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Supplementary Figure 2 from Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

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Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) α. We compared the effects of vanadate to PFTα and PFTμ, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis. Cancer Res; 70(1); 257–65

Supplementary Figure 1 from Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Epo-C12 is a synthetic derivative of epolactaene, isolated from Penicillium sp. BM 1689-P. Epo-C12 induces apoptosis in human acute lymphoblastoid leukemia BALL-1 cells. In our previous studies, seven proteins that bind to Epo-C12 were identified by a combination of pull-down experiments using biotinylated Epo-C12 (Bio-Epo-C12) and mass spectrometry. In the present study, the effect of Epo-C12 on peroxiredoxin 1 (Prx 1), one of the proteins that binds to Epo-C12, was investigated. Epo-C12 inhibited Prx 1 peroxidase activity. However, it did not suppress its chaperone activity. Binding experiments between Bio-Epo-C12 and point-mutated Prx 1s suggest that Epo-C12 binds to Cys52 and Cys83 in Prx 1. The present study revealed that Prx 1 is one of the target proteins through which Epo-C12 exerts an apoptotic effect in BALL-1 cells.

Retinoids are a family of vitamin A-derived molecules and include the biologically active metabolite, retinoic acid (RA). RA acts as a specific modulator of neuronal differentiation and proliferation. However, teratogenicity and a large excess of RA have been found in animal studies. Thus, development of effective and stable retinoids is desirable. In this study, we showed that treatment with novel synthetic retinobenzoic acids promotes neurite outgrowth in a selected subpopulation of the human neuroblastoma cell line SK-N-SH. Furthermore, we found that, although acting via a different mechanism, retinobenzoic acids have the same neurite outgrowth-inducing effect as RA. Retinoids, including RA, bind to nuclear retinoic acid receptors (RARs). Therefore, we examined the expression of RARs in retinobenzoic acid-treated cells. Similar to already known retinoids, novel synthetic retinobenzoic acids promote the upregulation of RARβ and have no effect on RARα or γ. These results suggest that retinobenzoic acids act via RARβ during neurite outgrowth. Moreover, stimulation with RA or retinobenzoic acids significantly increased the phosphorylation levels of both ERK1/2 and mTOR. ERK1/2 and mTOR inhibition blocked the retinobenzoic acid-induced increase in neurite outgrowth, suggesting that retinobenzoic acids promoted neurite outgrowth by activating the ERK1/2 and mTOR signaling pathways. Notably, the RA-induced increase in neurite outgrowth was blocked by the ERK1/2 inhibitor U0126, but not by the mTOR inhibitor rapamycin. In addition, ERK1/2 inhibition blocked the upregulation of RARβ promoted by RA and retinobenzoic acids. In contrast, mTOR inhibition had no effect on upregulation of RARβ. Our results show that novel synthetic retinobenzoic acids induce neurite outgrowth by a different mechanism than RA. These findings suggest that activation of both ERK1/2, which results in downstream regulation of RARβ, and mTOR, are responsible for the novel synthetic retinobenzoic acid-induced neurite outgrowth in human neuroblastoma cells.

// Akinori Morita 1,2 , Shinya Ariyasu 3 , Soichiro Ohya 4 , Ippei Takahashi 2 , Bing Wang 5 , Kaoru Tanaka 5 , Takatoshi Uchida 4 , Haruna Okazaki 4 , Kengo Hanaya 6 , Atsushi Enomoto 7 , Mitsuru Nenoi 5 , Masahiko Ikekita 4 , Shin Aoki 3,6 , Yoshio Hosoi 2 1 Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan 2 Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan 3 Center for Technologies against Cancer, Tokyo University of Science, Chiba, Japan 4 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba, Japan 5 Radiation Risk Reduction Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, Chiba, Japan 6 Department of Medicinal and Life Science, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan 7 Laboratory of Molecular Radiology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Correspondence: Akinori Morita, email: // Keywords : p53, zinc chelator, zinc binding site, radiation, apoptosis Received : October 23, 2013 Accepted : November 22, 2013 Published : November 24, 2013 Abstract In a previous study, we reported that sodium orthovanadate (vanadate) is the first known inhibitor that is capable of protecting mice from death from the radiation-induced gastrointestinal syndrome via its ability to block both transcription-dependent and transcription-independent p53 apoptotic pathways. In this paper, we report that vanadate has a unique activity for inducing the denaturation of p53 relative to other known radioprotective p53 inhibitors, pifithrin-α (PFTα) and pifithrin-µ (PFTµ). This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation. Based on the fact that p53 denaturation can be induced by the dissociation of a zinc ion, which is used as a structural factor of p53, we screened some zinc (II) chelators for the suppression of the DNA binding activity of p53 in vitro and the inhibition of radiation-induced p53-dependent apoptosis in MOLT-4 cells. The findings indicate that two of five zinc (II) chelators also suppressed apoptosis. Among the inhibitors tested, Bispicen ( N,N’ -Bis(2-pyridylmethyl)-1,2-ethanediamine) had the highest inhibition activity. A mechanistic study using cells bearing different p53 status or functions (i.e., p53-knockdown MOLT-4 transformant and its revertants, p53 mutant cells, p53-null cells), and p53-independent apoptotic stimuli revealed that the suppressive effect of Bispicen on apoptosis is specifically mediated through p53. Moreover, Bispicen, similar to vanadate, induces the denaturation of p53 as well as the blocking of both transcription-dependent and -independent apoptotic pathways. Our findings indicate that the use of zinc (II) chelators represent a new approach for protecting against radiation-induced p53-dependent apoptosis through the inhibition of p53-dependent apoptotic pathways.

