Sinomenine hydrochloride enhancement of the inhibitory effects of anti-transferrin receptor antibody-dependent on the COX-2 pathway in human hepatoma cells

Transferrin receptor (TfR) has been used as a target for the antibody-based therapy of cancer due to its higher expression in tumors relative to normal tissues. Great potential has been shown by anti-TfR antibodies combined with chemotherapeutic drugs as a possible cancer therapeutic strategy. In our study, we investigated the anti-tumor effects of anti-TfR monoclonal antibody (mAb) alone or in combination with sinomenine hydrochloride in vitro. Results suggested that anti-TfR mAb or sinomenine hydrochloride could induce apoptosis, inhibit proliferation, and affect the cell cycle. A synergistic effect was found in relation to tumor growth inhibition and the induction of apoptosis when anti-TfR mAb and sinomenine hydrochloride were used simultaneously. The expression of COX-2 and VEGF protein in HepG2 cells treated with anti-TfR mAb alone was increased in line with increasing dosage of the agent. In contrast, COX-2 expression was dramatically decreased in HepG2 cells treated with sinomenine hydrochloride alone. Furthermore, we demonstrated that the inhibitory effects of sinomenine hydrochloride and anti-TfR mAb administered in combination were more prominent than when the agents were administered singly. To sum up, these results showed that the combined use of sinomenine hydrochloride and anti-TfR mAb may exert synergistic inhibitory effects on human hepatoma HepG2 cells in a COX-2-dependent manner. This finding provides new insight into how tumor cells overcome the interference of iron intake to survive and forms the basis of a new therapeutic strategy involving the development of anti-TfR mAb combined with sinomenine hydrochloride for liver cancer.     

DNA methylation and MeCP2 regulation of PTCH1 expression during rats hepatic fibrosis

Hepatic stellate cell (HSC) activation plays an important role in liver fibrogenesis. Transdifferentiation of quiescent hepatic stellate cells into myofibroblastic-HSCs is a key event in liver fibrosis. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. MeCP2 binds to methylated CpG dinucleotides, which are abundant in the promoters of many genes. Treatment of HSCs with DNA methylation inhibitor 5-aza-2′- deoxycytidine (5-azadC) prevented proliferation and activation. Treatment with 5-azadC prevented loss of Patched (PTCH1) expression that occurred during HSCs activation. In a search for underlying molecular medchanisms, we investigated whether the targeting of epigenetic silencing mechanisms could be useful in the treatment of PTCH1-associated fibrogenesis. It was indicated that hypermethylation of PTCH1 is associated with the perpetuation of fibroblast activation and fibrosis in the liver. siRNA knockdown of MeCP2 increased the expressions of PTCH1 mRNA and protein in hepatic myofibroblasts. These data suggest that DNA methylation and MeCP2 may provide molecular mechanisms for silencing of PTCH1.     

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K⁺ channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1–4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12–17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19–21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.     

Development of a novel recombinant strain Zygosacharomyces rouxii JL2011 for 1,3-propanediol production from glucose

1,3-propanediol (1,3-PDO) is one of the most important industrial chemicals due to its highly desired properties and its wide applications as a key component of the emerging polymer industry. Biotechnology route has been one of the most interesting methods for 1,3-PDO production, whereas, the dha genes were essential to 1,3-PDO biosynthesis. In this study, we cloned and placed the dha cassettes under the control of a glyceraldehyde 3-phosphate dehydrogenase gene promoter pGAP and homologous ZrFPS1 gene promoter pZrfps1; these two promoters were further integrated into the chromosome of Z. rouxii JL2011 to generate recombinant strain JL2011-GZ and JL2011-ZZ, respectively. The results showed that the two strains could produce 1,3-PDO from glucose with a final yield of 6.9 and 10.3 g/l, respectively. The engineered strain JL2011-ZZ showed a 2.3- and 1.5-fold increase in the specific activities and final concentration of 1,3-PDO, respectively, with respect to JL2011-GZ. Batch fermentation with aerobic/micro-aerobic combined strategy of JL2011-ZZ resulted a titer of 17.1 g/l and a yield from glucose of 8.6 %. These results demonstrated that JL2011-ZZ would be a potential strain for 1,3-PDO production from glucose.     

