Volume 400, Issue 2 - Highlight: Mitochondria, Apoptosis & Cancer (MAC) 2017

The maintenance of protein homeostasis (proteostasis) is a fundamental aspect of cell physiology that is essential for the survival of organisms under a variety of environmental and/or intracellular stress conditions. Acute and/or persistent stress exceeding the capacity of the intracellular homeostatic systems results in protein aggregation and/or damaged organelles that leads to pathological cellular states often resulting in cell death. These events are continuously suppressed by a complex macromolecular machinery that uses different intracellular pathways to maintain the proteome integrity in the various subcellular compartments ensuring a healthy cellular life span. Recent findings have highlighted the role of the multifunctional enzyme type 2 transglutaminase (TG2) as a key player in the regulation of intracellular pathways, such as autophagy/mitophagy, exosomes formation and chaperones function, which form the basis of proteostasis regulation under conditions of cellular stress. Here, we review the role of TG2 in these stress response pathways and how its various enzymatic activities might contributes to the proteostasis control.

Mitochondria and oxidative phosphorylation (OXPHOS) are emerging as intriguing targets for the efficient elimination of cancer cells. The specificity of this approach is aided by the capacity of non-proliferating non-cancerous cells to withstand oxidative insult induced by OXPHOS inhibition. Recently we discovered that mitochondrial targeting can also be employed to eliminate senescent cells, where it breaks the interplay between OXPHOS and ATP transporters that appear important for the maintenance of mitochondrial morphology and viability in the senescent setting. Hence, mitochondria/OXPHOS directed pharmacological interventions show promise in several clinically-relevant scenarios that call for selective removal of cancer and senescent cells.

Oxidative stress has for a long time been associated with cell death, especially classical necrosis, however, its role in other cell death pathways is less clear. Here, we evaluated in a comparative way, the effect of four different reactive oxygen species (ROS) scavengers, N-acetyl-L-cysteine (NAC), α-tocopherol and two superoxide dismutase mimetics, n(III)tetrakis(4-benzoic acid)porphyrin chloride, and 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol), in four different cell death models, including menadione-triggered necrosis, staurosporine-induced apoptosis and tumor necrosis factor (TNF)-induced apoptosis and necroptosis. While menadione-triggered necrosis was completely prevented by the classical ROS scavenger NAC and to a substantial amount by the other scavengers, ROS targeting was found to have a marginal effect on the other cell death modalities investigated. Despite its side-effects at higher concentrations, Tempol was able to substantially prevent TNF-induced apoptosis and to a somewhat lesser extent TNF-induced necroptosis. However, this seems to be separated from its ROS-scavenging function.

Mitophagy, the selective degradation of mitochondria via the autophagic pathway, is a vital mechanism of mitochondrial quality control in cells. The removal of malfunctioning or damaged mitochondria is essential for normal cellular physiology and tissue development. Stimulation of mitochondrial permeabilization and release of proapoptotic factors from the intermembrane space is an essential step in triggering the mitochondrial pathway of cell death. In this study, we analyzed the extent to which mitophagy interferes with cell death, attenuating the efficiency of cancer therapy. We show that stimulation of mitophagy suppressed cisplatin-induced apoptosis, while mitophagy inhibition stimulates apoptosis and autophagy. Suppression of mitophagy involved production of reactive oxygen species, and the fate of cell was dependent on the interplay between endoplasmic reticulum stress and autophagy.

Smac mimetics (SMs) are considered promising cancer therapeutics. However, the mechanisms responsible for mediating cell death by SMs are still only partly understood. Therefore, in this study, we investigated signaling pathways upon treatment with the bivalent SM BV6 using two SM-sensitive breast cancer cell lines as models. Interestingly, genetic silencing of transforming growth factor (TGF)β activated kinase (TAK)1, an upstream activator of the nuclear factor-kappaB (NF-κB) subunit RelA (p65), increased BV6-induced cell death only in EVSA-T cells, although it reduced BV6-mediated upregulation of tumor necrosis factor (TNF)α in both EVSA-T and MDA-MB-231 cells. By comparison, genetic silencing of p65, a key component of canonical NF-κB signaling, blocked BV6-induced cell death in MDA-MB-231 but not in EVSA-T cells. Similarly, knockdown of NF-κB-inducing kinase (NIK) rescued MDA-MB-231 cells from BV6-induced cell death, while it failed to do so in EVSA-T cells. Consistently, silencing of p65 or NIK reduced BV6-stimulated upregulation of TNFα in MDA-MB-231 cells. In conclusion, TAK1, receptor-interacting kinase 1 (RIPK1) as well as canonical and non-canonical NF-κB signaling are differentially involved in SM-induced cell death in breast cancer cells. These findings contribute to a better understanding of SM-induced signaling pathways.

