects. Under sphere-forming conditions, when stemness genes are up-regulated, endogenous NEU3 expression was found to be significantly increased, whereas NEU3 silencing suppressed sphere-formation and in vivo tumor incidence in NOD-SCID mice. Increased ability of clonogenicity on soft agar and sphere formation by Wnt stimulation was abrogated by NEU3 silencing. Furthermore, NEU3 was found to regulate phosphorylation of ERK and Akt via EGF receptor and Ras cascades, thought to be additionally required for tumor progression. The results indicate an essential contribution of NEU3 to tumorigenic potential through maintenance of stem-like characteristics of colon cancer cells by regulating Wnt signaling at the receptor level, in addition to tumor progression via Ras/MAPK signaling.

Upregulation of sialidase NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis.

Regional lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) is a crucial event for its progression, associated with a high rate of mortality. Sialidase, a key enzyme for the regulation of cellular sialic acids through catalyzing the initial step of degradation of glycoproteins and glycolipids, has been implicated in cancer progression. To facilitate the development of novel treatments for HNSCC, we have investigated whether sialidase is involved in the progression of this cancer. We found plasma membrane-associated sialidase (NEU3) to be significantly upregulated in tumor compared to non-tumor tissues; particularly, an increase in its mRNA levels was significantly associated with lymph node metastasis. To understand the mechanisms, we analyzed the NEU3-mediated effects on the malignant phenotype using squamous carcinoma HSC-2 and SAS cells. NEU3 promoted cell motility and invasion, accompanied by the increased expression of MMP-9, whereas NEU3 silencing or the activity-null mutant did not. NEU3 enhanced phosphorylation of epidermal growth factor receptor (EGFR), and an EGFR inhibitor, AG1478, abrogated the NEU3-induced MMP9 augmentation. These findings identify NEU3 as a participant in HNSCC progression through the regulation of EGFR signaling and thus as a potential target for inhibiting EGFR-mediated tumor progression.

Increased sialidase activity in serum of cancer patients: Identification of sialidase and inhibitor activities in human serum.

Aberrant sialylation in glycoproteins and glycolipids is a characteristic feature of malignancy. Human sialidases, which catalyze the removal of sialic acid residues from glycoconjugates, have been implicated in cancer progression. They have been detected in a wide variety of human cells and tissues, but few studies have focused on their existence in human serum. Among the four types identified to date, we previously demonstrated that plasma membrane-associated ganglioside sialidase (NEU3) is markedly upregulated in various human cancers, including examples in the colon and prostate. Here, using a sensitive assay method, we found a significant increase of sialidase activity in the serum of patients with prostate cancer compared with that in healthy subjects having low activity, if any. Activity was apparent with gangliosides as substrates, but only to a very limited extent with 4-methylumbelliferyl sialic acid, a good synthetic substrate for sialidases other than human NEU3. The serum sialidase was also almost entirely immunoprecipitated with anti-NEU3 antibody, but not with antibodies for other sialidases. Interestingly, sera additionally contained inhibitory activity against the sialidase and also against recombinant human NEU3. The sialidase and inhibitor activities could be separated by exosome isolation and by hydrophobic column chromatography. The serum sialidase was assessed by a sandwich ELISA method using two anti-NEU3 antibodies. The results provide strong evidence that the serum sialidase is, in fact, NEU3, and this subtype may, therefore, be a potential utility for novel diagnosis of human cancers.

MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galß1-4Glc). MytiLec revealed ß-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt's lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

A galactose-binding lectin isolated from Aplysia kurodai (sea hare) eggs inhibits streptolysin-induced hemolysis.

A specific galactose-binding lectin was shown to inhibit the hemolytic effect of streptolysin O (SLO), an exotoxin produced by Streptococcus pyogenes. Commercially available lectins that recognize N-acetyllactosamine (ECA), T-antigen (PNA), and Tn-antigen (ABA) agglutinated rabbit erythrocytes, but had no effect on SLO-induced hemolysis. In contrast, SLO-induced hemolysis was inhibited by AKL, a lectin purified from sea hare (Aplysia kurodai) eggs that recognizes α-galactoside oligosaccharides. This inhibitory effect was blocked by the co-presence of d-galactose, which binds to AKL. A possible explanation for these findings is that cholesterol-enriched microdomains containing glycosphingolipids in the erythrocyte membrane become occupied by tightly stacked lectin molecules, blocking the interaction between cholesterol and SLO that would otherwise result in penetration of the membrane. Growth of S. pyogenes was inhibited by lectins from a marine invertebrate (AKL) and a mushroom (ABA), but was promoted by a plant lectin (ECA). Both these inhibitory and promoting effects were blocked by co-presence of galactose in the culture medium. Our findings demonstrate the importance of glycans and lectins in regulating mechanisms of toxicity, creation of pores in the target cell membrane, and bacterial growth.

