Inhibition of HAS2 induction enhances the radiosensitivity of cancer cells via persistent DNA damage

Hyaluronan synthase 2 (HAS2), a synthetic enzyme for hyaluronan, regulates various aspects of cancer progression, including migration, invasion and angiogenesis. However, the possible association of HAS2 with the response of cancer cells to anticancer radiotherapy, has not yet been elucidated. Here, we show that HAS2 knockdown potentiates irradiation-induced DNA damage and apoptosis in cancer cells. Upon exposure to radiation, all of the tested human cancer cell lines exhibited marked (up to 10-fold) up-regulation of HAS2 within 24 h. Inhibition of HAS2 induction significantly reduced the survival of irradiated radioresistant and -sensitive cells. Interestingly, HAS2 depletion rendered the cells to sustain irradiation-induced DNA damage, thereby leading to an increase of apoptotic death. These findings indicate that HAS2 knockdown sensitizes cancer cells to radiation via persistent DNA damage, further suggesting that the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. Thus, HAS2 could potentially be targeted for therapeutic interventions aimed at radiosensitizing cancer cells.     

HDAC6 controls autophagosome maturation essential for ubiquitin‐selective quality‐control autophagy

Autophagy is primarily considered a non‐selective degradation process induced by starvation. Nutrient‐independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin‐binding deacetylase, histone deacetylase‐6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin‐dependent, actin‐remodelling machinery, which in turn assembles an F‐actin network that stimulates autophagosome–lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build‐up, and neurodegeneration. Remarkably, HDAC6 and F‐actin assembly are completely dispensable for starvation‐induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome–lysosome fusion.     

Purification and biochemical characterization of a 17 kDa fibrinolytic enzyme from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods, including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.     

A novel nucleotide kinase encoded by gene 1.7 of bacteriophage T7

Gene 1.7 of bacteriophage T7 confers sensitivity of both phage T7 and its host Escherichia coli to dideoxythymidine (ddT). We have purified the product of gene 1.7, gp1.7. It exists in two forms of molecular weight 22 181 and 17 782. Only the C‐terminal half of the protein is required to confer ddT sensitivity. We show that gp1.7 catalyses the phosphorylation of dGMP and dTMP to dGDP and dTDP, respectively, by using either GTP, dGTP or dTTP as the phosphate donor. Either form of gp1.7 exhibit identical kinase activity as compared with wild‐type gp1.7 that contains a mixture of both forms. The K_m of 70 µM and Kcat of 4.3 s^?1 for dTMP are similar to those found for E. coli thymidylate kinase. However, unlike the host enzyme, gp1.7 efficiently catalyses the conversion of the chain‐terminating dideoxythymidylate (ddTMP) to ddTDP. This finding explains the sensitivity of phage T7 but not E. coli to exogenous ddT. Gp1.7 is unusual in that it has no sequence homology to any known nucleotide kinase, it has no identifiable nucleotide‐binding motif and its activity is independent of added metal ions. When coupled with nucleoside diphosphate kinase, gp1.7 exponentially converts dTMP to dTTP.     

Synthesis of aminoquinazoline derivatives and their antiproliferative activities against melanoma cell line

The synthesis of a novel series of aminoquinazoline derivatives 1a–r and their antiproliferative activities against A375 human melanoma cell line were described. Among them, six compounds showed superior antiproliferative activities to Sorafenib as a reference compound. In particular, the representative compound 1q bearing chromen-4-one moiety exhibited excellent antiproliferative activity (IC₅₀ = 0.006 μM) and good selectivity over HS27 fibroblast cell line.     

Asymmetric synthesis of (S)-ethyl-4-chloro-3-hydroxy butanoate using a Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme–substrate docking studies

Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced (S)-ECHB exclusively.     

Inhibition of P‐Glycoprotein‐Induced Multidrug Resistance by a Clerodane‐Type Diterpenoid from Sindora sumatrana

The aim of the present study was to investigate the effects of di‐ and sesquiterpenoids isolated from the pods of Sindora sumatrana Miq. (Leguminosae) on P‐glycoprotein (P‐gp) function in an adriamycin‐resistant human breast cancer cell line, MCF‐7/ADR. Over‐expression of P‐gp is known to be one of the mechanisms involved in multidrug resistance (MDR), which is a major obstacle in clinical cancer treatment. Among six di‐ and sesquiterpenoids extracted from S. sumatrana, (+)‐7β‐acetoxy‐15,16‐epoxycleroda‐3,13(16),14‐trien‐18‐oic acid ( 1 ) showed a strong P‐gp inhibitory effect, as great as that of verapamil, a representative P‐gp inhibitor. Compound 1 enhanced daunomycin accumulation more than fourfold and significantly decreased daunomycin efflux compared with control, resulting in a decrease in the IC₅₀ value for daunomycin. These results suggest that compound 1 inhibits the functioning of P‐gp and, therefore, can be developed as an MDR‐reversing agent.     

Differential expression of skeletal muscle proteins in high‐fat diet‐fed rats in response to capsaicin feeding

In this study, the effects of capsaicin on expression of skeletal muscle proteins in Sprague–Dawley rats fed with a high‐fat diet (HFD) were investigated. Rats were fed a HFD with or without capsaicin treatment for 8 wk. After HFD feeding, capsaicin‐treated rats weighed an average of 8% less than those of the HFD control group. Gastrocnemius muscle tissue from lean and obese rats with or without capsaicin treatment was arrayed using 2‐DE for detection of HFD‐associated markers. Proteomic analysis using 2‐DE demonstrated that 36 spots from a total of approximately 600 matched spots showed significantly different expression; 27 spots were identified as gastrocnemius muscle proteins that had been altered in response to capsaicin feeding, and 6 spots could not be identified by mass fingerprinting. Expression of various muscle proteins was determined by immunoblot analysis for the determination of molecular mechanisms, whereby capsaicin caused inhibition of adipogenesis. Immunoblot analysis revealed increased uncoupling protein 3 (UCP3) protein expression in HFD‐fed rats, whereas contents were reduced with capsaicin treatment. Compared with the HFD control group, capsaicin treatment increased phosphorylation of AMP‐activated protein kinase (AMPIC) CP3 and acetyl‐CoA carboxylase (ACC). To support this result, we also analyzed in vitro differential protein expression in L6 skeletal muscle cells. These data suggest that the AMPK‐ACC‐malonyl‐CoA metabolic signaling pathway is one of the targets of capsaicin action. To the best of our knowledge, this is the first proteomic study to report on analysis of diet‐induced alterations of protein expression that are essential for energy expenditure in rat muscle.