Pulsed Electromagnetic Field Enhances Caffeic Acid Phenethyl Ester-induced Death of MCF-7 Breast Cancer Cells.

BACKGROUND/AIM: Caffeic acid phenethyl ester (CAPE) exerts anticancer effects against several cancer types, including breast cancer. Pulsed electromagnetic field (PEMF) improves the efficiency of some chemotherapeutic drugs. In this study, we examined the effects of PEMF stimulation on the anticancer activity of CAPE in MCF-7 breast cancer cells and the underlying signal transduction pathways. MATERIALS AND METHODS: MCF-7 cells were seeded and incubated for 24 h. Each of the drugs (5-fluorouracil, paclitaxel, gefitinib, or CAPE) was added to the cells on day 0. Then, cells were immediately stimulated with a 60-min PEMF session thrice a day (with 4-h interval between sessions) for 1-3 days. Cell death and viability were assessed by flow cytometry and trypan blue dye exclusion assay. Molecular mechanisms involved in cell death were confirmed by western blot assay. RESULTS: Compared with treatment with CAPE alone, co-treatment with CAPE and PEMF more strongly reduced the viability of MCF-7 cells, further increased the percentage of the sub-G1 population, poly (ADP-ribose) polymerase (PARP) cleavage, activation of apoptotic caspases, up-regulation of pro-apoptotic proteins, such as Fas cell surface death receptor (FAS) and BCL2 associated X, apoptosis regulator (BAX), and reduced the expression of anti-apoptotic proteins, such as BCL-2 apoptosis regulator (BCL-2), MCL-1 apoptosis regulator, BCL-2 family member (MCL-1), and survivin. PEMF stimulation also increased CAPE-induced phosphorylation of p53, and inhibition of p53 partially restored the PEMF-reduced viability of CAPE-treated MCF-7 cells. CONCLUSION: PEMF stimulation enhanced CAPE-induced cell death by activating p53, which regulates the expression of apoptosis-related molecules, subsequently activating the caspase-dependent apoptotic pathway in MCF-7 cells, suggesting that PEMF can be utilized as an adjuvant to enhance the effect of CAPE on breast cancer cells.

Parkin Enhances Gefitinib-induced Anoikis in HeLa Cervical Cancer Cells.

BACKGROUND/AIM: Gefitinib exhibits anticancer activity against cervical cancer cells via anoikis, a type of apoptosis induced by cell detachment from the extracellular matrix. Previous studies have reported that Parkin expression affects the efficacy of anticancer drugs. However, the impact of Parkin expression on the therapeutic effects of gefitinib in human cervical cancer remains unclear. Thus, this study aimed to evaluate whether Parkin over-expression improves the therapeutic effects of gefitinib against HeLa cervical cancer cells. MATERIALS AND METHODS: Cell viability and apoptotic death of HeLa cells were measured by trypan blue dye exclusion assay and flow cytometry. Cell detachment, adhesion, spreading, and cell-cell interaction were observed by inverted microscopy. Alteration of adhesion-related molecules was evaluated by confocal microscopy and western blot assay. RESULTS: Parkin expression potentiated gefitinib-induced cell detachment by affecting the organization of the actin cytoskeleton. In addition, Parkin expression induced a further reduction in the reattachment of and interaction between detached cells. The therapeutic efficacy of low-dose gefitinib combined with Parkin expression was equivalent to that of high-dose gefitinib alone. CONCLUSION: Parkin expression promotes gefitinib-induced anoikis, consequently increasing the efficacy of gefitinib against HeLa human cervical cancer cells. Based on our results, we propose that Parkin can be used to increase the anti-cancer effect of gefitinib on cervical cancer cells.

Gefitinib induces anoikis in cervical cancer cells.