Binding profiles and cytokine-inducing effects of fish rhamnose-binding lectins on Burkitt's lymphoma Raji cells.

Rhamnose-binding lectin (RBL) is one of the animal lectin categories which take part in the innate immune responses of fish. Osmerus lanceolatus lectin (OLL) from shishamo smelt eggs is an RBL composed of two tandem-repeated domains, both of which are considered to be a carbohydrate-recognition domain. SAL, catfish (Silurus asotus) egg RBL composed of three domains, binds to Burkitt's lymphoma Raji cells through globotriaosylceramide (Gb3) carbohydrate chain and to reduce cell size and growth by altering membrane composition without causing cell death. In this experiment, we tried to compare the binding effects of these two RBLs on Raji cells. Flow cytometric and fluorescence microscopic analyses revealed that OLL also directly bound to and shrunk Raji cells with ten times less reactivity than SAL but reduced cell growth with decreasing cell viability. Anti-Gb3 antibody completely blocked the binding of SAL to Raji cells but not that of OLL. In addition, the direct bindings of OLL and SAL to Raji cells were comparably inhibited by melibiose, but lactose was more effective inhibitor for the binding of OLL than that of SAL. These results suggest that OLL has slightly different cell-binding property compared with SAL and binds not only to Gb3 but also to the other carbohydrate receptor-bearing ß-galactoside chains. The quantitative RT-PCR analysis revealed that SAL induced the expression of TNF-α but not of IFN-γ, IL-1ß, and IL-10. Thus, SAL-induced cytostatic effect on Raji cells might be partially caused by TNF-α-mediated signaling pathway.

Sialidase NEU3 Contributes to the Invasiveness of Bladder Cancer.

Bladder cancer is the 10th most commonly diagnosed cancer worldwide. The current standard treatment for advanced bladder cancer is neoadjuvant cisplatin (NAC)-based chemotherapy followed by cystectomy. However, the response rate to chemotherapy is only 50%, owing to cisplatin resistance, and there is a need for novel therapies. Because the invasiveness of bladder cancer greatly influences patient prognosis, a mechanistic analysis of the invasive function can lead to therapeutic targets. Sialidases, which remove sialic acid residues from the nonreducing ends of sugar chains and catalyze the initial reaction in the degradation of sugar chains, are predicted to be involved in cell invasion and motility. However, the involvement of sialidases in bladder cancer, especially their relationship with the invasive ability, remains unclear. Here, using patient tissues and multiple bladder cancer cell lines, we show that the sialidase NEU3 is highly expressed in bladder cancer. Analysis of NEU3's function using its siRNA-mediated knockdown revealed that NEU3 contributes to bladder cancer invasiveness. Mechanistic analysis showed that NEU3 activates ERK and PI3K signaling. Our results show that NEU3 is involved in the malignancy of bladder cancer, and its suppression may lead to novel treatments for bladder cancer.

Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons.

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.

A lectin from the mussel Mytilus galloprovincialis has a highly novel primary structure and induces glycan-mediated cytotoxicity of globotriaosylceramide-expressing lymphoma cells.

A novel lectin structure was found for a 17-kDa α-D-galactose-binding lectin (termed "MytiLec") isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45-52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1-4Galß1-4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.

Domain composition of rhamnose-binding lectin from shishamo smelt eggs and its carbohydrate-binding profiles.

Osmerus (Spirinchus) lanceolatus egg lectin (OLL) is a member of the rhamnose-binding lectin (RBL) family which is mainly found in aqueous beings. cDNA of OLL was cloned, and its genomic architecture was revealed. The deduced amino acid (aa) sequence indicated that OLL was composed of 213 aa including 95 aa of domain N and 97 aa of domain C. N and C showed 73 % sequence identity and contained both -ANYGR- and -DPC-KYL-peptide motifs which are conserved in most of the RBL carbohydrate recognition domains. The calculated molecular mass of mature OLL was 20,852, consistent with the result, and 20,677.716, from mass spectrometry. OLL was encoded by eight exons: exons 1 and 2 for a signal peptide; exons 3-5 and 6-8 for N- and C-domains, respectively. Surface plasmon resonance spectrometric analyses revealed that OLL showed comparable affinity for Galα- and ß-linkages, whereas Silurus asotus lectin (SAL), a catfish RBL, bound preferentially to α-linkages of neoglycoproteins. The Kd values of OLL and SAL against globotriaosylceramide (Gb3) were 1.69 × 10⁻5 M for and 2.81 × 10⁻6 M, respectively. Thus, the carbohydrate recognition property of OLL is slightly different from that of SAL. On the other hand, frontal affinity chromatography revealed that both OLL and SAL interacted with only glycolipid-type oligosaccharides such as Gb3 trisaccharides, not with N-linked oligosaccharides. The domain composition of these RBLs and an analytical environment such as the "cluster effect" of a ligand might influence the binding between RBL and sugar chains.

