Saffron and natural carotenoids: Biochemical activities and anti-tumor effects

Saffron, a spice derived from the flower of Crocus sativus, is rich in carotenoids. Two main natural carotenoids of saffron, crocin and crocetin, are responsible for its color. Preclinical studies have shown that dietary intake of some carotenoids have potent anti-tumor effects both in vitro and in vivo, suggesting their potential preventive and/or therapeutic roles in several tissues. The reports represent that the use of carotenoids without the potential for conversion to vitamin A may provide further protection and avoid toxicity. The mechanisms underlying cancer chemo-preventive activities of carotenoids include modulation of carcinogen metabolism, regulation of cell growth and cell cycle progression, inhibition of cell proliferation, anti-oxidant activity, immune modulation, enhancement of cell differentiation, stimulation of cell-to-cell gap junction communication, apoptosis and retinoid-dependent signaling. Taken together, different hypotheses for the antitumor actions of saffron and its components have been proposed such as a) the inhibitory effect on cellular DNA and RNA synthesis, but not on protein synthesis; b) the inhibitory effect on free radical chain reactions; c) the metabolic conversion of naturally occurring carotenoids to retinoids; d) the interaction of carotenoids with topoisomerase II, an enzyme involved in cellular DNA-protein interaction. Furthermore, the immunomodulatory activity of saffron was studied on driving toward Th1 and Th2 limbs of the immune system. In this mini-review, we briefly describe biochemical and immunological activities and chemo-preventive properties of saffron and natural carotenoids as an anticancer drug.     

Posttranscriptional regulation of angiotensin II type 1 receptor expression by glyceraldehyde 3-phosphate dehydrogenase

Regulation of angiotensin II type 1 receptor (AT1R) has a pathophysiological role in hypertension, atherosclerosis and heart failure. We started from an observation that the 3′-untranslated region (3′-UTR) of AT1R mRNA suppressed AT1R translation. Using affinity purification for the separation of 3′-UTR-binding proteins and mass spectrometry for their identification, we describe glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an AT1R 3′-UTR-binding protein. RNA electrophoretic mobility shift analysis with purified GAPDH further demonstrated a direct interaction with the 3′-UTR while GAPDH immunoprecipitation confirmed this interaction with endogenous AT1R mRNA. GAPDH-binding site was mapped to 1–100 of 3′-UTR. GAPDH-bound target mRNAs were identified by expression array hybridization. Analysis of secondary structures shared among GAPDH targets led to the identification of a RNA motif rich in adenines and uracils. Silencing of GAPDH increased the expression of both endogenous and transfected AT1R. Similarly, a decrease in GAPDH expression by H₂O₂ led to an increased level of AT1R expression. Consistent with GAPDH having a central role in H₂O₂-mediated AT1R regulation, both the deletion of GAPDH-binding site and GAPDH overexpression attenuated the effect of H₂O₂ on AT1R mRNA. Taken together, GAPDH is a translational suppressor of AT1R and mediates the effect of H₂O₂ on AT1R mRNA.     

beta cell cytoprotective strategies: establishing the relative roles for iNOS and ROS

Cytokine-induced beta cell destruction may be mediated by the generation of nitric oxide and/or reactive oxygen species. The relative importance of NO and ROS in cytokine-induced beta cell pathophysiology remains unclear. This investigation evaluates and contrasts the cytoprotective potential of antioxidant gene transfer, versus NF-kappaB inhibition, using a degradation-resistant mutant of IkappaBalpha. NF-kappaB inhibition conferred significant protection against cytokine-induced damage whereas antioxidant overexpression failed to provide protection. Conferred cytoprotection was associated with a suppression of iNOS activation and nitrite accumulation. Our data implicates iNOS, as opposed to ROS, as the pivotal player in cytokine-induced beta cell damage. From a therapeutic standpoint, strategies aimed at targeting the activation of iNOS may harbor therapeutic potential in preserving beta cell survival in the face of proinflammatory cytokine exposure.     

