Search Results (53)

Background: Colorectal cancer (CRC) is a prevalent global malignancy with particularly challenging treatment outcomes in advanced stages. Oxaliplatin (OXA) is a frontline chemotherapeutic agent for CRC. However, 15% to 50% of stage III patients experience recurrence due to drug resistance. Elucidating the molecular mechanisms underlying OXA resistance is, therefore, crucial for improving CRC prognosis. The role of DIRAS1, a RAS superfamily member with reported tumor-suppressive functions in various cancers, remains poorly defined in CRC. Methods: The effects of DIRAS1 on CRC cell proliferation and migration were evaluated using MTT, wound healing, and colony formation assays. Stable cell lines with knockdown or overexpression of DIRAS1 and PHB1 were established via plasmid and lentiviral systems. Drug sensitivity to OXA was assessed through cytotoxicity assays and IC₅₀ determination. Clinical relevance was validated through immunohistochemical analysis of CRC tissue samples. Transcriptomic sequencing was performed to explore downstream regulatory mechanisms. Results: DIRAS1 expression was positively correlated with OXA resistance and was significantly upregulated following prolonged chemotherapy exposure. Silencing DIRAS1 reduced the IC₅₀ of OXA in vitro and increased tumor sensitivity to OXA in vivo. Transcriptome analysis identified PHB1 as a downstream effector of DIRAS1. Functional studies revealed that PHB1 contributes to chemoresistance by maintaining mitochondrial stability. Conclusions: This study identifies DIRAS1 as a key contributor to OXA resistance in CRC by modulating PHB1 expression and mitochondrial function. Targeting the DIRAS1–PHB1 axis may offer a novel therapeutic strategy to overcome chemoresistance in CRC. Full article

Background: The limited regenerative capacity of adult mammalian cardiomyocytes (CMs) poses a significant challenge for cardiac repair following myocardial infarction. In contrast to adult mammals, CMs in zebrafish and newt hearts retain a lifelong capacity for proliferation and cardiac regeneration. Likewise, neonatal mice exhibit a brief postnatal period, during which CMs retain the ability to proliferate and contribute to myocardial repair, which markedly diminishes within the first week of life. Emerging evidence indicates that adult CM cell cycle progression is critically influenced by oxidative stress. Adult mammalian CMs possess a high mitochondrial content to meet their substantial energy demands. However, this also leads to elevated reactive oxygen species (ROS) production, resulting in DNA damage and subsequent cell cycle arrest. We hypothesize that reducing the mitochondrial content in adult CMs will mitigate ROS production, thereby facilitating cell cycle progression. Methods: Adult CMs were isolated from adult rats (≥12 weeks old). To induce mitophagy, adult CMs were transfected with parkin-expressing plasmid and then treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial protonophore, for 7 days. Post-treatment assessments included the quantification of adult CM proliferation, mitochondrial content, and ROS levels. Results: CCCP-treated adult CMs exhibited a significant increase in proliferation markers, including EdU incorporation, KI67, phospho-histone H3, and Aurora B. Furthermore, CCCP treatment significantly reduced the mitochondrial content, as evidenced by decreased MitoTracker, TMRM, and Tom20 staining compared to controls. This was accompanied by electron microscopy analysis, which showed a significant reduction in the mitochondrial number in the adult CM after CCCP treatment. Moreover, our results also demonstrate a marked reduction in oxidative stress, demonstrated by lower 123-dihydro-rhodamine (123-DHR), CellROX signals, and VDAC. Conclusions: Our findings demonstrate that CCCP-mediated mitochondrial depletion reduces oxidative stress and promotes cell cycle re-entry in adult CM. This study provides direct experimental evidence and substantiates the role of elevated mitochondria and ROS levels in adult CM cell cycle exit. Full article

