Search Results (60)

Difenoconazole (DIF) is a fungicide used to control various fungi. It is absorbed on the surface of different plants and contributes significantly to increased crop production. However, DIF is reported to exhibit toxicity to fungi and to aquatic plants, fish, and mammals, including humans, causing adverse effects. However, research on the impact of DIF on the mammary epithelial cells of herbivorous bovines is limited. DIF-induced damage and accumulation in the mammary glands can have direct and indirect effects on humans. Therefore, we investigated the effects and mechanisms of DIF toxicity in MAC-T cells. The current study revealed that DIF reduces cell viability and proliferation while triggering apoptotic cell death through the upregulation of pro-apoptotic proteins, including cleaved caspase 3 and Bcl-2-associated X protein (BAX), and the downregulation of leukemia type 2 (BCL-2). DIF also induced endoplasmic reticulum (ER) stress by increasing the expression of genes or proteins of Bip/GRP78, protein disulfide isomerase (PDI), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and endoplasmic reticulum oxidoreductase 1 Alpha (ERO1-Lα). We demonstrated that DIF induces mitochondria-mediated apoptosis in MAC-T cells by activating ER stress pathways. This cellular damage resulted in a significant increase in the expression of inflammatory response genes and proteins, including cyclooxygenase 2 (COX2), transforming growth factor beta 3 (TGFB3), CCAAT enhancer binding protein delta (CEBPD), and iNOS, in DIF-treated groups. In addition, spheroid formation by MAC-T cells was suppressed by DIF treatment. Our findings suggest that DIF exposure in dairy cows may harm mammary gland function and health and may indirectly affect human consumption of milk. Full article

Anticancer drugs induce apoptotic and non-apoptotic cell death in various cancer types. The signaling pathways for anticancer drug-induced apoptotic cell death have been shown to differ between drug-sensitive and drug-resistant cells. In atypical multidrug-resistant leukemia cells, the c-Jun/activator protein 1 (AP-1)/p53 signaling pathway leading to apoptotic death is altered. Cancer cells treated with anticancer drugs undergo c-Jun/AP-1–mediated apoptotic death and are involved in c-Jun N-terminal kinase activation and growth arrest- and DNA damage-inducible gene 153 (Gadd153)/CCAAT/enhancer-binding protein homologous protein pathway induction, regardless of the p53 genotype. Gadd153 induction is associated with mitochondrial membrane permeabilization after anticancer drug treatment and involves a coupled endoplasmic reticulum stress response. The induction of apoptosis by anticancer drugs is mediated by the intrinsic pathway (cytochrome c, Cyt c) and subsequent activation of the caspase cascade via proapoptotic genes (e.g., Bax and Bcl-xS) and their interactions. Anticancer drug-induced apoptosis involves caspase-dependent and caspase-independent pathways and occurs via intrinsic and extrinsic pathways. The targeting of antiapoptotic genes such as Bcl-2 enhances anticancer drug efficacy. The modulation of apoptotic signaling by Bcl-xS transduction increases the sensitivity of multidrug resistance-related protein-overexpressing epidermoid carcinoma cells to anticancer drugs. The significance of autophagy in cancer therapy remains to be elucidated. In this review, we summarize current knowledge of cancer cell death-related signaling pathways and their alterations during anticancer drug treatment and discuss potential strategies to enhance treatment efficacy. Full article

