Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation

Apoptotic cell death has been observed in many in vivo and in vitro models of ischemia. However, the molecular pathways involved in ischemia-induced apoptosis remain unclear. We have examined the role of Bcl-2 family of proteins in mediating apoptosis of PC12 cells exposed to the conditions of oxygen and glucose deprivation (OGD) or OGD followed by restoration of oxygen and glucose (OGD-restoration, OGD-R). OGD decreased mitochondrial membrane potential and induced necrosis of PC12 cells, which were both prevented by the overexpression of Bcl-2 proteins. OGD-R caused apoptotic cell death, induced cytochrome C release from mitochondria and caspase-3 activation, decreased mitochondrial membrane potential, and increased levels of pro-apoptotic Bax translocated to the mitochondrial membrane, all of which were reversed by overexpression of Bcl-2. These results demonstrate that the cell death induced by OGD and OGD-R in PC12 cells is potentially mediated through the regulation of mitochondrial membrane potential by the Bcl-2 family of proteins. It also reveals the importance of developing therapeutic strategies for maintaining the mitochondrial membrane potential as a possible way of reducing necrotic and apoptotic cell death that occurs following an ischemic insult.     

Synthesis of DNA interactive C3-trans-cinnamide linked β-carboline conjugates as potential cytotoxic and DNA topoisomerase I inhibitors

A series of new C3-trans-cinnamide linked β-carboline conjugates has been synthesized by coupling between various β-carboline amines and substituted cinnamic acids. Evaluation of their anti-proliferative activity against a panel of selected human cancer cell lines such as A549 (lung cancer), MCF-7 (breast cancer), B16 (melanoma), HeLa (cervical cancer) and a normal cell line NIH3T3 (mouse embryonic fibroblast cell line), suggested that the newly designed conjugates are considerably active against all the tested cancer cell lines with IC₅₀ values 13–45?nM. Moreover, the conjugates 8v and 8x were the most active against MCF-7 cells (14.05?nM and 13.84?nM respectively) and also even potent on other cell lines tested. Further, detailed investigations such as cell cycle analysis, apoptosis induction study, topoisomerase I inhibition assay, DNA binding affinity and docking studies revealed that these new conjugates are DNA interactive topoisomerase I inhibitors.     

Interaction of transducin with uncoordinated 119 protein (UNC119): implications for the model of transducin trafficking in rod photoreceptors

The key visual G protein, transducin undergoes bi-directional translocations between the outer segment (OS) and inner compartments of rod photoreceptors in a light-dependent manner thereby contributing to adaptation and neuroprotection of rods. A mammalian uncoordinated 119 protein (UNC119), also known as Retina Gene 4 protein (RG4), has been recently implicated in transducin transport to the OS in the dark through its interaction with the N-acylated GTP-bound transducin-α subunit (Gα(t1)). Here, we demonstrate that the interaction of human UNC119 (HRG4) with transducin is dependent on the N-acylation, but does not require the GTP-bound form of Gα(t1). The lipid specificity of UNC119 is unique: UNC119 bound the myristoylated N terminus of Gα(t1) with much higher affinity than a prenylated substrate, whereas the homologous prenyl-binding protein PrBP/δ did not interact with the myristoylated peptide. UNC119 was capable of interacting with Gα(t1)GDP as well as with heterotrimeric transducin (G(t)). This interaction of UNC119 with G(t) led to displacement of Gβ(1)γ(1) from the heterotrimer. Furthermore, UNC119 facilitated solubilization of G(t) from dark-adapted rod OS membranes. Consistent with these observations, UNC119 inhibited rhodopsin-dependent activation of G(t), but had no effect on the GTP-hydrolysis by Gα(t1). A model for the role of UNC119 in the IS→OS translocation of G(t) is proposed based on the UNC119 ability to dissociate G(t) subunits from each other and the membrane. We also found that UNC119 inhibited activation of G(o) by D2 dopamine receptor in cultured cells. Thus, UNC119 may play conserved inhibitory role in regulation of GPCR-G protein signaling in non-visual tissues.     

Spatiotemporal Characterization of a Fibrin Clot Using Quantitative Phase Imaging

Studying the dynamics of fibrin clot formation and its morphology is an important problem in biology and has significant impact for several scientific and clinical applications. We present a label-free technique based on quantitative phase imaging to address this problem. Using quantitative phase information, we characterized fibrin polymerization in real-time and present a mathematical model describing the transition from liquid to gel state. By exploiting the inherent optical sectioning capability of our instrument, we measured the three-dimensional structure of the fibrin clot. From this data, we evaluated the fractal nature of the fibrin network and extracted the fractal dimension. Our non-invasive and speckle-free approach analyzes the clotting process without the need for external contrast agents.     

A Fourteen Gene GBM Prognostic Signature Identifies Association of Immune Response Pathway and Mesenchymal Subtype with High Risk Group

Background

Recent research on glioblastoma (GBM) has focused on deducing gene signatures predicting prognosis. The present study evaluated the mRNA expression of selected genes and correlated with outcome to arrive at a prognostic gene signature.

