Increased ROS Production Research Articles

S. glabra exerts anti-lung cancer effects by inducing ferroptosis and anticancer immunity

BackgroundSarcandra glabra (S. glabra), a traditional Chinese medicine (TCM), has demonstrated significant anticancer activity; however, the underlying mechanisms have not yet been fully elucidated. PurposeThis study aimed to investigate the effects of S. glabra on lung cancer and to explore its underlying mechanisms. MethodsThe chemical profile of S. glabra was analyzed via ultrahigh-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). The effects of S. glabra on the viability, proliferation, apoptosis, migration, and invasion of lung cancer cells were assessed via CCK8, colony formation, flow cytometry, scratch, and Transwell assays. In vivo anticancer activity was evaluated in an LLC mouse model. Proteomic analysis was performed to identify key molecules and pathways in S. glabra-treated LLC cells. The expression of ferroptotic proteins and associated cellular events were examined via western blotting, ROS production, iron accumulation, and lipid peroxidation assays. Immune modulation in tumor-bearing mice was evaluated by detecting immune cells and cytokines in the peripheral blood and tumor tissue. ResultsOur analysis quantified 1997 chemical markers in S. glabra aqueous extracts. S. glabra inhibited the viability and proliferation of lung cancer cells and induced cell cycle arrest and apoptosis. Scratch and Transwell assays demonstrated that S. glabra suppressed the migration and invasion of lung cancer cells. Oral administration of S. glabra significantly inhibited tumor growth in LLC tumor-bearing mice. Proteomic analysis revealed that S. glabra upregulated the expression of the HMOX1 protein and activated the ferroptosis pathway. Consistent with these findings, we found that S. glabra triggered ferroptosis in lung cancer cells, as evidenced by the upregulation of HMOX1, downregulation of GPX4 and ferritin light chain proteins, iron accumulation, increased ROS production, and lipid peroxidation. Furthermore, S. glabra demonstrated immunostimulatory properties in LLC tumor-bearing mice, leading to increased populations of immune cells (NK cells) and elevated cytokine levels (IL-2). ConclusionThis study is the first to demonstrate that S. glabra induces ferroptosis in lung cancer cells by regulating HMOX1, GPX4, and FTL. These findings provide a robust scientific basis for the clinical application of S. glabra in lung cancer treatment.

Mitochondrial damage precedes the changes of glutathione metabolism in CdCl2 treated neuronal SH-SY5Y cells

Cadmium crosses the blood-brain barrier inducing damage to neurons. Cell impairment is predominantly linked to oxidative stress and glutathione (GSH) depletion. On the other hand, several reports have described an increase of GSH levels in neuronal cells after CdCl2 exposure. Therefore, the aim of the present report was to investigate the relation between changes in GSH levels and mitochondrial damage in neuronal cells after CdCl2 treatment. To characterize neuronal impairment after CdCl2 treatment (0–200 μM) for 1–48 h, we used the SH-SY5Y cell line. We analyzed GSH metabolism and determined mitochondrial activity using high-resolution respirometry. CdCl2 treatment induced both the decreases and increases of GSH levels in SH-SY5Y cells. GSH concentration was significantly increased in cells incubated with up to 50 μM CdCl2 but only 100 μM CdCl2 induced GSH depletion linked to increased ROS production. The overexpression of proteins involved in GSH synthesis increased in response to 50 and 100 μM CdCl2 after 6 h. Finally, strong mitochondrial impairment was detected even in 50 μM CdCl2 treated cells after 24 h. We conclude that a significant decrease in mitochondrial activity can be observed in 50 μM CdCl2 even without the occurrence of GSH depletion in SH-SY5Y cells.