The recent applications of a TiO2-modified Ti-mesh filter (TMiP™) for water purification are summarized with newly collected data including biological assays as well as sewage water treatment. The water purification reactors consist of the combination of a TMiP, a UV lamp, an excimer VUV lamp, and an ozonation unit. The water purification abilities of the reactor were evaluated by decomposition of organic contaminants, inactivation of waterborne pathogens, and treatment efficiency for sewage water. The UV-C/TMiP/O3 reactor disinfected E. coli in aqueous suspension in approximately 1 min completely, and also decreased the number of E. coli in sewage water in 15 min dramatically. The observed rate constants of 7.5 L/min and 1.3 L/min were calculated by pseudo-first-order kinetic analysis respectively. Although organic substances in sewage water were supposed to prevent the UV-C/TMiP/O3 reactor from purifying water, the reactor reduced E. coli in sewage water continuously. On the other hand, although much higher efficiencies for decomposition of organic pollutants in water were achieved in the excimer/TMiP reactor, the disinfection activity of the reactor for waterborne pathogens was not as effective as the other reactors. The difference of efficiency between organic pollutants and waterborne pathogens in the excimer/TMiP reactor may be due to the size, the structure, and the decomposition mechanism of the organic pollutants and waterborne pathogens. These results show that a suitable system assisted by synergy of photocatalysts and other technologies such as ozonation has a huge potential as a practical wastewater purification system.

A versatile photocatalyst-excimer-lamp hybrid unit for both air- and water-purification at practical scale was investigated. The unit consisted of a xenon filled quartz tube (20 mm i.d. × 300 mm length) and TiO2 nanoparticles-modified Ti-mesh sheet (TMiP™). Emission at 172 nm from a xenon excimer ( Xe 2 ∗ ) was able to produce reactive species by direct photolysis of water or air. At the same time, the emission was able to excite TiO2 on TMiP surface. Therefore, the unit decomposes organic contaminants by synergy of photocatalysis and photolysis. Air- and water-purification efficiency of the unit was examined by high concentration of acetaldehyde and phenol decomposition test, respectively. The disinfection activity of the units for waterborne pathogens was also investigated. For comparison, the purification units using excimer-lamp alone or UV lamps with TMiP and/or ozone treatment were evaluated in the same methods. The photocatalyst-excimer-lamp hybrid unit was able to decompose acetaldehyde and phenol effectively, compared with the other units. On the other hand, disinfection activity of the unit for waterborne pathogens was strongly affected by the kind of the waterborne pathogens. The difference of efficiency between organic pollutants and waterborne pathogens by the unit may be due to the molecular or bacterial size and the surface composition of bacteria and viruses, critical wavelength for disinfection, and the permeation ability of the outer wall of bacterial cells or virus particles for reactive species. Taken together, the photocatalyst-excimer-lamp hybrid unit has wide potentials for environmental purification by the synergy of photocatalysis and photolysis. These results would be attractive to develop a practical environmental purification system such as sewage water treatment.

Bacteria that cause serious food poisoning are known to sporulate under conditions of nutrient and water shortage. The resulting spores have much greater resistance to common sterilization methods, such as heating at 100 °C and exposure to various chemical agents. Because such bacteria cannot be inactivated with typical alcohol disinfectants, peroxyacetic acid (PAA) often is used, but PAA is a harmful agent that can seriously damage human health. Furthermore, concentrated hydrogen peroxide, which is also dangerous, must be used to prepare PAA. Thus, the development of a facile and safe sporicidal disinfectant is strongly required. In this study, we have developed an innovative sporicidal disinfection method that employs the combination of an aqueous ethanol solution, visible light irradiation and a photocatalyst. We successfully produced a sporicidal disinfectant one hundred times as effective as commercially available PAA, while also resolving the hazards and odor problems associated with PAA. The method presented here can potentially be used as a replacement for the general disinfectants employed in the food and health industries.

The inactivation of Escherichia coli and Qβ phage was examined following their photocatalytic treatment with TiO2 hollows and spheres that had been prepared by electrospray, hydrothermal treatment, and calcination. The crystal structures of the hollows and spheres were assigned to TiO2 anatase, and the surface areas of the hollows and spheres were determined to be 91 and 79 m(2) g(-1), respectively. Interestingly, TiO2 spheres exhibited higher anti-pathogen performance than TiO2 hollows, a difference we ascribe to the prevention of light multi-scattering by microorganisms covering the surfaces of the TiO2 particles. The photocatalytic decomposition of dimethyl sulfoxide (DMSO) in the presence of TiO2 hollows and spheres was examined in order to study the dependence of photocatalytic activity on TiO2 morphology for the size scale of the reactants. TiO2 hollows provided greater photocatalytic decomposition of DMSO than did TiO2 spheres, in contrast to the pattern seen for pathogen inactivation. Fabrication of photocatalysts will need to vary depending on what substance (e.g., organic compound or biological agent) is being targeted for environmental remediation.

β-Methoxyacrylate antibiotics are well known to inhibit the fungal and yeast mitochondrial respiratory chain. In addition, β-methoxyacrylates are reported to suppress the proliferation of mammalian cancer cells. Differentiation and cell-cycle arrest are closely related. The cell cycle of proliferating cells is suppressed before differentiation. In this study, we synthesized a β-methoxyacrylate analog and treated neuronal differential model cells with it. We then estimated β-methoxyacrylate's neurotrophic effect by inhibiting cell proliferation so as to orient neuronal differentiation. SUTAF-027-a novel β-methoxyacrylate derivative, arrested the cell cycle and thereby suppressed the proliferation of PC12 rat pheochromocytoma cells and mouse neuroblastoma Neuro2a cells at very low treatment doses, as low as 1nM. However, a single SUTAF-027 treatment did not affect neuritogenesis. Surprisingly, however, co-treatment of SUTAF-027 and nerve growth factor (NGF) significantly augmented the NGF-induced neurite outgrowth of PC12. On the other hand, a single treatment of 1nM SUTAF-027 induced neurite outgrowth in Neuro2a cells. Further signal transduction mechanism studies revealed that SUTAF-027 induced the phosphorylation of extracellular signal-regulated kinase (ERK) and slight phosphorylation of c-jun N-terminal kinase (JNK). Moreover, inhibition of ERK and JNK blocked SUTAF-027-augmented neurite outgrowth. These results suggested that the novel β-methoxyacrylate analog SUTAF-027 augmented neurite outgrowth by arresting the cell cycle and activating the ERK and JNK pathways.

