Cocaine-derived hippuric acid activates mtDNA-STING signaling in alcoholic liver disease: Implications for alcohol and cocaine co-abuse.

The simultaneous abuse of alcohol-cocaine is known to cause stronger and more unpredictable cellular damage in the liver, heart, and brain. However, the mechanistic crosstalk between cocaine and alcohol in liver injury remains unclear. The findings revealed cocaine-induced liver injury and inflammation in both marmosets and mice. Of note, co-administration of cocaine and ethanol in mice causes more severe liver damage than individual treatment. The metabolomic analysis confirmed that hippuric acid (HA) is the most abundant metabolite in marmoset serum after cocaine consumption and that is formed in primary marmoset hepatocytes. HA, a metabolite of cocaine, increases mitochondrial DNA leakage and subsequently increases the production of proinflammatory factors via STING signaling in Kupffer cells (KCs). In addition, conditioned media of cocaine-treated KC induced hepatocellular necrosis via alcohol-induced TNFR1. Finally, disruption of STING signaling in vivo ameliorated co-administration of alcohol- and cocaine-induced liver damage and inflammation. These findings postulate intervention of HA-STING-TNFR1 axis as a novel strategy for treatment of alcohol- and cocaine-induced excessive liver damage.

Targeting Mitochondrial Guanine Quadruplexes for Photoactivatable Chemotherapy in Hypoxic Environments.

A first example of a mitochondrial G-quadruplex (mitoG4s) targeted Ru(II) photooxidant complex is reported. The complex, Ru-TAP-PDC3 induces photodamage toward guanine quadruplexes (G4s) located in the mitochondrial genome under hypoxic and normoxic conditions. Ru-TAP-PDC3 shows high affinity for mitoG4s and localises within mitochondria of live HeLa cells. Immunolabelling with anti-G4 antibody, BG4, confirms Ru-TAP-PDC3 associates with G4s within the mitochondria of fixed cells. The complex induces depletion of mtDNA in live cells under irradiation at 405 nm, confirmed by loss of PicoGreen signal from mitochondria. Biochemical studies confirm this process induces apoptosis. The complex shows low dark toxicity and an impressive phototoxicity index (PI) of >89 was determined in Hela under very low intensity irradiation, 5 J/cm2. The phototoxicity is thought to operate through both Type II singlet oxygen and Type III pathways depending on normoxic or hypoxic conditions, from live cell assays and plasmid DNA cleavage. Overall, we demonstrate targeting mitoG4s and mtDNA with a photooxidant is a potent route to achieving apoptosis under hypoxic conditions that can be extended to phototherapy.

Vitamin C inhibits NLRP3 inflammasome activation and delays the development of age-related hearing loss in male C57BL/6 mice.

The efficacy of vitamin C in age-related hearing loss, i.e., presbycusis, remains debatable. On a separate note, inflammation induced by the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is involved in the progression of presbycusis. In this study, we investigated the effect of vitamin C on male C57BL/6 mice's presbycusis and NLRP3 inflammasome. The results showed that vitamin C treatment improved hearing, reduced the production of inflammatory factors, inhibited NLRP3 inflammasome activation, and decreased cytosolic mitochondrial DNA (mtDNA) in the C57BL/6 mouse cochlea, inferior colliculus, and auditory cortex. According to this study, vitamin C protects auditory function in male C57BL/6 presbycusis mice through reducing mtDNA release, inhibiting the NLRP3 inflammasome activation in the auditory pathway. Our study provides a theoretical basis for applying vitamin C to treat presbycusis.

Venturicidin A affects the mitochondrial membrane potential and induces kDNA loss in Trypanosoma brucei.

Neglected tropical diseases caused by trypanosomatid parasites have devastating health and economic consequences, especially in tropical areas. New drugs or new combination therapies to fight these parasites are urgently needed. Venturicidin A, a macrolide extracted from Streptomyces, inhibits the ATP synthase complex of fungi and bacteria. However, its effect on trypanosomatids is not fully understood. In this study, we tested venturicidin A on a panel of trypanosomatid parasites using Alamar Blue assays and found it to be highly active against Trypanosoma brucei and Leishmania donovani, but much less so against Trypanosoma evansi. Using fluorescence microscopy, we observed a rapid loss of the mitochondrial membrane potential in T. brucei bloodstream forms upon venturicidin A treatment. Additionally, we report the loss of mitochondrial DNA in approximately 40%-50% of the treated parasites. We conclude that venturicidin A targets the ATP synthase of T. brucei, and we suggest that this macrolide could be a candidate for anti-trypanosomatid drug repurposing, drug combinations, or medicinal chemistry programs.

