RajiおよびK562細胞に対するナマズ卵レクチンと抗腫瘍薬との併用効果

Silurus asotus(catfish)lectin(SAL)is a 32 kDa protein belonging to the rhamnose-binding lectin(RBL)family. SAL also recognizes galactosylα-linked carbohydrate chains of not only glycoproteins but also glycosphingolipids, especially globotriaosylceramide(Gb3). in the previous study, we revealed that SAL increased uptake of doxorubicin(Dox)into Raji cells, and consequently enhanced cytotoxic effect of Dox. we studied whether SAL also enhances the cytotoxic effect of vinblastine and irinotecan in Gb3-expressing Raji or Gb3-negative K562 cells. Treatment of Raji cells with vinblastine(2.5μg/mL)and irinotecan(5μM)for 48 h caused decrease of cell viability to 69% and 68%, respectively. although cytotoxic effects of vinblastine and irinotecan were significantly increased in SAL-pretreated Raji cells, only a slight increase was observed in SAL-pretreated K562 cells with vinblastine. These results indicate that SAL enhances the cytotoxic effect of vinblastine or irinotecan in raji but not K562 cells

レスベラトロール処理細胞における抗腫瘍薬の効果

Chemo-resistance to anti-cancer drugs is a major obstacle in efforts to develop a successful treatment of cancer.Resveratrol(RSV)facilitates the cellular uptake of doxorubicin through an induced down-regulation of multidrug resistance -associated protein-1(MRP1)expression.We studied whether RSV enhances the cytotoxic effect of vinblastine,vincristine,irinotecan,and etoposide in MRP1-expressing Burkitt\u27s lymphoma Raji cells.Treatment with RSV caused decrease of cell viability in a time-and dose-dependent manner,and down-regulation of MRP1 expression in Raji cells.The cytotoxic effects of vinblastine,vincristine,and etoposide increased in RSV-pretreated Raji cells,but the effect of irinotecan did not change in the cells.These results indicate that RSV enhances the cellular uptake of vinblastine,and etoposide in Raji cell

ナマズ卵レクチンのクラスター形成と細胞内輸送

Rhamnose-binding lectins are widely found in fush eggs,and Silurus asotus lectin (SAL) isolated from catfish eggs having three carbohydrate recognition domains preferentially recognizes non-reducing end Galα-linked sugar chain.In the previous study,we revealed that SRL binds to globotriaosylceramide (Gb3) by the surface plasmon resonance analysis.However,its biological effect on cultured cells is still unclear.To investigate localization and trafficking of SAL in the renel adenocarcinoma ACHN cells,which express Gb3 on the cell surface,we prepared HiLyte Fluor 555-labeled SAL (HL-SAL).When ACHN cells were treated with HL-SAL at 4℃ for 5min, and at 37℃ for 24h,HL-SAL was distributed on the cell membrane and in the intracellular compartment,respectively.To trace the trafficking route of HL-SAL from cell surface to the intracellular compartment,the images of HL-SAL-treated live cells were obtained using confocal scanning microscopy.HL-SAL was clustered on ACHN cell surface,and furthermore,partially co-localised with transferrin in intracellular compartment.These results suggest that SAL induces alteration of Gb3 distribution on the membrane and migrates from the cell surface to the intracellular vesicles

キアンコウ(Lophius litulon)におけるレクチン活性の探索

Fish eggs or roe often contain rhamnose-binding lectins (RBLs) that have unique properties among animal lectins in the carbohydrate recognition and domain structure. To investigate new lectin activity in deep-sea fishes,we attempted to extract proteins from eggs (GFE) and liver (GFL) of goose fish,Lophius litulon. The crude saline-extracted fraction (SEF) was obtained from acetone-dried powders of GFE and GFL.Each SEF was separated by DEAE-ion exchange chromatography. Contrary to our expectation,any DEAE fractions had no hemaggulatination activity on rabbit erythrocytes. On the other hand,S-180 mouse ascites tumor cells were strongly aggutinated by GFL D100,D200 and SC fractions with the final concentrations required minimum agglutination,65.0, 98.75 and 112.5ug/mL,respectively. Whereas any typical ligand molecules including carbohydrates and glycoconjugates showed no inhibitory effect for such tumor cell agglutination,several protease inhibitors including phenylmethanesufonyl fluoride and EDTA could do that. agglutination was caused by their proteolytic activity on the tumor cell surface components.The results from additional separation by hydroxyapatite column chromatography,SDS-PAGE and zymography indicated that putative candidates possessing proteolytic activity might be ca. 100 or 45 kDa proteins

First-line pembrolizumab vs chemotherapy in metastatic non-small-cell lung cancer: KEYNOTE-024 Japan subset