The diagnosis of renal cell carcinoma is currently based on imaging techniques, mainly because there is no blood marker available for its detection. Thus, there is still the need for the development of novel tumor markers. We examined plasma levels of eight proteins in 15 renal cell carcinoma patients before and after surgery, and in 51 healthy controls using enzyme-linked immunosorbent assay. Plasma levels of α-enolase, calnexin, galectin-1, galectin-3 and lectin mannose-binding 2 were significantly higher in renal cell carcinoma patients than in controls (P < 0.05). Among these proteins, the sensitivities for galectin-1 and galectin-3 were higher than those for calnexin and lectin mannose-binding 2 in the specificity range from 80% to 100%. A combined use of galectin-1 and galectin-3 showed 98% specificity and 47% sensitivity. In addition, the assays showed that plasma α-enolase levels decreased significantly 4 weeks after nephrectomy (P = 0.0034), and this tendency continued until 12 weeks after nephrectomy (P = 0.0156). These findings suggest that α-enolase could be used in the postoperative follow up of renal cell carcinoma patients, whereas the combined use of galectin-1 and galectin-3 might represent a useful tool for primary detection.

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized type I membrane glycoprotein, and promotes dendritic filopodia formation. Although we have determined the N-glycan structures of ICAM-5 in a previous report, their function is unknown. Here, we produced fifteen ICAM-5 gene constructs, in which each potential N-glycosylation site was mutated, to elucidate the function of the N-glycans of ICAM-5, and observed the effects of transfection of them on a neuronal cell line, Neuro-2a (N2a). Only the N54Q mutant, which is the mutant for the most N-terminal glycosylation site, failed to induce filopodia-like protrusions in N2a cells. Immunofluorescence staining and cell surface biotinylation revealed that N54Q ICAM-5 was confined to the ER and also could not be expressed on the cell surface. This is further supported by the biochemical evidence that almost all N-glycans of N54Q ICAM-5 were digested by Endo glycosidase H and peptide:N-glycanase, indicating that almost all of them retain high-mannose-type structures in ER. In additon, it also failed to form disulfide bonds or functional protein complexes. The stable transformants of N54Q ICAM-5 showed retarded cell growth, but it was interesting that there was no apparent ER stress, because the mutant was sequentially degraded via ER associated degradation pathway by comparing the susceptibilities of the responses to various inhibitors of this pathway in wild-type and N54Q ICAM-5 transfectants. Taken together, the Asn(54)-linked glycan is necessary for normal trafficking and function of ICAM-5, but is unassociated with ER-associated degradation of it.

Inulin is a polysaccharide that enhances various immune responses, mainly to T and B cells, natural killer cells, and macrophages in vivo and in vitro. Previous reports describe that inulin activates macrophages indirectly by affecting the alternative complement pathway. In this study, we examined the direct effect of inulin on PMA-treated THP-1 macrophages. Inulin treatment did not stimulate the proliferation of THP-1 macrophages at all. However, inulin treatment significantly increased phagocytosis of the polystyrene beads without the influence of serum. Doses of around 1 mg/mL had the maximal effect, and significant progression of phagocytosis occurred at times treated over 6 h. Inulin augmented phagocytosis not only with polystyrene beads but also with apoptotic cancer cells. The inulin-induced phagocytosis uptake was suppressed in Toll-like receptor (TLR) 4 mutated C3H/HeJ mice peritoneal macrophages. Moreover, inulin-induced THP-1 macrophage TNF-α secretion was inhibited using a blocking antibody specific to TLR4, suggesting that TLR4 is involved in the binding of inulin to macrophages. Furthermore, we used specific kinase inhibitors to assess the involvement of inulin-induced phagocytosis and revealed that phosphoinositide 3-kinase and mitogen-activated protein kinase, especially p38, participated in phagocytosis. These results suggest that inulin affects macrophages directly by involving the TLR4 signaling pathway and stimulating phagocytosis for enhancing immunomodulation.

3-(3-Phenoxybenzyl)amino-β-carboline 2h showed extremely-high activity; the IC 50 value was 0.074 μM. To verify 2h -induced cell death types, we observed the chromatin condensation, the DNA fragmentation and activated caspase-3 using Hoechst 33342, agarose electrophoresis and western blot, and suggesting 2h -induced cell death type was apoptosis. Flow cytometry showed that 2h -treated cell was induced SubG1 cell population after G2/M cell cycle arrest. In addition, using affinity chromatography and peptide mass fingerprinting, we found that interacting protein with this compound was α-tubulin protein.

Nobiletin is a citrus polymethoxyflavonoid that suppresses tumor growth and metastasis, both of which depend on angiogenesis. We recently identified nobiletin as a cell differentiation modulator. Because cell differentiation is a critical event in angiogenesis, it might be possible that nobiletin could exhibit antiangiogenic activity, resulting in suppression of these tumor malignant properties. To verify this possibility, we examined the antiangiogenic effects of nobiletin in vitro and in vivo. Nobiletin had concentration-dependent inhibitory effects on multiple functions of angiogenesis-related endothelial cells (EC); it suppressed the proliferation, migration and tube formation on matrigel of human umbilical vein EC (HUVEC) stimulated with endothelial cell growth supplement (ECGS), a mixture of acidic and basic fibroblast growth factors (FGFs). Gelatin zymography and northern blotting revealed that nobiletin suppressed pro-matrix metalloproteinase-2 (proMMP-2) production and MMP-2 mRNA expression in ECGS-stimulated HUVEC. Nobiletin also downregulated cell-associated plasminogen activator (PA) activity and urokinase-type PA mRNA expression. Furthermore, nobiletin inhibited angiogenic differentiation induced by vascular endothelial growth factor and FGF, an in vitro angiogenesis model. This inhibition was accompanied by downregulation of angiogenesis-related signaling molecules, such as extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase, and transcriptional factors (c-Jun and signal transducer and activator of transcription 3), and activation of the caspase pathway. In a chick embryo chorioallantoic membrane assay, nobiletin showed an antiangiogenic activity, the ID(50) value being 10μg (24.9nmol) per egg. These results indicate that nobiletin is a novel antiangiogenic compound that exhibits its activity through combined inhibition of multiple angiogenic EC functions.