Induction of apoptosis by selective BH3-mimetics is currently investigated as a novel strategy for cancer treatment. Here, we report that selective BH3-mimetics induce apoptosis in a variety of hematological malignancies. Apoptosis is accompanied by severe mitochondrial toxicities upstream of caspase activation. Specifically, the selective BH3-mimetics ABT-199, A-1331852 and S63845, which target BCL-2, BCL-X L and MCL-1, respectively, induce comparable ultrastructural changes including mitochondrial swelling, a decrease of mitochondrial matrix density and severe loss of cristae structure. These shared effects on mitochondrial morphology indicate a similar function of these anti-apoptotic BCL-2 proteins in maintaining mitochondrial integrity and function.

Aerobic glycolysis (‘Warburg effect’) is used by cancer cells to fuel tumor growth. Interestingly, metastatic melanoma cells rely on glutaminolysis rather than aerobic glycolysis for their bioenergetic needs through the tricarboxylic acid (TCA) cycle. Here, we compared the effects of glucose or glutamine on melanoma cell proliferation, migration and oxidative phosphorylation in vitro . We found that glutamine-driven melanoma cell’s aggressive traits positively correlated with increased expression of HIF1α and its pro-autophagic target BNIP3. BNIP3 silencing reduced glutamine-mediated effects on melanoma cell growth, migration and bioenergetics. Hence, BNIP3 is a vital component of the mitochondria quality control required for glutamine-driven melanoma aggressiveness.

In vitro evaluation of drugs for interaction with transporters is essential during drug development. As polyspecific organic cation transporters (OCTs) are critical for pharmacokinetics of many cationic drugs, in vitro testing of human OCT1 and human OCT2 is recommended. In the currently applied tests it is determined whether uptake of one model cation in stably transfected epithelial cells is inhibited using a substrate concentration in the micromolar range. In this review experimental evidence for the existence of low- and high-affinity cation binding sites in OCTs that may interact with drugs is compiled. Most data were obtained from studies performed with rat Oct1. Whereas overlapping low-affinity cation binding sites are directly involved in transport, the high-affinity cation binding sites may induce allosteric inhibition of transport. Remarkably, high-affinity inhibition is only observed when uptake is measured using nanomolar substrate concentrations far below the respective K m values. Affinities of inhibitors are dependent on molecular structure and concentration of the employed substrate. Because the currently applied in vitro tests for identification of interaction of novel drugs with OCTs do not consider the influence of substrate structure and are not capable of identifying high-affinity inhibition, more sophisticated testing protocols are proposed.

High-density lipoprotein (HDL), also known as antiatherogenic lipoprotein, consists of heterogeneous particles in terms of size, density and composition, suggesting differences among HDL subclasses in characteristics and functions. We investigated the role of apolipoprotein E (apoE)-containing HDL, a minor HDL subclass, in the cholesterol efflux capacity (CEC) of HDL, which is its predominant atheroprotective function. The CEC of apoE-containing HDL was similar to that of apoE-deficient HDL, but the former exhibited a greater rate increase (1.48-fold) compared to that of the latter (1.10-fold) by the stimulation of THP-1 macrophages with the Liver X Receptor (LXR) agonist. No difference in CEC was observed without the LXR agonist between apoA-I, the main apolipoprotein in HDL, and apoE, whereas the increase in CEC in response to treatment with the LXR agonist was greater for apoA-I (4.25-fold) than for apoE (2.22-fold). Furthermore, the increase in the CEC of apoE-containing HDL induced by the LXR agonist was significantly reduced by treatment with glyburide, an inhibitor of ATP-binding cassette transporter A1 (ABCA1). These results suggest that apoE-containing HDL, unlike apoE-deficient HDL, is involved in cholesterol efflux via ABCA1.