Alcohol exposure during pregnancy induces cardiac mitochondrial damage in offspring mice.

BACKGROUND: Prenatal alcohol exposure (PAE) has been linked to congenital heart disease and fetal alcohol syndrome. The heart primarily relies on mitochondria to generate energy, so impaired mitochondrial function due to alcohol exposure can significantly affect cardiac development and function. Our study aimed to investigate the impact of PAE on myocardial and mitochondrial functions in offspring mice. METHODS: We administered 30% alcohol (3 g/kg) to pregnant C57BL/6 mice during the second trimester. We assessed cardiac function by transthoracic echocardiography, observed myocardial structure and fibrosis through staining tests and electron transmission microscopy, and detected cardiomyocyte apoptosis with dUTP nick end labeling assay and real-time quantitative PCR. Additionally, we measured the reactive oxygen species content, ATP level, and mitochondrial DNA copy number in myocardial mitochondria. Mitochondrial damage was evaluated by assessing the level of mitochondrial membrane potential and the opening degree of mitochondrial permeability transition pores. RESULTS: Our findings revealed that PAE caused cardiac systolic dysfunction, ventricular enlargement, thinned ventricular wall, cardiac fibrosis in the myocardium, scattered loss of cardiomyocytes, and disordered arrangement of myocardial myotomes in the offspring. Furthermore, we observed a significant increase in mitochondrial reactive oxygen species content, a decrease in mitochondrial membrane potential, ATP level, and mitochondrial DNA copy number, and sustained opening of mitochondrial permeability transition pores in the heart tissues of the offspring. CONCLUSIONS: These results indicated that PAE had adverse effects on the cardiac structure and function of the newborn mice and could trigger oxidative stress in their myocardia and contribute to mitochondrial dysfunction.

Oxidative damage to mitochondrial DNA in maternal zebrafish (Danio rerio) exposed to dibutyl phthalate at environmentally relevant level.

Dibutyl phthalate (DBP) is a widely-used plasticizer that is dispersed in various environments, causing significant pollution and health risks. The toxic mechanism of DBP has been discussed in recent years, while the susceptibility of mitochondrial DNA (mtDNA) to DBP exposure and the resulting damage remain unclear. In this study, maternal zebrafish were exposed to environmentally relevant concentration of DBP for 0, 2, 4, and 6 weeks. Results showed that DBP exposure impaired health status, leading to the reduced body length and weight, condition factor, hepatosomatic index, and gonadosomatic index. Furthermore, DBP exposure induced oxidative stress and ATP deficiency in the gill and liver in a time-dependent manner. The oxidized mtDNA (ox-mtDNA) levels in the D-loop and ND1 regions were assessed in different tissues, showing distinct response patterns. The high energy-consuming tissues such as heart, brain, gill, and liver exhibited elevated susceptibility to mitochondrial damage, with a rapid increase in ox-mtDNA levels in the short term. Conversely, in muscle, ovary, eggs, and offspring, ox-mtDNA gradually accumulated over the exposure period. Notably, the ox-mtDNA levels in the D-loop region of blood showed a prompt response to DBP exposure, making it convenient for evaluation. Additionally, decreased hatching rates, increased mortality, lipoperoxidation, and depressed swimming performance were observed in offspring following maternal DBP exposure, suggesting the inherited impairments of maternal mtDNA. These findings highlight the potential for ox-mtDNA to serve as a convenient biomarker for environmental contamination, aiding in ecological risk assessment and forewarning systems in aquatic environment.

Evaluation of genotoxic and mitotoxic effects of TAF-loaded chitosan nanoparticles in HepG2 cells.