Purpose: Doxorubicin (DOX) is a broad-spectrum anti-tumor anthracycline drug. However, its clinical application is greatly limited due to the side effect of cardiotoxicity. Caffeic acid phenethyl ester (CAPE) is one of the major biologically active compounds isolated from propolis, which is effective in the treatment of cardiovascular diseases. The purpose of this study aimed to explore the possible mechanism of CAPE’s protective effect on DOX-induced cardiotoxicity (DIC). Methods: In vivo, a DIC model was established by the intraperitoneal injection of 3 mg/kg DOX. The cardiac function of mice was monitored by electrocardiograms. Histopathological changes in myocardial tissue were detected by H&E staining. Serum samples were tested for the level of markers of myocardial injury. In vitro, transmission electron microscopy was used to assess the mitochondrial damage. Oxidative stress was measured by flow cytometry and mitochondrial respiration analysis. Necroptosis pathway changes were detected by Western blotting. Furthermore, the overexpression plasmid of a key necroptosis gene, necroptosis inhibitor or ROS inducer/inhibitor was applied to H9c2 and AC16 cells to explore whether CAPE exerted a protective effect against DIC through the cross-talk mediated by ROS and MLKL. Results: CAPE could improve the cardiac function and protect against myocardial tissue. CAPE pre-administration treatment attenuated the DOX-induced generation of ROS, protected mitochondrial functions and inhibited necroptosis. Moreover, there was cross-talk between the ROS and necroptosis. CAPE could protect against DIC by inhibiting the ROS-MLKL signaling that regulated the cross-talk. Conclusions: CAPE alleviated the oxidative stress and necroptosis of DIC, indicating that the cross-talk mediated by ROS-MLKL signaling may be a potential therapeutic mechanism for clinical DIC. Full article

Carnitine palmitoyltransferase 1 (CPT1), which catalyzes the rate-limiting step of fatty acid oxidation, has been implicated in therapeutic approaches to several human diseases characterized by aberrant lipid metabolism. The isoform-specific quantification of CPT1 activity is essential in the characterization of small molecule inhibitors of CPT1, but several existing means to quantify enzymatic activity, including the use of radioisotope-labeled carnitine, are not amenable to scalable, high throughput screening. Here, we demonstrate that mitochondrial extracts from Expi293 cells transfected with a CPT1a plasmid are a reliable and robust source of catalytically active human CPT1. Moreover, with a source of catalytically active enzyme in hand, we modified a previously reported colorimetric method of coenzyme A (CoA) easily scalable to a 96-well format for the screening of CPT1a inhibitors. This assay platform was validated by two previously reported inhibitors of CPT1a: R-etomoxir and perhexiline. To further demonstrate the applicability of this method in small molecule screening, we prepared and screened a library of 87 known small molecule APIs, validating the inhibitory effect of chlorpromazine on CPT1. Full article

It was previously reported that tyrosine 97 (Y97) of cytochrome c is phosphorylated in cow heart tissue under physiological conditions. Y97 phosphorylation was shown to partially inhibit respiration in vitro in the reaction with purified cytochrome c oxidase. Here, we use phosphomimetic Y97E Cytc to further characterize the functional effects of this modification both in vitro and in cell culture models. In vitro, phosphomimetic Y97E Cytc showed lower activity in the reaction with purified cow heart cytochrome c oxidase (COX), decreased caspase-3 activity, and reduced rate of reduction. Additionally, the phosphomimetic Y97E Cytc tended to be resistant to heme degradation and showed an increased rate of oxidation. Intact mouse Cytc double knockout fibroblasts were transfected with plasmids coding for phosphomimetic Y97E Cytc and other variants. Compared to cells expressing wild-type Cytc, the cells expressing phosphomimetic Y97E Cytc showed reduced respiration, mitochondrial membrane potential, and reactive oxygen species production, and protection from apoptosis. In an oxygen–glucose deprivation/reoxygenation cell culture model of ischemia/reperfusion injury, mitochondrial membrane potential and reactive oxygen species production were decreased. These data show that Cytc phosphorylation controls the overall flux through the electron transport chain by maintaining optimal intermediate ΔΨm potentials for efficient ATP production while minimizing reactive oxygen species production, thus protecting the cell from apoptosis. Full article

