Inhibition of specific signaling pathways rather than epigenetic silencing of effector genes is the leading mechanism of innate tolerance.

Introduction: Macrophages activated through a pattern-recognition receptor (PRR) enter a transient state of tolerance characterized by diminished responsiveness to restimulation of the same receptor. Signaling-based and epigenetic mechanisms are invoked to explain this innate tolerance. However, these two groups of mechanisms should result in different outcomes. The epigenetic scenario (silencing of effector genes) predicts that activation of a PRR should broadly cross-tolerize to agonists of unrelated PRRs, whereas in the signaling-based scenario (inhibition of signaling pathways downstream of specific PRRs), cross-tolerization should occur only between agonists utilizing the same PRR and/or signaling pathway. Also, the so-called non-tolerizeable genes have been described, which acquire distinct epigenetic marks and increased responsiveness to rechallenge with the same agonist. The existence of such genes is well explained by epigenetic mechanisms but difficult to explain solely by signaling mechanisms. Methods: To evaluate contribution of signaling and epigenetic mechanisms to innate tolerance, we tolerized human macrophages with agonists of TLR4 or NOD1 receptors, which signal via distinct pathways, and assessed responses of tolerized cells to homologous restimulation and to cross-stimulation using different signaling, metabolic and transcriptomic read-outs. We developed a transcriptomics-based approach to distinguish responses to secondary stimulation from continuing responses to primary stimulation. Results: We found that macrophages tolerized with a NOD1 agonist lack responses to homologous restimulation, whereas LPS-tolerized macrophages partially retain the ability to activate NF-κB pathway upon LPS rechallenge, which allows to sustain low-level expression of a subset of pro-inflammatory genes. Contributing to LPS tolerance is blockade of signaling pathways required for IFN-ß production, resulting in 'pseudo-tolerization' of IFN-regulated genes. Many genes in NOD1- or TLR4-tolerized macrophages are upregulated as the result of primary stimulation (due to continuing transcription and/or high mRNA stability), but do not respond to homologous restimulation. Hyperresponsiveness of genes to homologous rechallenge is a rare and inconsistent phenomenon. However, most genes that have become unresponsive to homologous stimuli show unchanged or elevated responses to agonists of PRRs signaling via distinct pathways. Discussion: Thus, inhibition of specific signaling pathways rather than epigenetic silencing is the dominant mechanism of innate tolerance.

Mitochondrial sirtuin 3 and various cell death modalities.

Sirtuin 3, a member of the mammalian sirtuin family of proteins, is involved in the regulation of multiple processes in cells. It is a major mitochondrial NAD+-dependent deacetylase with a broad range of functions, such as regulation of oxidative stress, reprogramming of tumor cell energy pathways, and metabolic homeostasis. One of the intriguing functions of sirtuin 3 is the regulation of mitochondrial outer membrane permeabilization, a key step in apoptosis initiation/progression. Moreover, sirtuin 3 is involved in the execution of various cell death modalities, which makes sirtuin 3 a possible regulator of crosstalk between them. This review is focused on the role of sirtuin 3 as a target for tumor cell elimination and how mitochondria and reactive oxygen species (ROS) are implicated in this process.

Modeling hypoxia facilitates cancer cell survival through downregulation of p53 expression.

A hypoxic environment of rapidly growing tumor cells makes them resistant to antitumor drugs. Mimicking hypoxia with iron chelator deferoxamine, suppressed cell death induced by widely used anticancer drugs doxorubicin or cisplatin. Deferoxamine decreased the number of dead (detached) cells, the size of SubG1 population, the release of cytochrome c, and the processing of caspase-3 in HCT116 colon carcinoma cells treated with cisplatin or doxorubicin. Deferoxamine-mediated suppression of apoptosis correlated with the level of pro-apoptotic Bcl-2 family proteins Bax, Bid, and Puma, which stimulate mitochondrial apoptotic pathway through permeabilization of the outer mitochondrial membrane and cytochrome c release. Here we show that one of the reasons for apoptosis suppression is downregulation of p53 expression under hypoxic conditions, and, as a result, attenuation of the expression of pro-apoptotic Bcl-2 family proteins. Indeed, p53 knock-out did not affect the stabilization of hypoxia-inducible factor but made undetectable the expression of pro-apoptotic proteins.

Interplay between NOD1 and TLR4 Receptors in Macrophages: Nonsynergistic Activation of Signaling Pathways Results in Synergistic Induction of Proinflammatory Gene Expression.

