Lipopolysaccharide From E. coli Increases Glutamate-Induced Disturbances of Calcium Homeostasis, the Functional State of Mitochondria, and the Death of Cultured Cortical Neurons

Lipopolysaccharide (LPS), a fragment of the bacterial cell wall, specifically interacting with protein complexes on the cell surface, can induce the production of pro-inflammatory and apoptotic signaling molecules, leading to the damage and death of brain cells. Similar effects have been noted in stroke and traumatic brain injury, when the leading factor of death is glutamate (Glu) excitotoxicity too. But being an amphiphilic molecule with a significant hydrophobic moiety and a large hydrophilic region, LPS can also non-specifically bind to the plasma membrane, altering its properties. In the present work, we studied the effect of LPS from Escherichia coli alone and in combination with the hyperstimulation of Glu-receptors on the functional state of mitochondria and Ca2+ homeostasis, oxygen consumption and the cell survival in primary cultures from the rats brain cerebellum and cortex. In both types of cultures, LPS (0.1–10 μg/ml) did not change the intracellular free Ca2+ concentration ([Ca2+]i) in resting neurons but slowed down the median of the decrease in [Ca2+]i on 14% and recovery of the mitochondrial potential (ΔΨm) after Glu removal. LPS did not affect the basal oxygen consumption rate (OCR) of cortical neurons; however, it did decrease the acute OCR during Glu and LPS coapplication. Evaluation of the cell culture survival using vital dyes and the MTT assay showed that LPS (10 μg/ml) and Glu (33 μM) reduced jointly and separately the proportion of live cortical neurons, but there was no synergism or additive action. LPS-effects was dependent on the type of culture, that may be related to both the properties of neurons and the different ratio between neurons and glial cells in cultures. The rapid manifestation of these effects may be the consequence of the direct effect of LPS on the rheological properties of the cell membrane.

A Novel Small-Molecule Inhibitor Displays Its Therapeutic Potency Through Mitochondria-Mediated Apoptosis And Autophagy In Melanoma

Melanoma is the most aggressive skin cancer with high mortality. It is vital to develop novel low toxicity drugs with anti-proliferation activity and metastasis suppressive activity in melanoma. Here, we reported a novel anti-tumor drug SCZ0148, and then investigated its inhibition effect on melanoma. The anticancer efficacy of SCZ0148 was confirmed by using cytotoxicity test, colony formation assay, wound-healing assay, cell apoptosis detection, mitochondrial potential assay, reactive oxygen species (ROS) production and western-blot analysis. The cytotoxicity test showed that SCZ0148 inhibited melanoma cell lines proliferation in a dose- and time-dependent manner without obvious toxicity and side effects on normal cells. The results of the colony formation assay were in agreement with the cytotoxicity test. In addition, SCZ0148 induced melanoma cell apoptosis and promoted cell destructive autophagy through the ROS-mediated mitochondrial apoptosis pathway. Notably, SCZ0148 significantly inhibited the migration of melanoma cells through the matrix metalloprotein 9 (MMP-9) mediated pathway. In conclusion, these findings suggest that SCZ0148 may be a potential therapeutic drug to inhibit the proliferation and metastasis of melanoma.

Bicalutamide Exhibits Potential to Damage Kidney via Destroying Complex I and Affecting Mitochondrial Dynamics

Bicalutamide (Bic) is an androgen deprivation therapy (ADT) for treating prostate cancer, while ADT is potentially associated with acute kidney injury. Previously, we recognized Bic induced renal mitochondria dysfunction in vitro and in vivo via the ROS -HIF1α pathway. Whether OXPHOS complex, as well as mitochondrial dynamics, can be influenced by Bic via modulation of peroxisome proliferator-activated receptor coactivator 1α (PGC1α), NADPH oxidase 4 (Nox4), mitofusins 1/2 (MFN 1/2), optic atrophy 1 (OPA1), and sirtuins (SIRTs) has not been documented. Renal mesangial cell line was treated with Bic (30~60 μM) for the indicated time. SIRTs, complex I, mitochondrial dynamics- and oxidative stress-related proteins were analyzed. Bic dose-dependently reduced mitochondrial potential, but dose- and time-dependently suppressed translocase of the outer mitochondrial membrane member 20 (Tomm 20), complex I activity. Nox4 and glutathione lead to decreased NAD+/NADH ratio, with upregulated superoxide dismutase 2. SIRT1 was initially stimulated and then suppressed, while SIRT3 was time- and dose-dependently downregulated. PGC1α, MFN2, and OPA1 were all upregulated, with MFN1 and pro-fission dynamin-related protein I downregulated. Bic exhibits potential to damage mitochondria via destroying complex I, complex I activity, and mitochondrial dynamics. Long-term treatment with Bic should be carefully followed up.