Catfish Egg Lectin Enhances the Cytotoxicity of Sunitinib on Gb3-Expressing Renal Cancer Cells.

Metastatic renal cell carcinoma (RCC) is not sufficiently responsive to anticancer drugs, and thus, developing new drugs for advanced RCC remains vital. We previously reported that the treatment of globotriaosylceramide (Gb3)-expressing cells with catfish (Silurus asotus) egg lectin (SAL) increased the intracellular uptake of propidium iodide (PI) and sunitinib (SU). Herein, we investigated whether SAL pretreatment affects the intracellular uptake and cytotoxic effects of molecular-targeted drugs in RCC cells. We analyzed Gb3 expression in TOS1, TOS3, TOS3LN, and ACHN human RCC cells. Surface Gb3 expression was higher in TOS1 and TOS3 cells than in TOS3LN and ACHN cells. In the PI uptake assay, 41.5% of TOS1 cells and 21.1% of TOS3 cells treated with SAL were positive for PI. TOS1 cell viability decreased to 70% after treatment with 25 µM SU alone and to 48% after pretreatment with SAL (50 µg/mL). Time-series measurements of the intracellular fluorescence of SU revealed significantly enhanced SU uptake in SAL-treated TOS1 cells compared to control cells. SAL treatment did not increase PI uptake in normal renal cells. Our findings suggest that adequate cytotoxic activity may be achieved even when SU is administered at a sufficiently low dose not to cause side effects in combination with SAL.

Human cytosolic sialidase NEU2-low general tissue expression but involvement in PC-3 prostate cancer cell survival.

Human cytosolic sialidase (NEU2) has been identified and characterized using a NEU2 cDNA constructed from a genomic library of human skeletal muscle. However, the tissue distribution of NEU2 mRNA and the physiological functions of the enzyme remain unclear. In the present study, unlike other human sialidases, NEU2 expression as assessed by quantitative real-time PCR was found to be extremely low or undetectable in many human tissues and cells, with notable exceptions like the placenta and testis. The gene forms obtained by PCR with cDNAs synthesized from poly (A)(+) RNA of human brain and colon were verified to encode cytosolic sialidase with appropriate activity, regardless of the brain gene feature of SNPs. Among a series of human cancer cell lines examined, only prostate cancer PC-3 cells exhibited relatively high expression and NEU2-silencing with an siRNA resulted in decreased cell survival and motility. To gain insights into the significance of the high levels, transcription factors in the promoter region of the NEU2 gene were surveyed for involvement. PC-3 cells were characterized by high expression of Runx2 and Sp3, and their silencing reduced NEU2, suggesting regulatory roles.

Transcriptomic alterations in malignant pleural mesothelioma cells in response to long­term treatment with bullfrog sialic acid­binding lectin.

Malignant pleural mesothelioma (MPM) is a universally lethal type of cancer that is increasing in incidence worldwide; therefore, the development of new drugs for MPM is an urgent task. Bullfrog sialic acid­binding lectin (cSBL) is a multifunctional protein that has carbohydrate­binding and ribonuclease activities. cSBL exerts marked antitumor activity against numerous types of cancer cells, with low toxicity to normal cells. Although in vitro and in vivo studies revealed that cSBL was effective against MPM, the mechanism by which cSBL exerts antitumor effects is not fully understood. To further understand the mechanism of action of cSBL, the present study aimed to identify the key molecules whose expression was affected by cSBL. The present study established cSBL­resistant MPM cells. Microarray analyses revealed that there were significant pleiotropic changes in the expression profiles of several genes, including multiple genes involved in metabolic pathways in cSBL­resistant cells. Furthermore, the expression of some members of the aldo­keto reductase family was revealed to be markedly downregulated in these cells. Among these, it was particularly interesting that cSBL action reduced the level of AKR1B10, which has been reported as a biomarker candidate for MPM prognosis. These findings revealed novel aspects of the effect of cSBL, which may contribute to the development of new therapeutic strategies for MPM.