Intracellular Reactive Oxygen Species Generation Research Articles

Mechanistic evaluation of myristicin on apoptosis and cell cycle regulation in breast cancer cells.

The current study was focused on the anticancer activity of myristicin against MCF-7 human breast cancer (BC) cells. BC is the most common and leading malignant disease in women worldwide. Now-a-days, various conventional therapies are used against BC and still represent a chief challenge because those treatments fail to differentiate normal cells from malignant cells, and they have severe side effects also. So, there is a need develop new therapies to decrease BC-related morbidity and mortality. Myristicin, a 1‑allyl‑5‑methoxy‑3, 4‑methylenedioxybenzene, is a main active aromatic compound present in various spices, such as nutmeg, mace, carrot, cinnamon, parsely and some essential oils. Myristicin has a wide range of effects, including antitumor, antioxidative and antimicrobial activity. Nevertheless, the effects of myristicin on human BC cells remain largely unrevealed. The cytotoxicity effect of myristicin on MCF‑7 cells was increased dose dependently detected by (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Lactate Dehydrogenase assays. Myristicin was found to be significantly inducing the cell apoptosis, as compared to control, using acridine orange/ethidium bromide, Hoechst stain and annexin V. Moreover, it activates cell antimigration, intracellular reactive oxygen species generation and cell cycle arrest in the G1/S phase. In addition, myristicin induces the expression of apoptosis and cell cycle genes (Caspases8, Bax, Bid, Bcl2, PARP, p53, and Cdk1) was demonstrated by quantitative polymerase chain reaction and apoptosis proteins (c-PARP, Caspase 9, Cytochrome C, PDI) expression was also analyzed with western blot. Overall, we illustrated that myristicin could regulate apoptosis signaling pathways in MCF-7 BC cells.

Cellular Impact and Biodegradability of S‐ and N‐Doped Graphene Quantum Dots on Human Monocytes and Macrophages

AbstractGraphene quantum dots (GQDs), small graphene domains with lateral dimensions lower than 10 nm, are increasingly used in electronics, composites, and biomedicine. Chemical doping of GQDs allows tuning their optical properties. Immune cells are among the first cells exposed to nanomaterials entering a living body, rapidly triggering a downstream immune response. However, the assessment of the impact of chemically‐doped GQDs on the immune system remains rather limited if not absent. In this context, the effects and the biodegradability of sulfur‐doped and nitrogen‐doped GQDs (S‐GQDs and N‐GQDs) on human monocytes and macrophages are evaluated. The metabolic activity, membrane integrity, apoptosis, and intracellular reactive oxygen species (ROS) generation are studied. In parallel, the degradation of GQDs using human myeloperoxidase and a peroxynitrite‐mediated system is investigated in test tube. Their degradation in macrophages is also pursued. High‐resolution transmission electron microscopy (HRTEM), fluorescence spectroscopy, Raman, and flow cytometry are used to confirm the degradation. Overall, both GQDs exert little activation on monocytes and macrophages although they decrease the metabolic viability in a dose‐dependent manner. The loss of native GQD structure and crystal lattice provide evidence of their biodegradability. Both the safety and biodegradability of S‐GQDs and N‐GQDs ensure their potential in biomedical applications.

High glucose reduces Nrf2-dependent cRAGE release and enhances inflammasome-dependent IL-1β production in monocytes: the modulatory effects of EGCG

Soluble receptor for advanced glycation end products (sRAGE) acts as a decoy sequestering of RAGE ligands, thus preventing the activation of the ligand-RAGE axis linking human diseases. However, the molecular mechanisms underlying sRAGE remain unclear. In this study, THP-1 monocytes were cultured in normal glucose (NG, 5.5 mmol/L) and high glucose (HG, 15 mmol/L) to investigate the effects of diabetes-relevant glucose concentrations on sRAGE and interleukin-1β (IL-1β) secretion. The modulatory effects of epigallocatechin gallate (EGCG) in response to HG challenge were also evaluated. HG enhanced intracellular reactive oxygen species (ROS) generation and RAGE expression. The secretion of sRAGE, including esRAGE and cRAGE, was reduced under HG conditions, together with the downregulation of a disintegrin and metallopeptidase 10 (ADAM10) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation. Mechanistically, the HG effects were counteracted by siRAGE and exacerbated by siNrf2. Chromatin immunoprecipitation results showed that Nrf2 binding to the ADAM10 promoter and HG interfered with this binding. Our data reinforce the notion that RAGE and Nrf2 might be sRAGE-regulating factors. Under HG conditions, the treatment of EGCG reduced ROS generation and RAGE activation. EGCG-stimulated cRAGE release was likely caused by the upregulation of the Nrf2-ADAM10 pathway. EGCG inhibited HG-mediated NLRP3 inflammasome activation at least partly by stimulating sRAGE, thereby reducing IL-1β release.

