Microcystin-LR-induced epithelial-mesenchymal transition-like cells acquire resistance to multi-toxins.

The protein phosphatase inhibitor microcystin-LR (MC-LR), a hepatocyte-selective cyanotoxin, induces phenotypic changes in HEK293 OATP1B3-expressing (HEK293-OATP1B3) cells, which include cytoskeletal reorganization (HEK293-OATP1B3-AD) and anoikis resistance (HEK293-OATP1B3-FL) transformed cells, respectively. These cells acquire resistance to MC-LR and partial epithelial-mesenchymal transition (EMT) characteristics. In cancer cells, EMT is generally involved in multi-drug resistance. Here, we focused on the multi-drug resistance of HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. The MTT assay and immunoblotting were conducted to examine the responses of HEK293-OATP1B3, HEK293-OATP1B3-AD, and HEK293-OATP1B3-FL cells to multiple toxins and drugs that function as substrates for OATP1B3, including MC-LR, nodularin (Nod), okadaic acid (OA), and cisplatin (CDDP). HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells were more resistant to MC-LR, Nod, and OA than HEK293-OATP1B3 cells. Conversely, the three cell types were equivalently sensitive to CDDP. By using protein phosphatase assay, the reduction of the inhibitory effect of MC-LR and Nod on phosphatase activity might be one reason for the resistance to MC-LR and Nod in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Furthermore, the parental HEK293-OATP1B3 cells showed enhanced p53 phosphorylation and stabilization after MC-LR exposure, while p53 phosphorylation was attenuated in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Moreover, in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells, AKT phosphorylation was higher than that of the parental HEK293-OATP1B3 cell line. These results suggest that the multi-toxin resistance observed in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells is associated with AKT activation and p53 inactivation.

Acteoside from Conandron ramondioides Reduces Microcystin-LR Cytotoxicity by Inhibiting Intracellular Uptake Mediated by OATP1B3.

The hepatotoxin microcystin-LR is a strong inhibitor of serine/threonine protein phosphatase (PP) 1 and PP2A. The onset of its cytotoxicity depends on its selective uptake via the hepatocyte uptake transporters, organic anion transporting polypeptide (OATP) 1B1 and OATP1B3. Understanding and preventing the cytotoxicity of microcystin-LR is crucial to maintain human health. This chemoprevention study demonstrates that the herbal plant extract of iwajisha (20 µg/mL) reduced microcystin-LR cytotoxicity in OATP1B3-expressing cells by approximately six times. In addition, 20 µM acteoside, which is one of the major compounds in iwajisha, reduced microcystin-LR cytotoxicity by approximately 7.4 times. Acteoside could also reduce the cytotoxicity of other compounds, such as okadaic acid and nodularin, which are both substrates of OATP1B3 and inhibitors of PP1/PP2A. To investigate the mechanism by which the cytotoxicity of microcystin-LR is attenuated by acteosides, microcystin-LR and microcystin-LR-binding proteins in cells were examined after microcystin-LR and acteosides were co-exposed. Thus, acteoside noncompetitively inhibited microcystin-LR uptake by OATP1B3-expressing cells. Furthermore, acteoside inhibited the intracellular interaction of microcystin-LR with its binding protein(s), including the 22 kDa protein. Furthermore, using immunoblot analysis, acteoside induced the phosphorylation of extracellular signal-regulated kinase (ERK), which is one of the survival signaling molecules. These results suggest that acteoside reduces microcystin-LR cytotoxicity through several mechanisms, including the inhibition of microcystin-LR uptake via OATP1B3, and decreased interaction between microcystin-LR and its binding protein(s), and that ERK signaling activation contributes to the attenuation effect of acteoside against microcystin-LR cytotoxicity.

Neu1 deficiency induces abnormal emotional behavior in zebrafish.