Detailed analysis of Mdivi-1 effects on complex I and respiratory supercomplex assembly

Several human diseases, including cancer and neurodegeneration, are associated with excessive mitochondrial fragmentation. In this context, mitochondrial division inhibitor (Mdivi-1) has been tested as a therapeutic to block the fission-related protein dynamin-like protein-1 (Drp1). Recent studies suggest that Mdivi-1 interferes with mitochondrial bioenergetics and complex I function. Here we show that the molecular mechanism of Mdivi-1 is based on inhibition of complex I at the IQ site. This leads to the destabilization of complex I, impairs the assembly of N- and Q-respirasomes, and is associated with increased ROS production and reduced efficiency of ATP generation. Second, the calcium homeostasis of cells is impaired, which for example affects the electrical activity of neurons. Given the results presented here, a potential therapeutic application of Mdivi-1 is challenging because of its potential impact on synaptic activity. Similar to the Complex I inhibitor rotenone, Mdivi-1 may lead to neurodegenerative effects in the long term.

Polystyrene nanoplastics significantly facilitate largemouth bass ranavirus infection of host cells

Novel pollutants nanoplastics (NPs) are widely distributed in aquatic environments and may pose a health threat to aquatic organisms. Notably, the contribution of NPs to the occurrence of viral diseases in aquatic animals remains largely uncertain. In this study, the effects of polystyrene nanoplastics (PS-NPs) on Largemouth bass ranavirus (LMBV)-infected MsF cells were investigated. MsF cells took up PS-NPs in a time- and dose-dependent manner and significantly affect cell viability at an exposure concentration of 500 μg/mL. Western blot and qPCR assays indicated that exposure to PS-NPs accelerated LMBV replication in MsF cells. PS-NPs act synergistically with LMBV to disrupt the cellular antioxidant system, as evidenced by increased ROS production and decreased mRNA levels of antioxidant-associated genes. Furthermore, PS-NPs was found to exacerbate LMBV-induced inflammatory responses, as demonstrated by disturbed expression of inflammation-related factors. In addition, our results suggest that PS-NPs reduce IFN production by inhibiting the expression of molecules related to the cGAS-STING signaling pathway, thereby promoting viral replication. Collectively, our findings suggest the potential threat of NPs to infectious diseases caused by freshwater fish viruses and provide new insights for fish disease prevention and control.

α-amanitin induce inflammatory response by activating ROS/NF-κB-NLRP3 signaling pathway in human hepatoma HepG2 cells

α-amanitin (AMA) is a hepatotoxic mushroom toxin responsible for over 90% of mushroom poisoning fatalities worldwide, seriously endangering human life and health. Few evidences have indicated that AMA leads to inflammatory responses and inflammatory infiltration in vitro and in vivo. However, the molecular mechanism remains unknown. In this study, human hepatocellular carcinomas cells (HepG2) were exposed to AMA at various concentrations for short period of times. Results revealed that AMA increased ROS production and elevated the releases of malondialdehyde (MDA) and lactate dehydrogenase (LDH), resulting in oxidative damage in HepG2 cells. Also, AMA exposure significantly increased the secreted levels of inflammatory cytokines and activated the NLRP3 inflammasome. The inflammatory responses were reversed by NLRP3 inhibitor MCC950 and NF-κB inhibitor Bay11-7082. Additionally, N-acetylcysteine (NAC) blocked the upregulation of the NF-κB/NLRP3 signaling pathway and remarkably alleviated the inflammatory response. These results demonstrated that AMA could induce inflammation through activating the NLRP3 inflammasome triggered by ROS/NF-κB signaling pathway. Our research provides new insights into the molecular mechanism of AMA-induced inflammation damage and may contribute to establish new prevention strategies for AMA hepatotoxicity.

FOXO-regulated OSER1 reduces oxidative stress and extends lifespan in multiple species

FOXO transcription factors modulate aging-related pathways and influence longevity in multiple species, but the transcriptional targets that mediate these effects remain largely unknown. Here, we identify an evolutionarily conserved FOXO target gene, Oxidative stress-responsive serine-rich protein 1 (OSER1), whose overexpression extends lifespan in silkworms, nematodes, and flies, while its depletion correspondingly shortens lifespan. In flies, overexpression of OSER1 increases resistance to oxidative stress, starvation, and heat shock, while OSER1-depleted flies are more vulnerable to these stressors. In silkworms, hydrogen peroxide both induces and is scavenged by OSER1 in vitro and in vivo. Knockdown of OSER1 in Caenorhabditis elegans leads to increased ROS production and shorter lifespan, mitochondrial fragmentation, decreased ATP production, and altered transcription of mitochondrial genes. Human proteomic analysis suggests that OSER1 plays roles in oxidative stress response, cellular senescence, and reproduction, which is consistent with the data and suggests that OSER1 could play a role in fertility in silkworms and nematodes. Human studies demonstrate that polymorphic variants in OSER1 are associated with human longevity. In summary, OSER1 is an evolutionarily conserved FOXO-regulated protein that improves resistance to oxidative stress, maintains mitochondrial functional integrity, and increases lifespan in multiple species. Additional studies will clarify the role of OSER1 as a critical effector of healthy aging.

