Synthesis of 3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside (ODSG) as a lipase-resistant SQAP derivative.

Sulfoquynovosylacyl propanediol (SQAP; 1) has been developed as a radiosensitizer (anti-cancer agent) for solid tumors, but it was easily cleaved in vivo and had a problem of short residence time. We synthesized a novel compound of a SQAP derivative (3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside: ODSG; 2) to solve these problems not easily cleaved by lipase. ODSG (2) cytotoxicity was investigated in vitro, resulting in low toxicity like SQAP (1).

Epo-C12 inhibits peroxiredoxin 1 peroxidase activity.

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.

SQAP, an acyl sulfoquinovosyl derivative, suppresses expression of histone deacetylase and induces cell death of cancer cells under hypoxic conditions.

Sulfoglycolipid, SQAP, is a radiosensitizing agent that makes tumor cells more sensitive to radiation therapy. A previous study revealed that SQAP induced the degradation of hypoxia-inducible factor-1α (HIF-1α) and inhibited angiogenesis in a hepatoma model mouse. Herein, we examined the biological activities of SQAP against hepatocarcinoma cells under low oxygen conditions. Cell growth inhibition of SQAP under hypoxic conditions was significantly higher than that under normoxic conditions. In addition, SQAP was found to impair the expression of histone deacetylase (HDAC) under low oxygen conditions. Our present data suggested that SQAP induced the degradation of HIF-1α and then decreased the expression of HDAC1. Unlike known HDAC inhibitors, SQAP increased the acetylation level of histone in cells without inhibition of enzymatic activity of HDACs. Our data demonstrated hypoxia-specific unique properties of SQAP.

Distribution and metabolism of 14C-sulfoquinovosylacylpropanediol (14C-SQAP) after a single intravenous administration in tumor-bearing mice.

Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits angiogenesis in vivo and results in increased oxigenation and reduced tumor volume. We investigated the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a human pulmonary carcinoma, Lu65. For the metabolism analysis, a 2 mg (2.98 MBq)/kg of [glucose-U-14C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body. The degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-14C]-SQAP (CP-3635) was administered through intravenous injection of a 1 mg (3.48 MBq)/kg dose followed by whole body autoradiography of the mice. The autoradiography analysis demonstrated that SQAP rapidly distributed throughout the whole body and then quickly decreased within 4 hours except the tumor and excretion organs such as liver, kidney. Retention of SQAP was longer in tumor parts than in other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the tumor had also completely disappeared within 72 hours.

Promotion of crystalline cellulose degradation by expansins from Oryza sativa.

MAIN CONCLUSION: Enzymatic activities of Oryza sativa expansins, which were heterologously overexpressed in Escherichia coli , were analyzed. Results suggested that expansins promote degradation of cellulose by cellulase in a synergistic manner. Sustainable production of future biofuels is dependent on efficient saccharification of lignocelluloses. Expansins have received a lot of attention as proteins promoting biological degradation of cellulose using cellulase. The expansins are a class of plant cell wall proteins that induce cell wall loosening without hydrolysis. In this study, the expansins from Oryza sativa were classified using phylogenetic analysis and five proteins were selected for functional evaluation. At low cellulose loading, the cellulase in expansin mixtures was up to 2.4 times more active than in mixtures containing only cellulase, but at high cellulose loading the activity of cellulase in expansin mixtures and cellulase only mixtures did not differ. Furthermore, expansin activity was greater in cellulase mixtures compared with cellulase-deficient mixtures. Therefore, the expansins showed significant synergistic activity with cellulase. Expansin may play an important role in efficient saccharification of cellulose.

Sulfoquinovosylacylpropanediol is a novel potent radiosensitizer in prostate cancer.

OBJECTIVES: To examine the effects of combined treatment with sulfoquinovosylacylpropanediol and X-ray irradiation on the remodeling of the prostate cancer microenvironment, including angiogenic and hypoxic characteristics. METHODS: Human prostate cancer cells (DU145 and PC3) were implanted subcutaneously into the right hind legs of athymic nude mice. After the tumor volume reached 100-300mm(3) , 2mg/kg/day sulfoquinovosylacylpropanediol was given intravenously from day0 to day4, and cells were exposed to 4Gy X-ray irradiation on days0 and 3 (for a total of 8Gy). Tumors were fixed and stained for pathological analyses and immunohistochemical evaluations. To analyze vascular normalization, 60mg/kg pimonidazole dissolved in saline was injected intraperitoneally. RESULTS: Combined treatment with sulfoquinovosylacylpropanediol plus X-ray irradiation enhanced growth inhibition in DU145 xenografts. The tumor vessel density in DU145 cells significantly decreased after the combined treatment. Staining for αsmooth muscle actin in vessels was significantly increased. Pimonidazole staining, showing hypoxic lesions, was negative from 72h, but positive at 6 and 24h after the first combined treatment. In contrast, no enhancement of the microenvironment in PC3 xenografts was observed with sulfoquinovosylacylpropanediol plus X-ray irradiation. CONCLUSION: Sulfoquinovosylacylpropanediol could be a novel potent radiosensitizing agent targeting angiogenesis in prostate cancer.

A silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose functions as an efficient photosensitizer for photodynamic therapy.

We synthesized a new silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose (SGlc). Conjugation with SGlc improved A549 cellular uptake without significant changes in the photophysical and photochemical properties and subcellular localization. This improved cellular uptake led to enhanced photodynamic activity. Furthermore, conjugation with SGlc suppressed dark toxicity. These advantages were not observed for a conjugate with a glucose molecule. These results indicated that the conjugation with SGlc is a promising strategy for enhancing photodynamic efficacy.

Applications of biomaterials to liquid crystals.

Nowadays, chemically synthesized proteins and peptides are attractive building blocks and have potential in many important applications as biomaterials. In this review, applications of biomaterials to thermotropic liquid crystals are discussed. The review covers the improvement of the performance of liquid crystal displays using liquid crystal physical gels consisting of a liquid crystal and amino acid-based gelators, and also new functionalization of liquid crystals. Moreover, the influence of DNA, which is one of the more attractive biomaterials, dispersed in thermotropic liquid crystals and its potential use in the liquid crystal industry is described. In addition, we found interesting results during electrooptical measurements of liquid crystals doped with DNA, and explain them from the point of view of biological applications. These recent approaches suggest that these biomaterials may be applicable in the electronic device industry and should be considered as an interesting material with their physical properties having the potential to create or refine an industrial product.

Anti-angiogenesis effect of 3'-sulfoquinovosyl-1'-monoacylglycerol via upregulation of thrombospondin 1.

We previously reported that 3'-sulfoquinovosyl-1'-monoacylglycerol (SQMG) effectively suppresses the growth of solid tumors, likely via its anti-angiogenic activity. To investigate how SQMG affects angiogenesis, we performed DNA microarray analysis and quantitative real-time polymerase chain reaction. Consequently, upregulation of thrombospondin 1 (TSP-1) in SQMG-treated tumors in vitro and in vivo was confirmed. To address the mechanisms of TSP-1 upregulation by SQMG, we established stable TSP-1-knockdown transformants (TSP1-KT) by short hairpin RNA induction and performed reporter assay and in vivo assessment of anti-tumor assay. On the reporter assay, transcriptional upregulation of TSP-1 in TSP1-KT could not be induced by SQMG, thus suggesting that TSP-1 upregulation by SQMG occurred via TSP-1 molecule. In addition, growth of TSP1-KT xenografted tumors in vivo was not inhibited by SQMG, thus suggesting that anti-angiogenesis via TSP-1 upregulation induced by SQMG did not occur, as the SQMG target molecule TSP-1 was knocked down in TSP1-KT transformants. These data provide that SQMG is a promising candidate for the treatment of tumor-induced angiogenesis via TSP-1 upregulation.

The antitumor agent doxorubicin binds to Fanconi anemia group F protein.

Doxorubicin, a commonly used cancer chemotherapy agent, elicits several potent biological effects, including synergistic-antitumor activity in combination with cisplatin. However, the mechanism of this synergism remains obscure. Here, we employed an improved T7 phage display screening method to identify Fanconi anemia group F protein (FANCF) as a doxorubicin-binding protein. The FANCF-doxorubicin interaction was confirmed by pull-down assay and SPR analysis. FANCF is a component of the Fanconi anemia complex, which monoubiquitinates D2 protein of Fanconi anemia group as a cellular response against DNA cross-linkers such as cisplatin. We observed that the monoubiquitination was inhibited by doxorubicin treatment.

Exploration of the binding proteins of perfluorooctane sulfonate by a T7 phage display screen.

Perfluorooctane sulfonate (PFOS) is a pollutant widely found throughout nature and is toxic to animals. We created a PFOS analogue on a polyethylene glycol polyacrylamide copolymer and isolated peptides that preferentially bound the PFOS analogue using a T7 phage display system. Bioinformatic analysis using the FASTAskan program on the RELIC bioinformatics server showed several human proteins that likely bound PFOS. Among them, we confirmed binding between PFOS and a recombinant soluble form of monocyte differentiation antigen CD14 (sCD14) by a surface plasmon biosensor. Furthermore, PFOS inhibited TNF-α production induced by the sCD14 in mouse macrophage RAW264.7 cells.