The Epstein–Barr virus (EBV) is accepted as a primary risk factor for certain nasopharyngeal carcinoma (NPC) subtypes, where the virus persists in a latent stage which is thought to contribute to tumorigenesis. Current treatments are sub-optimal, and recurrence occurs in many cases. An alternative therapeutic concept is aimed at triggering the lytic cycle of EBV selectively in tumor cells as a means to add clinical benefit. While compounds able to stimulate the lytic cascade have been identified, their clinical application so far has been limited. We are developing a novel anticancer molecule, NEO212, that was generated by covalent conjugation of the alkylating agent temozolomide (TMZ) to the naturally occurring monoterpene perillyl alcohol (POH). In the current study, we investigated its potential to trigger the lytic cycle of EBV in NPC cells in vitro and in vivo. We used the established C666.1 cell line and primary patient cells derived from the brain metastasis of a patient with NPC, both of which harbored latent EBV. Upon treatment with NEO212, there was an increase in EBV proteins Zta and Ea-D, key markers of the lytic cycle, along with increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP), a marker of endoplasmic reticulum (ER) stress, followed by the activation of caspases. These effects could also be confirmed in tumor tissue from mice implanted with C666.1 cells. Towards a mechanistic understanding of these events, we used siRNA-mediated knockdown of CHOP and inclusion of anti-oxidant compounds. Both approaches blocked lytic cycle induction by NEO212. Therefore, we established a sequence of events, where NEO212 caused reactive oxygen species (ROS) production, which triggered ER stress and elevated the levels of CHOP, which was required to stimulate the lytic cascade of EBV. Inclusion of the antiviral agent ganciclovir synergistically enhanced the cytotoxic impact of NEO212, pointing to a potential combination treatment for EBV-positive cancers which should be explored further. Overall, our study establishes NEO212 as a novel agent able to stimulate EBV’s lytic cycle in NPC tumors, with implications for other virus-associated cancers. Full article

Obesity and metabolic syndrome involve chronic low-grade inflammation called metabolic inflammation as well as metabolic derangements from increased endotoxin and free fatty acids. It is debated whether the endoplasmic reticulum (ER) stress in monocytic cells can contribute to amplify metabolic inflammation; if so, by which mechanism(s). To test this, metabolic stress was induced in THP-1 cells and primary human monocytes by treatments with lipopolysaccharide (LPS), palmitic acid (PA), or oleic acid (OA), in the presence or absence of the ER stressor thapsigargin (TG). Gene expression of tumor necrosis factor (TNF)-α and markers of ER/oxidative stress were determined by qRT-PCR, TNF-α protein by ELISA, reactive oxygen species (ROS) by DCFH-DA assay, hypoxia-inducible factor 1-alpha (HIF-1α), p38, extracellular signal-regulated kinase (ERK)-1,2, and nuclear factor kappa B (NF-κB) phosphorylation by immunoblotting, and insulin sensitivity by glucose-uptake assay. Regarding clinical analyses, adipose TNF-α was assessed using qRT-PCR/IHC and plasma TNF-α, high-sensitivity C-reactive protein (hs-CRP), malondialdehyde (MDA), and oxidized low-density lipoprotein (OX-LDL) via ELISA. We found that the cooperative interaction between metabolic and ER stresses promoted TNF-α, ROS, CCAAT-enhancer-binding protein homologous protein (CHOP), activating transcription factor 6 (ATF6), superoxide dismutase 2 (SOD2), and nuclear factor erythroid 2-related factor 2 (NRF2) expression (p ≤ 0.0183),. However, glucose uptake was not impaired. TNF-α amplification was dependent on HIF-1α stabilization and p38 MAPK/p65 NF-κB phosphorylation, while the MAPK/NF-κB pathway inhibitors and antioxidants/ROS scavengers such as curcumin, allopurinol, and apocynin attenuated the TNF-α production (p ≤ 0.05). Individuals with obesity displayed increased adipose TNF-α gene/protein expression as well as elevated plasma levels of TNF-α, CRP, MDA, and OX-LDL (p ≤ 0.05). Our findings support a metabolic–ER stress cooperativity model, favoring inflammation by triggering TNF-α production via the ROS/CHOP/HIF-1α and MAPK/NF-κB dependent mechanisms. This study also highlights the therapeutic potential of antioxidants in inflammatory conditions involving metabolic/ER stresses. Full article