Activin A is a multifunctional homo-dimeric protein that belongs to the transforming growth factor (TGF)-beta superfamily. In neurons, activin has neuroprotective effects both in vitro and in vivo, but it inhibits neuronal differentiation in some cell lines. Here we report that activin A can promote neuronal differentiation in particular cases. We examined activin A-induced neuronal differentiation and survival in a selected subpopulation of a human neuroblastoma cell line, SK-N-SH, grown in low-serum (differentiation-inducing) conditions. Activin A caused dramatic neurite outgrowth, and increased the expression of neuronal markers and the transactivation of dopamine beta-hydroxylase. We demonstrated that the activin A signal is transduced through the activin A type 1 receptor, ALK4, and transactivates several TGF-beta target genes in a SMAD-independent manner. That is, activin A did not induce the phosphorylation of SMAD2/3, the interaction of SMAD2/3 with SMAD4, the binding of SMAD2/3 to the promoter of TGF-beta target genes, or the accumulation of SMAD2/3 in the nucleus. These results suggest that, in particular cases, activin A can induce neuronal differentiation and support neuronal survival in vitro. These findings may reflect previously unknown functions of activin A in neuronal cells in vivo.

We previously reported on a 8-quinolinol-pendant cyclen (L(5)) as a Zn(2+) fluorophore (cyclen = 1,4,7,10-tetraazacyclododecane) and its caged derivative, 8-(benzenesulfonyloxy)-5-(N,N-dimethylaminosulfonyl)quinolin-2-ylmethyl-pendant cyclen (BS-caged-L(5)), which can be reactivated by hydrolysis of benzenesulfonyl group upon complexation with Zn(2+) at neutral pH to give a 1:1 Zn(2+)-L(5) complex (Zn(H(-1)L(5))). We report herein on the synthesis of 5,7-bis(N,N-dimethylaminosulfonyl)-8-hydroxyquinolin-2-ylmethyl-pendant cyclen (L(6)) and its caged derivative (BS-caged-L(6)) for more sensitive and more efficient cell-membrane permeability than those of L(5) and BS-caged-L(5). By potentiometric pH, (1)H NMR, and UV-vis spectroscopic titrations, the deprotonation constants pK(a1)-pK(a6) of H(5)L(6) were determined to be

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized membrane glycoprotein expressed in tissues distinct from those expressing other ICAMs. Here, we determined the N-glycan structure of ICAM-5 purified from adult rat brain and compared it with that of other ICAMs. N-glycans were released by N-glycosidase F digestion and labeled with p-amino benzoic octylester (ABOE). ABOE-labeled glycans were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. The N-glycans obtained from rat brain ICAM-5 consisted of approximately 85% neutral, 10.2% sialylated-only, 2.8% sulfated-only, and 1.2% sialylated and sulfated glycans. Compared with the N-glycan structures of human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells, rat brain ICAM-5 had less highly branched glycans, sialylated glycans, and N-acetyllactosamine structures. In contrast, high-mannose-type N-glycans and Lewis X were more commonly found in rat brain ICAM-5 than in human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells. In addition, sulfated glycans contained GlcNAc 6-O-sulfate on the non-reducing terminal side. Our data will be important for the elucidation of the roles of the N-glycans expressed in neural cells, including those present on ICAM-5.

Background Many fibroblast growth factor family proteins (FGFs) bind to the heparan sulfate/heparin (HP) subtypes of sulfated glycosaminoglycans (GAGs), and a few have recently been reported to also interact with chondroitin sulfate (CS), another sulfated GAG subtype. Methods To gain additional insight into this interaction, we prepared all currently known FGFs (i.e., FGF1–FGF23) and assessed their affinity for HP, CS-B, CS-D and CS-E. In addition, midkine, hepatocyte growth factor and pleiotrophin were studied as other known HP-binding proteins. Results We found that members of the FGF19 subfamily (i.e., FGF15, 19, 21 and 23) had little or no affinity for HP; all of the other secretable growth factors tested had strong affinities for HP, as was indicated by the finding that their elution from HP-Sepharose columns required 1.0–1.5 M NaCl. We also found that FGF3, 6, 8 and 22 had strong affinities for CS-E, while FGF5 had a moderate affinity for CS-D. The interactions between FGFs and GAGs thus appear to be more diverse than previously understood. General significance This is noteworthy, as the differential interactions of these growth factors with GAGs may be key determinants of their specific biological activities.

Epolactaene, isolated from cultured Penicillium sp. BM 1689-P mycelium, induces neurite outgrowth and arrests the cell cycle of the human neuroblastoma cell line, SH-SY5Y, at the G1 phase. We have found that epolactaene and its derivatives induce apoptosis in the human leukemia B-cell line, BALL-1. In this study, we prepared fluorescent and biotinylated epolactaene derivatives. We characterized the cellular location and the identification of BALL-1 proteins that reacted with these compounds. The results obtained from the reaction of epolactaene or its derivative with N -acetylcysteine methyl ester indicate that these compounds induce the disulfide formation and the α-position of the epoxylactam core is the reactive site.

Background Recently, enzyme enhancement therapy (EET) for Pompe disease involving imino sugars, which act as potential inhibitors of acid α-glucosidases in vitro , to improve the stability and/or transportation of mutant acid α-glucosidases in cells was studied and attracted interest. However, the mechanism underlying the molecular interaction between the imino sugars and the enzyme has not been clarified yet. Methods We examined the inhibitory and binding effects of four imino sugars on a recombinant human acid α-glucosidase, alglucosidase alfa, by means of inhibition assaying and isothermal titration calorimetry (ITC). Furthermore, we built structural models of complexes of the catalytic domain of the enzyme with the imino sugars bound to its active site by homology modeling, and examined the molecular interaction between them. Results All of the imino sugars examined exhibited a competitive inhibitory action against the enzyme, 1-deoxynojirimycin (DNJ) exhibiting the strongest action among them. ITC revealed that one compound molecule binds to one enzyme molecule and that DNJ most strongly binds to the enzyme among them. Structural analysis revealed that the active site of the enzyme is almost completely occupied by DNJ. Conclusion These biochemical and structural analyses increased our understanding of the molecular interaction between a human acid α-glucosidase and imino sugars.

Four pseudo -symmetrical tamoxifen derivatives, RID-B ( 13 ), RID-C ( 14 ), RID-D ( 15 ), and bis(dimethylaminophenetole) ( 16 ), were synthesized via the novel three-component coupling reaction, and the structure–activity relationships of these pseudo -symmetrical tamoxifen derivatives were examined. It was discovered that 13 and 16 strongly inhibit the viability of the HL-60 human acute promyelocytic leukemia cell line, whereas 14 possesses a medium activity against the same cell line and 15 has no effect on the cell viability. The global anti-tumor activity of 13 – 16 against a variety of human cancer cells was assessed using a panel of 39 human cancer cell lines (JFCR 39), and it was shown that RID-B ( 13 ) strongly inhibited the growth of several cancer cell lines at concentrations of less than 1 μM (at 0.38 μM for SF-539 [central nervous system], at 0.58 μM for HT-29 [colon], at 0.20 μM for DMS114 [lung], at 0.21 μM for LOX-IMVI [melanoma], and at 0.23 μM for MKN74 [stomach]).