Gefitinib exerts anticancer effects on various types of cancer, such as lung, ovarian, breast, and colon cancers. However, the therapeutic effects of gefitinib on cervical cancer and the underlying mechanisms remain unclear. Thus, this study aimed to explore whether gefitinib can be used to treat cervical cancer and elucidate the underlying mechanisms. Results showed that gefitinib induced a caspase-dependent apoptosis of HeLa cells, which consequently became round and detached from the surface of the culture plate. Gefitinib induced the reorganization of actin cytoskeleton and downregulated the expression of p-FAK, integrin ß1 and E-cadherin, which are important in cell-extracellular matrix adhesion and cell-cell interaction, respectively. Moreover, gefitinib hindered cell reattachment and spreading and suppressed interactions between detached cells in suspension, leading to poly (ADP-ribose) polymerase cleavage, a hallmark of apoptosis. It also induced detachment-induced apoptosis (anoikis) in C33A cells, another cervical cancer cell line. Taken together, these results suggest that gefitinib triggers anoikis in cervical cancer cells. Our findings may serve as a basis for broadening the range of anticancer drugs used to treat cervical cancer. [BMB Reports 2024; 57(2): 104-109].

TET3 plays a critical role in white adipose development and diet-induced remodeling.

Maintaining healthy adipose tissue is crucial for metabolic health, requiring a deeper understanding of adipocyte development and response to high-calorie diets. This study highlights the importance of TET3 during white adipose tissue (WAT) development and expansion. Selective depletion of Tet3 in adipose precursor cells (APCs) reduces adipogenesis, protects against diet-induced adipose expansion, and enhances whole-body metabolism. Transcriptomic analysis of wild-type and Tet3 knockout (KO) APCs unveiled TET3 target genes, including Pparg and several genes linked to the extracellular matrix, pivotal for adipogenesis and remodeling. DNA methylation profiling and functional studies underscore the importance of DNA demethylation in gene regulation. Remarkably, targeted DNA demethylation at the Pparg promoter restored its transcription. In conclusion, TET3 significantly governs adipogenesis and diet-induced adipose expansion by regulating key target genes in APCs.

Tumor suppressor Parkin induces p53-mediated cell cycle arrest in human lung and colorectal cancer cells.

Dysregulation of the E3 ubiquitin ligase Parkin has been linked to various human cancers, indicating that Parkin is a tumor suppressor protein. However, the mechanisms of action of Parkin remain unclear to date. Thus, we aimed to elucidate the mechanisms of action of Parkin as a tumor suppressor in human lung and colorectal cancer cells. Results showed that Parkin overexpression reduced the viability of A549 human lung cancer cells by inducing G2/M cell cycle arrest. In addition, Parkin caused DNA damage and ATM (Ataxia telangiectasia mutated) activation, which subsequently led to p53 activation. It also induced the p53-mediated upregulation of p21 and downregulation of cyclin B1. Moreover, Parkin suppressed the proliferation of HCT-15 human colorectal cancer cells by a mechanism similar to that in A549 lung cancer cells. Taken together, our results suggest that the tumor-suppressive effects of Parkin on lung and colorectal cancer cells are mediated by DNA damage/p53 activation/cyclin B1 reduction/cell cycle arrest. [BMB Reports 2023; 56(10): 557-562].

Alpha-synuclein fibrils amplified from multiple system atrophy and Parkinson's disease patient brain spread after intracerebral injection into mouse brain.

Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB) are neurodegenerative disorders with alpha-synuclein (α-syn) aggregation pathology. Different strains of α-syn with unique properties are suggested to cause distinct clinical and pathological manifestations resulting in PD, MSA, or DLB. To study individual α-syn spreading patterns, we injected α-syn fibrils amplified from brain homogenates of two MSA patients and two PD patients into the brains of C57BI6/J mice. Antibody staining against pS129-α-syn showed that α-syn fibrils amplified from the brain homogenates of the four different patients caused different levels of α-syn spreading. The strongest α-syn pathology was triggered by α-syn fibrils of one of the two MSA patients, followed by comparable pS129-α-syn induction by the second MSA and one PD patient material. Histological analysis using an antibody against Iba1 further showed that the formation of pS129-α-syn is associated with increased microglia activation. In contrast, no differences in dopaminergic neuron numbers or co-localization of α-syn in oligodendrocytes were observed between the different groups. Our data support the spreading of α-syn pathology in MSA, while at the same time pointing to spreading heterogeneity between different patients potentially driven by individual patient immanent factors.