Novel role of dual oxidase 2 as a mediator of podocyte injury in the diabetic environment

Glomerular injury is a prominent pathological feature of diabetic nephropathy (DN). In glomerular epithelial cells, or podocytes, hyperglycemia alters slit diaphragm proteins and causes foot process effacement, apoptosis and cell detachment. Oxidative stress has emerged as an important pathogenic mechanism in the development of glomerular injury in DN. However, the mechanisms by which these factors exert their action remain poorly understood. We provide evidence that the NADPH oxidase of the Nox family, Dual oxidase 2 (Duox2), is present in cultured glomerular cells, including podocytes. Exposure of cultured podocytes to high concentrations of glucose (HG) elicited a rapid upregulation of Duox2 protein expression. Inhibition of Duox2 with specific siRNA prevented the HG-induced increase in intracellular reactive oxygen species (ROS) generation and hydrogen peroxide (H2O2) production in cultured podocytes. In additional experiments, we established a functional link between Duox2-derived ROS generation and podocyte injury in response to HG. Impairment of Duox2 function nearly abolished HG-mediated DNA fragmentation, apoptosis and decrease in/reorganization of slit diaphragm protein expression in podocytes, indicating that Duox2 is required for the deleterious effects of glucose in podocytes. Our study demonstrates, for the first time, that Duox2 is responsible for increased ROS generation and subsequent alteration of podocyte function in response to HG. Our work serves as proof of concept to demonstrate the utility of targeting Duox2 as a future therapeutic intervention to reduce diabetes-mediated glomerular lesions. Research reported in this poster was supported by the University of the Incarnate Word Offce of Research and Graduate Studies and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC2GM136569. The content is solely the responsibility of the authors and does not necessarily represent the offcial views of the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Potato protein hydrolysate inhibits RANKL-induced osteoclast development by inhibiting osteoclastogenic genes via the NF-κB/MAPKs signaling pathways.

In recent times, there has been growing attention towards exploring the nutritional and functional aspects of potato protein, along with its diverse applications. In the present study, we examined the anti-osteoclast properties of potato protein hydrolysate (PP902) in vitro. Murine macrophages (RAW264.7) were differentiated into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL), and PP902 was examined for its inhibitory effect. Initially, treatment with PP902 was found to significantly prevent RANKL-induced morphological changes in macrophage cells, as determined by tartrate-resistant acid phosphatase (TRAP) staining analysis. This notion was further supported by F-actin analysis using a confocal microscope. Furthermore, PP902 treatment effectively and dose-dependently down-regulated the expression of RANKL-induced osteoclastogenic marker genes, including TRAP, CTR, RANK, NFATc1, OC-STAMP, and c-Fos. These inhibitory effects were associated with suppressing NF-κB transcriptional activation and subsequent reduced nuclear translocation. The decrease in NF-κB activity resulted from reduced activation of its upstream kinases, including I-κBα and IKKα. Moreover, PP902 significantly inhibited RANKL-induced p38MAPK and ERK1/2 activities. Nevertheless, PP902 treatment prevents RANKL-induced intracellular reactive oxygen species generation via increased HO-1 activity. The combined antioxidant and anti-inflammatory effects of PP902 resulted in significant suppression of osteoclastogenesis, suggesting its potential as an adjuvant therapy for osteoclast-related diseases.

Molecular mechanisms of mitochondria-mediated ferroptosis: a potential target for antimalarial interventions.