To examine the uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts from a patient with mucopolysaccharidosis I (MPS I) and its effect on the cleavage of accumulated substrates, we performed enzymological, Western blotting, immunocytochemical and morphological studies. Laronidase was incorporated into the MPS I cells dose-dependently mainly via mannose 6-phosphate (M6P) receptors. Then the incorporated enzyme was transported to lysosomes and processed to the mature form, the pathological changes of the cells being improved. Furthermore, we compared the uptake of laronidase by cultured mouse osteoblasts with that by cultured mouse fibroblasts. The enzyme was incorporated into the cultured mouse osteoblasts mainly via M6P receptors, although mannose (Man) receptors were partially involved in the uptake of the enzyme, as in the cultured fibroblasts. But the uptake by the former was apparently lower than that by the latter. The administration of a high dose of the enzyme or development of a recombinant alpha-L-iduronidase containing many M6P residues is required for further improvement of enzyme replacement therapy for skeletal disorders caused by MPS I.

Human diploid fibroblastic cell line, TIG-3, has a finite life span of about 80 population doubling levels (PDL), and is used for in vitro aging studies. Young cells (PDL 23) grew to higher cell densities at a higher growth rate than aged cells (PDL 77). When the electrophoretic mobility of cells was determined, the negative surface charge of the aged cells decreased significantly when compared to that of young cells. Lectin blot analysis of membrane glycoproteins showed that the alpha-2-6-sialylation but not the alpha-2-3-sialylation of N-glycans decreases markedly in the aged cells when compared to the young cells. In support of this observation, the cDNA microarray assay and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the gene expression of the alpha-2,6-sialyltransferase I (ST6Gal I), which transfers sialic acid to galactose residues of N-glycans, decreases in the aged cells. These results indicate that the concordant decrease of the alpha-2,6-sialylation of N-glycans with the ST6Gal I gene expression is induced in TIG-3 cells by in vitro aging.

Coenzyme Q functions as an electron carrier and reversibly changes to either an oxidized (CoQ), intermediate (CoQ· − ), or reduced (CoQH 2 ) form within a biomembrane. The CoQH 2 form also acts as an antioxidant and prevents cell death, and thus has been successfully used as a supplement. On the other hand, the value of the CoQ/CoQH 2 ratio has been shown to increase in a number of diseases, presumably due to an anti-proliferative effect involving CoQ. In the present study, we examined the effect of CoQ and its isoprenoid side chain length variants on the growth of cells having different p53 statuses. Treatment with CoQs having shorter isoprenoid chains, especially CoQ 2 , induced apoptosis in p53-point mutated BALL-1 cells, whereas treatment with longer isoprenoid chains did not. However, CoQ 2 did not induce apoptosis in either a p53 wild-type cell line or a p53 null mutant cell line. These results indicated that the induction of apoptosis by CoQ 2 was dependent on p53 protein levels. Moreover, CoQ 2 induced reactive oxygen species (ROS) and the phosphorylation of p53. An antioxidant, l -ascorbic acid, inhibited CoQ 2 -induced p53 phosphorylation and further apoptotic stimuli. Overall, these results suggested that short tail CoQ induces ROS generation and further p53-dependent apoptosis.

A 48-kDa β- N -acetylglucosamine (GlcNAc)-binding protein was isolated from mouse brain by GlcNAc-agarose column chromatography. The N-terminal amino acid residues showed the protein to be a mouse Na + /K + -ATPase β1-subunit. When the recombinant FLAG-β1-subunit expressed in Sf-9 cells was applied to a GlcNAc-agarose column, only the glycosylated 38- and 40-kDa proteins bound to the column. In the absence of KCl, little of the proteins bound to a GlcNAc-agarose column, but the 38- and 40-kDa proteins bound in the presence of KCl at concentrations above 1 mM. Immunohistochemical study showed that the β1-subunit and GlcNAc-terminating oligosaccharides are at the cell contact sites. Inclusion of anti-β1-subunit antibody or chitobiose in cell aggregation assays using mouse neural cells resulted in inhibition of cell aggregation. These results indicate that the Na + /K + -ATPase β1-subunit is a potassium-dependent lectin that binds to GlcNAc-terminating oligosaccharides: it may be involved in neural cell interactions.

Although adenosine or ATP is known to control various physiological functions in the brain, including synaptic transmission, neuronal cell death, and neurite outgrowth via P1 or P2 purinergic receptors in the nervous system, little is known about the functions of many other purine derivatives. We examined the effects of various purines on survival in the cerebellar cortex of Purkinje cells with large cell bodies and highly branched dendrites, and it was found that some purine and pyrimidine derivatives influence Purkinje cell survival. Treatment with adenine, guanine, guanosine, guanine nucleotides, and uracil nucleotides protected Purkinje cells from cell death in the cerebellar primary cultures. Among the effective compounds, adenine had the most potent survival activities on Purkinje cells. Other adenine-based purines such as adenosine, AMP, ADP, and ATP did not promote Purkinje cell survival. Furthermore, metabolic inhibitors of adenine had no effect on the protective ability of adenine for Purkinje cells, suggesting that adenine itself, not adenine metabolites, maintains Purkinje cell survival. These results suggest that adenine is involved in the control of Purkinje cell survival in cerebellar primary cultures via a novel adenine-dependent mechanism.

The purpose of this work is to know the effect of surface properties of liposomes on their phagocytic uptake by macrophages. For this, liposomes were prepared by the Bangham technique from the mixture of phosphatidylcholine (PC) and cholesterol (Chol) incorporated either with phosphatidylserine (PS), phosphatidylethanolamine (PE) or phosphatidic acid (PA). The liposomes thus prepared had diameters in the range between 150 and 260 nm. Electric surface properties of the liposomes and the macrophages differentiated from HL-60RG cells were determined by measuring their electrophoretic mobilities. The phagocytic uptake of liposomes with different contents of PS, PE and PA by macrophage-like HL-60RG cells was investigated by measuring oxygen consumption associated with phagocytic uptake. The phagocytic activity was found to be the highest with the PC–Chol liposomes containing 7 mol% PS, but no significant effects were observed with PA- and PE-containing PC–Chol liposomes. As the uptake was independent of the electric surface property of liposomes, PS was concluded to be specifically important for phagocytic activity of macrophages.