Statins suppress cell-to-cell propagation of α-synuclein by lowering cholesterol.

Cell-to-cell propagation of protein aggregates has been implicated in the progression of neurodegenerative diseases. However, the underlying mechanism and modulators of this process are not fully understood. Here, we screened a small-molecule library in a search for agents that suppress the propagation of α-synuclein and mutant huntingtin (mHtt). These screens yielded several molecules, some of which were effective against both α-synuclein and mHtt. Among these molecules, we focused on simvastatin and pravastatin. Simvastatin administration in a transgenic model of synucleinopathy effectively ameliorated behavioral deficits and α-synuclein accumulation, whereas pravastatin had no effect. Because only simvastatin enters the brain effectively, these results suggest that inhibition of brain cholesterol synthesis is important in simvastatin effects. In cultured cells, accumulation of intracellular cholesterol, induced by genetic ablation of the NPC1 gene or by pharmacological treatment with U18666A, increased α-synuclein aggregation and secretion. In contrast, lowering cholesterol using methyl-ß-cyclodextrin or statins reversed α-synuclein aggregation and secretion in NPC1-knockout cells. Consistent with these observations, feeding a high-fat diet aggravated α-synuclein pathology and behavioral deficits in the preformed fibril-injected mouse model, an effect that was also reversed by simvastatin administration. These results suggest that statins suppress propagation of protein aggregates by lowering cholesterol in the brain.

Triglyceride induces DNA damage leading to monocyte death by activating caspase-2 and caspase-8.

Monocytes are peripheral leukocytes that function in innate immunity. Excessive triglyceride (TG) accumulation causes monocyte death and thus can compromise innate immunity. However, the mechanisms by which TG mediates monocyte death remain unclear to date. Thus, this study aimed to elucidate the mechanisms by which TG induces monocyte death. Results showed that TG induced monocyte death by activating caspase-3/7 and promoting poly (ADP-ribose) polymerase (PARP) cleavage. In addition, TG induced DNA damage and activated the ataxia telangiectasia mutated (ATM)/checkpoint kinase 2 and ATM-and Rad3-related (ATR)/checkpoint kinase 1 pathways, leading to the cell death. Furthermore, TG-induced DNA damage and monocyte death were mediated by caspase-2 and -8, and caspase-8 acted as an upstream molecule of caspase-2. Taken together, these results suggest that TG-induced monocyte death is mediated via the caspase-8/caspase-2/DNA damage/executioner caspase/PARP pathways. [BMB Reports 2023; 56(3): 166-171].

Inflammation promotes synucleinopathy propagation.

The clinical progression of neurodegenerative diseases correlates with the spread of proteinopathy in the brain. The current understanding of the mechanism of proteinopathy spread is far from complete. Here, we propose that inflammation is fundamental to proteinopathy spread. A sequence variant of α-synuclein (V40G) was much less capable of fibril formation than wild-type α-synuclein (WT-syn) and, when mixed with WT-syn, interfered with its fibrillation. However, when V40G was injected intracerebrally into mice, it induced aggregate spreading even more effectively than WT-syn. Aggregate spreading was preceded by sustained microgliosis and inflammatory responses, which were more robust with V40G than with WT-syn. Oral administration of an anti-inflammatory agent suppressed aggregate spreading, inflammation, and behavioral deficits in mice. Furthermore, exposure of cells to inflammatory cytokines increased the cell-to-cell propagation of α-synuclein. These results suggest that the inflammatory microenvironment is the major driver of the spread of synucleinopathy in the brain.