NEU1 sialidase hydrolyzes sialic acids from glycoconjugates in lysosomes. Deficiency of NEU1 causes sialidosis with symptoms including facial dysmorphism, bone dysplasia, and neurodegeneration. However, the effects of NEU1 deficiency on emotional activity have not been explored. Here, we conducted the behavioral analysis using Neu1-knockout zebrafish (Neu1-KO). Neu1-KO zebrafish showed normal swimming similar to wild-type zebrafish (WT), whereas shoaling was decreased and accompanied by greater inter-fish distance than WT zebrafish. The aggression test showed a reduced aggressive behavior in Neu1-KO zebrafish than in WT zebrafish. In the mirror and 3-chambers test, Neu1-KO zebrafish showed more interest toward the opponent in the mirror and multiple unfamiliar zebrafish, respectively, than WT zebrafish. Furthermore, Neu1-KO zebrafish also showed increased interaction with different fish species, whereas WT zebrafish avoided them. In the black-white preference test, Neu1-KO zebrafish showed an abnormal preference for the white region, whereas WT zebrafish preferred the black region. Neu1-KO zebrafish were characterized by a downregulation of the anxiety-related genes of the hypothalamic-pituitary-adrenal axis and upregulation of lamp1a, an activator of lysosomal exocytosis, with their brains accumulating several sphingoglycolipids. This study revealed that Neu1 deficiency caused abnormal emotional behavior in zebrafish, possibly due to neuronal dysfunction induced by lysosomal exocytosis.

Conserved and Divergent Mechanisms That Control TORC1 in Yeasts and Mammals.

Target of rapamycin complex 1 (TORC1), a serine/threonine-protein kinase complex highly conserved among eukaryotes, coordinates cellular growth and metabolism with environmental cues, including nutrients and growth factors. Aberrant TORC1 signaling is associated with cancers and various human diseases, and TORC1 also plays a key role in ageing and lifespan, urging current active research on the mechanisms of TORC1 regulation in a variety of model organisms. Identification and characterization of the RAG small GTPases as well as their regulators, many of which are highly conserved from yeast to humans, led to a series of breakthroughs in understanding the molecular bases of TORC1 regulation. Recruitment of mammalian TORC1 (mTORC1) by RAGs to lysosomal membranes is a key step for mTORC1 activation. Interestingly, the RAG GTPases in fission yeast are primarily responsible for attenuation of TORC1 activity on vacuoles, the yeast equivalent of lysosomes. In this review, we summarize our current knowledge about the functions of TORC1 regulators on yeast vacuoles, and illustrate the conserved and divergent mechanisms of TORC1 regulation between yeasts and mammals.

Identification of novel fish sialidase genes responsible for KDN-cleaving activity.

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a minor component of sialic acids detected in vertebrates, such as human cancer cells, rat liver, and fish tissues. Although the enzyme activity of KDN-cleaving sialidase (KDN-sialidase) has been detected in rainbow trout, the gene responsible for its expression has not been identified in vertebrates. We evaluated sialidases in human and various fish for their KDN-cleaving activity using an artificial substrate, methylumbelliferyl-KDN (MU-KDN). Four of the human sialidases tested (NEU1, NEU2, NEU3, and NEU4) did not hydrolyze MU-KDN. Although most fish Neu1s showed negligible KDN-sialidase activity, two Neu1b sialidases from Oreochromis niloticus and Astyanax mexicanus, a paralog of Neu1, exhibited a potent KDN-sialidase activity. Further, O. niloticus and Oryzias latipes Neu3a exhibited a drastically high KDN-sialidase activity, while Danio rerio Neu3.1 showed moderate activities and other Neu3 proteins exhibited little activity. All the Neu4 sialidases tested in fish cleaved KDN and Neu5Ac from MU-KDN and MU-Neu5Ac, respectively, with equivalent potential. To our knowledge, this is the first report to identify KDN-sialidase genes in vertebrates and we believe that KDN-sialidase activity could be conserved among fish Neu4s.

Establishment and characterization of Neu1-knockout zebrafish and its abnormal clinical phenotypes.