1. Our previous studies revealed that the immunosuppressive agent, FTY720, mainly induces mitochondria-involved apoptosis in some types of cancer cells, since Bcl-2 overexpression prevents the FTY720-induction of apoptotic stimuli. Furthermore, FTY720 induces G0/G1 cell cycle arrest. The present study further examines the correlation between intracellular signaling kinases with FTY720-induced mitochondria-involved apoptosis. 2. Human T cell leukemia Jurkat was exposed to FTY720. Dephosphorylation of Akt occurred in a time- and concentration-dependent manner. FTY720 also induced Bad (Ser(136)) and ribosomal p70S6 kinase (p70(S6k)) (Thr(389)) dephosphorylation. 3. FTY720-induced Akt dephosphorylation was not because of Akt upstream phosphatidylinositol 3'-kinase (PI 3-kinase) pathway inhibition. 4. FTY720 also induced Akt dephosphorylation in human B cell leukemia BALL-1. BALL-1 cells were resistant to FTY720-induced apoptosis. 5. Okadaic acid (OA) inhibited the FTY720-induced dephosphorylation of Akt and p70(S6k), suggesting that FTY720 promotes Ser/Thr protein phosphatase (PP) activity. 6. OA partially inhibited FTY720-induced caspase-3 activation. 7. PP2A or PP2A-like phosphatase was temporarily activated in cells exposed to FTY720. In addition, FTY720 activated purified PP2A (ABC). 8. Overall, the results suggest that FTY720 activated PP2A or PP2A-like phosphatase and dephosphorylated Akt pathway factors resulting in the enhancement of apoptosis via mitochondria.

Our previous study revealed that rhizoxin ([1 S -[1 R *,3 R *,5 S *,8 R *(1 R *,2 S *,3 E ,5 E ,7 E ),10 R *,11 S *,13 S *,14 E ,16 S *,17 S *]]-10-hydroxy-8-[2-methoxy-1,3,7-trimethyl-8-(2-methyl-4-oxazolyl)-3,5,7-octatrienyl]-11,16-dimethyl-4,7,12,18-tetraoxatetracyclo[15.3.1.03,5.011,13]heneicos-14-ene-6,19-dione) has a potent inhibitory effect on in vivo angiogenesis. However, little is known regarding the mechanism by which rhizoxin exhibits antiangiogenic activity. In this study, we examined its effects on the functions of endothelial cells associated with neovascular formation in vivo, using cultured vascular endothelial cells. Rhizoxin concentration-dependently inhibited the proliferation of bovine carotid artery endothelial cells, human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the IC 50 values being 7, 5 and 0.4 nM, respectively. In addition, it reduced the extracellular plasminogen activator level in bovine vascular endothelial cells in the low nM range, and suppressed the migration of human dermal microvascular endothelial cells in the pM range. Furthermore, it blocked the tubular morphogenesis of human umbilical vein endothelial cells and human dermal microvascular endothelial cells on Matrigel in a concentration-dependent manner; the IC 50 values being 40 and 130 pM, respectively. These results suggest that rhizoxin exhibits antiangiogenic activity through the combined inhibition of some functions of endothelial cells responsible for induction of in vivo angiogenesis.

An ozone-assisted photocatalytic water-purification unit using a TiO2 modified titanium-mesh sheet (TMiP) was investigated. Significant decomposition of biological and chemical contaminants has been achieved by highly active intermediates formed by catalytic decomposition and photocatalysis.