Tenofovir alafenamide (TAF) is a new drug from the nucleotide reverse transcriptase inhibitor group approved for the treatment of chronic Hepatitis B in 2016. With this study, we aimed to test whether possible cellular toxicity can be reduced by controlled drug release as a result of loading with chitosan nanoparticles (CHS). We investigated the genotoxic and mitotoxic effects of 45 µM TAF-loaded CHS and TAF-only on HepG2 cells by micronucleus (MN), comet assay, determination of mtDNA quantification, mitochondrial membrane potential (ΔΨm), and ROS levels. Additionally, we compared the samples by RNAseq analyses to reveal the transcriptional responses to each regimen. In terms of genotoxic tests, although MN and comet were found higher in all experimental treatment conditions, the encapsulation of CHS reduced the genotoxicity of TAF. MtDNA level was found to be lower in the TAF treatment, whereas it was higher in CHS and CHS-TAF treatments. The TAF-loaded CHS and TAF treatments had an impaired ΔΨm value. Cellular ROS levels were higher in all treatment conditions. According to the analyses of gene expression patterns; CHS-only changed the expression of relatively few genes (187 genes), while TAF changed the expression of the 1974 genes and TAF-loaded CHS changed the expression of 734 genes. Considering the gene expression numbers, CHS encapsulation of TAF significantly reduced the number of genes that were differentially expressed by TAF-only. Overall, we observed that TAF has genotoxic and mitotoxic effects on HepG2 cells, and upon encapsulation with CHS, its genotoxic and mitotoxic effects were decreased.

mtDNA amplifies beryllium sulfate-induced inflammatory responses via the cGAS-STING pathway in 16HBE cells.

Beryllium sulfate (BeSO4) can cause inflammation through the mechanism, which has not been elucidated. Mitochondrial DNA (mtDNA) is a key contributor of inflammation. With mitochondrial damage, released mtDNA can bind to specific receptors (e.g., cGAS) and then activate related pathway to promote inflammatory responses. To investigate the mechanism of mtDNA in BeSO4-induced inflammatory response in 16HBE cells, we established the BeSO4-induced 16HBE cell inflammation model and the ethidium bromide (EB)-induced ρ016HBE cell model to detect the mtDNA content, oxidative stress-related markers, mitochondrial membrane potential, the expression of the cGAS-STING pathway, and inflammation-related factors. Our results showed that BeSO4 caused oxidative stress, decline of mitochondrial membrane potential, and the release of mtDNA into the cytoplasm of 16HBE cells. In addition, BeSO4 induced inflammation in 16HBE cells by activating the cGAS-STING pathway. Furthermore, mtDNA deletion inhibited the expression of cGAS-STING pathway, IL-10, TNF-α, and IFN-ß. This study revealed a novel mechanism of BeSO4-induced inflammation in 16HBE cells, which contributes to the understanding of the molecular mechanism of beryllium and its compounds-induced toxicity.

Biochemical and molecular evaluation of oxidative stress and mitochondrial damage in fruit fly exposed to carmoisine.

BACKGROUND: In today's world, appearance is an important factor in almost all areas of our lives. Therefore, it has become common to use dyes to color foods to make them look appetizing and visually appealing. However, food additives have negative effects on biochemical processes in cells at both high and low doses. METHODS AND RESULTS: This study investigated the effect of carmoisine, a commonly used food coloring, on oxidative stress and damage parameters in Drosophila melanogaster in terms of both enzymatic and gene expression. The change in mitochondrial DNA copy number (mtDNA-CN), a marker of oxidative stress, was also examined. When the data obtained were analyzed, it was observed that carmoisine caused a significant decrease in GSH levels depending on the increase in dose. SOD, CAT, GPx, and AChE enzyme activities and gene expression levels were also found to be significantly decreased. All groups also showed a significant decrease in mtDNA-CN. The effect of carmoisine on Drosophila melanogaster morphology was also investigated in our study. However, no significant change was observed in terms of morphological development in any group. CONCLUSIONS: When all the findings were evaluated together, it was observed that carmoisin triggered oxidative stress and these effects became more risky at high doses. Therefore, we believe that the consumer should be made more aware of the side effects of azo dyes in food and that the type and concentration of each substance added to food should be specified.

Lack of detectable sex differences in the mitochondrial function of Caenorhabditis elegans.