Mammalian sialidase Neu1 is involved in various physiological functions, including cell adhesion, differentiation, cancer metastasis, and diabetes through lysosomal catabolism and desialylation of glycoproteins at the plasma membrane. Various animal models have been established to further explore the functions of vertebrate Neu1. The present study focused on zebrafish (Danio rerio) belonging to Cypriniformes as an experimental animal model with neu1 gene deficiency. The results revealed that the zebrafish Neu1 desialyzed both α2-3 and α2-6 sialic acid linkages from oligosaccharides and glycoproteins at pH 4.5, and it is highly conserved with other fish species and mammalian Neu1. Furthermore, Neu1-knockout zebrafish (Neu1-KO) was established through CRISPR/Cas9 genome editing. Neu1-KO fish exhibited slight abnormal embryogenesis with the accumulation of pleural effusion; however, no embryonic lethality was observed. Although Neu1-KO fish were able to be maintained as homozygous, they showed smaller body length and weight than the wild-type (WT) fish, and muscle atrophy and curvature of the vertebra were observed in adult Neu1-KO fish (8 months). The expression patterns of myod and myog transcription factors regulating muscle differentiation varied between Neu1-KO and WT fish embryo. Expression of lysosomal-related genes, including ctsa, lamp1a, and tfeb were up-regulated in adult Neu1-KO muscle as compared with WT. Furthermore, the expression pattern of genes involved in bone remodeling (runx2a, runx2b, and mmp9) was decreased in Neu1-KO fish. These phenotypes were quite similar to those of Neu1-KO mice and human sialidosis patients, indicating the effectiveness of the established Neu1-KO zebrafish for the study of vertebrate Neu1 sialidase.

Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge.

The human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.

Biological and Pathological Roles of Ganglioside Sialidases.

Sialidases are glycosidases responsible for the removal of α-glycosidically linked sialic acid residues from carbohydrate portions of glycoproteins and glycolipids, this process being the initial step in the degradation of such glycoconjugates. Sialic acids are considered to play important roles in various biological processes largely in two ways, one related to their hydrophilic and acidic properties exerting physicochemical effects on the glycoconjugates to which they are attached, and the other as recognition sites or in an opposing fashion as masking sites. The removal of sialic acids catalyzed by a sialidase, therefore greatly influences many biological processes through changing the conformation of glycoproteins and through recognition and masking of biological sites of functional molecules. Sialidases are found widely distributed in metazoan animals, from echinoderms to mammals, and are also present in viruses and other microorganisms, including fungi, protozoa, and bacteria even mostly lacking sialic acids. In mammals, there are four forms of sialidase (Neu1, Neu2, Neu3, and Neu4), differing in their major subcellular localization and enzymatic properties. They have been implicated in regulation of various cellular activities, such as cell differentiation, cell growth, and cell adhesion and motility, depending on their particular properties. In contrast, in microorganisms the enzymes appear to play roles limited to nutrition and pathogenesis. In this chapter, the focus is on mammalian sialidases preferentially hydrolyzing gangliosides, mostly Neu3 and Neu4, with an attempt to provide a brief overview of their physiological and pathological roles.

Unique nuclear localization of Nile tilapia (Oreochromis niloticus) Neu4 sialidase is regulated by nuclear transport receptor importin α/ß.

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Different from Neu1 and Neu3 sialidases, Neu4 enzymatic properties such as substrate specificity and subcellular localization are not well-conserved among vertebrates. In fish only zebrafish and medaka neu4 genes have been cloned and their polypeptides have been characterized so far. Thus, characterization of Neu4 from other fish species is necessary to evaluate Neu4 physiological functions. Here, Nile tilapia was chosen for the characterization of Neu4 polypeptide considering that it is one of the major cultured fish all over the world and that its genomic sequences are now available. Coding DNA sequence of tilapia Neu4 was identified as 1,497 bp and its recombinant protein showed broad substrate specificity and optimal sialidase enzyme activity pH at 4.0. Neu4 activity was sustained even in neutral and alkali pH. Interestingly, immunofluorescence analysis revealed that major subcellular localization of tilapia Neu4 was nuclear, quite distinct from zebrafish (ER) and medaka Neu4 (lysosome). Bioinformatic analysis showed the existence of putative nuclear localization signal (NLS) in tilapia Neu4. In general, it is known that importin families bind to several proteins via NLS and transfer them into nucleus. Therefore, to determine the involvement of putative NLS in Neu4 nuclear localization, Neu4 mutant deleting NLS was constructed and expressed in cultured cells. As a result, NLS deletion significantly diminished the nuclear localization. Furthermore, treatment of importazole, interrupter of binding importin ß and RanGTP, significantly suppressed Neu4 nuclear localization. In summary, tilapia Neu4 is a unique sialidase localized at nucleus and its transport system into nucleus is regulated by importin.

