Ascorbic Acid Promotes Arsenic-induced Cytotoxicity in Human Hepatocarcinoma Cells and Their Underlying Mechanisms

Objective: To study synergistic effect with Ascorbic acid(AA) on arsenic trioxide inducing human Hepatocarcinoma cell apoptosis, and provide theoretical basis for promoting human Hepatocarcinoma cell apoptosis induced by arsenic trioxide(AT). Methods: Human Hepatocarcinoma cell line BEL-7402 being cultured in vitro, the effect of AT and (or) AA on its growth inhibition and its two intracellular signal molecules was evaluated separately using MTT and Western blot. Results: AT at a few μmol/L concentration could suppress abnormal proliferation of human hepatocarcinoma cells, and initiate their apoptosis by activation of caspase-3, and activate extracellular-signal regulated kinases (ERKs), which were dependent on the dosage of AT conspicuously. The effect of AA on BEL-7402 was not significant; However, AA could effectively enhance AT-induced hepatocarcinoma cell apoptosis and lesion severity through activation of caspase-3 but not ERKs. Conclusion: Caspase-3 and ERKs proteins could involve in arsenic-induced hepatocarcinoma cell apoptosis and differentiation respectively as intracellular signaling molecules; The effect between AT and AA on hepatocarcinoma is synergistic, which further inhibits cell growth and induces apoptosis in human hepatocarcinoma cells through activation of caspase-3 but not ERKs.

Ohmefentanyl stereoisomers induce changes of CREB phosphorylation in hippocampus of mice in conditioned place preference paradigm

The present study was designed to determine the changes of phosphorylation of cAMP- response ele-ment binding protein (CREB) in hippocampus induced by ohmefentanyl stereoisomers (F9202 and F9204)in conditioned place preference (CPP) paradigm. The results showed that mice receiving F9202 and F9204displayed obvious CPP. They could all significantly stimulate CREB phosphorylation and maintained for along time without affecting total CREB protein levels. The effect of F9204 was similar to morphine whicheffect was more potent and longer than F9202. We also examined the effects of ketamine, a noncompetitiveN-mthyl-D-aspartate receptor (NR) antagonist, on morphine-, F9202- and F9204- induced CPP and phos-phorylation of CREB in hippocampus. Ketamine could suppress not only the place preference but also thephosphorylation of CREB produced by morphine, F9202 and F9204. These findings suggest that alterationsin the phosphorylation of CREB be relevant to opiates signaling and the development of opiates dependence.NR antagonists may interfere with opiates dependence and may have potential therapeutic implications.

Bile acids:Chemistry,physiology,and pathophysiology

The family of bile acids includes a group of molecular species of acidic steroids with very peculiar physical-chemical and biological characteristics.They are synthesized by the liver from cholesterol through several complementary pathways that are controlled by mechanisms involving finetuning by the levels of certain bile acid species.Although their best-known role is their participation in the digestion and absorption of fat,they also play an important role in several other physiological processes.Thus,genetic abnormalities accounting for alterations in their synthesis,biotransformation and/or transport may result in severe alterations,even leading to lethal situations for which the sole therapeutic option may be liver transplantation.Moreover,the increased levels of bile acids reached during cholestatic liver diseases are known to induce oxidative stress and apoptosis,resulting in damage to the liver parenchyma and,eventually,extrahepatic tissues.When this occurs during pregnancy,the outcome of gestation may be challenged.In contrast,the physical-chemical and biological properties of these compounds have been used as the bases for the development of drugs and as pharmaceutical tools for the delivery of active agents.

The mitochondrial Na^＋/Ca^2＋ exchanger may reduce high glucose-induced oxidative stress and nucleotide-binding oligomerization domain receptor 3 inflammasome activation in endothelial cells

Background The mitochondrial Na^＋/Ca^2＋ exchanger, NCLX, plays an important role in the balance between Ca2. influx and efflux across the mitochondrial inner membrane in endothelial ceils. Mitochondrial metabolism is likely to be affected by the activity of NCLX because Ca^2＋ activates several enzymes of the Krebs cycle. It is currently believed that mitochondria are not only centers of energy produc- tion but are also important sites of reactive oxygen species （ROS） generation and nucleotide-binding oligomerization domain receptor 3 （NLRP3） inflammasome activation. Methods ＆ Results This study focused on NCLX function, in rat aortic endothelial cells （RAECs）, induced by glucose. First, we detected an increase in NCLX expression in the endothelia of rats with diabetes mellitus, which was induced by an injection of streptozotocin. Next, colocalization of NCLX expression and mitochondria was detected using confocal analysis. Suppression of NCLX expression, using an siRNA construct （siNCLX）, enhanced mitochondrial Ca^2＋ influx and blocked efflux induced by glucose. Unexpectedly, silencing of NCLX expression induced increased ROS generation and NLRP3 inflammasome activation. Conclusions These findings suggest that NCLX affects glucose-dependent mitochondrial Ca^2＋ signaling, thereby regulating ROS generation and NLRP3 in- flammasome activation in high glucose conditions. In the early stages of high glucose stimulation, NCLX expression increases to compensate in order to self-protect mitochondrial maintenance, stability, and function in endothelial cells.

Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gabl without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3（N） or the regulatory Y221 residue of CrkII, is critical for the induction of Gabl- Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gabl. In GST pull-down assay, Crk-SH2 bound to wild-type Gahl, whereas Crk-SH3（N） interacted with the Gabl mutant, which lacks the clus- tered tyrosine region （residues 242-410）. Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gabl-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory- lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

TNF-α induces apoptosis of Molt-4 cells and cell cycle specificity of Bcl-2 phosphorylation

Objective： The aim of the study was to observe the expression of Bcl-2 and its phosphorylation in Molt-4 cells induced by tumor necrosis factor-α （TNF-α）, and to investigate the possible mechanism of cell cycle specificity of apoptosis. Methods： Exponentially growing Molt-4 cells were treated with TNF-α. Apoptosis was detected by DNA fragmentation assay. API method was applied to illustrate the cell cycle specificity of apoptotic cells. Cells of sub-phases were sorted by FACSvan- tage flow cytometer and then submitted to immunoblot. Results： Molt-4 cells which were treated with TNF-α went to apoptosis and showed a DNA ladder pattern. Most apoptosis happened in Gl-phase of cell cycle. Bcl-2 expression increased for the Molt-4 cells treated with TNF-α. The phosphorylation state of Bcl-2 was only presented in Gl-phase cells, in accordance with the specified time and cell cycle phase of apoptosis. Conclusion： The phosphorylation of Bcl-2 in the Molt-4 cells treated with TNF-α happened with the same cell cycle specificity as cell apoptosis. The cell cycle specificity of Bcl-2 phosphorylation was one of the mechanisms of receptor-mediated apoptosis. The cell cycle machine can trigger the apoptosis program.

Role of Acetylated p53 in Regulating the Expression of map2 in Retinoic Acid-induced P19 Cells

Objective To investigate the regulatory mechanisms of acetylated p53 in the expression of microtubule-associated protein-2(MAP2) in neuronal differentiation of P19 cells induced by all-trans retinoic acid(RA).Methods Neuronal differentiation of P19 cells was initiated with 4-day RA treatment.Immunofluorescence,real-time reverse transcription-polymerase chain reaction(RT-PCR) assay,and map2 promoter driven luciferase assay were performed to detect the expression and relative promoter activity of MAP2 in those RA-treated cells.Real-time PCR-based chromatin immunoprecipitation assay(ChIP) was carried out to reveal the specific recruitment of acetylated p53 onto its binding sites on map2 promoter.Results The expression of MAP2 was markedly increased in RA-induced P19 cells.The map2 mRNA increased 34-fold after 4 days of RA treatment and 730-fold 2 days after the treatment,compared with the cells without RA treatment(control).p53 was recruited to the promoter of map2 gene in acetylated form and thereby enhanced its promoter activity.p300/CBP associated factor(PCAF) was found induced in RA-treated cells and enriched in the nucleus,which might contribute to the acetylation of p53 in the regulation of map2 gene.Conclusions Acetylated p53 may participate in regulating the expression of map2 in RA-induced differentiation of P19 cells.PCAF is possibly involved in this process by mediating the acetylation of p53.

Coronatine Induces an Accumulation of Anthocyanin and Starch in Purple-fleshed Sweetpotato(Ipomoea batatas Lam.)

[Objective] The objective of this research was to examine the effects of COR on anthocyanin and starch content in storage roots of two PFS genotypes, and to explore the relationships between anthocyanin synthesis and starch accumula- tion. [Method] A field experiment was carried out to determine the changes in yielc components, yield, contents of anthocyanin and starch, activities of phenylalanine ammonia-lyase （PAL） and adenosine 5-diphosphate glucose pyrophosphorylase （AG- Pase） in two genotypes of PFS （Ipomoea batatas L., var. ＇Ayamurasaki＇ and ＇Jishu18＇）. [Result] The application of COR significantly increased starch and antho- cyanin content in storage roots of Jishu18 across developmental stages by inducing the activities of PAL and AGPase, and finally enhanced yield by promoting fresh weight of storage roots. Ayamurasaki was insensitive to treatment with COR al- though its PAL activity temporally increased. The starch and anthocyanin content of Aya, and the anthocyanin content of Jishu18 increased progressively across devel- opmental stages with or without COR application, but the starch content of Jishu18 increased initially, then decreased before increasing again without application of COR. Treatment with COR reduced downward trend of starch accumulation in Jishu18. Thus, the effect of COR on accumulation of anthocyanin and starch in storage roots of PFS differs according to genotypes. [Conclusion] The application of 0.05 μmol/L COR may increase starch and anthocyanin content in PFS genotypes with lower starch and anthocyanin content in storage roots.