BACKGROUND: Sex differences in mitochondrial function have been reported in multiple tissue and cell types. Additionally, sex-variable responses to stressors including environmental pollutants and drugs that cause mitochondrial toxicity have been observed. The mechanisms that establish these differences are thought to include hormonal modulation, epigenetic regulation, double dosing of X-linked genes, and the maternal inheritance of mtDNA. Understanding the drivers of sex differences in mitochondrial function and being able to model them in vitro is important for identifying toxic compounds with sex-variable effects. Additionally, understanding how sex differences in mitochondrial function compare across species may permit insight into the drivers of these differences, which is important for basic biology research. This study explored whether Caenorhabditis elegans, a model organism commonly used to study stress biology and toxicology, exhibits sex differences in mitochondrial function and toxicant susceptibility. To assess sex differences in mitochondrial function, we utilized four male enriched populations (N2 wild-type male enriched, fog-2(q71), him-5(e1490), and him-8(e1498)). We performed whole worm respirometry and determined whole worm ATP levels and mtDNA copy number. To probe whether sex differences manifest only after stress and inform the growing use of C. elegans as a mitochondrial health and toxicologic model, we also assessed susceptibility to a classic mitochondrial toxicant, rotenone. RESULTS: We detected few to no large differences in mitochondrial function between C. elegans sexes. Though we saw no sex differences in vulnerability to rotenone, we did observe sex differences in the uptake of this lipophilic compound, which may be of interest to those utilizing C. elegans as a model organism for toxicologic studies. Additionally, we observed altered non-mitochondrial respiration in two him strains, which may be of interest to other researchers utilizing these strains. CONCLUSIONS: Basal mitochondrial parameters in male and hermaphrodite C. elegans are similar, at least at the whole-organism level, as is toxicity associated with a mitochondrial Complex I inhibitor, rotenone. Our data highlights the limitation of using C. elegans as a model to study sex-variable mitochondrial function and toxicological responses.

Mitochondrial DNA Stress-Mediated Health Risk to Dibutyl Phthalate Contamination on Zebrafish (Danio rerio) at Early Life Stage.

Plastics contaminations are found globally and fit the exposure profile of the planetary boundary threat. The plasticizer of dibutyl phthalate (DBP) leaching has occurred and poses a great threat to human health and the ecosystem for decades, and its toxic mechanism needs further comprehensive elucidation. In this study, environmentally relevant levels of DBP were used for exposure, and the developmental process, oxidative stress, mitochondrial ultrastructure and function, mitochondrial DNA (mtDNA) instability and release, and mtDNA-cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway with inflammatory responses were measured in zebrafish at early life stage. Results showed that DBP exposure caused developmental impairments of heart rate, hatching rate, body length, and mortality in zebrafish embryo. Additionally, the elevated oxidative stress damaged mitochondrial ultrastructure and function and induced oxidative damage to the mtDNA with mutations and instability of replication, transcription, and DNA methylation. The stressed mtDNA leaked into the cytosol and activated the cGAS-STING signaling pathway and inflammation, which were ameliorated by co-treatment with DBP and mitochondrial reactive oxygen species (ROS) scavenger, inhibitors of cGAS or STING. Furthermore, the larval results suggest that DBP-induced mitochondrial toxicity of energy disorder and inflammation were involved in the developmental defects of impaired swimming capability. These results enhance the interpretation of mtDNA stress-mediated health risk to environmental contaminants and contribute to the scrutiny of mitochondrial toxicants.

YiQi GuBen capsule alleviates OVA-induced asthma through improving mitochondrial dysfunction.