A novel immunosuppressant FTY720 caused a significant decrease in peripheral T lymphocytes, but not in B lymphocytes upon oral administration. This decrease was mainly a result of FTY720-induced apoptosis. In this study, we confirmed FTY720-induced T cell selective apoptosis using lymphoma cell lines in vitro. Viability loss, DNA fragmentation, Annexin V binding, and caspases activation (caspase-3, -8, and -9) were observed in Jurkat cells (T lymphoma cells), but not significantly in BALL-1 cells (B lymphoma cells). These results indicated that FTY720 selectively induced apoptosis in T cell lymphoma to a greater extent than in B cell lymphoma, a finding that is similar to the result observed when FTY720 was treated with T lymphocytes and B lymphocytes in vitro. FTY720 released cytochrome c from mitochondria in Jurkat intact cells as well as from isolated Jurkat mitochondria directly, but not from mitochondria in BALL-1 cells nor from isolated BALL-1 mitochondria. BALL-1 cells and B cells had more abundant mitochondria-localized anti-apoptotic protein Bcl-2 than did Jurkat cells and T cells. FTY720-induced apoptosis is inhibited by the overexpression of Bcl-2, suggesting that the cellular Bcl-2 level regulates the sensitivity to FTY720. Keywords: FTY720, immunosuppressant, apoptosis, mitochondria, lymphoma cells, Bcl-2 Introduction Bcl-2 is localized mainly to the membranes of the mitochondria, and possesses anti-apoptotic activity (Krajewski et al., 1993). Bcl-2 prevents the opening of an apoptosis-related pore opening called mitochondrial permeability transition pore complex (PTPC), which is the basis for the mitochondria-mediated apoptosis pathway (Decaudin et al., 1997; Zamzami et al., 1998). This complex consists of several proteins, including hexokinase, cyclophilin D, and adenine nucleotide translocator, and a voltage-dependent anion channel (Zoratti, & Szabo, 1995). PTPC opening by pro-apoptotic members of the Bcl-2 family of proteins, e.g., Bax, or through the modulation of PTPC by chemicals results in a rapid loss of mitochondrial membrane potential (ΔΨm) and organelle swelling (Fulda et al., 1998; Narita et al., 1998; Zamzami et al., 1998). PTPC opening also releases cytochrome c. Cytochrome c, with the involvement of apaf-1, activates the initiator caspase, caspase-9, and activated caspase-9 in turn activates the effector caspase, caspase-3. Activated caspase-3 initiates further apoptotic processes (Li et al., 1997; Liu et al., 1996; Zou et al., 1997). However, the release of cytochrome c can also occur in the absence of ΔΨm loss, suggesting that PTPC may not be the only target of the Bcl-2 family proteins to release cytochrome c (Bossy-Wetzel et al., 1998; Eskes et al., 1998). In some types of apoptotic cell death, the anti-apoptotic effect of Bcl-2 is abrogated. Death receptors, e.g., Fas, aggregate and form a complex. This complex recruits through several adapter proteins and multiple procaspase-8, resulting in caspase-8 activation through induced proximity (Muzio et al., 1996; 1998). Activated caspase-8 in turn activates caspase-3, bypassing the mitochondria-mediated apoptotic pathway (Scaffidi et al., 1998). FTY720, 2-amino-2- [2-(4-octylphenyl) ethyl]-1,3-propanediol hydrochloride, was screened from synthesized analogues of ISP-1, which is an immunosuppressive metabolite of Isaria sinclairii (Fujita et al., 1994; 1996). Unlike other conventional immunosuppressants that are currently used, the action mechanism of FTY720 to suppress graft rejection is thought to be a significant decrease in the number of peripheral blood lymphocytes, especially T cells (Enosawa et al., 1996). FTY720 does not interfere with cytokine production as do cyclosporine A and FK506 (Dumont et al., 1990; Nelson, 1984), nor does it inhibit DNA biosynthesis as do azathioprine, mizoribine and RS61443 (Behrend, 1996; Koyama & Tsuji, 1983; van Furth et al., 1975), suggesting that this drug has a unique immunosuppressive action. We have previously demonstrated that the blood lymphocyte decrease was mainly a result of FTY720-induced apoptosis (Nagahara et al., 2000a). FTY720 also induces apoptosis significantly and rapidly in lymphoma cells, demonstrating a potent ability for apoptosis induction (Suzuki et al., 1996b). The FTY720-induced cell death mechanism has been studied. FTY720-induced apoptosis is observed in splenocytes from MRL lpr/lpr mice, and is blocked by Bcl-2 overexpression, demonstrating that FTY720-induced apoptosis might be involved with the mitochondria-mediated pathway (Suzuki et al., 1996b; 1997). Recently, we clarified that FTY720 directly perturbed mitochondria function, which resulted in a decrease of mitochondrial permeability transition and a release of cytochrome c from the mitochondria to cytosol, followed by the activation of caspase-3 (Nagahara et al., 2000b). Allograft rejection occurred by intragraft immune response, including the infiltration of mature T cells into the graft and the activation of these T cells by Th1-associated cytokines (Josien et al., 1995; Kupiec-Weglinski et al., 1993). Thus, modulating and investigating the apoptosis mechanisms of T cells would be greatly beneficial to the field of organ transplantation. In this study, we examined the sensitivity to FTY720-induced apoptosis using T cells and B cells in vitro. We also described how these sensitivity differences occurred using T cell and B cell lymphomas.

We have developed a suitable radiolabeling method for our new type of glycoprotein-liposome conjugate (GCL), in order to investigate its potential utility as a drug carrier that can target the cellular functions of carbohydrate-binding proteins. In order to obtain radiolabeled GCL with high labeling efficiency, we introduced p-hydroxyphenylpropyl groups into the liposome membrane through the amine moiety of a constitutive phospholipid, dipalmitoylphosphatidylethanolamine (DPPE) by using Bolton-Hunter reagent (BHR). Radioiodination of the introduced tyrosyl groups was performed by the Chloramine-T method. The labeling efficiency of the BHR-treated liposome conjugate was high in comparison with that of the BHR-untreated liposome conjugate. An in vitro inhibition study showed that the binding affinity of 125I-labeled BHR-treated GCL (125I-F3S-BH) with lectin was twice as high as that of untreated conjugate (125I-F3S). The biodistribution of 125I-F3S-BH in mice was considerably different from that of 125I-F3S. 125I-F3S-BH was more rapidly taken up by the liver and was more rapidly excreted from the liver than 125I-F3S. Moreover, 125I-F3S-BH accumulated more rapidly into the kidneys, which resulted a lower radioactivity in the blood circulation at an earlier time point than in the case of 125I-F3S. The characteristics of tumor accumulation of 125I-F3S-BH and 125I-F3S were similar to those in blood. If F3S is to be employed as an in vivo targeting ligand in biodistribution studies, BHR would be a suitable tool for radiolabeling because it allows GCL to retain the biological activity and characteristics of the unmodified conjugate.

A novel lipid compound, epolactaene, was isolated from the culture supernatant of Penicillium sp. 1689-P and it has already been reported that it induced neurite outgrowth in a human neuroblastoma cell line. In this study, we first investigated the effects of epolactaene on a human leukemia B-cell line, BALL-1 cells, and clarified that epolactaene induces apoptosis in BALL-1 cells in a dose- and time-dependent manner. Furthermore, we focused on the side chain structure of epolactaene, and chemically synthesized epolactaene derivatives. One derivative, which has a straight long alkyl chain as its side chain, induced apoptosis more effectively than epolactaene. On the other hand, other derivatives with a short alkyl side chain had weaker apoptosis-inducing actions. A good correlation was found between the apoptosis-inducing action of these compounds and their octanol/water partition coefficients (log P). These results suggested that the apoptosis-inducing activities of epolactaene and its derivatives were related to the hydrophobicity of these compounds; so that side chain structure of epolactaene is very important for its apoptosis-inducing activities. These apoptosis-inducing actions of epolactaene and its derivatives were also observed in various blood tumor cell lines and normal lymphocytes.

One of the major goals of tissue engineering is to establish an integrated organ in vivo. We have previously shown that transfection of vascular endothelial growth factor (VEGF) gene into hepatocytes promotes tissue formation by engrafted cells. Here we show that tissue growth was significantly enhanced by co-transplantation of hepatocyte growth factor (HGF) and tumor necrosis factor-alpha (TNF alpha) gene transfected hepatocytes with VEGF-gene transfected cells, but tissue islands were scattered nonspecifically in the abdomen of mice. The result brought us forward to the next step to establish an integrated mass and structural formation of liver tissue. We entrapped VEGF gene transfected hepatocytes in a nylon mesh bag and intraperitoneally engrafted close to the liver. Three weeks later, the bag was covered by a thick network of blood vessels, compared to the control. Histological examination showed that the blood vessels penetrated the parenchyma of the engrafted bag and formed a well-developed vessel network in the region. The use of hepatocytes from lacZ transgenic mice and PCR analysis demonstrated survival and albumin production by hepatocytes in the engrafted bag. Our model can potentially be developed into a heterotropic artificial liver with direct access to the host blood circulation.