Interactions between pattern-recognition receptors shape innate immune responses to pathogens. NOD1 and TLR4 are synergistically interacting receptors playing a pivotal role in the recognition of Gram-negative bacteria. However, mechanisms of their cooperation are poorly understood. It is unclear whether synergy is produced at the level of signaling pathways downstream of NOD1 and TLR4 or at more distal levels such as gene transcription. We analyzed sequential stages of human macrophage activation by a combination of NOD1 and TLR4 agonists (N-acetyl-d-muramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid [M-triDAP] and LPS, respectively). We show that events preceding or not requiring activation of transcription, such as activation of signaling kinases, rapid boost of glycolysis, and most importantly, nuclear translocation of NF-κB, are regulated nonsynergistically. However, at the output of the nucleus, the combination of M-triDAP and LPS synergistically induces expression of a subset of M-triDAP- and LPS-inducible genes, particularly those encoding proinflammatory cytokines (TNF, IL1B, IL6, IL12B, and IL23A). This synergistic response develops between 1 and 4 h of agonist treatment and requires continuous signaling through NOD1. The synergistically regulated genes have a lower basal expression and higher inducibility at 4 h than those regulated nonsynergistically. Both gene subsets include NF-κB-inducible genes. Therefore, activation of the NF-κB pathway does not explain synergistic gene induction, implying involvement of other transcription factors. Inhibition of IKKß or p38 MAPK lowers agonist-induced TNF mRNA expression but does not abolish synergy. Thus, nonsynergistic activation of NOD1- and TLR4-dependent signaling pathways results in the synergistic induction of a proinflammatory transcriptional program.

Glycolytic reprogramming of macrophages activated by NOD1 and TLR4 agonists: No association with proinflammatory cytokine production in normoxia.

Upon activation with pathogen-associated molecular patterns, metabolism of macrophages and dendritic cells is shifted from oxidative phosphorylation to aerobic glycolysis, which is considered important for proinflammatory cytokine production. Fragments of bacterial peptidoglycan (muramyl peptides) activate innate immune cells through nucleotide-binding oligomerization domain (NOD) 1 and/or NOD2 receptors. Here, we show that NOD1 and NOD2 agonists induce early glycolytic reprogramming of human monocyte-derived macrophages (MDM), which is similar to that induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide. This glycolytic reprogramming depends on Akt kinases, independent of mTOR complex 1 and is efficiently inhibited by 2-deoxy-d-glucose (2-DG) or by glucose starvation. 2-DG inhibits proinflammatory cytokine production by MDM and monocyte-derived dendritic cells activated by NOD1 or TLR4 agonists, except for tumor necrosis factor production by MDM, which is inhibited initially, but augmented 4 h after addition of agonists and later. However, 2-DG exerts these effects by inducing unfolded protein response rather than by inhibiting glycolysis. By contrast, glucose starvation does not cause unfolded protein response and, in normoxic conditions, only marginally affects proinflammatory cytokine production triggered through NOD1 or TLR4. In hypoxia mimicked by treating MDM with oligomycin (a mitochondrial ATP synthase inhibitor), both 2-DG and glucose starvation strongly suppress tumor necrosis factor and interleukin-6 production and compromise cell viability. In summary, the requirement of glycolytic reprogramming for proinflammatory cytokine production in normoxia is not obvious, and effects of 2-DG on cytokine responses should be interpreted cautiously. In hypoxia, however, glycolysis becomes critical for cytokine production and cell survival.

Distinct effects of etoposide on glutamine-addicted neuroblastoma.

The majority of anticancer drugs are DNA-damaging agents, and whether or not they may directly target mitochondria remains unclear. In addition, tumors such as neuroblastoma exhibit addiction to glutamine in spite of it being a nonessential amino acid. Our aim was to evaluate the direct effect of widely used anticancer drugs on mitochondrial activity in combination with glutamine withdrawal, and possible apoptotic effects of such interaction. Our results revealed that etoposide inhibits mitochondrial respiratory chain Complex I causing the leakage of electrons and the superoxide radical formation. However, it was not sufficient to induce apoptosis, and apoptotic manifestation was detectable only alongside the withdrawal of glutamine, a precursor for antioxidant glutathione. Thus, the simultaneous depletion of glutathione and destabilization of mitochondria by ROS can compromise the barrier properties of the mitochondrial membrane, leading to cytochrome c release and the activation of the mitochondrial apoptotic pathway. Thus, the depletion of antioxidants or the inhibition of the pathways responsible for cellular antioxidant response can enhance mitochondrial targeting and strengthen antitumor therapy.

Biodegradable Porous Silicon Nanocontainers as an Effective Drug Carrier for Regulation of the Tumor Cell Death Pathways.

Nanocontainers based on solid materials have great potential for drug delivery applications. However, since nanocontainer-mediated delivery can alter the drug internalization pathways and metabolism, it is important to find out what are the mechanisms of cancer cell death induced by nanocontainers and, moreover, is it possible to regulate them. Here, we report on the detailed investigation of the internalization kinetics and intracellular spatial distribution of porous silicon nanoparticles (PSi NPs) loaded with doxorubicin (DOX) and response of cancer cells to treatment with DOX-PSi NPs as well as studies of nanocontainer biodegradation by applying various microscopy methods, Raman microspectroscopy and biological experiments with cancer cells of different etiology. The obtained results revealed the absence of toxicity of unloaded PSi NPs to cancer cells up to a concentration of 700 µg/mL during the prolonged incubation time. Thus, given the fact that the nanocontainers themselves are not toxic, it is easy to adjust the dose of the drug that they deliver to the cells. It is shown, that the treatment with DOX-loaded PSi NPs more efficiently eliminates cancer cells in comparison with the free DOX. At the same time, the obtained results demonstrate the possibility of regulating the initiation of apoptosis or necrosis in tumor cells after treatment with different concentrations of DOX-PSi NPs, as revealed by the analysis of the caspase-3 processing, the accumulation of sub-G1 cell fraction, and morphological changes determined by electron and light microscopy. The obtained results are important for future applications of porous silicon nanocontainers in drug delivery for apoptotic pathway-targeted cancer therapy.