Objective: This study aims to explore the effect of YiQi GuBen capsule on improving mitochondrial dysfunction in an animal model of asthma.Methods: The mice (n = 8) were divided into four groups including control (NC), ovalbumin (OVA), dexamethasone (OVA + DEX), and YiQi GuBen (OVA + YQGB) groups. Firstly, we established an OVA-induced mouse asthma model except for the NC group, which then were treated with dexamethasone and YiQi GuBen capsule. Subsequently, HE staining and Masson staining were used for pathological analysis of mice lung tissues. Next, we used transmission electron microscopy (TEM) to observe the effect of the Yiqi Guben capsule on the ultrastructure of mitochondria. Flow cytometry was used to analyze the ROS level, membrane potential, and the number of mitochondria in lung tissue. Moreover, we analyzed the copy number of mitochondrial DNA (mtDNA) and the expression levels of activator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM).Results: The results of the pathological analysis showed that after treatment with the YiQi GuBen capsule, the lung tissue damage was significantly reduced. In addition, we observed that the ultrastructural damage of mitochondria was improved. Flow cytometry proved that after treatment with the YiQi GuBen capsule, the level of ROS in the mitochondria was effectively reduced, while the mitochondrial membrane potential decreased and the number increased significantly. Moreover, we found that the copy number of mtDNA was significantly increased and the expression levels of PGC-1α and TFAM were significantly upgraded.Conclusion: This study suggests YiQi GuBen capsule can effectively improve mitochondrial dysfunction in the OVA-induced mouse model.

Diterpenoid honatisine overcomes temozolomide resistance in glioblastoma by inducing mitonuclear protein imbalance through disruption of TFAM-mediated mtDNA transcription.

BACKGROUND: Glioblastoma (GBM) represents as the most formidable intracranial malignancy. The systematic exploration of natural compounds for their potential applications in GBM therapy has emerged as a pivotal and fruitful avenue of research. PURPOSE: In the present study, a panel of 96 diterpenoids was systematically evaluated as a repository of potential antitumour agents. The primary objective was to discern their potency in overcoming resistance to temozolomide (TMZ). Through an extensive screening process, honatisine, a heptacyclic diterpenoid alkaloid, emerged as the most robust candidate. Notably, honatisine exhibited remarkable efficacy in patient-derived primary and recurrent GBM strains. Subsequently, we subjected this compound to comprehensive scrutiny, encompassing GBM cultured spheres, GBM organoids (GBOs), TMZ-resistant GBM cell lines, and orthotopic xenograft mouse models of GBM cells. RESULTS: Our investigative efforts delved into the mechanistic underpinnings of honatisine's impact. It was discerned that honatisine prompted mitonuclear protein imbalance and elicited the mitochondrial unfolded protein response (UPRmt). This effect was mediated through the selective depletion of mitochondrial DNA (mtDNA)-encoded subunits, with a particular emphasis on the diminution of mitochondrial transcription factor A (TFAM). The ultimate outcome was the instigation of deleterious mitochondrial dysfunction, culminating in apoptosis. Molecular docking and surface plasmon resonance (SPR) experiments validated honatisine's binding affinity to TFAM within its HMG-box B domain. This binding may promote phosphorylation of TFAM and obstruct the interaction of TFAM bound to heavy strand promoter 1 (HSP1), thereby enhancing Lon-mediated TFAM degradation. Finally, in vivo experiments confirmed honatisine's antiglioma properties. Our comprehensive toxicological assessments underscored its mild toxicity profile, emphasizing the necessity for a thorough evaluation of honatisine as a novel antiglioma agent. CONCLUSION: In summary, our data provide new insights into the therapeutic mechanisms underlying honatisine's selective inducetion of apoptosis and its ability to overcome chemotherapy resistance in GBM. These actions are mediated through the disruption of mitochondrial proteostasis and function, achieved by the inhibition of TFAM-mediated mtDNA transcription. This study highlights honatisine's potential as a promising agent for glioblastoma therapy, underscoring the need for further exploration and investigation.

SS-31 inhibits mtDNA-cGAS-STING signaling to improve POCD by activating mitophagy in aged mice.

BACKGROUND: Neuroinflammation is crucial in the development of postoperative cognitive dysfunction (POCD), and microglial activation is an active participant in this process. SS-31, a mitochondrion-targeted antioxidant, is widely regarded as a potential drug for neurodegenerative diseases and inflammatory diseases. In this study, we sought to explore whether SS-31 plays a neuroprotective role and the underlying mechanism. METHODS: Internal fixation of tibial fracture was performed in 18-month-old mice to induce surgery-associated neurocognitive dysfunction. LPS was administrated to BV2 cells to induce neuroinflammation. Neurobehavioral deficits, hippocampal injury, protein expression, mitophagy level and cell state were evaluated after treatment with SS-31, PHB2 siRNA and an STING agonist. RESULTS: Our study revealed that SS-31 interacted with PHB2 to activate mitophagy and improve neural damage in surgically aged mice, which was attributed to the reduced cGAS-STING pathway and M1 microglial polarization by decreased release of mitochondrial DNA (mtDNA) but not nuclear DNA (nDNA). In vitro, knockdown of PHB2 and an STING agonist abolished the protective effect of SS-31. CONCLUSIONS: SS-31 conferred neuroprotection against POCD by promoting PHB2-mediated mitophagy activation to inhibit mtDNA release, which in turn suppressed the cGAS-STING pathway and M1 microglial polarization.