C/EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducible protein 153 (GADD153), belongs to the CCAAT/enhancer-binding protein (C/EBP) family. CHOP expression is induced by unfolded protein response (UPR), and sustained CHOP activation acts as a pivotal trigger for ER stress-induced apoptosis. MicroRNA-616 is located within an intron of the CHOP gene. However, the regulation of miR-616 expression during UPR and its function in breast cancer is not clearly understood. Here we show that the expression of miR-616 and CHOP (host gene of miR-616) is downregulated in human breast cancer. Both miR-5p/-3p arms of miR-616 are expressed with levels of the 5p arm higher than the 3p arm. During conditions of ER stress, the expression of miR-616-5p and miR-616-3p arms was concordantly increased primarily through the PERK pathway. Our results show that ectopic expression of miR-616 significantly suppressed cell proliferation and colony formation, whereas knockout of miR-616 increased it. We found that miR-616 represses c-MYC expression via binding sites located in its protein coding region. Furthermore, we show that miR-616 exerted growth inhibitory effects on cells by suppressing c-MYC expression. Our results establish a new role for the CHOP locus by providing evidence that miR-616 can inhibit cell proliferation by targeting c-MYC. In summary, our results suggest a dual function for the CHOP locus, where CHOP protein and miR-616 can cooperate to inhibit cancer progression. Full article

Poxviruses have been associated with humans for centuries. From smallpox to mpox to lumpy skin disease virus (LSDV), members of the poxvirus family have continued to threaten the lives of humans and domestic animals. A complete understanding of poxvirus-mediated cellular processes will aid in the response to challenges from the viruses. In this study, we demonstrate that LSDV infection results in an abnormal ultrastructure of the endoplasmic reticulum (ER) lumen in primary bovine embryonic fibroblast (BEF) cells, and we further show that an ER imbalance occurs in LSDV-infected BEF cells. Additionally, we believe that ER stress-related apoptosis plays a role in the late apoptosis of BEF cells infected with LSDV, primarily through the activation of the CCAAT/enhancer binding protein homologous protein (CHOP)-Caspase-12 signal. In addition to cell apoptosis, a further investigation showed that LSDV could also activate autophagy in BEF cells, providing additional insight into the exact causes of LSDV-induced BEF cell death. Our findings suggest that LSDV-induced BEF cell apoptosis and autophagy may provide new avenues for laboratory diagnosis of lumpy skin disease progression and exploration of BEF cell processes. Full article

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a poor clinical prognosis and unsatisfactory treatment options. We previously found that the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) is lowly expressed in PDAC compared to healthy pancreas duct cells, and that patient survival and lymph node involvement in PDAC is correlated with the expression of C/EBPδ in primary tumor cells. C/EBPδ shares a homologous DNA-binding sequence with other C/EBP-proteins, leading to the presumption that other C/EBP-family members might act redundantly and compensate for the loss of C/EBPδ. This implies that patient stratification could be improved when expression levels of multiple C/EBP-family members are considered simultaneously. In this study, we assessed whether the quantification of C/EBPβ or C/EBPγ in addition to that of C/EBPδ might improve the prediction of patient survival and lymph node involvement using a cohort of 68 resectable PDAC patients. Using Kaplan–Meier analyses of patient groups with different C/EBP-expression levels, we found that both C/EBPβ and C/EBPγ can partially compensate for low C/EBPδ and improve patient survival. Further, we uncovered C/EBPβ as a novel predictor of a decreased likelihood of lymph node involvement in PDAC, and found that C/EBPβ and C/EBPδ can compensate for the lack of each other in order to reduce the risk of lymph node involvement. C/EBPγ, on the other hand, appears to promote lymph node involvement in the absence of C/EBPδ. Altogether, our results show that the redundancy of C/EBP-family members might have a profound influence on clinical prognoses and that the expression of both C/EPBβ and C/EBPγ should be taken into account when dichotomizing patients according to C/EBPδ expression. Full article