Quaternary structure of patient-homogenate amplified α-synuclein fibrils modulates seeding of endogenous α-synuclein.

Parkinson's disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases that are neuropathologically characterized by α-synuclein inclusions. Increasing evidence supports the aggregation of α-synuclein in specific brain areas early in the disease course, followed by the spreading of α-synuclein pathology to multiple brain regions. However, little is known about how the structure of α-synuclein fibrils influence its ability to seed endogenous α-synuclein in recipient cells. Here, we aggregated α-synuclein by seeding with homogenates of PD- and MSA-confirmed brain tissue, determined the resulting α-synuclein fibril structures by cryo-electron microscopy, and characterized their seeding potential in mouse primary oligodendroglial cultures. The combined analysis shows that the two patient material-amplified α-synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface and their ability to recruit endogenous α-synuclein. Our study indicates that the quaternary structure of α-synuclein fibrils modulates the seeding of α-synuclein pathology inside recipient cells. It thus provides an important advance in the quest to understand the connection between the structure of α-synuclein fibrils, cellular seeding/spreading, and ultimately the clinical manifestations of different synucleinopathies.

TNF-α promotes α-synuclein propagation through stimulation of senescence-associated lysosomal exocytosis.

Cell-to-cell propagation of α-synuclein is thought to be the underlying mechanism of Parkinson's disease progression. Recent evidence suggests that inflammation plays an important role in the propagation of protein aggregates. However, the mechanism by which inflammation regulates the propagation of aggregates remains unknown. Here, using in vitro cultures, we found that soluble factors secreted from activated microglia promote cell-to-cell propagation of α-synuclein and further showed that among these soluble factors, TNF-α had the most robust stimulatory activity. Treatment of neurons with TNF-α triggered cellular senescence, as shown by transcriptomic analyses demonstrating induction of senescence-associated genes and immunoanalysis of senescence phenotype marker proteins. Interestingly, secretion of α-synuclein was increased in senescent neurons, reflecting acquisition of a senescence-associated secretory phenotype (SASP). Using vacuolin-1, an inhibitor of lysosomal exocytosis, and RNAi against rab27a, we demonstrated that the SASP was mediated by lysosomal exocytosis. Correlative light and electron microscopy and immunoelectron microscopy confirmed that propagating α-synuclein aggregates were present in electron-dense lysosome-like compartments. TNF-α promoted the SASP through stimulation of lysosomal exocytosis, thereby increasing the secretion of α-synuclein. Collectively, these results suggest that TNF-α is the major inflammatory factor that drives cell-to-cell propagation of α-synuclein by promoting the SASP and subsequent secretion of α-synuclein.

AIFM2 Is Required for High-Intensity Aerobic Exercise in Promoting Glucose Utilization.

Skeletal muscle is a major regulator of glycemic control at rest, and glucose utilization increases drastically during exercise. Sustaining a high glucose utilization via glycolysis requires efficient replenishment of NAD+ in the cytosol. Apoptosis-inducing mitochondrion-associated factor 2 (AIFM2) was previously shown to be a NADH oxidoreductase domain-containing flavoprotein that promotes glycolysis for diet and cold-induced thermogenesis. Here, we find that AIFM2 is selectively and highly induced in glycolytic extensor digitorum longus (EDL) muscle during exercise. Overexpression (OE) of AIFM2 in myotubes is sufficient to elevate the NAD+-to-NADH ratio, increasing the glycolytic rate. Thus, OE of AIFM2 in skeletal muscle greatly increases exercise capacity, with increased glucose utilization. Conversely, muscle-specific Aifm2 depletion via in vivo transfection of hairpins against Aifm2 or tamoxifen-inducible haploinsufficiency of Aifm2 in muscles decreases exercise capacity and glucose utilization in mice. Moreover, muscle-specific introduction of NDE1, Saccharomyces cerevisiae external NADH dehydrogenase (NDE), ameliorates impairment in glucose utilization and exercise intolerance of the muscle-specific Aifm2 haploinsufficient mice. Together, we show a novel role for AIFM2 as a critical metabolic regulator for efficient utilization of glucose in glycolytic EDL muscles.