Ferroptosis is an iron-dependent form of regulated cell death characterized by glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4) inactivation, and the build-up of lipotoxic reactive species. Ferroptosis-targeted induction is a promising therapeutic approach for addressing antimalarial drug resistance. In addition to being the primary source of intracellular energy supply and reactive oxygen species (ROS) generation, mitochondria actively participate in diverse forms of regulated cell death, including ferroptosis. Altered mitochondrial morphology and functionality are attributed to ferroptosis. Diverse mitochondria-related proteins and metabolic activities have been implicated in fine-tuning the action of ferroptosis inducers. Herein, we review recent progress in this evolving field, elucidating the numerous mechanisms by which mitochondria regulate ferroptosis and giving an insight into the role of the organelle in ferroptosis. Additionally, we present an overview of how mitochondria contribute to ferroptosis in malaria. Furthermore, we attempt to shed light on an inclusive perspective on how targeting malaria parasites' mitochondrion and attacking redox homeostasis is anticipated to induce ferroptosis-mediated antiparasitic effects.

High Singlet Oxygen Generating Benzothienyl‐[b]‐fused BODIPY Nanoaggregates and Photoinduced Apoptosis in Human Carcinoma

AbstractHeavy atom free organic materials absorbing in red to near infrared (NIR) region are potential candidates as photosensitizers (PS) in photodynamic therapy (PDT). PDT relies on many factors and one of them is singlet oxygen generation ability of PS in aqueous medium. In this contribution, we report the synthesis of a new PS i. e., 8‐thienyl bisbenzothieno‐[b]‐fused‐boron‐dipyrromethene (BT‐[b]‐fused BODIPY). This PS shows absorption (λabs) at ~650 nm in toluene while in its water‐dispersible nanoaggregates it is found at ~764 nm. Nanoaggregates of BT‐[b]‐fused BODIPY demonstrated dual advantages, (i) facilitating PS release in the cellular system and (ii) sharply enhanced singlet oxygen efficiency ( ~48 %) in aqueous medium. Intracellular reactive oxygen species (ROS) generation was confirmed by 2′,7′‐dichlorodihydrofluorescein diacetate (DCFDA) assay in A549 cells. In vitro photocytotoxic studies of BT‐[b]‐fused BODIPY are carried out in different carcinomas i. e., MCF‐7 and A549 cells. It has zero dark cell toxicity while half maximal inhibitory concentration (IC50) in MCF‐7 is found to be low (6.08±1.1) μM and 7.41±1.9 μM in A549 on 15 min light exposure. These nanoaggregates induce apoptosis in cancer cells on light irradiation thereby proving it to be a potential PS for PDT.

Antioxidant Activity and Antitumor Potential of Hedyotis chrysotricha Extract in Human Colorectal Cancer and Breast Cancer Cell Lines

Background: Cancer is a serious disease severely endangering human health and life in the world in the world, and conventional anticancer drugs have frequently suffered from severe side effects and resistance. Therefore, developing novel and effective therapeutic strategies is urgently needed. As a Chinese herbal medicine with important medical values, Hedyotis chrysotricha has been suggested to be useful for the treatment of many diseases, especially cancer. Objective: Our aim was to investigate the antioxidant and antitumor activities of the phytochemical extracts of H. chrysotricha. Methods: 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay was applied to determine antioxidant property. 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay, wound healing assay, transwell matrix assay, flow cytometry and cell morphology were established to observe anti-proliferative, anti-migration, anti-invasion and apoptosis induction effects. The intracellular reactive oxygen species generation was detected by reactive oxygen species detection assay. Results: The ethyl acetate fraction showed promising antioxidant activity with IC50 of 98.67±0.49 μg/ml, and petroleum ether fraction exhibited the most potent antiproliferative effect on various human cancer cell lines, especially MDA-MB-231 (IC50 = 8.90±1.23 μg/ml) and HCT-116 (IC50 = 9.69±3.69 μg/ml) cancer cells. Further investigation revealed that the petroleum ether fraction suppressed the proliferation, migration and invasion of the cancer cells significantly. Additionally, it promoted cell apoptosis by increasing reactive oxygen species levels. Conclusion: H. chrysotricha extract possessed excellent antioxidant and antitumor activities. Therefore, it could be useful as a source of antioxidants and compounds for cancer therapy.

