Variety Of Intracellular Mechanisms Research Articles

Abstract B087: A systematic approach to JAK/STAT pathway shut-down

Abstract Janus Kinase Protein (JAK) plays a crucial role in a variety of intracellular mechanisms, and has been extensively studied for its role in proliferation and survival of cancer cells. One of the unique characteristics of this kinase is the variety of cell membrane receptors with which it is associated, and can trigger JAK activation process. Family of Signal Transducer and Activators of Transcription (STATs) are the direct downstream proteins that further transfer JAK signaling to the nucleus, which results in modification of expression level of a wide range of proteins. Triple-negative breast cancer (TNBC) represents one of the most aggressive and challenging subtypes of human breast cancer, due to lack of estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2) receptors, which renders the cells nonresponsive to hormone therapy. Several studies have confirmed that IL-6/gp130/JAK2/STAT3 signaling pathway is one of the crucial pathways that is implicated in TNBC development. However, monotherapies targeting JAK2 or STAT3 with molecularly targeted drugs have faced several challenges, including inherent and acquired resistance. Therefore, we aimed to determine feasibility and efficacy of therapy of TNBC using combinatorial silencing of different components in gp130/JAK2/STAT3 pathway. The downregulatory effects of siRNA targeting gp130, JAK2, STAT3, Importin α-3 alone or in combination were evaluated in human TNBC cells MDA 231 and MDA 468, and their effect on downstream targets involved in cell apoptosis and cell cycle were determined by real-time PCR and Western blot. The effect of individual and combinatorial silencing of the pathway components on cell proliferation and cell migration were also examined using CCK assay and Scratch assay, respectively. The siRNA treatment effectively reduced gene expression of gp130/JAK2/STAT pathway components at the mRNA level. Simultaneous inhibition of selected components significantly decreased TNBC cell proliferation rate. Moreover, combinatorial siRNA silencing inhibited the migration of TNBC cells. In conclusion, simultaneous inhibition of well-selected components of JAK/STAT pathway could represent an effective therapy for human TNBC. This warrants further in vivo study in animal models. Citation Format: Hamidreza Montazeri Aliabadi, Emira Bousoik, Parvin Mahdipoor. A systematic approach to JAK/STAT pathway shut-down [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B087.

The frequency of yeast [PSI+] prion formation is increased during chronological ageing.

Ageing involves a time-dependent decline in a variety of intracellular mechanisms and is associated with cellular senescence. This can be exacerbated by prion diseases which can occur in a sporadic manner, predominantly during the later stages of life. Prions are infectious, self-templating proteins responsible for several neurodegenerative diseases in mammals and several prion-forming proteins have been found in yeast. We show here that the frequency of formation of the yeast [PSI+] prion, which is the altered form of the Sup35 translation termination factor, is increased during chronological ageing. This increase is exacerbated in an atg1 mutant suggesting that autophagy normally acts to suppress age-related prion formation. We further show that cells which have switched to [PSI+] have improved viability during chronological ageing which requires active autophagy. [PSI+] stains show increased autophagic flux which correlates with increased viability and decreased levels of cellular protein aggregation. Taken together, our data indicate that the frequency of [PSI+] prion formation increases during yeast chronological ageing, and switching to the [PSI+] form can exert beneficial effects via the promotion of autophagic flux.

The ROS-generating oxidase Nox1 is required for epithelial restitution following colitis.

Accumulating evidence suggests that reactive oxygen species (ROS) generated by endogenous metabolic enzymes are involved in a variety of intracellular mechanisms. In particular, superoxide-generating NADPH oxidase (Nox) 1 is highly expressed in the colon and has been implicated in physiological and pathophysiological states of colon tissues. However, its role in tissue repair following colitis has not been fully elucidated. Our study using experimental colitis in mice showed that repair of the mucosal layer did not occur in Nox1-deficient mice following dextran sulfate sodium-induced colitis. This was accompanied by inhibition of proliferation, cell survival, migration, and terminal differentiation (generation of goblet cells) of crypt progenitor cells, as determined by histochemical analyses. Furthermore, Nox1 expression as well as ROS production in the colon crypt was increased during the repair process, and Nox1 deficiency suppressed these events. The results suggest that Nox1 promotes colon mucosal wound repair by sustaining the bioactivity of crypt progenitor cells and plays a crucial role in the epithelial restitution in the case of damage associated with colitis.

Mechanisms activating PGC-1α and consequential transcriptional mechanisms following exercise: A mini review

Skeletal muscle has the ability to adapt in response to exercise to increase the oxidative potential of the tissue. The adaptive response contributes to a favourable phenotype, increasing the ability of the tissue to augment superior fat oxidation and glucose uptake. In this respect, exercise, particularly endurance modes of exercise, forms a strong preventative and treatment strategy for metabolic diseases. The acute disturbance of homeostasis in response to muscle contraction during exercise stimulates a variety of intra-cellular mechanisms that signal to a putative stimulator of mitochondrial biogenesis, PGC-1α. The activation of these mechanisms is explored in this mini-review, drawing upon in vivo, ex vivo and in vitro data. Additionally, the effect of PGC-1α activation and the consequential transcriptional regulatory network is discussed in relation to stimulating mitochondrial biogenesis. Our understanding of these mechanisms has been hindered by the complex nature of the coordination of both nuclear and mitochondrial genomes. Further, the nature of in vivo experimentation with respect to exercise modalities and nutritional manipulation has often presented conflicting findings. Delineating the mechanisms further with highly controlled in vivo experiments, along with targeted in vitro experiments will define targets for potential genetic and pharmacological therapies in the future.

Involvement of SNARE proteins in thrombin-induced platelet aggregation: Evidence for the relevance of Ca 2+ entry

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca 2+ mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca 2+ entry, the main mechanism for Ca 2+ influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca 2+ entry for full activation. Depletion of the intracellular Ca 2+ stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca 2+ concentration reached a critical level (around 1.5 μM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca 2+ mobilization in human platelets.

Muscle fatigue: the role of intracellular calcium stores.

Force declines when muscles are used repeatedly and intensively and a variety of intracellular mechanisms appear to contribute to this muscle fatigue. Intracellular calcium release declines during fatigue and has been shown to contribute to the reduction in force. Three new approaches have helped to define the role of calcium stores to this decline in calcium release. Skinned fibre experiments show that when intracellular phosphate is increased the amount of Ca2+ released from the sarcoplasmic reticulum (SR) declines. Intact fibre experiments show that the size of the calcium store declines during fatigue and recovers on rest. Intact muscles which lack the enzyme creatine kinase, do not exhibit the usual rise of phosphate during fatigue and, under these conditions, the decline of Ca2+ release is absent or delayed. These results can be explained by the "calcium phosphate precipitation" hypothesis. This proposes that if phosphate in the myoplasm rises, it enters the SR and binds to Ca2+ as Ca2+ phosphate. The resultant reduction in free Ca2+ within the SR contributes to the reduced Ca2+ release during fatigue.