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.
Conjugation of native triterpenoids, namely, betulinic and ursolic acids, with a lypophilic triphenylphosphonium cation led to the dramatic enhancement, as compared to betulinic acid, of their ability to trigger the mitochondrial apoptosis pathway in various types of cancer cells.
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.
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 IKKβ 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.
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.
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