Influence of dual-cure resin-cement curing modes on gingival cytotoxicity and inflammatory responses

Background/purposeDual-cure resin-cements are used for various dental restorations. However, whether the curing modes of these resin-cements influence gingival inflammation remains unclear. Hence, herein, we evaluated the effects of dual-cure resin-cement curing modes on gingival cytotoxicity and inflammatory responses. Materials and methodsSpecimens were prepared using two dual-cure resin-cements—RelyX Unicem 2 (RU) and G-CEM ONE (GO)—by light-cure or self-cure modes. Degree of conversion (DC) and monomer elution of the resin-cements were measured using Fourier-transform infrared spectroscopy and high-performance liquid chromatography, respectively. Human gingival fibroblasts (GFs) and macrophages were cultured on resin-cements, and inflammatory cytokine levels, intracellular reactive oxygen species (ROS) generation, and mitogen-activated protein (MAP) kinase activation were evaluated. ResultsLight-cured (LC) resin-cements exhibited significantly higher DC and lower monomer elution than did self-cured (SC) resin-cements. Triethyleneglycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) were substantially eluted from RU and GO, respectively. Neither LC resin-cement exhibited cytotoxicity and enhanced pro-inflammatory cytokine expression in GFs and macrophages. However, both SC resin-cements significantly decreased cell numbers and promoted cellular inflammatory responses. SC generated higher intracellular ROS levels compared to that seen with LC, and different patterns of MAP kinase activation were observed between SC–RU and SC–GO. ConclusionCompared with LC dual-cure resin-cements, SC dual-cure resin-cements show stronger cytotoxicity and elicit greater inflammatory responses in gingival cells owing to residual monomers (e.g., TEGDMA and UDMA) by activating MAP kinases in GFs and macrophages. Clinicians should ensure adequate light irradiation during prosthesis cementation and make efforts to remove the excess cement.

Protective effect of 3-bromo-4,5-dihydroxybenzaldehyde against PM2.5-induced cell cycle arrest and autophagy in keratinocytes

Particulate matter 2.5 (PM2.5) poses a serious threat to human health and is responsible for respiratory disorders, cardiovascular diseases, and skin disorders. 3-Bromo-4,5- dihydroxybenzaldehyde (3-BDB), abundant in marine red algae, exhibits anti-inflammatory, antioxidant, and anti-diabetic activities. In this study, we investigated the protective mechanisms of 3-BDB against PM2.5-induced cell cycle arrest and autophagy in human keratinocytes. Intracellular reactive oxygen species (ROS) generation, DNA damage, cell cycle arrest, intracellular Ca2+ level, and autophagy activation were tested. 3-BDB was found to restore cell proliferation and viability which were reduced by PM2.5. Furthermore, 3-BDB reduced PM2.5-induced ROS levels, DNA damage, and attenuated cell cycle arrest. Moreover, 3-BDB ameliorated the PM2.5-induced increases in cellular Ca2+ level and autophagy activation. While PM2.5 treatment reduced cell growth and viability, these were restored by the treatment with the autophagy inhibitor bafilomycin A1 or 3-BDB. The findings indicate that 3-BDB ameliorates skin cell death caused by PM2.5 via inhibiting cell cycle arrest and autophagy. Hence, 3-BDB can be exploited as a preventive/therapeutic agent for PM2.5-induced skin impairment.

Tumor targeted nanohybrid for dual stimuli responsive and NIR amplified photothermal/photo-induced thermodynamic/chemodynamic combination therapy