Photoaging of polystyrene-based microplastics amplifies inflammatory response in macrophages

The continuous release of municipal and industrial products into the environment poses a growing concern for public health. Among environmental pollutants, polystyrene (PS) stands out as a primary constituent of environmental plastic waste, given its widespread use and high production rates owing to its durability and user-friendly properties. The detection of polystyrene microparticles (PS-MPs) in various living organisms has been well-documented, posing a serious threat due to their potential passage into the human ecosystem. In this manuscript, we aimed to study the toxicological effects of low concentrations of pristine and photoaged PS-MPs in a murine macrophage cell line. To this purpose, PS-MPs were photoaged by indoor exposure to visible light to simulate environmental weathering due to solar irradiation (PS-MPs3h). Physical characterization revealed that the irradiation treatment results in particle degradation and the possible release of nanoparticles. Monocultures of the RAW264.7 cell line were then exposed to PS-MPs and PS-MPs3h at concentrations comparable to experimental measurements from biological samples, to assess cytotoxicity, intracellular oxidative stress, primary genotoxicity, and inflammatory effects. Significant toxicity-related outcomes were observed in cells treated with both pristine PS-MPs and PS-MPs3h even at low concentrations (0,10 μg/ml and 1 μg/ml). PS-MPs3h exhibited greater adverse effects compared to PS-MPs, including reduced cell viability, increased ROS production, elevated DNA damage, and upregulation of IL-6 and NOS2 gene expression. Therefore, we can conclude that changes induced by environmental aging in the physicochemical composition of PS microplastics play a crucial role in the adverse health outcomes associated with microplastic exposure.

Mitochondrial chaperon TNF-receptor- associated protein 1 as a novel apoptotic regulator conferring susceptibility to Pneumocystis jirovecii pneumonia.

Molecular chaperons stabilize protein folding and play a vital role in maintaining tissue homeostasis. To this intent, mitochondrial molecular chaperons may be involved in the regulation of oxidative phosphorylation and apoptosis during stress events such as infections. However, specific human infectious diseases relatable to defects in molecular chaperons have yet to be identified. To this end, we performed whole exome sequencing and functional immune assessment in a previously healthy Asian female, who experienced severe respiratory failure due to Pneumocystis jiroveci pneumonia and non-HIV-related CD4 lymphocytopenia. This revealed that a chaperon, the mitochondrial paralog of HSP90, TRAP1, may have been involved in the patient's susceptibility to an opportunistic infection. Two rare heterozygous variants in TRAP1, E93Q, and A64T were detected. The patient's peripheral blood mononuclear cells displayed diminished TRAP1 expression, but had increased active, cleaved caspase-3, caspase-7, and elevated IL-1β production. Transfection of A64T and E93Q variants in cell lines yielded decreased TRAP1 compared to transfected wildtype TRAP1 and re-capitulated the immunotypic phenotype of enhanced caspase-3 and caspase-7 activity. When infected with live P. jiroveci, the E93Q or A64T TRAP1 mutant expressing cells also exhibited reduced viability. Patient cells and cell lines transfected with the TRAP1 E93Q/A64T mutants had impaired respiration, glycolysis, and increased ROS production. Of note, co-expression of E93Q/A64T double mutants caused more functional aberration than either mutant singly. Taken together, our study uncovered a previously unrecognized role of TRAP1 in CD4+ lymphocytopenia, conferring susceptibility to opportunistic infections.