Effects of Cadmium Exposure on Oxidative Stress in Atherosclerotic Rats by Downregulating TopBP1 Expression to Induce Mitochondrial DNA Damage.

Objective: To explore the mechanism of the effect of cadmium exposure on TopBP1-induced mitochondrial DNA damage in atherosclerotic rats to affect oxidative stress. Methods: 50 rats were established atherosclerotic model, and they were divided into model control group (MC group), low-dose cadmium exposure group (LD group), medium-dose cadmium exposure group (MD group), high-dose cadmium exposure group (HD group), and positive control group, with 10 rats in each group. Rats in the LD group, MD group, and HD group were intraperitoneally injected with different doses of cadmium acetate solution for intervention, rats in the PC group were intraperitoneally injected with oxidized banking solution, and those in the MC group were injected with normal saline. 10 rats were taken as the normal control group (NC group). Human umbilical vein endothelial cells were taken for cell experiments, normal saline was added as the blank control group (group A), cadmium acetate solution was added (group B), oxidized bankning solution was added (Group C), and oxidized bankning solution and cadmium acetate solution were added (Group D). Western blot and fluorescence quantitative PCR were used to detect the protein and mRNA expressions respectively. ROS, MDA, and SOD were detected by ELISA, apoptosis of endothelial cells was detected by flow cytometry, and arterial plaque damage was observed by oil red O staining. Results: The relative expressions of TopBP, Bax, and Bcl-2 proteins in rat aortic tissues in each group were significantly different (all P < .05). The relative expressions of TopBP1 and Bcl-2 proteins in the aortic tissues of rats in NC group, MC group, LD group, MD group, HD group, and PC group decreased (all P < .05), while the relative expressions of Bax protein in those groups were increased (all P < .05). Similarly, the relative expression levels of Topbp1mRNA, BaxmRNA, and Bcl-2mRNA in the aortic tissues of rats in each group were significantly different (all P < .05). There were statistically significant differences in the expression levels of ROS, MDA, SOD, and mtDNA expression levels in the aortic tissues of rats in each group. There were statistically significant differences in TopBP1, Topbp1mRNA, and mtDNA among groups (all P < .05); while the relative expression of TopBP1 and Topbp1mRNA in groups A, B, C, and D decreased (all P < .05), the expression levels of mtDNA in those group increased (all P < .05), and the apoptosis rates of endothelial cells were also increased (all P < .05). Conclusion: Cadmium exposure can down-regulate the expression of TopBP1 in atherosclerotic rats, aggravate mitochondrial DNA damage, promote oxidative stress response, and then induce the development of atherosclerosis.

Exploiting DNA Ligase III addiction of multiple myeloma by flavonoid Rhamnetin.

BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.

Movement of Mitochondria with Mutant DNA through Extracellular Vesicles Helps Cancer Cells Acquire Chemoresistance.

Triple negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer with the worst prognosis after chemo- or radiation therapy. This is mainly due to the development of cancer chemoresistance accompanied by tumor recurrence. In this work, we investigated a new mechanism of acquired chemoresistance of TNBC cells. We showed that extracellular vehicles (EVs) of chemoresistant TNBC cells can transfer mitochondria to sensitive cancer cells, thus increasing their chemoresistance. Such transfer, but with less efficiency, can be carried out over short distances using tunneling nanotubes. In addition, we showed that exosome fractions carrying mitochondria from resistant TNBC cells contribute to acquired chemoresistance by increasing mtDNA levels with mutations in the mtND4 gene responsible for tumorigenesis. Blocking mitochondrial transport by exosome inhibitors, including GW4869, reduced acquired TNBC chemoresistance. These results could lead to the identification of new molecular targets necessary for more effective treatment of this type of cancer.