Disruption of ShcA signaling halts cell proliferation--characterization of ShcC residues that influence signaling pathways using yeast

Shc adapter proteins are thought to regulate cellular proliferation, differentiation and apoptosis by activating the SOS-Grb2-RAS-MAPK signaling cascade. Using the small hairpin RNA (shRNA) technique, we found that decreasing ShcA mRNA reduced the proliferative ability of HEK293 mammalian culture cells. We then recapitulated phosphorylation-dependent Shc-Grb2 complex formation in Saccharomyces cerevisiae. Immunoprecipitation followed by Western analysis demonstrated that activated TrkB, composed of the intracellular domain of TrkB fused to glutathione S-transferase (GST-TrkB(ICD)), promoted the association of ShcC and Grb2 in yeast. The Ras-recruitment system (RRS), in which a myristoylated (Myr)-bait and son of sevenless (hSOS)-prey are brought together to complement the defective Ras-cAMP pathway in a thermosensitive cdc25H mutant yeast strain, was used to validate a phenotypic assay. Yeast cells transformed with both Myr-ShcC and hSOS-Grb2 (referred to as scheme 1) or Myr-Grb2 and hSOS-ShcC (scheme 2) did not grow at non-permissive temperature; the additional transformation of GST-TrkB(ICD) enabled growth. GST-TrkB(ICD) also enabled growth with hSOS-Grb2 and either Myr-ShcA or Myr-SHP2. Mutational analysis of TrkB showed that its kinase activity was essential for complementation, while its docking site for Shc proteins was not. Mutational analysis of ShcC showed that the PTB and SH2 domains were not essential for complementation but phosphorylation at Y304 in the CH1 domain was. Phosphorylation at Y304 could not be substituted by an acidic amino acid. The RRS provides a genetic system to probe Shc proteins and potentially identify member specific protein partners and pharmacological reagents.     

Dexamethasone inhibits apoptosis in C6 glioma cells through increased expression of Bcl-XL

The glucocorticoid dexamethasone (Dex) has been reported to modulate a number of signaling pathways and physiological processes, including apoptosis. This study was carried out to investigate the cytoprotective mechanism of Dex in C6 glioma cells. Pre-treatment of cells with Dex inhibited apoptosis induced by staurosporine, etoposide and thapsigargin. Apoptosis inhibition correlated with blockade of mitochondrial cytochrome c release, abolition of caspase-3 activity along with inhibition of caspase-9 and PARP cleavage. Dex-mediated cytoprotection coincided with the induction of the anti-apoptotic protein, Bcl-X_L. The specific glucocorticoid receptor antagonist, RU486, reversed the anti-apoptotic effect of Dex and prevented Bcl-X_L induction. Here, we show for the first time that knockdown of Bcl-X_L expression with siRNA reversed the protective effects of the glucocorticoid in glioma cells. We conclude that Dex-mediated inhibition of apoptosis in C6 glioma cells is through induction of Bcl-X_L.     

Concomitant blockade of A2AR and CTLA-4 by siRNA-loaded polyethylene glycol-chitosan-alginate nanoparticles synergistically enhances antitumor T-cell responses

Inhibitory immune checkpoint (ICP) molecules are important immunosuppressive factors in a tumor microenvironment (TME). They can robustly suppress T-cell-mediated antitumor immune responses leading to cancer progression. Among the checkpoint molecules, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is one of the critical inhibitors of anticancer T-cell responses. Besides, the expression of adenosine receptor (A2AR) on tumor-infiltrating T cells potently reduces their function. We hypothesized that concomitant silencing of these molecules in T cells might lead to enhanced antitumor responses. To examine this assumption, we purified T cells from the tumor, spleen, and local lymph nodes of CT26 colon cancer-bearing mice and suppressed the expression of A2AR and CTLA-4 using the small interfering RNA (siRNA)-loaded polyethylene glycol-chitosan-alginate (PCA) nanoparticles. The appropriate physicochemical properties of the produced nanoparticles (NPs; size of 72 nm, polydispersive index [PDI] < 0.2, and zeta potential of 11 mV) resulted in their high efficiency in transfection and suppression of target gene expression. Following the silencing of checkpoint molecules, various T-cell functions, including proliferation, apoptosis, cytokine secretion, differentiation, and cytotoxicity were analyzed, ex vivo. The results showed that the generated nanoparticles had optimal physicochemical characteristics and significantly suppressed the expression of target molecules in T cells. Moreover, a concomitant blockade of A2AR and CTLA-4 in T cells could synergistically enhance antitumor responses through the downregulation of PKA, SHP2, and PP2Aα signaling pathways. Therefore, this combination therapy can be considered as a novel promising anticancer therapeutic strategy, which should be further investigated in subsequent studies.     