The combination of photodynamic (PDT) and chemodynamic therapy (CDT) for cancer treatment has gathered a lot of attention in recent years. However, its efficacy is severely limited by elevated levels of hypoxia and glutathione (GSH) in the tumor microenvironment (TME). Multifunctional nanoparticles that can help remodel the TME while facilitating PDT/CDT combination therapy are the need of the hour. To this effect, we have developed O2 self-supplying, free radical generating nanohybrids that exhibit near infra-red (NIR) triggered photothermal (PTT)/photo-induced thermodynamic (P-TDT) and CDT for efficient breast cancer treatment. The surface of nanohybrids has been further modified by biointerfacing with cancer cell membrane. The biomimetic nanohybrids have been comprehensively characterized and found to exhibit high 2,2′-azobis-[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) loading, GSH depletion, oxygen self-supply with TME responsive AIPH release. Biological activity assays demonstrate efficient cellular uptake with homotypic targeting, excellent hemo- and cytocompatibility as well as high intracellular reactive oxygen species generation with synergistic cytotoxicity against tumor cells. The multifunctional nanohybrid proposed in the present study provides an attractive strategy for achieving NIR responsive, tumor targeted PTT/P-TDT/CDT combination therapy for breast cancer treatment.

Abstract 5743: Liposome platform enables X-ray induced photodynamic therapy treatment against human triple negative breast cancer cells

Abstract In this study, we employed X-ray induced photodynamic therapy (X-PDT) for the treatment on triple negative breast cancer (TNBC) cells. To do this, we rationally developed a liposome delivery system co-loaded with protoporphyrin IX (PPIX) and perfluorooctyl bromide (PFOB). Low-dose X-ray at 2Gy was employed to activate PPIX for reactive oxygen species (ROS) generation, and the co-loading of PFOB provided additional oxygen to augment ROS production. The highly toxic ROS triggered TNBC cell death. In vitro X-PDT effects including intracellular ROS generation, cytotoxicity, cell viability and apoptosis/necrosis assay in TNBC cells were studied. Our results indicate that the nanocarriers effectively induced X-PDT effect with very low dose radiation, which makes it possible to damage cancer cells. Our strategy may offer a paradigm-shifting treatment alternative for TNBC patients who need neoadjuvant radiotherapy but wish to avoid long term detrimental effect on functional outcome by undergoing X-PDT using only a fraction of the conventional radiotherapy. Citation Format: Biyao Yang, Rui Sang, Yi Li, Ewa Goldys, Wei Deng. Liposome platform enables X-ray induced photodynamic therapy treatment against human triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5743.

Efficient Combination Chemo-Sonodynamic Cancer Therapy Using Mitochondria-Targeting Sonosensitizer-Loaded Polysorbate-Based Micelles.

Sonodynamic therapy (SDT), utilizing ultrasound (US) and sonosensitizers, holds immense potential as a noninvasive and targeted treatment for a variety of deep-seated tumors. However, the clinical translation of SDT is hampered by several key limitations in sonosensitizers, especially their low aqueous stability and poor cellular uptake. In this study, non-ionic polysorbate (Tween 80, T80) was adopted to formulate effective nanocarriers for the safe and efficient delivery of sonosensitizers to cancer cells. Mitochondria-targeting triphenylphosphonium (TPP)-conjugated chlorin e6 (Ce6) sonosensitizer was loaded into T80-based micelles for efficient SDT. Pro-oxidant piperlongumine (PL) was co-encapsulated with TPP-conjugated Ce6 (T-Ce6) in T80 micelles to enable combination chemo-SDT. T80 micelles substantially enhanced the cellular internalization of T-Ce6. As a result, T80 micelles loaded with T-Ce6 and PL [T80(T-Ce6/PL)] significantly elevated intracellular reactive oxygen species (ROS) generation in MCF-7 human breast cancer cells upon US exposure. Moreover, T-Ce6 exhibited selective accumulation within the mitochondria, leading to efficient cell death under US irradiation. Importantly, T80(T-Ce6/PL) micelles caused cancer-specific cell death by selectively triggering apoptosis in cancer cells through PL. This study demonstrated the feasibility of using T80(T-Ce6/PL) micelles for efficient and cancer-specific combination chemo-SDT.

Unraveling Multicopper [Cu3] and [Cu6] Clusters with Rare μ3-Sulfato and Linear μ2-Oxido-Bridges as Potent Antibiofilm Agents against Multidrug-Resistant Staphylococcus aureus.