The effects of Acyclovir administration to NCI-H1975 non-small cell lung cancer cells.

The biochemical mechanisms by which the antiviral drug Acyclovir (ACV) may induce anticancer effects even without detecting human herpesviruses (HHVs) are still poorly understood. Herein, we investigated for the first time how NCI-H1975 non-small cell lung cancer cells responded in vitro to ACV administration by exploring mitochondrial damage and apoptosis induction. We confirmed ACV ability to cause the inhibition of cancer cell growth even without detecting intracellular HHVs; the drug also significantly inhibited the colony formation capacity of NCI-H1975 cells. Cell cycle analysis revealed an increase of the sub-G1 hypodiploid peak after ACV treatment; the activation of caspase-3 and the presence of DNA laddering sustained the capacity of the drug to induce apoptotic cell death. Regarding mitochondrial toxicity, a reduction of mitochondrial membrane potential, altered mitochondrial size and shape, and mtDNA damage were found after ACV administration. Furthermore, an increment of intracellular reactive oxygen species levels as well as the upregulation of NudT3 involved in DNA repair mechanisms were observed. Altogether, these findings suggest that mitochondria may be possible initial targets and/or sites of ACV cytotoxicity within cancer cells in the absence of intracellular HHVs.

Nuclear Genome-Encoded Long Noncoding RNAs and Mitochondrial Damage in Diabetic Retinopathy.

Retinal mitochondria are damaged in diabetes-accelerating apoptosis of capillary cells, and ultimately, leading to degenerative capillaries. Diabetes also upregulates many long noncoding RNAs (LncRNAs), including LncMALAT1 and LncNEAT1. These RNAs have more than 200 nucleotides and no open reading frame for translation. LncMALAT1 and LncNEAT1 are encoded by nuclear genome, but nuclear-encoded LncRNAs can also translocate in the mitochondria. Our aim was to investigate the role of LncMALAT1 and LncNEAT1 in mitochondrial homeostasis. Using human retinal endothelial cells, the effect of high glucose on LncMALAT1 and LncNEAT1 mitochondrial localization was examined by RNA fluorescence in situ hybridization. The role of these LncRNAs in mitochondrial membrane potential (by JC-I staining), mtDNA integrity (by extended length PCR) and in protective mtDNA nucleoids (by SYBR green staining) was examined in MALAT1- or NEAT1-siRNA transfected cells. High glucose increased LncMALAT1 and LncNEAT1 mitochondrial expression, and MALAT1-siRNA or NEAT1-siRNA ameliorated glucose-induced damage to mitochondrial membrane potential and mtDNA, and prevented decrease in mtDNA nucleoids. Thus, increased mitochondrial translocation of LncMALAT1 or LncNEAT1 in a hyperglycemic milieu plays a major role in damaging the mitochondrial structural and genomic integrity. Regulation of these LncRNAs can protect mitochondrial homeostasis, and ameliorate formation of degenerative capillaries in diabetic retinopathy.

TNF leads to mtDNA release and cGAS/STING-dependent interferon responses that support inflammatory arthritis.

Tumor necrosis factor (TNF) is a key driver of several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, in which affected tissues show an interferon-stimulated gene signature. Here, we demonstrate that TNF triggers a type-I interferon response that is dependent on the cyclic guanosine monophosphate-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. We show that TNF inhibits PINK1-mediated mitophagy and leads to altered mitochondrial function and to an increase in cytosolic mtDNA levels. Using cGAS-chromatin immunoprecipitation (ChIP), we demonstrate that cytosolic mtDNA binds to cGAS after TNF treatment. Furthermore, TNF induces a cGAS-STING-dependent transcriptional response that mimics that of macrophages from rheumatoid arthritis patients. Finally, in an inflammatory arthritis mouse model, cGAS deficiency blocked interferon responses and reduced inflammatory cell infiltration and joint swelling. These findings elucidate a molecular mechanism linking TNF to type-I interferon signaling and suggest a potential benefit for therapeutic targeting of cGAS/STING in TNF-driven diseases.