Iris setosa Pall. ex Link Extract Reveals Amoebicidal Activity against Acanthamoeba castellanii and Acanthamoeba polyphaga with Low Toxicity to Human Corneal Cells.

Acanthamoeba keratitis (AK) is a sight-threatening and difficult-to-treat ocular infection. The significant side effects of current AK treatments highlight the urgent need to develop a safe and effective AK medication. In this study, the amoebicidal activity of Iris setosa Pall. ex Link extract (ISE) against Acanthamoeba was examined and its specific amoebicidal mechanism was explored. ISE induced significant morphological changes in Acanthamoeba trophozoites and exhibited amoebicidal activity against A. castellanii and A. polyphaga. ISE was further fractionated into five subfractions by sequential extraction with n-hexane, chloroform, ethyl acetate, n-butanol, and water, and their amoebicidal activities and underlying amoebicidal mechanisms were investigated. The n-butanol subfraction of ISE (ISE-BuOH) displayed selective amoebicidal activity against the Acanthamoeba species with minimal cytotoxicity in human corneal cells (HCE-2). ISE-BuOH triggered apoptosis-like programmed cell death (PCD) in amoebae, characterized by DNA fragmentation, increased ROS production, and caspase-3 activity elevation. ISE-BuOH also demonstrated a partial cysticidal effect against the amoeba species. ISE-BuOH could be a promising candidate in the development of therapeutic drugs for AK.

Endolysin CHAP domain-carbosilane metallodendrimer complexes with triple action on Gram-negative bacteria: Membrane destabilization, reactive oxygen species production and peptidoglycan degradation

Bacterial resistance to antibiotics is a significant challenge that is associated with increased morbidity and mortality. Gram-negative bacteria are particularly problematic due to an outer membrane (OM). Current alternatives to antibiotics include antimicrobial peptides or proteins and multifunctional systems such as dendrimers. Antimicrobial proteins such as lysins can degrade the bacterial cell wall, whereas dendrimers can permeabilize the OM, enhancing the activity of endolysins against gram-negative bacteria. In this study, we present a three-stage action of endolysin combined with two different carbosilane (CBS) silver metallodendrimers, in which the periphery is modified with N-heterocyclic carbene (NHC) ligands coordinating a silver atom. The different NHC ligands contained hydrophobic methyl or N-donor pyridyl moieties. The effects of these endolysin/dendrimer combinations are based on OM permeabilization, peptidoglycan degradation, and reactive oxygen species production. The results showed that CBS possess a permeabilization effect (first action), significantly reduced bacterial growth at higher concentrations alone and in the presence of endolysin, increased ROS production (second action), and led to bacterial cell damage (third action). The complex formed between the CHAP domain of endolysin and a CBS silver metallodendrimer, with a triple mechanism of action, may represent an excellent alternative to other antimicrobials with only one resistance mechanism.

Sex Differences in Hepatic Inflammation, Lipid Metabolism, and Mitochondrial Function Following Early Lipopolysaccharide Exposure in Epileptic WAG/Rij Rats.

Among the non-communicable neurological diseases, epilepsy is characterized by abnormal brain activity with several peripheral implications. The role of peripheral inflammation in the relationship between seizure development and nonalcoholic fatty liver disease based on sex difference remains still overlooked. Severe early-life infections lead to increased inflammation that can aggravate epilepsy and hepatic damage progression, both related to increased odds of hospitalization for epileptic patients with liver diseases. Here, we induced a post-natal-day 3 (PND3) infection by LPS (1 mg/kg, i.p.) to determine the hepatic damage in a genetic model of young epileptic WAG/Rij rats (PND45). We evaluated intra- and inter-gender differences in systemic and liver inflammation, hepatic lipid dysmetabolism, and oxidative damage related to mitochondrial functional impairment. First, epileptic rats exposed to LPS, regardless of gender, displayed increased serum hepatic enzymes and altered lipid profile. Endotoxin challenge triggered a more severe inflammatory and immune response in male epileptic rats, compared to females in both serum and liver, increasing pro-inflammatory cytokines and hepatic immune cell recruitment. Conversely, LPS-treated female rats showed significant alterations in systemic and hepatic lipid profiles and reduced mitochondrial fatty acid oxidation. The two different sex-dependent mechanisms of LPS-induced liver injury converge in increased ROS production and related mitochondrial oxidative damage in both sexes. Notably, a compensatory increase in antioxidant defense was evidenced only in female rats. Our study with a translational potential demonstrates, for the first time, that early post-natal infections in epileptic rats induced or worsened hepatic disorders in a sex-dependent manner, amplifying inflammation, lipid dysmetabolism, and mitochondrial impairment.