The objective of this study was to clarify the effects of benzalkonium chloride (BAC) on two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells, which are in vitro models replicating the epithelial barrier and the stromal supportive functions of the human conjunctiva. The cultured HconF cells were subjected to the following analyses in the absence and presence of 10⁻⁵% or 10⁻⁴% concentrations of BAC; (1) the barrier function of the 2D HconF monolayers, as determined by trans-endothelial electrical resistance (TEER) and FITC dextran permeability, (2) real-time metabolic analysis using an extracellular Seahorse flux analyzer, (3) the size and stiffness of 3D HconF spheroids, and (4) the mRNA expression of genes that encode for extracellular matrix (ECM) molecules including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), ER stress related genes including the X-box binding protein-1 (XBP1), the spliced XBP1 (sXBP1) glucose regulator protein (GRP)78, GRP94, and the CCAAT/enhancer-binding protein homologous protein (CHOP), hypoxia inducible factor 1α (HIF1α), and Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α). In the presence of BAC, even at low concentrations at 10⁻⁵% or 10⁻⁴%, the maximal respiratory capacity, mitochondrial respiratory reserve, and glycolytic reserve of HconF cells were significantly decreased, although the barrier functions of 2D HconF monolayers, the physical properties of the 3D HconF spheroids, and the mRNA expression of the corresponding genes were not affected. The findings reported herein highlight the fact that BAC, even such low concentrations, may induce unfavorable adverse effects on the cellular metabolic capacity of the human conjunctiva. Full article

Cisplatin is a prevalent chemotherapeutic agent used for non-small cell lung cancer (NSCLC) that is difficult to treat by targeted therapy, but the emergence of resistance severely limits its efficacy. Thus, an effective strategy to combat cisplatin resistance is required. This study demonstrated that, at clinically achievable concentrations, the combination of selenium yeast (Se-Y) and fish oil (FO) could synergistically induce the apoptosis of cancer stem cell (CSC)-like A549 NSCLC sphere cells, accompanied by a reversal of their resistance to cisplatin. Compared to parental A549 cells, sphere cells have higher cisplatin resistance and possess elevated CSC markers (CD133 and ABCG2), epithelial–mesenchymal transition markers (anexelekto (AXL), vimentin, and N-cadherin), and cytoprotective endoplasmic reticulum (ER) stress marker (glucose-regulated protein 78) and increased oncogenic drivers, such as yes-associated protein, transcriptional coactivator with PDZ-binding motif, β-catenin, and cyclooxygenase-2. In contrast, the proapoptotic ER stress marker CCAAT/enhancer-binding protein homologous protein and AMP-activated protein kinase (AMPK) activity were reduced in sphere cells. The Se-Y and FO combination synergistically counteracted the above molecular features of A549 sphere cells and diminished their elevated CSC-like side population. AMPK inhibition by compound C restored the side population proportion diminished by this nutrient combination. The results suggest that the Se-Y and FO combination can potentially improve the outcome of cisplatin-treated NSCLC with phenotypes such as A549 cells. Full article

We report herein on the effects of brimonidine (BRI), an α2-adrenergic agonist, on two-dimensional (2D) and three-dimensional (3D) cell-cultured TGF-β2-untreated and -treated human trabecular meshwork (HTM) cells. In the presence of TGF-β2 (5 ng/mL), (1) the effects of BRI on (1) the 2D HTM monolayers’ barrier function were investigated as estimated using trans-endothelial electrical resistance (TEER) measurement and FITC dextran permeability; (2) real-time analyses of cellular metabolism using a Seahorse Bioanalyzer; (3) the largeness and hardness of 3D spheroids; and (4) the expression of genes that encode extracellular matrix (ECM) proteins, including collagens (COL) 1, 4, and 6; fibronectin (FN) and α-smooth muscle actin (α-SMA); ECM modulators, including a tissue inhibitor of matrix proteinase (TIMP) 1–4; matrix metalloproteinase (MMP) 2, 9, and 14; and several endoplasmic reticulum (ER) stress-related genes, including the X-box-binding protein 1 (XBP1), the spliced XBP1 (sXBP1), glucose-regulated protein (GRP)78, GRP94, and CCAAT-enhancer-binding protein homologous protein (CHOP). BRI markedly inhibited the TGF-β2-induced increase in the values of TEER of the 2D cell monolayer and the hardness of the 3D spheroids, although it had no effect on their sizes. BRI also cancelled the TGF-β2-induced reduction in mitochondrial maximal respiration but had no effect on the glycolytic capacity. In addition, the gene expression of these molecules was quite different between the 2D and 3D cultures of HTM cells. The present observations found in this study indicate that BRI may beneficially affect TGF-β2-induced changes in both cultures, 2D and 3D, of HTM cells, although their structural and functional properties that were altered varied significantly between both cultures of HTM cells. Full article