Methods

Patients with GBM (n = 123) were prospectively recruited, treated with a uniform protocol and followed up. Expression of 175 genes in GBM tissue was determined using qRT-PCR. A supervised principal component analysis followed by derivation of gene signature was performed. Independent validation of the signature was done using TCGA data. Gene Ontology and KEGG pathway analysis was carried out among patients from TCGA cohort.

Results

A 14 gene signature was identified that predicted outcome in GBM. A weighted gene (WG) score was found to be an independent predictor of survival in multivariate analysis in the present cohort (HR = 2^.507; B = 0^.919; p<0^.001) and in TCGA cohort. Risk stratification by standardized WG score classified patients into low and high risk predicting survival both in our cohort (p = <0^.001) and TCGA cohort (p = 0^.001). Pathway analysis using the most differentially regulated genes (n = 76) between the low and high risk groups revealed association of activated inflammatory/immune response pathways and mesenchymal subtype in the high risk group.

Conclusion

We have identified a 14 gene expression signature that can predict survival in GBM patients. A network analysis revealed activation of inflammatory response pathway specifically in high risk group. These findings may have implications in understanding of gliomagenesis, development of targeted therapies and selection of high risk cancer patients for alternate adjuvant therapies.     

Functional androgen receptor confers sensitization of androgen-independent prostate cancer cells to anticancer therapy via caspase activation

Therapeutic resistance remains an unresolved problem in the clinical management of human prostate cancer (PC). Despite initial positive response to androgen ablation therapy (AAT), virtually all PC patients will relapse due to acquisition of hormone refractory disease and selective outgrowth of tumor cells with multidrug resistance phenotype. We here provide the first experimental evidence that restoring a functional androgen receptor (AR) in the androgen-independent prostate cancer PC3 cells enhances their sensitivity to growth arrest and suppresses their colony-forming ability in response to paclitaxel and gamma-irradiation. Furthermore, functional AR increases the susceptibility of these cells to the apoptotic potentials of therapeutic agents, as evidenced by an increase in caspase activity, annexin V binding, and internucleosomal DNA fragmentation, by inducing caspase activation. The abrogation of the cytotoxic effects by 4-hydroxyflutamide suggests a crucial role for AR activation in enhancing the therapeutic sensitivity of these cells in a ligand-independent fashion. Our data thus demonstrate that a functional AR is a prerequisite for effective therapeutic response and that aberrant expression or blockade by AAT may trigger pathways leading to emergence of PC cells with therapeutic resistance phenotype. Since the mainstay of primary therapy for PC has been AAT by pharmaco-therapeutic or surgical means, this study thus provides a new frontier for revising the AAT therapeutic strategy in conjunction with radiation and/or chemotherapeutic agents.     

Bacteriophage T4 gp2 Interferes with Cell Viability and with Bacteriophage Lambda Red Recombination

The T4 head protein, gp2, promotes head-tail joining during phage morphogenesis and is also incorporated into the phage head. It protects the injected DNA from degradation by exonuclease V during the subsequent infection. In this study, we show that recombinant gp2, a very basic protein, rapidly kills the cells in which it is expressed. To further illustrate the protectiveness of gp2 for DNA termini, we compare the effect of gp2 expression on Red-mediated and Int-mediated recombination. Red-mediated recombination is nonspecific and requires the transient formation of double-stranded DNA termini. Int-mediated recombination, on the other hand, is site specific and does not require chromosomal termini. Red-mediated recombination is inhibited to a much greater extent than is Int-mediated recombination. We conclude from the results of these physiological and genetic experiments that T4 gp2 expression, like Mu Gam expression, kills bacteria by binding to double-stranded DNA termini, the most likely mode for its protection of entering phage DNA from exonuclease V.     

Neo-tanshinlactone D-ring modified novel analogues induce apoptosis in human breast cancer cell via DNA damage

Neo-tanshinlactone (NTL) a natural product is known for its specificity and selectivity towards the breast cancer cells. By NTL D-ring modification approach, 13 new analogues were synthesized (1A–1M). Among them 1J showed the best anticancer activity in MCF-7 (ER+, PR+/?, HER2?), SKBR3 (ER?, PR?, HER2+) and MDA-MB-231 (ER?, PR?, HER2?) cells lines with IC₅₀ value 11.98 nM, 23.71 nM, and 62.91 nM respectively. 1J showed minor grove binding interaction with DNA at AT-rich region and induced DNA double strand breaks (DDSBs). This had triggered several key molecular events involving, activation of ATM, Chk2 and p53, reduction in mitochondrial potential (Δψ_m) leading to caspase-3 and PARP cleavage mediated apoptosis. These results along with other biochemical studies strongly suggest that novel NTL analogue 1J caused DNA cleavage mediated apoptosis in the breast cancer cells and this may serve as potential lead for future breast cancer treatment.