Alliin mitigates the acute kidney injury by suppressing ferroptosis via regulating the Nrf2/GPX4 axis.

Acute kidney injury (AKI) is a clinical syndrome that is characterized by a sudden loss of kidney function, leading to severe metabolic disorders, multiple organ failure, and even death. Recent studies have strengthened the evidence for ferroptosis in AKI development. Alliin, a sulfur-containing amino acid with multiple pharmacological functions, was claimed with promising antioxidant and anti-inflammation effects in protecting organ damages. Herein, Alliin's potential in AKI treatment was investigated by exploring its impact on ferroptosis, providing a new strategy for clinical AKI treatments. Cecal ligation and puncture (CLP) modeling was performed on rats, followed by treated with 7.5 and 15mg/kg/day of alliin for 6days. A declined survival rate, severe renal pathological changes, renal dysfunction, and enhanced inflammatory state were observed in CLP-treated rats, which were remarkably alleviated by alliin. Moreover, increased MDA levels, declined SOD activity, and downregulated Nrf2, GPX4, and xCT in CLP-treated rats were notably reversed by alliin. To explore potential mechanisms of alliin, NRK-52E cells were stimulated with 1μg/mL LPS for 24h, followed by culturing with 30 and 100μM of alliin for 24h. Reduced cell viability, enhanced apoptosis, increased ROS production, boosted MDA level, and declined SOD activity were observed in LPS-stimulated NRK-52E cells, accompanied by downregulated Nrf2, GPX4, and xCT, which were strikingly ameliorated by alliin. Additionally, the influence of alliin on cell viability, oxidative stress (OS), and ferroptosis in LPS-stimulated NRK-52E cells were markedly abolished by silencing Nrf2. Collectively, alliin mitigated AKI by suppressing ferroptosis via regulating the Nrf2/GPX4 axis.

CKIP-1 silencing suppresses OSCC via mitochondrial homeostasis-associated TFAM/cGAS-STING signalling axis.

Limited effective targets have challenged the treatment of oral squamous cell carcinoma (OSCC). Casein kinase 2 interacting protein 1 (CKIP-1) is a scaffold protein involved in various diseases. However, the role of CKIP-1 in OSCC remains unclear. The aim of this study was to explore the regulatory role of CKIP-1 in OSCC, as well as the involved mechanism. First, higher expression of CKIP-1 in OSCC tissues and cell lines were found. Series of gain- and loss-of-function experiments demonstrated suppressed malignant behaviours and enhanced apoptosis of OSCC cells when CKIP-1 was silenced. Also, inhibited tumour growth in CKIP-1-silenced group were proved. Further, mitochondrial transcription factor A (TFAM) downregulation, increased ROS production, decreased mitochondrial membrane potential and cGAS-STING activation in CKIP-1-silenced group were observed. The involvement of mitochondrial homeostasis-related TFAM/cGAS-STING axis in CKIP-1-silenced OSCC cells was finally demonstrated by tetramethylpyrazine (TMP) that inhibits TFAM degradation. Taken together, our study demonstrated that CKIP-1 silencing could significantly antagonize OSCC via TFAM/cGAS-STING axis, which may provide a candidate target for OSCC treatment.