This prespecified subanalysis of the global, randomized controlled phase Ill KEYNOTE-024 study of pembrolizumab vs chemotherapy in previously untreated metastatic non-small-cell lung cancer without EGFR/ALK alterations and a programmed death-ligand 1 (PD-L1) tumor proportion score of 50% or greater evaluated clinical outcomes among patients enrolled in Japan. Treatment consisted of pembrolizumab 200 mg every 3 weeks (35 cycles) or platinum-based chemotherapy (four to six cycles). The primary end-point was progression-free survival; secondary end-points included overall survival and safety. Of 305 patients randomized in KEYNOTE-024 overall, 40 patients were enrolled in Japan (all received treatment: pembrolizumab, n = 21; chemotherapy, n = 19). The hazard ratio (HR) for progression-free survival by independent central review (data cut-off date, 10 July 2017) was 0.25 (95% confidence interval [CI], 0.10-0.64; one-sided, nominal P = .001). The HR for overall survival (data cut-off date, 15 February 2019) was 0.39 (95% CI, 0.17-0.91; one-sided, nominal P = .012). Treatment-related adverse events occurred in 21/21 (100%) pembrolizumab-treated and 18/19 (95%) chemotherapy-treated patients; eight patients (38%) and nine patients (47%), respectively, had grade 3-5 events. Immune-mediated adverse events and infusion reactions occurred in 11 patients (52%) and four patients (21%), respectively; four patients (19%) and one patient (5%), respectively, had grade 3-5 events. Consistent with results from KEYNOTE-024 overall, first-line pembrolizumab improved progression-free survival and overall survival vs chemotherapy with manageable safety among Japanese patients with metastatic non-small-cell lung cancer without EGFRIALK alterations and a PD-L1 tumor proportion score of 50% or greater

First-line pembrolizumab vs chemotherapy in metastatic non-small-cell lung cancer: KEYNOTE-024 Japan subset

This prespecified subanalysis of the global, randomized controlled phase III KEYNOTE‐024 study of pembrolizumab vs chemotherapy in previously untreated metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a programmed death ligand 1 (PD‐L1) tumor proportion score of 50% or higher evaluated clinical outcomes among patients enrolled in Japan. Treatment consisted of pembrolizumab 200 mg every 3 weeks (35 cycles) or platinum‐based chemotherapy (four to six cycles). The primary end‐point was progression‐free survival; secondary end‐points included overall survival and safety. Of 305 patients randomized in KEYNOTE‐024 overall, 40 patients were enrolled in Japan (all received treatment: pembrolizumab, n = 21; chemotherapy, n = 19). Median progression‐free survival was 41.4 (95% confidence interval [CI], 4.2‐42.5) months with pembrolizumab and 4.1 (95% CI, 2.8‐8.3) months with chemotherapy (hazard ratio [HR], 0.27 [95% CI, 0.11‐0.65]; one‐sided, nominal P = .001). Median overall survival was not reached (NR) (95% CI, 22.9‒NR) and 21.5 (95% CI, 5.2‐35.0) months, respectively (HR, 0.39 [95% CI, 0.17‐0.91]; one‐sided, nominal P = .012). Treatment‐related adverse events occurred in 21/21 (100%) pembrolizumab‐treated and 18/19 (95%) chemotherapy‐treated patients; eight patients (38%) and nine patients (47%), respectively, had grade 3‐5 events. Immune‐mediated adverse events and infusion reactions occurred in 11 pembrolizumab‐treated patients (52%) and four chemotherapy‐treated patients (21%), respectively; four patients (19%) and one patient (5%), respectively, had grade 3‐5 events. Consistent with results from KEYNOTE‐024 overall, first‐line pembrolizumab improved progression‐free survival and overall survival vs chemotherapy with manageable safety among Japanese patients with metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a PD‐L1 tumor proportion score of 50% or higher. The trial is registered with Clinicaltrials.gov: NCT02142738

Glycosphingolipid-enriched microdomain中でGb3と会合する分子の探索

Silurus asotus (catfish) egg lectin (SAL) is a member of rhamnose-binding lectin family, and binds to globotriaosylceramide (Gb3) localized in a glycosphingolipid-enriched microdomain (GEM) on several tumor cell lines. It is known that GEM is involved in cell adhesion and has the capability of sending signals. We report here about proteins involved in GEM associating with Gb3 and mediating signal transduction system in Burkitt\u27s lymphoma Raji cells via SAL binding to Gb3. Although Gb3 molecule was sprinkled over the surface of Raji cells, SAL uniformly bound to the Raji cell surface. Sucrose density-gradient centrifugation method and fluorescence microscopic analysis revealed that not only CD20 and CD81 but also Lyn and Csk were distributed in GEM of Raji cells. The distribution of these molecules was not altered by the treatment with SAL at 4℃ for 30 ruin. Because CD20 was not co-precipitated with the anti-Gb3 antibody, CD20 was not associated with Gb3 on Raji cells. However, there was a 47kDa protein, which was specifically co-precipitated with anti-Gb3 antibody. These results suggest that the 47kDa protein is a candidate for Gb3-associating protein in Raji cells

イイダコ(Octopus ocellatus)卵由来の赤血球凝集素について

Hemagglutinating activity was found in extract from Octopus ocellatus eggs. The hemagglutinin was partially purified by ammonium sulfate precipitation and DEAE anion exchange chromatography. DT300, a 300 mM NaCl-eluted fraction from DEAE column, possessed Ca^-dependent hemagglutinating activity on rabbit erythrocytes at concentrations from 0.47 to 30 μg/mL. Protein bands over 200 kDa were observed in DT300 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. On the other hand, ruthenium red staining gave an obvious single band on a cellulose acetate membrane. The protein and carbohydrate contents of DT300 were 24% and 2%, respectively. DT300-induced hemagglutination was inhibited by fetuin, asialofetuin, heparin, and DNA but not by heparan sulfate and mono- or oligosaccharides tested. This activity was resistant to heat and protease digestion. These results indicate that the active component having C-type lectin-like activity from octopus eggs may not be composed of protein