β-Estradiol Induces Mitochondrial Apoptosis in Cervical Cancer Through the Suppression of AKT/NF-κB Signaling Pathway

Background: Cervical cancer is the fourth most prevalent gynecological cancer worldwide, which threatens women's health and causes cancer-related mortality. In the search for effective anticervical cancer drugs, we discovered that β-estradiol (E2), a patent drug for estrogen deficiency syndrome treatment, displays the most potent cytotoxicity against HeLa cells. Objective: This study aims to evaluate the growth inhibitory effect of β-estradiol on HeLa cells and explore its underlying mechanisms. Methods: CCK-8 assay was used to evaluate the cytotoxicity of 6 compounds against HeLa cells. Flow cytometric analysis and Hoechst 33258 staining assay were performed to detect cell cycle arrest and apoptosis induction. The collapse of the mitochondrial potential was observed by the JC-1 staining assay. The expression levels of proteins were examined by western blotting. Results: β-Estradiol, at high concentration, displays potent cytotoxicity against HeLa cells with an IC50 value of 18.71 ± 1.57 μM for 72 h treatment. β-Estradiol induces G2/M cell cycle arrest through downregulating Cyclin B1 and p-CDK1. In addition, β-estradiol-induced apoptosis is accompanied by the loss of mitochondrial potential, activation of the Caspase family, and altered Bax/Bcl-2 ratio. β-Estradiol markedly decreased the expression level of p-AKT and p-NF-κB. Conclusion: This study demonstrated that β-estradiol induces mitochondrial apoptosis in cervical cancer through the suppression of the AKT/NF-κB signaling pathway, indicating that β-estradiol may serve as a potential agent for cervical cancer treatment.

Ecological aspects of bivalve adaptation to salinity fluctuations on the example of Anadara Kagoshimensis

Impact of salinity stress on the ark clam (Anadara kagoshimensis (Tokunaga, 1906)) hemocyte functions were investigated using flow cytometry and light scattering technique. In control group water salinity was 18 ppm and experimental groups were carried at 14 ppm, 8 ppm, 35 ppm and 45 ppm. Hemolymph osmolarity decreased at hypoosmotic conditions and increased after hyperosmotic treatment. Osmotic stress induced changes in osmotic fragility of the ark clam hemocytes. Salinity 14 ppm did not affect the functional parameters of hemocytes. Incubation of ark clams at salinity and 35 ppm did not influence on the mitochondrial membrane potential of hemocytes but led to a decrease in hemocyte reactive oxygen species (ROS) production by 30 % compared to control. An increase in water salinity to 45 ppm and its decrease to 8 ppm induced substantial changes in the ROS production and mitochondrial membrane potential of hemocytes. Hyposalinity (8 ppm) led to an increase in ROS production by hemocytes (up to 2.4 times) and mitochondrial membrane potential (up to 1.3 times). An increase of salinity level from 18 ppm to 45 ppm decreased the total ability of hemocytes to produce ROS by 11% and increased mitochondrial potential of hemocytes by 150%.

Male Infertility: Pathogenetic Significance of Oxidative Stress and Antioxidant Defence (Review)

The basis of the pathogenesis of male infertility is the processes of peroxide oxidation of biological substrates, especially lipids and proteins. By destroying the sperm membrane, toxic peroxidation products reduce its motility and ability to fertilize the egg, which is determined by a decrease in the number of motile sperm in the ejaculate. These changes lead to complete or partial male infertility. The authors of the review found that is accompanied by a damaging effect on the structural and functional activity of the gonads and is manifested, in particular, by an imbalance in the hormonal background of the male body. Similar effects are characteristic of an increase in the content of reactive Nitrogen species and its metabolites, which cause nitrosative stress, which is also the cause of male hypofertility and is inseparable from the state of oxidative stress. In scientific work it is determined that the accumulation of harmful peroxidation products leads to damage and destruction of sperm DNA, reduced activity of acrosomal enzymes and mitochondrial potential of sperm, reduced overall antioxidant activity. This makes it impossible for an adequate response of the body. Multi component antioxidant defense system resists stress. It is represented by enzymatic and non-enzymatic links, which can neutralize harmful radicals and peroxidation products. It contributes to the full manifestation of reproductive function. The presence of powerful antioxidant properties of catalase, superoxide dismutase, and enzymes of the thiol-disulfide system, which form the enzymatic system of antioxidant protection, as well as selenium, zinc, copper, other trace elements, retinol, tocopherol, ascorbic acid, and vitamins as parts of the non-enzymatic system is shown. The efficiency of registration is substantiated thin biochemical shift detectors or complex methods, such as total antioxidant status of sperm or sperm plasma, mitochondrial membrane potential, etc along with simple markers of oxidative stress, such as diene conjugates, malonic dialdehyde, and metabolites of the Nitrogen Oxide cycle. Given the leading role of oxidative stress in the development of male hypofertility, the prospect of further research is the search for modern means for correction, especially among substances with pronounced redox activity