Background: Liver cancer treatment encounters considerable therapeutic challenges, especially because hypoxic microenvironments markedly reduce sensitivity to chemotherapeutic agents. TFAM (mitochondrial transcription factor A) plays a crucial role in maintaining mitochondrial function. Oroxylin A (OA), a flavonoid with potential therapeutic properties, demonstrated prospects in cancer treatment. However, the mechanism of the sensitizing effect of OA on cancer cells has not been elucidated. Methods: MTT assays were utilized to evaluate a hypoxia-induced resistance model. Plate colony formation assays, TEM, and JC-1 staining were used to examine the effects of siTFAM on proliferation and mitochondrial damage of HepG2 cells. Cox8-EGFP-mCherry plasmid transfection, LysoTracker and MitoTracker colocalization analysis, and WB were conducted to evaluate the influence of OA on mitophagy. The effect of OA on p53 ubiquitination levels was investigated by Co-IP and the CHX chase assay. A mouse xenograft tumor model was utilized to assess the therapeutic effect of OA on HepG2 cells in vivo. Results: OA significantly improved the inhibitory effect of sorafenib by inhibiting mitophagy on HepG2 cells in in vitro and in vivo models. Notably, the molecular docking and thermal shift assays indicated a clear binding of OA and TFAM. Further research revealed that OA suppressed p53 acetylation and promoted its degradation by downregulating TFAM expression, which ultimately inhibited mitophagy in hypoxia. Conclusions: OA has demonstrated the potential to enhance the efficacy of sorafenib treatment for liver cancer, and TFAM may be one of its targets. Full article

Background/Objectives: Mitochondrial oxidative phosphorylation (OXPHOS) has been exploited as a therapeutic target in cancer treatments because of its crucial role in tumorigenesis. CR6-interacting factor 1 (CRIF1), a mitochondrial ribosomal subunit protein, is essential for the regulation of mitochondrial OXPHOS capacity. However, the mechanism of CRIF1 in triple-negative breast cancer (TNBC) cells remains unclear. Methods/Results: We showed that the downregulation of CRIF1 reduced cell proliferation in the TNBC cell lines MDA-MB-468, MDA-MB-231, and, especially, BT549. In addition, wound scratch and Transwell assays showed that CRIF1 deficiency inhibited the migration and invasion of BT549 cells. CRIF1 downregulation resulted in the suppression of mitochondrial bioenergetics in BT549 cells, specifically affecting the inhibition of OXPHOS complexes I and II. This was evidenced by a decrease in the mitochondrial oxygen consumption rate and the depolarization of the mitochondrial membrane potential. Damage to mitochondria resulted in a lower adenosine triphosphate level and an elevated production of mitochondrial reactive oxygen species. In addition, CRIF1 deficiency decreased hypoxia-inducible factor 1α accumulation, NADPH synthesis, and TP53-induced glycolysis and apoptosis regulator (TIGAR) expression in BT549 cells. These events contributed to G0/G1-phase cell cycle inhibition and the upregulation of the cell cycle protein markers p53, p21, and p16. Transfection with a TIGAR overexpression plasmid reversed these effects and prevented CRIF1 downregulation-induced proliferation and migration reduction. Conclusions: These results indicate that blocking mitochondrial OXPHOS synthesis via CRIF1 may have a therapeutic antitumor effect in BT549 TNBC cells. Full article

Neural precursor cells contain two types of intermediate filaments (IFs): neurofilaments consisting of three IV type proteins and vimentin belonging to the type III IF proteins that disappear at the later stages of differentiation. The involvement of vimentin in neurogenesis was demonstrated earlier; however, the role of its temporary expression in neurons is not clear. We showed that the vimentin IFs that interacted with mitochondria maintained their membrane potential at the appropriate level, and thus, ensured their proper function. We examined the dependence of the mitochondrial membrane potential on the expression of vimentin in a CAD catecholaminergic neuronal cell line that was actively dividing in full culture media but stopped growing and started developing neurites when the serum was removed. Using the CRISPR Cas9 system to knock out the vimentin gene in these cells, we investigated the impact of this on the mitochondrial membrane potential. Our data show that the deletion of the vimentin IFs led to a decrease in the level of the mitochondrial potential. When the vimentin network in these cells was reconstituted by transfection with a plasmid that encoded human protein, the level of the potential was restored. Interestingly, mutated vimentin with a disrupted mitochondria-binding site had no such effect. Our data point to vimentin as a possible target in some neurological pathologies. Full article

Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway. Full article

A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G mutant-type plasmids were constructed, respectively. In this study, a PCR method based on the TaqMan amplification refractory mutation system was proposed to detect mtDNA 1555A>G. A common upstream primer, a common TaqMan probe, and two downstream allele-specific primers with mismatched bases were designed. One-step amplification and detection of the wild-type and mutant type at the 1555 site were realized for the deafness-related gene through two reactions. Based on this detection method, the minimum detection limit of the wild-type and mutant type detection systems for plasmids was 50 copies/μL. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/μL. In the normal DBS DNA sample, the detection limit of the mutation abundance reached 0.78%. The specificity of the detection method was 100%, and the coefficient of variation was less than 3.36%. This approach was validated using clinical DNA extracted from 113 DBS samples of newborns. Additionally, it showed 100% agreement with bi-directional Sanger sequencing. It can be used as an optional method for the clinical detection of deafness-related genes. Full article

Nitroxides, stable synthetic free radicals, are promising antioxidants, showing many beneficial effects both at the cellular level and in animal studies. However, the cells are usually treated with high millimolar concentrations of nitroxides which are not relevant to the concentrations that could be attained in vivo. This paper aimed to examine the effects of low (≤10 μM) concentrations of three nitroxides, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), 4-hydroxy-TEMPO (TEMPOL) and 4-amino-TEMPO (TEMPAMINE), in pure chemical systems and on SH-SY5Y cells transfected with the human tau protein (TAU cells), a model of chronic cellular oxidative stress, and transfected with the empty plasmid (EP cells). All nitroxides were active in antioxidant-activity tests except for the 2,2′-azinobis-(3-ethylbenzthiazolin-6-sulfonate) radical (ABTS^•) decolorization assay and reduced Fe³⁺, inhibited autoxidation of adrenalin and pyrogallol and oxidation of dihydrorhodamine123 by 3-morpholino-sydnonimine SIN-1. TEMPO protected against fluorescein bleaching from hypochlorite, but TEMPAMINE enhanced the bleaching. Nitroxides showed no cytotoxicity and were reduced by the cells to non-paramagnetic derivatives. They decreased the level of reactive oxygen species, depleted glutathione, and increased mitochondrial-membrane potential in both types of cells, and increased lipid peroxidation in TAU cells. These results demonstrate that even at low micromolar concentrations nitroxides can affect the cellular redox equilibrium and other biochemical parameters. Full article

Mitochondrial dysfunction is a common occurrence in the aging process and is observed in diseases such as age-related macular degeneration (AMD). Increased levels of reactive oxygen species lead to damaged mitochondrial DNA (mtDNA), resulting in dysfunctional mitochondria, and, consequently, mtDNA causes further harm in the retinal tissue. However, it is unclear whether the effects are locally restricted to the high-energy-demanding retinal pigment epithelium or are also systematically present. Therefore, we measured mtDNA copy number (mtDNA-CN) in peripheral blood using a qPCR approach with plasmid normalization in elderly participants with and without AMD from the AugUR study (n = 2262). We found significantly lower mtDNA-CN in the blood of participants with early (n = 453) and late (n = 170) AMD compared to AMD-free participants (n = 1630). In regression analyses, we found lower mtDNA-CN to be associated with late AMD when compared with AMD-free participants. Each reduction of mtDNA-CN by one standard deviation increased the risk for late AMD by 24%. This association was most pronounced in geographic atrophy (OR = 1.76, 95% CI 1.19–2.60, p = 0.004), which has limited treatment options. These findings provide new insights into the relationship between mtDNA-CN in blood and AMD, suggesting that it may serve as a more accessible biomarker than mtDNA-CN in the retina. Full article

A major route for the influx of calcium ions into neurons uses the STIM-Orai1 voltage-independent channel. Once cytosolic calcium ([Ca²⁺]i) elevates, it activates mitochondrial and endoplasmic calcium stores to affect downstream molecular pathways. In the present study, we employed a novel drug, carbonyl cyanide chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, to explore the role of mitochondria in cultured neuronal morphology. CCCP caused a sustained elevation of [Ca²⁺]i and, quite surprisingly, a massive increase in the density of dendritic filopodia and spines in the affected neurons. This morphological change can be prevented in cultures exposed to a calcium-free medium, Orai1 antagonist 2APB, or cells transfected with a mutant Orai1 plasmid. It is suggested that CCCP activates mitochondria through the influx of calcium to cause rapid growth of dendritic processes. Full article