The natural sulfoglycolipid derivative SQAP improves the therapeutic efficacy of tissue factor-targeted radioimmunotherapy in the stroma-rich pancreatic cancer model BxPC-3.

α-Sulfoquinovosylacyl-1,3-propanediol (SQAP) is a semi-synthetic derivative of natural sulfoglycolipid that sensitizes tumors to external-beam radiotherapy. How SQAP affects internal radiotherapy, however, is not known. Here, we investigated the effects of SQAP for radioimmunotherapy (RIT) targeting tissue factor (TF) in a stroma-rich refractory pancreatic cancer mouse model, BxPC-3. A low dose of SQAP (2 mg/kg) increased tumor uptake of the 111In-labeled anti-TF antibody 1849, indicating increased tumor perfusion. The addition of SQAP enhanced the growth-inhibitory effect of 90Y-labeled 1849 without leading to severe body weight changes, allowing for the dose of 90Y-labeled 1849 to be reduced to half that when used alone. Histologic analysis revealed few necrotic and apoptotic cells, but Ki-67-positive proliferating cells and increased vascular formation were detected. These results suggest that the addition of a low dose of SQAP may improve the therapeutic efficacy of TF-targeted RIT by increasing tumor perfusion, even for stroma-rich refractory pancreatic cancer.

Anticancer agent α-sulfoquinovosyl-acylpropanediol enhances the radiosensitivity of human malignant mesothelioma in nude mouse models.

Malignant pleural mesothelioma (MPM) is a highly malignant disease that develops after asbestos exposure. Although the number of MPM cases is predicted to increase, no effective standard therapies have been established. The novel radiosensitizer α-sulfoquinovosyl-acylpropanediol (SQAP) enhances the effects of γ-radiation in human lung and prostate cancer cell lines and in animal models. In this study, we explored the radiosensitizing effect of SQAP and its mechanisms in MPM. The human MPM cell lines MSTO-211H and MESO-4 were implanted subcutaneously into the backs and thoracic cavities of immunodeficient KSN/Slc mice, then 2 mg/kg SQAP was intravenously administered with or without irradiation with a total body dose of 8 Gy. In both the orthotopic and ectopic xenograft murine models, the combination of irradiation plus SQAP delayed the implanted human MSTO-211H tumor growth. The analysis of the changes in the relative tumor volume of the MSTO-211H indicated a statistically significant difference after 8 Gy total body combined with 2 mg/kg SQAP, compared to both the untreated control (P = 0.0127) and the radiation treatment alone (P = 0.0171). After the treatment in each case, immunostaining of the harvested tumors revealed decreased cell proliferation, increased apoptosis and normalization of tumor blood vessels in the SQAP- and irradiation-treated group. Furthermore, hypoxia-inducible factor (HIF) 1 mRNA and protein expression were decreased, indicating reoxygenation in this group. In conclusion, SQAP improved hypoxic conditions in tumor tissue and may elicit a radiosensitizing effect in malignant mesothelioma models.

Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins.

While mammalian DNA polymerase ß (Pol ß), which is a member of the Pol X family, play important roles in base excision repair (BER) that efficiently removes DNA base lesions arising from both endogenous and exogenous agents, this protein has been found only a subset of animals. To understand natural evolution of this enzyme, we isolated and characterized Pol ß from jellyfish Aurelia sp.1. (AsPol ß). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol ß were very similar to those of a mammalian counterpart. We also searched two other homologs of mammalian genes that were involved in short patch (sp) BER in the nucleotide sequence database, and found that both of these homologs were encoded in the genomes of a lineage from Cnidarians through mammals and Arthropods. This study suggests that a DNA repair mechanism resembling mammalian sp-BER may be largely limited to a subset of animals. On the basis of our findings and previous reports, we discuss possible evolutional model of Pol ß and the other members of the Pol X family.

Foam fractionation of protein: correlation of protein adsorption onto bubbles with a pH-induced conformational transition.