Aloe and coconut extracts mediated CuInS2 nanoparticles induce apoptosis in non-small lung cancer cells (A549)

The most prevalent type of cancer found in the world is lung cancer. Furthermore, chemotherapeutic intervention for lung carcinomas is frequently associated with severe off-target effects. As a result, there is a worldwide push to investigate alternative medicines with superior tolerance profiles, such as natural compounds, to substitute routinely used chemotherapeutics. The purpose of this study is to examine the anticancer potential of aloe (S1) and coconut (S2) extract-mediated CuInS2 (CIS) nanoparticles against non-small cell lung carcinoma (A549) cells. S1 and S2 both efficiently showed a dose-proportionate cytotoxic effect on A549 cells, while causing negligible toxic effects on normal healthy lung cells (WI-38), indicating their safety against healthy cells. Reduced cell viability was validated by acridine orange and ethidium bromide double staining, demonstrating that apoptosis was induced in A549 cells treated with both nanoparticles. In addition, the mitochondrial membrane potential was reduced by both nanoparticles, leading to an increase in ROS generation. Both nanoparticles increased caspase-3, caspase-9, and caspase-8 levels in A549 cells. To summarize, both nanoparticles induced apoptotic cell damage in A549 cells by targeting mitochondria, causing increased ROS production, activating the caspase cascade, and inducing apoptosis. Aloe (S1) and coconut (S2) extract-mediated CIS nanoparticles may represent viable therapeutic options for lung cancer management.

The Barrier Disruption and Pyroptosis of Intestinal Epithelial Cells Caused by Perfringolysin O (PFO) from Clostridium perfringens.

Clostridium perfringens (C. perfringens), a Gram-positive bacterium, produces a variety of toxins and extracellular enzymes that can lead to disease in both humans and animals. Common symptoms include abdominal swelling, diarrhea, and intestinal inflammation. Severe cases can result in complications like intestinal hemorrhage, edema, and even death. The primary toxins contributing to morbidity in C. perfringens-infected intestines are CPA, CPB, CPB2, CPE, and PFO. Amongst these, CPB, CPB2, and CPE are implicated in apoptosis development, while CPA is associated with cell death, increased intracellular ROS levels, and the release of the inflammatory factor IL-18. However, the exact mechanism by which PFO toxins exert their effects in the infected gut is still unidentified. This study demonstrates that a C. perfringens PFO toxin infection disrupts the intestinal epithelial barrier function through in vitro and in vivo models. This study emphasizes the notable influence of PFO toxins on intestinal barrier integrity in the context of C. perfringens infections. It reveals that PFO toxins increase ROS production by causing mitochondrial damage, triggering pyroptosis in IPEC-J2 cells, and consequently resulting in compromised intestinal barrier function. These results offer a scientific foundation for developing preventive and therapeutic approaches against C. perfringens infections.

The Survival of Human Intervertebral Disc Nucleus Pulposus Cells under Oxidative Stress Relies on the Autophagy Triggered by Delphinidin.

Delphinidin (Delp), a natural antioxidant, has shown promise in treating age-related ailments such as osteoarthritis (OA). This study investigates the impact of delphinidin on intervertebral disc degeneration (IVDD) using human nucleus pulposus cells (hNPCs) subjected to hydrogen peroxide. Various molecular and cellular assays were employed to assess senescence, extracellular matrix (ECM) degradation markers, and the activation of AMPK and autophagy pathways. Initially, oxidative stress (OS)-induced hNPCs exhibited notably elevated levels of senescence markers like p53 and p21, which were mitigated by Delp treatment. Additionally, Delp attenuated IVDD characteristics including apoptosis and ECM degradation markers in OS-induced senescence (OSIS) hNPCs by downregulating MMP-13 and ADAMTS-5 while upregulating COL2A1 and aggrecans. Furthermore, Delp reversed the increased ROS production and reduced autophagy activation observed in OSIS hNPCs. Interestingly, the ability of Delp to regulate cellular senescence and ECM balance in OSIS hNPCs was hindered by autophagy inhibition using CQ. Remarkably, Delp upregulated SIRT1 and phosphorylated AMPK expression while downregulating mTOR phosphorylation in the presence of AICAR (AMPK activator), and this effect was reversed by Compound C, AMPK inhibitor. In summary, our findings suggest that Delp can safeguard hNPCs from oxidative stress by promoting autophagy through the SIRT1/AMPK/mTOR pathway.