In this research article, two multicopper [Cu3] and [Cu6] clusters, [Cu3(cpdp)(μ3-SO4)(Cl)(H2O)2]·3H2O (1) and [Cu6(cpdp)2(μ2-O)(Cl)2(H2O)4]·2Cl (2) (H3cpdp = N,N'-bis[2-carboxybenzomethyl]-N,N'-bis[2-pyridylmethyl]-1,3-diaminopropan-2-ol), have been explored as potent antibacterial and antibiofilm agents. Their molecular structures have been determined by a single-crystal X-ray diffraction study, and the compositions have been established by thermal and elemental analyses, including electrospray ionization mass spectrometry. Structural analysis shows that the metallic core of 1 is composed of a trinuclear [Cu3] assembly encapsulating a μ3-SO42- group, whereas the structure of 2 represents a hexanuclear [Cu6] assembly in which two trinuclear [Cu3] motifs are exclusively bridged by a linear μ2-O2- group. The most striking feature of the structure of 2 is the occurrence of an unusual linear oxido-bridge, with the Cu3-O6-Cu3' bridging angle being 180.00°. Whereas 1 can be viewed as an example of a copper(II)-based compound displaying a rare μ3:η1:η1:η1 bridging mode of the SO42- group, 2 is the first example of any copper(II)-based compound showing an unsupported linear Cu-O-Cu oxido-bridge. Employing variable-temperature SQUID magnetometry, the magnetic susceptibility data were measured and analyzed exemplarily for 1 in the temperature range of 2-300 K, revealing the occurrence of antiferromagnetic interactions among the paramagnetic copper centers. Both 1 and 2 exhibited potent antibacterial and antibiofilm activities against methicillin-resistant Staphylococcus aureus (MRSA BAA1717) and the clinically isolated culture of methicillin-resistant S. aureus (MRSA CI1). The mechanism of antibacterial and antibiofilm activities of these multicopper clusters was investigated by analyzing and determining the intracellular reactive oxygen species (ROS) generation, lipid peroxidation, microscopic observation of cell membrane disruption, membrane potential, and leakage of cellular components. Additionally, 1 and 2 showed a synergistic effect with commercially available antibiotics such as vancomycin with enhanced antibacterial activity. However, 1 possesses higher antibacterial, antibiofilm, and antivirulence actions, making it a potent therapeutic agent against both MRSA BAA1717 and MRSA CI1 strains.

Rhizoma Paridis saponins attenuate Gram-negative bacteria-induced inflammatory acne by binding to KEAP1 and modulating Nrf2 and MAPK pathways.

Acne vulgaris represents a chronic inflammatory condition, the pathogenesis of which is closely associated with the altered skin microbiome. Recent studies have implicated a profound role of Gram-negative bacteria in acne development, but there is a lack of antiacne agents targeting these bacteria. Polyphyllins are major components of Rhizoma Paridis with great anti-inflammatory potential. In this study, we aimed to evaluate the antiacne effects and the underlying mechanisms of PPH and a PPH-enriched Rhizoma Paridis extract (RPE) in treating the Gram-negative bacteria-induced acne. PPH and RPE treatments significantly suppressed the mRNA and protein expressions of interleukin (IL)-1β and IL-6 in lipopolysaccharide (LPS)-induced RAW 264.7 and HaCaT cells, along with the intracellular reactive oxygen species (ROS) generation. Furthermore, PPH and RPE inhibited the nuclear translocation of nuclear factor kappa-B (NF-κB) P65 in LPS-induced RAW 264.7 cells. Based on molecular docking, PPH could bind to kelch-like ECH-associated protein 1 (KEAP1) protein. PPH and RPE treatments could activate nuclear factor erythroid 2-related factor 2 (NRF2) and upregulate haem oxygenase-1 (HO-1). Moreover, RPE suppressed the mitogen-activated protein kinase (MAPK) pathway. Therefore, PPH-enriched RPE showed anti-inflammatory and antioxidative effects invitro, which is promising for alternative antiacne therapeutic.

Naringenin Induces Cellular Apoptosis in Melanoma Cells via Intracellular ROS Generation.