Substrate specificity of TOR complex 2 is determined by a ubiquitin-fold domain of the Sin1 subunit.

The target of rapamycin (TOR) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT.

Overexpression of carboxylesterase contributes to the attenuation of cyanotoxin microcystin-LR toxicity.

Microcystin-LR is a hepatotoxin produced by several cyanobacteria. Its toxicity is mainly due to a inhibition of protein phosphatase, PP1 and PP2A. Previously, we used a cell line stably expressing uptake transporter for microcystin-LR, OATP1B3 (HEK293-OATP1B3 cells). In this study, to determine whether overexpression of carboxylesterase (CES), which degrades ester-group and amide-group, attenuates the cytotoxicity of microcystin-LR, we generated the HEK293-OATP1B3/CES2 double-transfected cells. HEK293-OATP1B3/CES2 cells showed high hydrolysis activity of p-nitrophenyl acetate (PNPA), which is an authentic substrate for esterase. CES activity in HEK293-OATP1B3/CES2 cells was approximately 3-fold higher than that in the HEK293-OATP1B3 cells. HEK293-OATP1B3/CES2 cells (IC50: 25.4±7.7nM) showed approximately 2.1-fold resistance to microcystin-LR than HEK293-OATP1B3 cells (IC50: 12.0±1.5nM). Moreover, the CES inhibition assay and microcystin-agarose pull down assay showed the possibility of the interaction between CES2 and microcystin-LR. Our results indicated that the overexpression of CES2 attenuates the cytotoxicity of microcystin-LR via interaction with microcystin-LR.

NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation.

Naringin, which is one of the flavonoids contained in citrus fruits, is well known to possess various healthy functions to humans. It has been reported that naringin suppresses cancer cell growth in vitro and in vivo, although the underlying mechanisms are not fully understood. Recently, the roles of glycoconjugates, such as gangliosides, in cancer cells have been focused because of their regulatory effects of malignant phenotypes. Here, to clarify the roles of naringin in the negative-regulation of cancer cell growth, the alteration of glycoconjugates induced by naringin exposure and its significance on cell signaling were investigated. Human cancer cells, HeLa and A549, were exposed to various concentrations of naringin. Naringin treatment induced the suppression of cell growth toward HeLa and A549 cells accompanied with an increase of apoptotic cells. In naringin-exposed cells, GM3 ganglioside was drastically increased compared to the GM3 content prior to the treatment. Furthermore, naringin inhibited NEU3 sialidase, a GM3 degrading glycosidase. Similarly, NEU3 inhibition activities were also detected by other flavanone, such as hesperidin and neohesperidin dihydrocalcone, but their aglycones showed less inhibitions. Naringin-treated cancer cells showed suppressed EGFR and ERK phosphorylation levels. These results suggest a novel mechanism of naringin in the suppression of cancer cell growth through the alteration of glycolipids. NEU3 inhibitory effect of naringin induced GM3 accumulation in HeLa and A549 cells, leading the attenuation of EGFR/ERK signaling accompanied with a decrease in cell growth.

Lysosomal localization of Japanese medaka (Oryzias latipes) Neu1 sialidase and its highly conserved enzymatic profiles with human.