Triple Isozyme Lactic Acid Dehydrogenase Inhibition in Fully Viable MDA-MB-231 Cells Induces Cytostatic Effects That Are Not Reversed by Exogenous Lactic Acid

A number of aggressive human malignant tumors are characterized by an intensified glycolytic rate, over-expression of lactic acid dehydrogenase A (LDHA), and subsequent lactate accumulation, all of which contribute toward an acidic peri-cellular immunosuppressive tumor microenvironment (TME). While recent focus has been directed at how to inhibit LDHA, it is now becoming clear that multiple isozymes of LDH must be simultaneously inhibited in order to fully suppress lactic acid and halt glycolysis. In this work we explore the biochemical and genomic consequences of an applied triple LDH isozyme inhibitor (A, B, and C) (GNE-140) in MDA-MB-231 triple-negative breast cancer cells (TNBC) cells. The findings confirm that GNE-140 does in fact, fully block the production of lactic acid, which also results in a block of glucose utilization and severe impedance of the glycolytic pathway. Without a fully functional glycolytic pathway, breast cancer cells continue to thrive, sustain viability, produce ample energy, and maintain mitochondrial potential (ΔΨM). The only observable negative consequence of GNE-140 in this work, was the attenuation of cell division, evident in both 2D and 3D cultures and occurring in fully viable cells. Of important note, the cytostatic effects were not reversed by the addition of exogenous (+) lactic acid. While the effects of GNE-140 on the whole transcriptome were mild (12 up-regulated differential expressed genes (DEGs); 77 down-regulated DEGs) out of the 48,226 evaluated, the down-regulated DEGS collectively centered around a loss of genes related to mitosis, cell cycle, GO/G1–G1/S transition, and DNA replication. These data were also observed with digital florescence cytometry and flow cytometry, both corroborating a G0/G1 phase blockage. In conclusion, the findings in this work suggest there is an unknown element linking LDH enzyme activity to cell cycle progression, and this factor is completely independent of lactic acid. The data also establish that complete inhibition of LDH in cancer cells is not a detriment to cell viability or basic production of energy.

Accelerated apoptosis, oxidative stress, and cholinergic inflammation in blood of metalworkers

Metalworkers are exposed to numerous chemicals in their workplace environment, such as solvents, heavy metals, and metalworking fluids, that have a negative impact on their health. Furthermore, there is an increase in the prevalence of chronic diseases among metalworkers; however, the molecular mechanisms involved in this increased predisposition to chronic diseases are unclear. Considering that occupational exposure represents a potential risk for metalworkers, the aim of this study was to measure biomarkers of oxidative stress, inflammation, and cytotoxicity in the peripheral blood of metalworkers from Southern Brazil. The study included 40 metalworkers and 20 individuals who did not perform activities with any recognized exposure to chemical substances, such as those working in administration, commerce, and education, as controls. Cellular and molecular biomarkers as leukocyte viability, intracellular production of reactive species, mitochondrial mass and membrane potential and plasma lipid peroxidation, sulfhydryl groups, total antioxidant capacity, and butyrylcholinesterase activity were evaluated in the blood of metalworkers and controls. Metalworkers were found to have higher rates of apoptosis, increased production of reactive species, and increased mitochondrial potential and mass in leukocytes associated with decreased antioxidant defenses and increased activity of the butyrylcholinesterase enzyme in their plasma. It can be concluded that cytotoxicity, oxidative stress, and inflammation are involved in the multiplicity of health outcomes related to chemical exposure in the metalworking industry.

Differential Effects of Oleic and Palmitic Acids on Lipid Droplet-Mitochondria Interaction in the Hepatic Cell Line HepG2