Hydrogen sulfide protects SH-SY5Y neuronal cells against d-galactose induced cell injury by suppression of advanced glycation end products formation and oxidative stress

d-Galactose is widely used as an agent to cause aging effects in experimental animals. The present study aims to investigate the effects of hydrogen sulfide (H₂S) in human neuroblastoma SH-SY5Y cells exposed to d-galactose. Cells were pretreated with NaHS, an H₂S donor, and then exposed to d-galactose (25–400 mM for 48 h). We found that NaHS pretreatment significantly reversed the d-galactose-induced cell death and cellular senescence. MTT assay shows that NaHS significantly increased cell viability from 62.31 ± 1.29% to 72.34 ± 0.46% compared with d-galactose (200 mM) treatment group. The underlying mechanism appeared to involve a reduction by NaHS in the formation of advanced glycation end products (AGEs), which are known to contribute to the progression of age-related diseases. In addition, NaHS decreased the elevation of reactive oxygen species from 151.17 ± 2.07% to 124.8 ± 2.89% and malondialdehyde from 1.72 ± 0.07 to 1.10 ± 0.08 (nmol/mg protein) in SH-SY5Y cells after d-galactose exposure. NaHS also stimulated activities of superoxide dismutase from 0.42 ± 0.05 to 0.73 ± 0.04 (U/mg protein) and glutathione peroxidase from 3.98 ± 0.73 to 14.73 ± 0.77 (nmol/min/mg protein) and upregulated the gene expression levels of copper transport protein ATOX1, glutathione synthetase (GSS) and thioredoxin reductase 1 (TXNRD1) while down-regulated aldehyde oxidase 1 (AOX1). In summary, our data indicate that H₂S may have potentially anti-aging effects through the inhibition of AGEs formation and reduction of oxidative stress.     

Dynamic expression of miR-126 and its effects on proliferation and contraction of hepatic stellate cells

In our previous study, miR-126 was identified as one of the leading miRNAs that is downregulated during activation of hepatic stellate cells (HSCs). However, the roles and related mechanisms of miR-126 in HSCs are not understood. In this study, we compared expression of miR-126 during HSC activation both in vitro and in vivo. We also applied RNA interference to analyze the role and mechanism of miR-126^∗ in the activation of HSCs. Restoring HSCs with Lv-miR-126^∗ resulted in decreased proliferation, accumulation of extracellular matrix components, and cell contraction, while also negatively regulating the vascular endothelial growth factor (VEGF) signal transduction pathways by partially targeted VEGF-A. Thus, we postulate that miR-126 may be a biological marker for the activation of HSCs, and useful for reducing intrahepatic vascular resistance and improving the sinusoidal microcirculation in chronic liver diseases.     

Myostatin induces mitochondrial metabolic alteration and typical apoptosis in cancer cells

Myostatin, a member of the transforming growth factor-β superfamily, regulates the glucose metabolism of muscle cells, while dysregulated myostatin activity is associated with a number of metabolic disorders, including muscle cachexia, obesity and type II diabetes. We observed that myostatin induced significant mitochondrial metabolic alterations and prolonged exposure of myostatin induced mitochondria-dependent apoptosis in cancer cells addicted to glycolysis. To address the underlying mechanism, we found that the protein levels of Hexokinase II (HKII) and voltage-dependent anion channel 1 (VDAC1), two key regulators of glucose metabolisms as well as metabolic stress-induced apoptosis, were negatively correlated. In particular, VDAC1 was dramatically upregulated in cells that are sensitive to myostatin treatment whereas HKII was downregulated and dissociated from mitochondria. Myostatin promoted the translocation of Bax from cytosol to mitochondria, and knockdown of VDAC1 inhibited myostatin-induced Bax translocation and apoptosis. These apoptotic changes can be partially rescued by repletion of ATP, or by ectopic expression of HKII, suggesting that perturbation of mitochondrial metabolism is causally linked with subsequent apoptosis. Our findings reveal novel function of myostatin in regulating mitochondrial metabolism and apoptosis in cancer cells.     

MicroRNA Cluster miR-17-92 Regulates Neural Stem Cell Expansion and Transition to Intermediate Progenitors in the Developing Mouse Neocortex

1. Download : Download full-size image

     

Methylation State of the EDA Gene Promoter in Chinese X-Linked Hypohidrotic Ectodermal Dysplasia Carriers

Introduction

Hypodontia, hypohidrosis, sparse hair and characteristic faces are the main characters of X-linked hypohidrotic ectodermal dysplasia (XLHED) which is caused by genetic ectodysplasin A (EDA) deficiency. Heterozygous female carriers tend to have mild to moderate XLHED phenotype, even though 30% of them present no obvious symptom.

Methods

A large Chinese XLHED family was reported and the entire coding region and exon–intron boundaries of EDA gene were sequenced. To elucidate the mechanism for carriers’ tempered phenotype, we analyzed the methylation level on four sites of the promoter of EDA by the pyrosequencing system.

Results

A known frameshift mutation (c.573–574 insT) was found in this pedigree. Combined with the pedigrees we reported before, 120 samples comprised of 23 carrier females from 11 families and 97 healthy females were analyzed for the methylation state of EDA promoter. Within 95% confidence interval (CI), 18 (78.26%) carriers were hypermethylated at these 4 sites.

Conclusion

Chinese XLHED carriers often have a hypermethylated EDA promoter.