The N-linked oligosaccharides were released from purified human intercellular adhesion molecule (ICAM)-3 by hydrazinolysis. Approximately 6 mol of oligosaccharides were released from 1 mol of ICAM-3. The oligosaccharides reduced with NaB[3H]4 were separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endo-glycosidase digestion and by methylation analysis revealed that N-linked oligosaccharides of ICAM-3 are mainly of tri- and tetra-antennary complex-type, about 60% of which contain two to three poly N-acetyllactosamine chains terminated with the type 1 structure and those without the type 1 structure per oligosaccharide. In addition, a small amount of the high mannose-type oligosaccharide with six alpha-mannose residues, which could act as a ligand for the dendritic cell-specific ICAM-3 grabbing nonintegrin, was detected.

The immunosuppressant FTY720 induces a drastic decrease in blood lymphocytes, especially T cells; a decrease which is assumed to be the immunosuppressive mechanism of this drug. FTY720 causes cell death in vitro in lymphocytes and leukemia cells. However, the deletion mechanism of blood lymphocytes in vivo remains unclear. We investigated whether administration of FTY720 induced lymphocyte apoptosis in blood circulation. A marked decrease in the number of blood lymphocytes was observed within an hour after a single oral administration of FTY720 at doses of 5–10 mg/kg in rats and mice. Experiments using fluorescein isothiocyanate (FITC)-Annexin V and APO-BRDU™ methods revealed that FTY720 induced blood lymphocyte apoptosis in a dose-dependent manner. On the other hand, lymphocyte homing to Peyer's patches was proposed as the mechanism underlying the blood lymphocyte decrease at these doses. However, similar results were obtained when using aly / aly mice, which lack Peyer's patches and lymph nodes. Thus, we concluded that apoptosis of blood lymphocytes was induced immediately after administration of FTY720, and the cells could be immediately scavenged by phagocytes or the reticuloendothelial system in addition to Peyer's patches homing. We also concluded that T cells were highly sensitive to FTY720, which induced apoptosis in vivo.

Complementary DNA encoding mouse alpha-1,6-fucosyltransferase (FUT) was cloned. The deduced primary structure consisted of 575 amino acids and had 96.0% and 93.0% identity with alpha-1,6-FUT of human and porcine origin, respectively. Quantitative analysis of alpha-1,6-FUT mRNA expression during selected developmental stages of the cerebrum showed that the expression increased during later embryonic stages and was highest in the early postnatal stages (P1 to P7), after which it declined somewhat but still remained relatively high in the mature adult. The expression profile suggests important roles of FUT in the developing central nervous system.

The changes in Asn-linked oligosaccharide composition in the murine cerebrum during development have been examined by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). The oligosaccharides, obtained from murine cerebrum in several developmental stages, were separated by HPLC on anion-exchange and reverse-phase columns. We found that two Asn-linked oligosaccharides, designated oligosaccharide I and oligosaccharide II, had their expression changed during postnatal development. Whereas oligosaccharide I was reduced during brain development, oligosaccharide II was increased. The structures of oligosaccharides I and II were analyzed by ESI-MS and sequential exoglycosidase digestions. Judging from the molecular and fragment ions in each oligosaccharide, the oligosaccharide I was composed of 5Hex+2HexNAc+ABOE (MW 1467.2) and the oligosaccharide II was 3Hex+4HexNAc+DoHex+ABOE (MW 1695.2). The results of sequential exoglycosidase digestion indicated that the oligosaccharide I was an oligomannose type saccharide and the oligosaccharide II was a biantennary complex type saccharide including fucose. The proposed structures are shown below. These results offer an important clue to the role of Asn-linked oligosaccharides associated with development of the central nervous system. Download : Download full-size image

Cancer cells often develop drug resistance. In cisplatin-resistant HeLa cisR cells, fibroblast growth factor 13 (FGF13/FHF2) gene and protein expression was strongly upregulated and intracellular platinum concentrations were kept low. When the FGF13 expression was suppressed, both the cells' resistance to platinum drugs and their ability to keep intracellular platinum low were abolished. Overexpression of FGF13 in parent cells led to greater resistance to cisplatin and reductions in the intracellular platinum concentration. These cisplatin-resistant cells also showed increased resistance to copper. In preoperative cervical cancer biopsy samples from poor prognoses patients after cisplatin chemoradiotherapy, FGF13-positive cells were detected more abundantly than in the biopsy samples from patients with good prognoses. These results suggest that FGF13 plays a pivotal role in mediating resistance to platinum drugs, possibly via a mechanism shared by platinum and copper. Our results point to FGF13 as a novel target and useful prognostic guide for cancer therapy.

Autophagy is a self-proteolysis process in eukaryotic cells that results in the sequestering of intracellular proteins and organelles in autophagosomes. Activation of autophagy progress continued growth of some tumors, instead extensive autophagy induces cell death. In a previous study, we synthesized a novel tamoxifen derivative, Ridaifen (RID)-B. RID-B induced mitochondria-involved apoptosis even in estrogen receptor (ER)-negative cells. Since tamoxifen induces autophagy other than apoptosis, we treated ER-negative Jurkat cells with RID-B in the present study. RID-B treatment induced apoptosis and LC3 and lysosome colocalization, which results in the formation of autolysosomes. Western blotting revealed that LC3 was converted to LC3-I to LC3-II with RID-B treatment, suggesting that RID-B induced autophagy without ER involvement. Moreover, overexpression of the anti-apoptotic protein Bcl-2 suppressed the RID-B-induced cell death, but not the induction of autophagy. These results presumed that RID-B-induced autophagy is independent of Bcl-2, making RID-B-induced autophagy different from RID-B-induced apoptosis. Since Beclin 1 level is unchanged during RID-B treatment, RID-B induced autophagy pathway is Bcl-2/Beclin1 independent noncanonical pathway.