Melanoma is a prevalent malignant tumor that arises from melanocytes. The treatment of malignant melanoma has become challenging due to the development of drug resistance. It is, therefore, imperative to identify novel therapeutic drug candidates for controlling malignant melanoma. Naringenin is a flavonoid abundant in oranges and other citrus fruits and recognized for its numerous medicinal benefits. The objective of the study was to assess the anti-carcinogenic potential of naringenin by evaluating its ability to regulate the cellular production of reactive oxygen species (ROS) and its effect on mitochondrial function and apoptosis in melanoma cells. Cell viability, intracellular ROS levels, cell apoptosis, and mitochondrial functions were evaluated. Naringenin decreased melanoma cell viability and triggered generation of ROS, leading to cell apoptosis. In addition, it stimulated mitochondrial damage in melanoma cells by elevating the levels of Ca2+ and ROS in the mitochondria and decreasing cellular ATP. Naringenin stimulated the expression of proapoptotic proteins, including phospho p53, B-cell lymphoma-2 (Bcl-2)-associated X protein, cleaved caspase-3, and cleaved caspase-9, in melanoma cells in a time-dependent manner. Furthermore, it reduced the expression of the anti-apoptotic protein Bcl-2. Naringenin triggered cell apoptosis by phosphorylating c-Jun N-terminal kinase and stimulating cellular autophagy. Naringenin caused oxidative stress and mitochondrial damage, and activated autophagy in melanoma cells, leading to cell apoptosis. These findings indicate the potential of naringenin as a new therapeutic candidate for melanoma.

Surface property and in vitro toxicity effect of insoluble particles given by protein corona: Implication for PM cytotoxicity assessment

In vitro toxicological assessment helps explore key fractions of particulate matter (PM) in association with the toxic mechanism. Previous studies mainly discussed the toxicity effects of the water-soluble and organic-soluble fractions of PM. However, the toxicity of insoluble fractions is relatively poorly understood, and the adsorption of proteins is rarely considered. In this work, the formation of protein corona on the surface of insoluble particles during incubation in a culture medium was investigated. It was found that highly abundant proteins in fetal bovine serum were the main components of the protein corona. The adsorbed proteins increased the dispersion stability of insoluble particles. Meanwhile, the leaching concentrations of some metal elements (e.g., Cu, Zn, and Pb) from PM increased in the presence of proteins. The toxicity effects and potential mechanisms of the PM insoluble particle–protein corona complex on macrophage cells RAW264.7 were discussed. The results revealed that the PM insoluble particle–protein corona complex could influence the phagosome pathway in RAW264.7 cells. Thus, it promoted the intracellular reactive oxygen species generation and induced a greater degree of cell differentiation, significantly altering cell morphology. Consequently, this work sheds new light on the combination of insoluble particles and protein corona in terms of PM cytotoxicity assessment.

A novel MTORC2-AKT-ROS axis triggers mitofission and mitophagy-associated execution of colorectal cancer cells upon drug-induced activation of mutant KRAS