2-Deoxy-D-glucose has distinct and cell line-specific effects on the survival of different cancer cells upon antitumor drug treatment.

The dependence of tumors on glycolysis for ATP generation offers a rationale for therapeutic strategies aimed at selective inhibition of the glycolytic pathway. Analysis of tumor cell responses to anticancer drugs revealed that inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) generally augmented the apoptotic response; however, in HCT116 human colon carcinoma cells, apoptosis was suppressed. A comparison of neuroblastoma SK-N-BE(2) and HCT116 cells revealed, that in contrast to HCT116, in SK-N-BE(2) cells 2-DG alone was able to induce cell death. In SK-N-BE(2) cells the decrease in ATP levels upon treatment with 2-DG was more prominent because in HCT116 cells mitochondria compensated for the loss of ATP caused by glycolysis suppression. In both cells lines 2-DG triggered endoplasmic reticulum (ER) stress, assessed by the accumulation of the marker protein GRP78/BiP. Suppression of ER stress by mannose attenuated the 2-DG-induced apoptotic response in SK-N-BE(2) cells, implying that apoptosis in these cells is a consequence of ER stress induction. In HCT116 cells, ER stress stimulated autophagy, assessed by the accumulation of the lipidated form of LC3. The inhibitor of ER stress mannose attenuated autophagy and reversed 2-DG-mediated suppression of cisplatin-induced apoptosis. When autophagy in HCT116 cells was suppressed by bafilomycin, cisplatin-induced apoptosis was decreased. At the same time, stimulation of autophagy in SK-N-BE(2) cells suppressed cell death. Thus, successful treatment of tumors with conventionally used anticancer drugs should be combined with targeting metabolic pathways involved in the regulation of apoptosis, autophagy, and cellular bioenergetics.

Mitochondria-targeted betulinic and ursolic acid derivatives: synthesis and anticancer activity.

A series of new betulinic and ursolic acid conjugates with a lipophilic triphenylphosphonium cation, meant to enhance the bioavailability and mitochondriotropic action of natural triterpenes, have been synthesized. The in vitro experiments on three human cancer cell lines (MCF-7, HCT-116 and TET21N) revealed that all the obtained triphenylphosphonium triterpene acid derivatives not only showed higher cytotoxicity as compared to betulinic acid but were also markedly superior in triggering mitochondria-dependent apoptosis, as assessed using a range of apoptosis markers such as cytochrome c release, stimulation of caspase-3 activity, and cleavage of poly(ADP-ribose) polymerase, which is one of the targets of caspase 3. The IC50 was much lower for all triphenylphosphonium derivatives when compared to betulinic acid. Out of the tested group of conjugates, the most potent toxicity was exhibited by the betulinic acid conjugate 9 (for 9, the IC50 values against MCF-7 and TET21N cells were 0.70 µM and 0.74 µM; for betulinic acid (BA), IC50 > 25 µM against MCF-7 cells).

Contrasting effects of glutamine deprivation on apoptosis induced by conventionally used anticancer drugs.

Tumor cells dependence on glutamine offers a rationale for their elimination via targeting of glutamine metabolism. The aim of this work was to investigate how glutamine deprivation affects the cellular response to conventionally used anticancer drugs. To answer this question, neuroblastoma cells were pre-incubated in a glutamine-free medium and treated with cisplatin or etoposide. Obtained results revealed that glutamine withdrawal affected cellular response to therapeutic drugs in a different manner. Glutamine deprivation suppressed etoposide-induced, but markedly stimulated cisplatin-induced apoptosis. Suppression of etoposide-induced cell death correlated with a downregulation of p53 expression, which, among other functions, regulates the expression of death receptor 5, one of the activators of caspase-8. In contrast, stimulation of cisplatin-induced cell death involved reactive oxygen species-mediated downregulation of FLIP-S, an inhibitor of caspase-8. As a result, the activity of caspase-8 was stimulated causing cleavage of the pro-apoptotic protein Bid, which is involved in the permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic factors, such as cytochrome c from mitochondria. Thus, suppression of glutamine metabolism can sensitize tumor cells to treatment and could be utilized for anti-cancer therapy. However, it should be done cautiously, since adverse effects may occur when combined with an inappropriate therapeutic drug.