Study the effect of 24-epibrassinolide application on the Cu/Zn-SOD expression and tolerance to drought stress in common bean

Physiology and Molecular Biology of Plants - To study the possibility of increasing the drought tolerance of common bean with the exogenous application of 24-epibrassinolide (EBL), an experiment...     

Conformational Transitions Provoked by Organic Solvents in Chicken Egg Ovalbumin: Mimicking the Local Environment

Glycoprotein ovalbumin is an important protein to study helix/sheet transitions as it possess almost equal amount of α-helix and β-sheet. Conformational changes on ovalbumin at various concentrations of glyoxal, ethylene glycol (EG) and polyethylene glycol-400 (PEG-400) were investigated by fluorescence spectroscopy, circular dichroism, attenuated total reflection Fourier transform infra red spectroscopy, 8-anilino-1-naphthalenesulfonic acid and thioflavin T assay. A partially folded state of ovalbumin at 50 % v/v glyoxal was detected that preceded the onset of the aggregation process at the maximum concentration (90 % v/v) of this aldehyde. Aggregates of ovalbumin in the presence EG and PEG-400 were deduced at 70 and 80 % v/v respectively. Maximum aggregation of ovalbumin was observed at 80 % v/v PEG-400, followed by 70 % v/v EG and 90 % v/v glyoxal. Our study confirms that protein aggregation is influenced by the chemistry of organic solvent used thus follows an order of solvent effectiveness (PEG > EG > glyoxal) in inducing the transition. These results provide valuable information on the mechanisms involved in the pathogenesis of some conformational diseases. The α-helix to β-sheet conversion is a diagnostic feature of protein aggregation and has been considered as a general characteristic of amyloid fibrillogenesis in vitro.     

Genetic structure and differentiation of four populations of Afghan Pika (Ochotona rufescens) in Iran based on mitochondrial cytochrome b gene

The population genetics of the Afghan Pika (Ochotona rufescens) was studied in Northern Khorasan Province, Iran. For prediction of the genetic differentiation of four populations, the DNA of mitochondrial cytochrome b of 32 individuals from four areas was sequenced and a Bayesian analysis based on the HKY model was constructed. In total, 15 polymorphic sites, 1125 conserved sites (98.7%) and 14 different haplotypes were found. The phylogenetic tree resulting from the Bayesian analysis and network analysis showed that all samples were clustered in two major groups and the haplotypes of the four populations did not separate geographically. An analysis of molecular variance (AMOVA) indicated that a large majority of the genetic variance was due to the variance within populations. The results of fixation indices showed significant genetic structure among populations in both methods. The pairwise F_st revealed that two northern populations have a significant genetic differentiation from two southern populations, but no significance pairwise F_st value was demonstrated between the closed populations. Nei's genetic distances between closed populations were not significant, while significant values occurred between distant populations. It seems that there is not a major discontinuity between populations of Afghan Pika based on cyt-b mitochondrial gene. However, phylogenetic analysis did not separate populations and a large majority of the genetic variance was found within populations. However, AMOVA analysis showed a significant level of genetic structure among populations (p?<?0.001) and between groups (p?<?0.5). It seems that these results suggest shallow genetic differentiation between populations of different geographic groups.     

New directions in ER stress-induced cell death

Endoplasmic reticulum (ER) stress has been implicated in the pathophysiology of many diseases including heart disease, cancer and neurodegenerative diseases such as Alzheimer’s and Huntington’s. Prolonged or excessive ER stress results in the initiation of signaling pathways resulting in cell death. Over the past decade much research investigating the onset and progression of ER stress-induced cell death has been carried out. Owing to this we now have a better understanding of the signaling pathways leading to ER stress-mediated cell death and have begun to appreciate the importance of ER localized stress sensors, IRE1α, ATF6 and PERK in this process. In this article we provide an overview of the current thinking and concepts concerning the various stages of ER stress-induced cell death, focusing on the role of ER localized proteins in sensing and triggering ER stress-induced death signals with particular emphasis on the contribution of calcium signaling and Bcl-2 family members to the execution phase of this process. We also highlight new and emerging directions in ER stress-induced cell death research particularly the role of microRNAs, ER-mitochondria cross talk and the prospect of mitochondria-independent death signals in ER stress-induced cell death.