ABSTRACT RAS is one of the most commonly mutated oncogenes associated with multiple cancer hallmarks. Notably, RAS activation induces intracellular reactive oxygen species (ROS) generation, which we previously demonstrated as a trigger for autophagy-associated execution of mutant KRAS-expressing cancer cells. Here we report that drug (merodantoin; C1)-induced activation of mutant KRAS promotes phospho-AKT S473-dependent ROS-mediated S616 phosphorylation and mitochondrial localization of DNM1L/DRP1 (dynamin 1 like) and cleavage of the fusion-associated protein OPA1 (OPA1 mitochondrial dynamin like GTPase). Interestingly, accumulation of the outer mitochondrial membrane protein VDAC1 (voltage dependent anion channel 1) is observed in mutant KRAS-expressing cells upon exposure to C1. Conversely, silencing VDAC1 abolishes C1-induced mitophagy, and gene knockdown of either KRAS, AKT or DNM1L rescues ROS-dependent VDAC1 accumulation and stability, thus suggesting an axis of mutant active KRAS-phospho-AKT S473-ROS-DNM1L-VDAC1 in mitochondrial morphology change and cancer cell execution. Importantly, we identified MTOR (mechanistic target of rapamycin kinsase) complex 2 (MTORC2) as the upstream mediator of AKT phosphorylation at S473 in our model. Pharmacological or genetic inhibition of MTORC2 abrogated C1-induced phosphorylation of AKT S473, ROS generation and mitophagy induction, as well as rescued tumor colony forming ability and migratory capacity. Finally, increase in thermal stability of KRAS, AKT and DNM1L were observed upon exposure to C1 only in mutant KRAS-expressing cells. Taken together, our work has unraveled a novel mechanism of selective targeting of mutant KRAS-expressing cancers via MTORC2-mediated AKT activation and ROS-dependent mitofission, which could have potential therapeutic implications given the relative lack of direct RAS-targeting strategies in cancer. Abbreviations: ACTB/ß-actin: actin beta; AKT: AKT serine/threonine kinase; C1/merodantoin: 1,3-dibutyl-2-thiooxo-imidazoldine-4,5-dione; CAT: catalase; CETSA: cellular thermal shift assay; CHX: cycloheximide; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H2O2: hydrogen peroxide; HSPA1A/HSP70-1: heat shock protein family A (Hsp70) member 1A; HSP90AA1/HSP90: heat shock protein 90 alpha family class A member 1; KRAS: KRAS proto-oncogene, GTPase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; LC3B-I: unlipidated form of LC3B; LC3B-II: phosphatidylethanolamine-conjugated form of LC3B; MAPKAP1/SIN1: MAPK associated protein 1; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MFI: mean fluorescence intensity; MiNA: Mitochondrial Network Analysis; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; O2.−: superoxide; OMA1: OMA1 zinc metallopeptidase; OPA1: OPA1 mitochondrial dynamin like GTPase; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR/raptor: regulatory associated protein of MTOR complex 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; VDAC1: voltage dependent anion channel 1; VDAC2: voltage dependent anion channel 2.

"Multi-in-One" Yolk-Shell Structured Nanoplatform Inducing Pyroptosis and Antitumor Immune Response Through Cascade Reactions.

Pyroptosis, a new mode of regulatory cell death, holds a promising prospect in tumor therapy. The occurrence of pyroptosis can trigger the release of damage-associated molecular patterns (DAMPs) and activate the antitumor immune response. Moreover, enhancing intracellular reactive oxygen species (ROS) generation can effectively induce pyroptosis. Herein, an integrated nanoplatform (hCZAG) based on zeolitic imidazolate framework-8 (ZIF-8) with Cu2+ and Zn2+ as active nodes and glucose oxidase (GOx) loading is constructed to evoke pyroptosis. GOx can effectively elevate intracellular hydrogen peroxide (H2 O2 ) levels to regulate the unfavorable tumor microenvironment (TME). Cu2+ can be reduced to Cu+ by endogenous overexpressed GSH and both Cu2+ and Cu+ can exert Fenton-like activity to promote ROS generation and amplify oxidative stress. In addition, the accumulation of Cu2+ leads to the aggregation of lipoylated dihydrolipoamide S-acetyltransferase (DLAT), thus resulting in cuproptosis. Notably, the outburst of ROS induced by hCZAG activates Caspase-1 proteins, leads to the cleavage of gasdermin D (GSDMD), and induces pyroptosis. Pyroptosis further elicits an adaptive immune response, leading to immunogenic cell death (ICD). This study provides effective strategies for triggering pyroptosis-mediated immunotherapy and achieving improved therapeutic effects.

Ultrasound-Responsive Nanodroplet-Based Targeted Therapy via Conversion to Microbubbles.

Ultrasound-based therapy is appealing as it can be used via a wireless approach at remote parts of the body including the brain. Microbubbles are commonly used in such therapy due to their highly sound-responsive property. However, the larger size of microbubbles limits selective targeting in vitro/in vivo. Here, we report the design of nanodroplets of 70-130 nm in size that can be easily converted to microbubbles via ultrasound exposure. The advantage of this approach is that smaller nanodroplets can be used for cell/subcellular targeting, and next, they can be used for therapy by converting to microbubbles. More specifically, folate/dopamine-terminated perfluorohexane nanodroplets are designed that are loaded with a molecular drug. These nanodroplets are used for selective cell targeting, followed by ultrasound-induced microbubble conversion that is associated with drug release and intracellular reactive oxygen species generation. This approach has been used for selective cell therapy applications. The designed nanodroplet and approach can be used for the enhanced therapeutic performance of existing drugs.