Desialylation in the lysosome is a crucial step for glycoprotein degradation. The abnormality of lysosomal desialylation by NEU1 sialidase is involved in diseases of mammals such as sialidosis and galactosialidosis. Mammalian Neu1 sialidase is also localized at plasma membrane where it regulates several signaling pathways through glycoprotein desialylation. In fish, on the other hand, the mechanism of desialylation in the lysosome and functions of Neu1 sialidase are still unclear. Here, to understand the significance of fish Neu1 sialidase, neu1 gene was cloned from medaka brain and the profiles of its polypeptides were analyzed. Open reading frame of medaka neu1 consisted 1,182 bp and the similarity of its deduced amino acids with human NEU1 was 57%. As this recombinant polypeptide did not show significant sialidase activity, medaka cathepsin A, known in mammals as protective protein activating Neu1, was cloned and then co-expressed with medaka Neu1 to examine whether medaka cathepsin A activates Neu1 activity. As a result, Neu1/cathepsin A showed a drastic increase of sialidase activity toward MU-NANA. Major substrate of medaka Neu1 was 3-sialyllactose and its optimal pH was 4.0. With immunofluorescence analysis, signal of overexpressed medaka Neu1 was found to coincide with Lysotracker signals (organelle marker of lysosome) and co-localized with medaka cathepsin A in fish hepatic Hepa-T1 cells. Furthermore, part of medaka Neu1 was also detected at plasma membrane. Medaka Neu1 possessed signal peptide sequence at N-terminal and incomplete lysosomal targeting sequence at C-terminus. Medaka neu1 gene was ubiquitously expressed in various medaka tissues, and its expression level was significantly higher than other sialidase genes such as neu3a, neu3b and neu4. The present study revealed the profiles of fish Neu1 sialidase and indicated its high conservation with human NEU1 for the first time, suggesting the presence of similar desialylation system in the medaka lysosome to human. Moreover, the present study showed the possibility of medaka as a model animal of human NEU1 sialidase.

Okadaic acid is taken-up into the cells mediated by human hepatocytes transporter OATP1B3.

Okadaic acid is known as a diarrheal shellfish poison. It is thought that there is no specific target organ for okadaic acid after it has been absorbed into the body. However, the details of its pharmacokinetics are still unknown. In this study, we demonstrated that okadaic acid was more toxic to the hepatocyte-specific uptake transporter OATP1B1- or OATP1B3-expressing cells than control vector-transfected cells. In addition, PP2A activity, which is a target molecule of okadaic acid, was more potently inhibited by okadaic acid in OATP1B1- or OATP1B3-expressing cells compared with control vector-transfected cells. The cytotoxicity of okadaic acid in OATP1B1- or OATP1B3-expressing cells was attenuated by known substrates of OATP1B1- and OATP1B3, but not in control vector-transfected cells. Furthermore, after uptake inhibition study using OATP1B3-expressing cells, Dixon plot showed that okadaic acid inhibited the uptake of hepatotoxin microcystin-LR, which is a substrate for OATP1B1 and OATP1B3, in a competitive manner. These results strongly suggested that okadaic acid is a substrate for OATP1B3 and probably for OATP1B1, and could be involved in unknown caused liver failure and liver cancer. Since okadaic acid possesses cytotoxicity and cell proliferative activity by virtue of its known phosphatase inhibition activity.

Naringin attenuates the cytotoxicity of hepatotoxin microcystin-LR by the curious mechanisms to OATP1B1- and OATP1B3-expressing cells.

Microcystin-LR, which is an inhibitor of serine/threonine protein phosphatase (PP)1 and PP2A, induces liver injury by its selective uptake system into the hepatocyte. It is also thought that microcystin-LR induces reactive oxygen species (ROS). We tried to establish the chemical prevention of microcystin-LR poisoning. We investigated the effect of grapefruit flavanone glycoside naringin on cytotoxicity of microcystin-LR using human hepatocyte uptake transporter OATP1B3-expressing HEK293-OATP1B3 cells. We found cytotoxicity of microcystin-LR was attenuated by naringin in a dose dependent manner. The inhibition magnitude of total cellular serine/threonine protein phosphatase activity induced by microcystin-LR was suppressed by naringin. In addition, uptake of microcystin-LR into HEK293-OATP1B3 cells was inhibited by naringin. Furthermore, microcystin-LR induced phosphorylation of p53 was inhibited by naringin. Regardless of the difference in the exposure pattern of pre-processing and post-processing of naringin, the toxicity of microcystin-LR was comparable. These results suggested that naringin is promising remedy as well as preventive medicine for liver damage with microcystin-LR. In addition, involvement of ROS production after exposure to the sublethal concentrations of microcystin-LR in the onset of cytotoxicity was negligible. Therefore, inhibition of microcystin-LR uptake and the pathway other than ROS production would be involved in the effect of naringin on the attenuation of microcystin-LR toxicity.