Conformation-specific Antibodies Targeting Aggregated Forms of α-synuclein Block the Propagation of Synucleinopathy.

Abnormal aggregation of α-synuclein is a key element in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. α-synuclein aggregation spreads through various brain regions during the course of disease progression, a propagation that is thought to be mediated by the secretion and subsequent uptake of extracellular α-synuclein aggregates between neuronal cells. Thus, aggregated forms of this protein have emerged as promising targets for disease-modifying therapy for PD and related diseases. Here, we generated and characterized conformation-specific antibodies that preferentially recognize aggregated forms of α-synuclein. These antibodies promoted phagocytosis of extracellular α-synuclein aggregates by microglial cells and interfered with cell-to-cell propagation of α-synuclein. In an α-synuclein transgenic model, passive immunization with aggregate-specific antibodies significantly ameliorated pathological phenotypes, reducing α-synuclein aggregation, gliosis, inflammation, and neuronal loss. These results suggest that conformation-specific antibodies targeting α-synuclein aggregates are promising therapeutic agents for PD and related synucleinopathies.

Pulsed electromagnetic field potentiates etoposide-induced MCF-7 cell death.

Etoposide is a chemotherapeutic medication used to treat various types of cancer, including breast cancer. It is established that pulsed electromagnetic field (PEMF) therapy can enhance the effects of anti-cancer chemotherapeutic agents. In this study, we investigated whether PEMFs influence the anti-cancer effects of etoposide in MCF-7 cells and determined the signal pathways affected by PEMFs. We observed that co-treatment with etoposide and PEMFs led to a decrease in viable cells compared with cells solely treated with etoposide. PEMFs elevated the etoposide- induced PARP cleavage and caspase-7/9 activation and enhanced the etoposide-induced down-regulation of survivin and up-regulation of Bax. PEMF also increased the etoposideinduced activation of DNA damage-related molecules. In addition, the reactive oxygen species (ROS) level was slightly elevated during etoposide treatment and significantly increased during co-treatment with etoposide and PEMF. Moreover, treatment with ROS scavenger restored the PEMF-induced decrease in cell viability in etoposide-treated MCF-7 cells. These results combined indicate that PEMFs enhance etoposide-induced cell death by increasing ROS induction-DNA damage-caspase-dependent apoptosis. [BMB Reports 2022; 55(3): 148-153].

Lewy Body-like Inclusions in Human Midbrain Organoids Carrying Glucocerebrosidase and α-Synuclein Mutations.

OBJECTIVE: We utilized human midbrain-like organoids (hMLOs) generated from human pluripotent stem cells carrying glucocerebrosidase gene (GBA1) and α-synuclein (α-syn; SNCA) perturbations to investigate genotype-to-phenotype relationships in Parkinson disease, with the particular aim of recapitulating α-syn- and Lewy body-related pathologies and the process of neurodegeneration in the hMLO model. METHODS: We generated and characterized hMLOs from GBA1-/- and SNCA overexpressing isogenic embryonic stem cells and also generated Lewy body-like inclusions in GBA1/SNCA dual perturbation hMLOs and conduritol-b-epoxide-treated SNCA triplication hMLOs. RESULTS: We identified for the first time that the loss of glucocerebrosidase, coupled with wild-type α-syn overexpression, results in a substantial accumulation of detergent-resistant, ß-sheet-rich α-syn aggregates and Lewy body-like inclusions in hMLOs. These Lewy body-like inclusions exhibit a spherically symmetric morphology with an eosinophilic core, containing α-syn with ubiquitin, and can also be formed in Parkinson disease patient-derived hMLOs. We also demonstrate that impaired glucocerebrosidase function promotes the formation of Lewy body-like inclusions in hMLOs derived from patients carrying the SNCA triplication. INTERPRETATION: Taken together, the data indicate that our hMLOs harboring 2 major risk factors (glucocerebrosidase deficiency and wild-type α-syn overproduction) of Parkinson disease provide a tractable model to further elucidate the underlying mechanisms for progressive Lewy body formation. ANN NEUROL 2021;90:490-505.