All-trans Retinoic Acid Induced the Differentiation of Human Glioma Cells

OBJECTIVE To observe the effect of all-trans retinoic acid （ATRA） on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms.METHODS The expression of glial fibrillary acidic protein （GFAP） was detected by immunocytochemistry staining. The mRNA levels of GFAP, retinoid X receptor α（RXRα）, p21 were examined by semi-quantitative RT-PCR analysis. Luciferase activity assay was performed in the COS-7, MO59K cells to measure p21 promoter transcription activity.RESULTS ATRA could significantly enhance the expression and mRNA level of GFAP by immunostaining and RT-PCR （P〈0.05）. Simultaneously, the mRNA levels of RXRα and p21 were remarkably increased in dose-dependent manner by RT-PCR （P〈0.05）. Furthermore, luciferase assay confirmed that ATRA and RXRα could transactivate p21 promoter in COS-7 and glioma cells （P〈0.05）.CONCLUSION ATRA can induce differentiation of human glioma cells. The RXRα and p21 were activated during ATRAinduced differentiation process. This effect may be caused by directly RXRα-induced p21 gene transactivation. Our findings provide novel evidence for the future studies to explore the molecular mechanism of transcriptional regulation for glioma cell differentiation and cellular therapeutic approaches for glioblastoma.

Astilbic Acid Induced COLO 205 cell Apoptosis by Regulating Bcl-2 and Bax Expression and Activating Caspase-3

To investigate the effect of astilbic acid (3β,6β-dihydroxyolean-12-en-27-oic acid, AA) on human colorectal carcinoma COLO 205 cell proliferation and apoptosis.Methods Proliferation of COLO 205 cells was measued by MTT assay. Content of DNA in COLO 205 cell was measued by modified diphenylamine assay. AA-induced morphological changes was observed with fluorescence microscope and transmission electron microscope.DNA fragmentation was visualized by agarose gel electrophoresis.Apoptosis rate and cell cycle distribution were deter-mined by flow cytometric analysis.Expressions of Bcl-2 and Bax proteins were visioned by immunohistochemical analysis.The change of relative mitochondral transmembrane potential (MTP) in COLO 205 cell was analyzed with FCM after rhodamine 123 staining. Results The IC50 (96h) of AA for inhibiting COLO 205 cell proliferation was 61.56±0.34 μmol/L.AA induced a marked concentration- and time-dependent inhibition of COLO 205 cell proliferation and reduced the DNA content in COLO 205 cell. Cells treated with AA 64 μmol/L showed typical morphological changes of apoptosis and DNA “ladder” pattern. The cell cycle was arrested in G0/G1 phase, and the apoptosis rate was 28.25% for COLO 205 cells treated with AA 64 μmol/L for 48h. Meanwhile the expression of Bcl-2 protein was decreased while that of Bax was increased and relative MTP was decreased as well. DEVD-CHO 1μmol/L could increase the viability of COLO 205 cells treated with AA for 48h.Conclusion AA showed potent inhibitory activity on COLO 205 cells proliferation,and could induce COLO 205 cells apoptosis through disturbing DNA replication, down-regulating Bcl-2 expression, and up-regulating Bax expression, lowering relative MTP, and activating caspase-3 pathway.

Synthesis of aziridines by visible-light induced decarboxylative cyclization of N-aryl glycines and diazo compounds

A visible-light induced decarboxylative aza-Darzens reaction between N-aryl glycines and diazo compounds was developed, which affords various mono-substituted aziridines in good yields.

Photoinduced directional domain sliding motion in peptide hydrogels promotes ectodermal differentiation of embryonic stem cells

Mechanical cues present in the stem cell niche resulting from intracellular processes or external force sources significantly affect the basic functions of stem cells such as self-renewal and differentiation.Creation of artificial cellular matrices exhibiting intrinsic mechanical cues generated by mechanical movements remains scarce.Herein,we reported on mechanically dynamic hydrogel matrices undergoing photo-induced directional domain sliding movement and their role in regulating embryonic stem cell(ESC)differentiation.The mechanically dynamic hydrogels were prepared via the self-assembly of an alternating hydrophilic and hydrophobic peptide with a photocaged cysteine residue.Upon light irradiation,the assemblies of the caged peptide were converted to non-equilibrated non-caged peptide bilayers that underwent the directional domain sliding motion induced by the thermodynamically favorable hydrophobic collapse transition.Culturing murine ESCs on the mechanically dynamic hydrogels resulted in biased differentiation toward the ectodermal lineage.We further showed that the mechanically dynamic hydrogels stimulated the translocation of a mechanotransduction protein Yes-associated protein(YAP)into the nucleus,implicating a potential mechanotransduction mechanism for the biased differentiation of ESCs.The finding of the biased ectodermal differentiation of ESCs induced by the mechanically dynamic hydrogels implies the great potency of the mechanically dynamic hydrogels as biomaterials for disease therapy and tissue regeneration in the future.