Fatty acid overload, either of the saturated palmitic acid (PA) or the unsaturated oleic acid (OA), causes triglyceride accumulation into specialized organelles termed lipid droplets (LD). However, only PA overload leads to liver damage mediated by mitochondrial dysfunction. Whether these divergent outcomes stem from differential effects of PA and OA on LD and mitochondria joint dynamics remains to be uncovered. Here, we contrast how both fatty acids impact the morphology and interaction between both organelles and mitochondrial bioenergetics in HepG2 cells. Using confocal microscopy, we showed that short-term (2–24 h) OA overload promotes more and bigger LD accumulation than PA. Oxygen polarography indicated that both treatments stimulated mitochondrial respiration; however, OA favored an overall build-up of the mitochondrial potential, and PA evoked mitochondrial fragmentation, concomitant with an ATP-oriented metabolism. Even though PA-induced a lesser increase in LD-mitochondria proximity than OA, those LD associated with highly active mitochondria suggest that they interact mainly to fuel fatty acid oxidation and ATP synthesis (that is, metabolically “active” LD). On the contrary, OA overload seemingly stimulated LD-mitochondria interaction mainly for LD growth (thus metabolically “passive” LDs). In sum, these differences point out that OA readily accumulates in LD, likely reducing their toxicity, while PA preferably stimulates mitochondrial oxidative metabolism, which may contribute to liver damage progression.

Elucidating the Mechanisms of Leukemogenesis Driven By FBXO11 Depletion

Mutations in SKP1 and CUL1 (Zhang et. al. Oncol Lett 2018), which encode components of the SKP1-CUL1-F-BOX (SCF) ubiquitin E3-ligase complex, have previously been reported or characterized in AML. FBXO11, which encodes the substrate recognizing component, however, has not been studied in AML. We performed whole exome sequencing and RNA-seq on140 clinical AML samples and identified recurrent inactivating mutations in FBXO11. Of the components of the SCF FBXO11 complex, FBXO11 transcript expression is most significantly reduced in AML samples compared to normal. We show that loss of FBXO11 drives leukemogenesis through dysregulation of the novel target, LONP1, by reducing mitochondrial potential and promoting self-renewal. We found that UPS mutations co-occur with AML1-ETO (RUNX1-RUNX1T1) fusions and RAS mutations. Fbxo11 knockdown in mouse hematopoietic stem/progenitor cells (HSPC) cooperated with AML1-ETO to generate serially transplantable AML in mice. FBXO11 depletion in human cord-blood derived CD34+ cells (CD34+ CB), combined with AML1-ETO and a KRAS mutant, promoted stem cell maintenance and myeloid malignancy in a human xenotransplant model. Mass spectrometry analysis of FLAG-FBXO11 co-immunoprecipitating proteins in K562 cells identified mitochondrial protease, LONP1, as a top target. LONP1 protein expression did not vary with FBXO11 loss or overexpression, suggesting that LONP1 is not a degradation target of the SCF FBXO11complex. Knockdown of either FBXO11 or LONP1 resulted in myeloid bias in CD34+ CB in vitro, pointing to an activating role of FBXO11 on LONP1. Both FBXO11 and LONP1 depletion reduced mitochondrial membrane potential (MMP) in CD34+ CB and myeloid cell lines, aligning with the stemness phenotypes observed with FBXO11 depletion, as long-term hematopoietic stem cells (LT-HSCs) are characterized by low MMP (Mansell et. al. Cell Stem Cell 2021), and disruption of MMP promotes self-renewal in HSCs (Vannini et. al. Nat Commun 2016). As FBXO11 neddylates p53 to regulate transcription (Abida et. al. J. Biol. Chem 2007), we examined protein neddylation, and detected increased neddylation in immunoprecipitated LONP1 from FLAG-FBXO11-expressing K562 cells. As, neddylation regulates protein activation (Wu et. al. Nature 2005), our findings suggest that FBXO11 neddylation of LONP1 activates LONP1 to maintain mitochondrial function. Consequently, loss of FBXO11 function primes HSPC for self-renewal by reduction of MMP. To clarify the regulatory relationship between FBXO11 and LONP1, we performed RNA-seq on CD34+ CB cells expressing combinations of shRNAs targeting FBXO11 or LONP1, with overexpression of FLAG -FBXO11 or LONP1. Unsupervised clustering revealed that LONP1-overexpressing samples clustered with controls, suggesting that LONP1 requires modification by FBXO11 for functional effects. Using gene set enrichment analysis, we found that both FBXO11 and LONP1 depletion enriched for HSC and LSC (leukemic stem cell) gene sets. Knockdown of LONP1 reversed the effect of FLAG-FBXO11 overexpression, supporting a model of LONP1 being a downstream mediator of FBXO11 function. Both FBXO11 and LONP1 depletion enriched for a gene set composed of mitochondrial electron transport chain complex (ETC) genes, potentially reflecting a transcriptional response to loss of functional ETC activity, as suggested by accumulation of misfolded ETC proteins with knockdown of LONP1 (Ghosh et. al. Oncogene 2019). In this work, we demonstrate the leukemogenic effects of FBXO11 loss. We draw a novel connection between the UPS and the mitochondrial protease system with the identification of LONP1 as an FBXO11 target that regulates hematopoiesis. Disclosures No relevant conflicts of interest to declare.