The kinase inhibitor sorafenib induces cell death through a process involving induction of endoplasmic reticulum stress

Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1alpha markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1alpha or XBP1, disruption of PERK activity, or inhibition of eIF2alpha phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib.     

Complete Genome Sequence and Genome Analysis of Eggplant mottled dwarf virus‐Iranian Isolate

The full‐length nucleotide sequence of the Iranian isolate of Eggplant mottled dwarf virus (EMDV), a phytorhabdovirus, was determined using the random polymerase chain reaction method (rPCR) followed by PCR with specific primers to fill in the gaps. The negative‐sense RNA genome of the Iranian isolate of EMDV contains 13154 nucleotides and seven open‐reading frames (ORFs) in the order 3′‐leader‐N‐X‐P‐Y‐M‐G‐L‐trailer‐5′. These ORFs encode the nucleocapsid, X protein (of unknown function), phosphoprotein, Y protein (putative movement protein), matrix protein, glycoprotein and RNA‐dependent RNA polymerase, respectively. EMDV has a 199 nt 3′ leader RNA and a 151 nt 5′ trailer, and the ORFs are separated by conserved intergenic sequences. Phylogenetic analyses indicate that EMDV is most closely related to Potato yellow dwarf virus, which has a distinctly different geographical distribution.     

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha‐amylase: a comparative study on bacterial,fungal, and mammalian enzymes

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha‐amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha‐amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha‐amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha‐amylase surface in domain B. This domain shows differences in various alpha‐amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

Structural features of pectic polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits

After removal of the mucilage with water at room temperature, pectic polysaccharides were solubilized from Opuntia ficus-indica fruit skin, by sequential extraction with water at 60 degrees C (WSP) and EDTA solution at 60 degrees C (CSP). Polysaccharides with neutral sugar content of 0.48 and 0.36 mol/mol galacturonic acid residue were obtained, respectively, in the WSP and CSP extracts. These pectic polysaccharides were de-esterified and fractionated by anion-exchange chromatography, yielding for each extract five fractions, which were thereafter purified by size-exclusion chromatography. Two of these purified fractions were characterized by sugar analysis combined with methylation and reduction-methylation analysis. The study was then supported by (1)H and (13)C NMR spectroscopy. The results showed that the water-soluble fraction WSP3 and the EDTA soluble fraction CSP3, consisted of a disaccharide repeating unit -->2)-alpha-l-Rhap-(1-->4)-alpha-d-GalpA-(1--> backbone, with side chains attached to O-4 of the rhamnosyl residues. The side chains contained highly branched alpha-(1-->5)-linked arabinan and short linear beta-(1-->4)-linked galactan.     

High expression of IMPACT protein promotes resistance to indoleamine 2,3‐dioxygenase‐induced cell death

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO‐induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non‐derepressible‐2(GCN2) kinase stress‐responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO‐induced low tryptophan environment. IDO‐expressing human fibroblasts were co‐cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO‐expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO‐induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co‐cultured with IDO‐expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT‐expressing vector, was rescued significantly in tryptophan‐deficient but not IDO‐expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO‐mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO‐generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non‐immune cells such as skin cells acts as a protective mechanism against IDO‐induced GCN2 activation, therefore, makes them resistant to the amino acid‐deprived environment caused by IDO. J. Cell. Physiol. 225: 196–205, 2010. © 2010 Wiley‐Liss, Inc.     

Fibrillation of α‐lactalbumin: Effect of crocin and safranal,two natural small molecules from Crocus sativus

Formation of toxic amyloid structures is believed to be associated with various late‐onset neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The fact that many proteins in addition to those that are associated with clinical conditions have the potential to form amyloid fibrils in vitro provides opportunities for studying the fundamentals of protein aggregation and amyloid formation in model systems. Accordingly, considerable interest and effort has been directed toward developing small molecules to inhibit the formation of fibrillar assemblies and their associated toxicities. In the present study, we investigated the inhibitory effect of crocin and safranal, two principal components of saffron, on fibrillation of apo‐α‐lactalbumin (a‐α‐LA), used as a model protein, under amyloidogenic conditions. In the absence of any ligand, formation of soluble oligomers became evident after 18 h of incubation, followed by subsequent appearance of mature fibrils. Upon incubation with crocin or safranal, while transition phase to monomeric beta structures was not significantly affected, formation of soluble oligomers and following fibrillar assemblies were inhibited. While both safranal and crocin had the ability to bind to hydrophobic patches provided in the intermediate structures, and thereby inhibit protein aggregation, crocin was found more effective, possibly due to its simultaneous hydrophobic and hydrophilic character. Cell viability assay indicated that crocin could diminish toxicity while safranal act in reverse order. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 854–865, 2010.     

R-carrying genotypes of serum paraoxonase (PON1) 192 polymorphism and higher activity ratio are related to susceptibility against ischemic stroke

The polymorphic gene of serum paraoxonase (PON1) and its activity involved in atherosclerosis. The purpose of the study was to analyze PON1 192 Q/R polymorphism and the enzyme activities in ischemic stroke. The polymorphism as the most common polymorphism in PON1 gene coding sequence is associated with variation in the enzyme activity and vascular disease. The study included 85 stroke patients and 71 control subjects. PON1 192 polymorphism was genotyped using PCR protocol. Paraoxonase activity (Para) and arylesterase activity (Aryl) were determined spectrophotometrically using paraoxon and phenylacetate as the substrates. The QR and RR genotypes were more frequent in stroke population compared to controls, resulting in a higher frequency of the R allele in patients (0.24 vs 0.18, OR?=?1.41). Patients had significantly higher Para/Aryl ratio than that of controls (P?=?0.016). In stroke patients, Para/Aryl and Para/HDL ratios increased with this order: QQ?<?QR?<?RR. Hypertension significantly increased the risk of ischemic stroke by 15-fold among R-containing people, while this was significantly increased 4-fold for QQ homozygotes. Smoking increased the risk of having ischemic stroke in both QQ homozygote and QR?+?RR group (OR?=?2.84 and OR?=?2.33, respectively). In conclusion, these data highlight the importance of PON1 192 R allele and high Para/Aryl ratio in susceptibility to ischemic stroke in the population. The presence of the 192 R allele potentiates the risk of stroke especially in hypertensive people. Decreased Aryl and increased Para/Aryl, Para/HDL and Aryl/HDL ratios may be markers indicated the increased susceptibility to ischemic stroke in the population.     

A new sesquiterpenoid from Sclerorhachis platyrachis (Boiss.) Podlech ex Rech.f

A new eudesmane-type sesquiterpenoid together with two known flavonoids were isolated from the chloroform extract of the aerial part of Sclerorhachis platyrachis. The structure of the new compound was deduced from its comprehensive spectroscopic analysis including IR, EI-MS, ¹H NMR, ¹³C NMR, DEPT, COSY, HMBC and HMQC and was shown to be 4R^*-hydroxy-6S^*-tigloyloxyeudesma-7S^*-11 (13)-en-12-oic acid (1). Finally, the structure of the new compound was unambiguously confirmed by single-crystal X-ray analysis. The structure of known compounds 2 and 3 were identified by comparison of their spectral data with those reported in the literature.