Epigenetic regulation of inflammatory factors in adipose tissue.

Obesity is a strong risk factor for insulin resistance. Chronic low-grade tissue inflammation and systemic inflammation have been proposed as major mechanisms that promote insulin resistance in obesity. Adipose tissue has been recognized as a nexus between inflammation and metabolism, but how exactly inflammatory gene expression is orchestrated during the development of obesity is not well understood. Epigenetic modifications are defined as heritable changes in gene expression and cellular function without changes to the original DNA sequence. The major epigenetic mechanisms include DNA methylation, histone modification, noncoding RNAs, nucleopositioning/remodeling and chromatin reorganization. Epigenetic mechanisms provide a critical layer of gene regulation in response to environmental changes. Accumulating evidence supports that epigenetics plays a large role in the regulation of inflammatory genes in adipocytes and adipose-resident immune cell types. This review focuses on the association between adipose tissue inflammation in obesity and major epigenetic modifications.

A necessary role of DNMT3A in endurance exercise by suppressing ALDH1L1-mediated oxidative stress.

Exercise can alter the skeletal muscle DNA methylome, yet little is known about the role of the DNA methylation machinery in exercise capacity. Here, we show that DNMT3A expression in oxidative red muscle increases greatly following a bout of endurance exercise. Muscle-specific Dnmt3a knockout mice have reduced tolerance to endurance exercise, accompanied by reduction in oxidative capacity and mitochondrial respiration. Moreover, Dnmt3a-deficient muscle overproduces reactive oxygen species (ROS), the major contributors to muscle dysfunction. Mechanistically, we show that DNMT3A suppresses the Aldh1l1 transcription by binding to its promoter region, altering its epigenetic profile. Forced expression of ALDH1L1 elevates NADPH levels, which results in overproduction of ROS by the action of NADPH oxidase complex, ultimately resulting in mitochondrial defects in myotubes. Thus, inhibition of ALDH1L1 pathway can rescue oxidative stress and mitochondrial dysfunction from Dnmt3a deficiency in myotubes. Finally, we show that in vivo knockdown of Aldh1l1 largely rescues exercise intolerance in Dnmt3a-deficient mice. Together, we establish that DNMT3A in skeletal muscle plays a pivotal role in endurance exercise by controlling intracellular oxidative stress.

Activation of pannexin-1 mediates triglyceride-induced macrophage cell death.

The accumulation of triglycerides (TGs) in macrophages induces cell death, a risk factor in the pathogenesis of atherosclerosis. We had previously reported that TG-induced macrophage death is triggered by caspase-1 and -2, therefore we investigated the mechanism underlying this phenomenon. We found that potassium efflux is increased in TG-treated THP-1 macrophages and that the inhibition of potassium efflux blocks TG-induced cell death as well as caspase-1 and -2 activation. Furthermore, reducing ATP concentration (known to induce potassium efflux), restored cell viability and caspase-1 and -2 activity. The activation of pannexin-1 (a channel that releases ATP), was increased after TG treatment in THP-1 macrophages. Inhibition of pannexin-1 activity using its inhibitor, probenecid, recovered cell viability and blocked the activation of caspase-1 and -2 in TG-treated macrophages. These results suggest that TG-induced THP-1 macrophage cell death is induced via pannexin- 1 activation, which increases extracellular ATP, leading to an increase in potassium efflux. [BMB Reports 2020; 53(11): 588-593].