Stevioside, the primary sweetener in stevia, is a glycoside with numerous beneficial biological activities. However, its anti-adipogenic effects on tissue differentiation and adipose tissues remain to be thoroughly investigated. In this study, the anti-adipogenic effects of stevioside during the differentiation of 3T3-L1 cells and epididymal adipose tissues of db/db mice were investigated by measuring the lipid droplets stained with Oil Red O and an immunoblot assay. Immunoblot analysis revealed that stevioside downregulated the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), sterol regulatory element-binding protein-1c (SREBP-1c), CCAAT/enhancer-binding protein alpha (C/EBPα), and fatty acid synthase (FAS). Additionally, the protein expression of carnitine palmitoyltransferase 1 (CPT1), silent mating type information regulation 2 homolog 1 (SIRT1), and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) increased following treatment with stevioside. Furthermore, stevioside increased the phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), both in vitro and in vivo. The activity of AMPK in stevioside-treated 3T3-L1 cells was further confirmed using agonists and antagonists of AMPK signaling. Our data indicate that stevioside ameliorates anti-adipogenic effects and promotes β-oxidation in adipocytes by activating AMPK-mediated signaling. The results of this study clearly demonstrated the inhibitory effect of stevioside on the differentiation of adipocytes and the reduction of lipid accumulation in the epididymal adipose tissues of db/db mice. Full article

Kirsten rat sarcoma 2 viral oncogene homolog (Kras) is a proto-oncogene that encodes the small GTPase transductor protein KRAS, which has previously been found to promote cytokine secretion, cell survival, and chemotaxis. However, its effects on preadipocyte differentiation and lipid accumulation are unclear. In this study, the effects of KRAS inhibition on proliferation, autophagy, and adipogenic differentiation as well as its potential mechanisms were analyzed in the 3T3-L1 and C2C12 cell lines. The results showed that KRAS was localized mainly in the nuclei of 3T3-L1 and C2C12 cells. Inhibition of KRAS altered mammalian target of rapamycin (Mtor), proliferating cell nuclear antigen (Pcna), Myc, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein beta (C/ebp-β), diacylglycerol O-acyltransferase 1 (Dgat1), and stearoyl-coenzyme A desaturase 1 (Scd1) expression, thereby reducing cell proliferation capacity while inducing autophagy, enhancing differentiation of 3T3-L1 and C2C12 cells into mature adipocytes, and increasing adipogenesis and the capacity to store lipids. Moreover, during differentiation, KRAS inhibition reduced the levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), p38, and phosphatidylinositol 3 kinase (PI3K) activation. These results show that KRAS has unique regulatory effects on cell proliferation, autophagy, adipogenic differentiation, and lipid accumulation. Full article