HIV-associated cognitive dysfunction during combination antiretroviral therapy (cART) involves mitochondrial dysfunction, but the impact of contemporary cART on chronic metabolic changes in the brain and in latent HIV infection is unclear. We interrogated mitochondrial function in a human microglia (hμglia) cell line harboring inducible HIV provirus and in SH-SY5Y cells after exposure to individual antiretroviral drugs or cART, using the MitoStress assay. cART-induced changes in protein expression, reactive oxygen species (ROS) production, mitochondrial DNA copy number, and cellular iron were also explored. Finally, we evaluated the ability of ROS scavengers or plasmid-mediated overexpression of the antioxidant iron-binding protein, Fth1, to reverse mitochondrial defects. Contemporary antiretroviral drugs, particularly bictegravir, depressed multiple facets of mitochondrial function by 20–30%, with the most pronounced effects in latently infected HIV+ hμglia and SH-SY5Y cells. Latently HIV-infected hμglia exhibited upregulated glycolysis. Increases in total and/or mitochondrial ROS, mitochondrial DNA copy number, and cellular iron accompanied mitochondrial defects in hμglia and SH-SY5Y cells. In SH-SY5Y cells, cART reduced mitochondrial iron–sulfur-cluster-containing supercomplex and subunit expression and increased Nox2 expression. Fth1 overexpression or pre-treatment with N-acetylcysteine prevented cART-induced mitochondrial dysfunction. Contemporary cART impairs mitochondrial bioenergetics in hμglia and SH-SY5Y cells, partly through cellular iron accumulation; some effects differ by HIV latency. Full article

Human corneal-endothelial cells (hCEnCs) are located on the inner layer of the cornea. Injury to CEnCs leads to permanent corneal edema, requiring corneal transplantation. NADPH oxidase 4 (NOX4) has been reported to be implicated in the pathogenesis of CEnCs diseases. Thus, we investigated the role of NOX4 in CEnCs in this study. In an animal study, siRNA for NOX4 (siNOX4) or plasmid for NOX4 (pNOX4) was introduced into the corneal endothelium of rats by electroporation, using a square-wave electroporator (ECM830, Havard apparatus) to decrease or increase the expression of NOX4, respectively, and the rat corneas were cryoinjured through contact with a metal rod of 3 mm diameter frozen in liquid nitrogen for 10 min. The immunofluorescence staining of NOX4 and 8-OHdG showed that the levels of NOX4 and 8-OHdG were decreased in the siNOX4 group compared to the siControl, and increased in the pNOX4 group compared to the pControl at one week after treatment. Without cryoinjury, corneal opacity was more severe, and the density of CEnCs was lower, in pNOX4-treated rats compared to pControl. After cryoinjury, the corneas were more transparent, and the CEnC density was higher, in siNOX4-treated rats. The hCEnCs were cultured and transfected with siNOX4 and pNOX4. The silencing of NOX4 in hCEnCs resulted in a normal cell shape, higher viability, and higher proliferation rate than those transfected with the siControl, while NOX4 overexpression had the opposite effect. NOX4 overexpression increased the number of senescent cells and intracellular oxidative stress levels. NOX4 overexpression increased ATF4 and ATF6 levels, and nuclear translocation of XBP-1, which is the endoplasmic reticulum (ER) stress marker, while the silencing of NOX4 had the opposite effect. Additionally, the mitochondrial membrane potential was hyperpolarized by the silencing of NOX4, and depolarized by NOX4 overexpression. The LC3II levels, a marker of autophagy, were decreased by the silencing of NOX4, and increased by NOX4 overexpression. In conclusion, NOX4 plays a pivotal role in the wound-healing and senescence of hCEnCs, by modulating oxidative stress, ER stress, and autophagy. The regulation of NOX4 may be a potential therapeutic strategy for regulating the homeostasis of CEnCs, and treating corneal-endothelial diseases. Full article