The balance between protein synthesis and degradation regulates the amount of expressed proteins. This protein turnover is usually quantified as the protein half-life time. Several studies suggest that protein degradation decreases with age and leads to increased deposits of damaged and non-functional proteins. Glycation is an age-dependent, non-enzymatic process leading to posttranslational modifications, so-called advanced glycation endproducts (AGE), which usually damage proteins and lead to protein aggregation. AGE are formed by the Maillard reaction, where carbonyls of carbohydrates or metabolites react with amino groups of proteins. In this study, we quantified the half-life time of two important receptors of the immunoglobulin superfamily, the neural cell adhesion molecule (NCAM) and the receptor for advanced glycation end products (RAGE) before and after glycation. We found, that in two rat PC12 cell lines glycation leads to increased turnover, meaning that glycated, AGE-modified proteins are degraded faster than non-glycated proteins. NCAM is the most prominent carrier of a unique enzymatic posttranslational modification, the polysialylation. Using two PC12 cell lines (a non-polysialylated and a polysialylated one), we could additionally demonstrate, that polysialylation of NCAM has an impact on its turnover and that it significantly increases its half-life time.

Myocardial infarction (MI) is an unsolved health problem which seriously affects human health around the world. miR-34a-5p acting as a tumor-suppressor is associated with left ventricular remodeling. We aimed to explore the functional roles of miR-34a-5p in cardiomyocytes. Hypoxia-induced cell injury in H9c2, HL-1 and human cardiac myocytes was analyzed according to the decrease of cell viability and increase of apoptosis. Expression of miR-34a-5p was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) when the concentration of O 2 was decreased. Then, the effects of aberrantly expressed miR-34a-5p on proliferation and apoptosis of cardiomyocytes incubated under hypoxia were assessed. Finally, the downstream protein and signaling pathways of miR-34a-5p were explored. The hypoxic model was successfully constructed after incubation under hypoxia for 48 h. When the concentration of O 2 decreased, the miR-34a-5p level was increased significantly. Then, we found miR-34a-5p aggravated hypoxia-induced alterations of proliferation and apoptosis in cardiomyocytes. Zinc finger E-box binding homeobox 1 (ZEB1) was identified as a target of miR-34a-5p , and miR-34a-5p conferred its function via targeting ZEB1. Finally, miR-34a-5p inhibition reversed hypoxia-induced decreases of phosphorylated kinases in the JAK/STAT and PI3K/AKT pathways through up-regulating ZEB1 . Our study revealed that miR-34a-5p inhibition protected cardiomyocytes against hypoxia-induced cell injury through activating the JAK/STAT and PI3K/AKT pathways by targeting ZEB1.

This study aimed to investigate the role of miRNA-1225-5p (miR-1225) in laryngeal carcinoma (LC). We found that the expression of miR-1225 was suppressed in human LC samples, while CDC14B (cell division cycle 14B) expression was reinforced in comparison with surrounding normal tissues. We also demonstrated that enhanced expression of miR-1225 impaired the proliferation and survival of LC cells, and resulted in G1/S cell cycle arrest. In contrast, reduced expression of miR-1225 promoted cell survival. Moreover, miR-1225 resulted in G1/S cell cycle arrest and enhanced cell death. Further, miR-1225 targets CDC14B 3′-UTR and recovery of CDC14B expression counteracted the suppressive influence of miR-1225 on LC cells. Thus, these findings offer insight into the biological and molecular mechanisms behind the development of LC.

Circular RNA (circRNA) play a crucial role in many biological processes and have been proved as potential biomarkers and therapeutic targets in many diseases. Manipulation of their expression is a critical task. In this study, we developed a new strategy for circRNA suppression with DNAzyme. Data showed single-digestion DNAzymes cleaved circRNA efficiently in vitro but not in cell culture. However, tandem DNAzymes for double digestion showed higher cleavage efficacy both in vitro and in cell culture. Functional study demonstrated that double-digestion DNAzymes suppressed the miRNA sponge function of circRNA and changed the proliferation and migration rates of HCC cells.