Copper-Based Complexes with Adamantane Ring-Conjugated bis(3,5-Dimethyl-pyrazol-1-yl)acetate Ligand as Promising Agents for the Treatment of Glioblastoma.

The new ligand L2Ad, obtained by conjugating the bifunctional species bis(3,5-dimethylpyrazol-1-yl)-acetate and the drug amantadine, was used as a chelator for the synthesis of new Cu complexes 1-5. Their structures were investigated by synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS), near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and by combining X-ray absorption fine structure (XAFS) spectroscopy techniques and DFT modeling. The structure of complex 3 was determined by single-crystal X-ray diffraction analysis. Tested on U87, T98, and U251 glioma cells, Cu(II) complex 3 and Cu(I) complex 5 decreased cell viability with IC50 values significantly lower than cisplatin, affecting cell growth, proliferation, and death. Their effects were prevented by treatment with the Cu chelator tetrathiomolybdate, suggesting the involvement of copper in their cytotoxic activity. Both complexes were able to increase ROS production, leading to DNA damage and death. Interestingly, nontoxic doses of 3 or 5 enhanced the chemosensitivity to Temozolomide.

#245 Deficiency of GADD45α expression mediated tubulointerstitial injury in diabetic nephropathy

Abstract Background and Aims The tubulointerstitial injury plays crucial roles in the development of diabetic nephropathy (DN). Recently, some studies have reported that disorder of mitochondrial fatty acid β-oxidation in proximal tubules is involved in tubulointerstitial injury of DN. The growth arrest and DNA damage-inducible gene 45 (GADD45) family proteins, especially, GADD45α, plays a crucial role in various cellular processes, such as mitochondrial biogenesis and lipid metabolism. Recent studies have demonstrated that GADD45α facilitates gene activation by inducing DNA demethylation. Therefore, this study aimed to investigate the role of GADD45α in tubulointerstitial injury of DN and its underlying mechanisms. Methods Bioinformatics and machine learning techniques were employed to identify differentially expressed genes between healthy individuals and DN patients. Experiments were conducted by using high glucose-treated HK-2 cells and streptozotocin (STZ)-induced mice. Lentivirus carrying the GADD45α gene was used to induce GADD45α overexpression in HK-2 cells. Adeno-Associated Virus vectors carrying LoxP-flanked GADD45α were intravenously injected into γGT-Cre transgenic mice expressing Cre recombinase in renal proximal tubular cells, specifically leading to GADD45α overexpression in proximal tubules. Results Through bioinformatics and machine learning techniques, GADD45α was identified and found to exhibit a strong association with tubulointerstitial injury in DN. Further analysis revealed that the expression of GADD45α was decreased in the kidneys of DN patients, diabetic mice, and high glucose-treated HK-2 cells. Notably, GADD45α overexpression ameliorated kidney damage and mitochondrial injury, leading to improved fatty acid β-oxidation, restored ATP production, and reduced ROS production. Transcriptomics analyses further demonstrated that GADD45α overexpression enhanced the expression of genes related to mitochondrial biogenesis, dynamics, and electron transport chain, with six transmembrane epithelial antigen of prostate 4 (STEAP4) being a key protein in this process. Knocking down STEAP4 using siRNA in HK-2 cells exacerbated the damage induced by high glucose. In addition, our further results indicated that high glucose increased methylation of the STEAP4 promoter, while GADD45α overexpression decreased methylation and increased STEAP4 expression. Conclusions Deficiency of GADD45α expression mediated tubulointerstitial injury in DN, which was mainly through the decreased STEAP4 expression by demethylating CpG motifs in the STEAP4 promoter region, thereby resulting in reduced fatty acid oxidation, energy synthesis, and increased ROS production, and finally accelerated tubular injuries. These findings suggested that targeting the GADD45α-STEAP4 pathway could be a potential therapeutic strategy for DN.