Potentiation of epidermal growth factor-mediated oncogenic transformation by sialidase NEU3 leading to Src activation.

We previously demonstrated that sialidase NEU3, a key glycosidase for ganglioside degradation, is up-regulated in various human cancers, leading to increased cell invasion, motility and survival of cancer cells possibly through activation of EGF signaling. Its up-regulation is also important for promotion of the stage of colorectal carcinogenesis in vivo in human NEU3 transgenic mice treated with azoxymethane for the induction of aberrant crypt foci in the colon mucosa, accompanied by enhanced phosphorylation of EGF receptor (EGFR). To address whether the activation of EGF signaling by the sialidase is associated with oncogenic transformation, we here analyzed the effects of overexpression of NEU3 and EGFR in NIH-3T3 cells. When NEU3 was stably transfected with or without EGFR, it was associated with significant increases in clonogenic growth, clonogenicity on soft agar and in vivo tumor growth in nude mice either with or without the receptor overexpression in the presence of EGF, compared with the levels in their vector controls. Despite the fact that the endogenous level of EGFR is known to be extremely low in these cells, NEU3 significantly enhanced the phosphorylation of Akt and ERK, as well as that of the receptor. The NEU3-mediated activation was largely abrogated by the EGFR inhibitor AG1478 or PD153035, but significant clonogenic growth still remained. NEU3 was then found to activate Src kinase, and the clonogenicity was completely suppressed by an Src inhibitor, PP2. The activity-null mutants failed to activate Src and EGFR, indicating that ganglioside modulation by NEU3 may be necessary for the activation. NEU3 and Src were co-immunoprecipitated with EGFR in NEU3- and EGFR- transfected cells. These findings identify NEU3 as an essential participant in tumorigenesis through the EGFR/Src signaling pathway and a potential target for inhibiting EGFR-mediated tumor progression.

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.

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.

Microcystin-LR induces anoikis resistance to the hepatocyte uptake transporter OATP1B3-expressing cell lines.

Microcystin-LR is a cyclic peptide released by several bloom-forming cyanobacteria. Understanding the mechanism of microcystin-LR toxicity is important, because of the both potencies of its acute cytotoxicity and tumor-promoting activity in hepatocytes of animals and humans. Recently, we have reported that the expression of human hepatocyte uptake transporter OATP1B3 was critical for the selective uptake of microcystin-LR into hepatocytes and for induction of its fatal cytotoxicity. In this study, we demonstrated a novel function of microcystin-LR which induced bipotential changes including anoikis resistance and cytoskeleton reorganization to OATP1B3-transfected HEK293 cells (HEK293-OATP1B3). After exposure to microcystin-LR, HEK293-OATP1B3 cells were divided to the floating cells and remaining adherent cells. After collection and reseeding the floating cells into a fresh flask, cells were confluently proliferated (HEK293-OATP1B3-FL) under the microcystin-LR-free condition. Both the proliferated HEK293-OATP1B3-FL and remaining adherent HEK293-OATP1B3-AD cells changed the character with down- and up-regulation of E-cadherin, respectively. Additionally, these cells acquired resistance to microcystin-LR. These results suggest that microcystin-LR could be associated with not only tumor promotion, but also epithelial-mesenchymal transition-mediated cancer metastasis. Furthermore, microcystin-LR might induce the cytoskeleton reorganization be accompanied epithelial-mesenchymal transition.