A foam fractionation apparatus was prepared to aid protein separation at the gas-liquid interface. Using lysozyme as a model protein, we investigated the alteration of enzymatic and optical activities through foaming. The lysozyme transferred to the gaseous nitrogen phase after 5 min of bubbling with no exogenous detergent. The bacteriolytic and optical activities of lysozyme from the foamate were nearly equivalent to those of the original lysozyme. This result indicated that lysozyme did not irreversibly denature during foam fractionation. We then performed protein separation using binary mixtures of lysozyme and α-amylase. When the two proteins were dissolved in bulk solution of pH 10.5, which is close to the isoelectric point (pI) of lysozyme (10.7), selective fractionation of lysozyme from the foam was observed. Indeed, this fractionation was identical to that from a single component solution of lysozyme. Similarly, selective fractionation of α-amylase was achieved in pH 3.0 buffer. Furthermore, circular dichroism (CD) and subsequent model fitting revealed that the protein had a reduced or nearly complete absence of α-helical content, whereas the amount of ß-sheet structure and random coil was elevated in the buffer conditions that promoted protein adsorption. These results indicate that a pH-induced conformational transition might correlate with protein foaming.

Fluorescence kinetics in HeLa cells after treatment with cell cycle arrest inducers visualized with Fucci (fluorescent ubiquitination-based cell cycle indicator).

Fucci (fluorescent ubiquitination-based cell cycle indicator) is able to visualize dynamics of cell cycle progression in live cells; G1- and S-/G2-/M-phase cells expressing Fucci emit red and green fluorescence, respectively. This system could be applied to cell kinetic analysis of tumour cells in the field of cancer therapy; however, it is still unclear how fluorescence kinetics change after various treatments, including exposure to anticancer agents. To explore this, we arrested live HeLa cells expressing the Fucci probes at various cell cycle stages and observed the fluorescence, in conjunction with flow cytometric analysis. X-irradiation, HU (hydroxyurea) and nocodazole arrest cells at G2/M boundary, early S-phase and early M-phase, respectively. Although X-irradiation and HU treatment induced similar accumulation kinetics of green fluorescent cells, nocodazole treatment induced an abnormal red fluorescence at M phase, followed by accumulation of both red and green fluorescent cells with 4N DNA content. We conclude that certain agents that disrupt normal cell cycle regulation could cause unexpected fluorescence kinetics in the Fucci system.

Transformation of thiols to disulfides by epolactaene and its derivatives.

In this paper we report a disulfide formation of thiols induced by epolactaene and its derivatives. We previously reported the disulfide formation of N-acetylcysteine methyl ester by epolactaene in a 1:1 MeOH/0.5M NaHCO(3) aq solution. The present studies reveal that the disulfide formation proceeds under mild conditions such as in PBS at pH 7.3, suggesting that epolactaene may induce disulfide formation of cellular thiols. This compound induces the disulfide formation of several thiols in a 1:1 MeOH/0.5M NaHCO(3) aq solution at room temperature. Moreover, our results show that the acyl side-chain of epolactaene greatly influences the products of the reaction. We analyzed the reaction mechanism by using thiolysis products of epolactaene derivatives and propose a new reaction mechanism.

Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site.

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix.

Camptothecin (CPT) directly binds to human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and inhibits the hnRNP A1/topoisomerase I interaction.

Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a K(D) value of 82.7 nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (K(D): 260 nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5-50 µM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster.

A screening of a library of T7 phage-displayed peptide identifies E2F-4 as an etoposide-binding protein.

Etoposide (VP-16) is an anti-tumor compound that targets topoisomerase II (top II). In this study, we have identified an alternative binding protein of etoposide by screening a library of T7 phage-displayed peptides. After four rounds of selection using a biotinylated etoposide derivative immobilized on a streptavidin-coated plate, T7 phage particles that display a 16-mer peptide NSSASSRGNSSSNSVY (ETBP16) or a 10-mer NSLRKYSKLK (ETBP10) were enriched with the ratio of 40 or 11 out of the 69 clones, respectively. Binding of etoposide to these peptides was confirmed by surface plasmon resonance (SPR) analysis, which showed ETBP16 and ETBP10 to have a kinetic constant of 4.85 × 10⁻5 M or 6.45 × 10⁻5 M, respectively. ETBP16 displays similarity with the ser-rich domain in E2F-4, a transcription factor in cell cycle-regulated genes, suggesting that etoposide might interact with E2F-4 via this domain. SPR analysis confirmed the specific binding of etoposide to recombinant E2F-4 is in the order of 10⁻5 M. Furthermore, etoposide was shown to inhibit luciferase reporter gene expression mediated by the heterodimeric E2F-4/DP complex. Taken together, our results suggest that etoposide directly binds to E2F-4 and inhibits subsequent gene transcription mediated by heterodimeric E2F-4/DP complexes in the nucleus.