Rotenone activates the LKB1-AMPK-ULK1 signaling pathway to induce autophagy and apoptosis in rat thoracic aortic endothelial cells

BackgroundThe specific mechanism by which rotenone impacts thoracic aortic autophagy and apoptosis is unknown. We aimed to investigate the regulatory effects of rotenone on autophagy and apoptosis in rat thoracic aortic endothelial cells (RTAEC) via activation of the LKB1-AMPK-ULK1 signaling pathway and to elucidate the molecular mechanisms of rotenone on autophagy and apoptosis in vascular endothelial cells.MethodsIn vivo, 60 male SD rats were randomly selected and divided into 5 groups: control (Con), DMSO, 1, 2, and 4 mg/kg groups, respectively. After 28 days of treatment, histopathological and ultrastructural changes in each group were observed using HE and transmission electron microscopy; Autophagy, apoptosis, and LKB1-AMPK-ULK1 pathway-related proteins were detected by Western blot; Apoptosis levels in the thoracic aorta were detected by TUNEL. In vitro, RTAEC were cultured and divided into control (Con), DMSO, 20, 100, 500, and 1000 nM groups. After 24 h of intervention, autophagy, apoptosis, and LKB1-AMPK-ULK1 pathway-related factors were detected by Western blot and qRT-PCR; Flow cytometry to detect apoptosis levels; Autophagy was inhibited with 3-MA and CQ to detect apoptosis levels, and changes in autophagy, apoptosis, and downstream factors were detected by the AMPK inhibitor CC intervention.ResultsGavage in SD rats for 28 days, some degree of damage was observed in the thoracic aorta and heart of the rotenone group, as well as the appearance of autophagic vesicles was observed in the thoracic aorta. TUNEL analysis revealed higher apoptosis in the rotenone group’s thoracic aorta; RTAEC cultured in vitro, after 24 h of rotenone intervention, showed increased ROS production and significantly decreased ATP production. The flow cytometry data suggested an increase in the number of apoptotic RTAEC. The thoracic aorta and RTAEC in the rotenone group displayed elevated levels of autophagy and apoptosis, and the LKB1-AMPK-ULK1 pathway proteins were activated and expressed at higher levels. Apoptosis and autophagy were both suppressed by the autophagy inhibitors 3-MA and CQ. The AMPK inhibitor CC reduced autophagy and apoptosis in RTAEC and suppressed the production of the AMPK downstream factors ULK1 and P-ULK1.ConclusionsRotenone may promote autophagy in the thoracic aorta and RTAEC by activating the LKB1-AMPK-ULK1 signaling pathway, thereby inducing apoptosis.

LC3A-mediated autophagy elicits PERK-eIF2α-ATF4 axis activation and mitochondrial dysfunction: Exposing vulnerability in aggresome-positive cancer cells

The unfolded protein response pathways (UPR), autophagy, and compartmentalization of misfolded proteins into inclusion bodies are critical components of the protein quality control network. Among inclusion bodies, aggresomes are particularly intriguing due to their association with cellular survival, drug resistance, and cancer-aggressive behavior. Aggresomes are molecular condensates formed when collapsed vimentin cages encircle misfolded proteins before final removal by autophagy. Yet significant gaps persist in the mechanisms governing aggresome formation and elimination in cancer cells. Understanding these mechanisms is crucial, especially considering the involvement of LC3A, a member of the MAP1LC3 family, which plays a unique role in autophagy regulation and has been reported to be epigenetically silenced in many cancers. Herein, we utilized tetracycline-inducible expression of LC3A to investigate its role in choroid plexus carcinoma cells, which inherently exhibit the presence of aggresomes. Live cell imaging was employed to demonstrate the effect of LC3A expression on aggresome-positive cells, while SILAC-based proteomics identified LC3A-induced protein and pathway alterations. Our findings demonstrate that extended expression of LC3A is associated with cellular senescence. However, the obstruction of lysosomal degradation in this context has a deleterious effect on cellular viability. In response to LC3A-induced autophagy, we observed significant alterations in mitochondrial morphology, reflected by mitochondrial dysfunction and increased ROS production. Furthermore, LC3A expression elicited the activation of the PERK-eIF2α-ATF4 axis of the UPR, underscoring a significant change in protein quality control network. In conclusion, our results elucidate that LC3A-mediated autophagy alters the protein quality control network, exposing a vulnerability in aggresome-positive cancer cells.