Fine particulate matter (PM_2.5) originates from the combustion of coal and is found in the exhaust of fumes of diesel vehicles. PM_2.5 readily penetrates the skin via the aryl hydrocarbon receptor, causing skin senescence, inflammatory skin diseases, DNA damage, and carcinogenesis. In this study, we investigated whether fisetin, a bioactive flavonoid, prevents PM_2.5-induced apoptosis in HaCaT human keratinocytes. The results demonstrated that fisetin significantly downregulated PM_2.5-induced apoptosis at concentrations below 10 μM. Fisetin strongly inhibited the production of reactive oxygen species (ROS) and the expression of pro-apoptotic proteins. The PM_2.5-induced apoptosis was associated with the induction of the endoplasmic reticulum (ER) stress response, mediated via the protein kinase R-like ER kinase (PERK)–eukaryotic initiation factor 2α (eIF2α)–activating transcription factor 4 (ATF4)–CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP) axis. Additionally, the cytosolic Ca²⁺ levels were markedly increased following exposure to PM_2.5. However, fisetin inhibited the expression of ER stress-related proteins, including 78 kDa glucose-regulated protein (GRP78), phospho-eIF2α, ATF4, and CHOP, and reduced the cytosolic Ca²⁺ levels. These data suggest that fisetin inhibits PM_2.5-induced apoptosis by inhibiting the ER stress response and production of ROS. Full article

We have evaluated the role of mitochondrial oxidative stress and its association with endoplasmic reticulum (ER) stress activation in the progression of obesity-related cardiovascular fibrosis. MitoQ (200 µM) was orally administered for 7 weeks to male Wistar rats that were fed a high-fat diet (HFD, 35% fat) or a control diet (CT, 3.5% fat). Obese animals presented cardiovascular fibrosis accompanied by increased levels of extracellular matrix proteins and profibrotic mediators. These alterations were associated with ER stress activation characterized by enhanced levels (in heart and aorta vs. CT group, respectively) of immunoglobulin binding protein (BiP; 2.1-and 2.6-fold, respectively), protein disulfide-isomerase A6 (PDIA6; 1.9-fold) and CCAAT-enhancer-binding homologous protein (CHOP; 1.5- and 1.8-fold, respectively). MitoQ treatment was able to prevent (p < 0.05) these modifications at cardiac and aortic levels. MitoQ (5 nM) and the ER stress inhibitor, 4-phenyl butyric acid (4 µM), were able to block the prooxidant and profibrotic effects of angiotensin II (Ang II, 10⁻⁶ M) in cardiac and vascular cells. Therefore, the data show a crosstalk between mitochondrial oxidative stress and ER stress activation, which mediates the development of cardiovascular fibrosis in the context of obesity and in which Ang II can play a relevant role. Full article

The objective of the current study was to perform a screening of the drug-induced effects of the prostaglandin F2α (PGF2α) and EP2 agonist, omidenepag (OMD), using two- and three-dimensional (2D and 3D) cultures of dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells. The drug-induced effects on 2D monolayers were characterized by measuring the transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)–dextran permeability, the physical properties of 3D spheroids, and the gene expression of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4 and 6, and fibronectin (FN), α smooth muscle actin (αSMA), a tissue inhibitor of metalloproteinase (TIMP) 1–4, matrix metalloproteinase (MMP) 2, 9 and 14 and endoplasmic reticulum (ER) stress-related factors. DEX induced a significant increase in TEER values and a decrease in FITC–dextran permeability, respectively, in the 2D HTM monolayers, and these effects were substantially inhibited by PGF2α and OMD. Similarly, DEX also caused decreased sizes and an increased stiffness in the 3D HTM spheroids, but PGF2α or OMD had no effects on the stiffness of the spheroids. Upon exposure to DEX, the following changes were observed: the upregulation of COL4 (2D), αSMA (2D), and TIMP4 (2D and 3D) and the downregulation of TIMP1 and 2 (3D), MMP2 and 14 (3D), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) (2D), and glucose regulator protein (GRP)78 (3D). In the presence of PGF2α or OMD, the downregulation of COL4 (2D), FN (3D), αSMA (2D), TIMP3 (3D), MMP9 (3D) and the CCAAT/enhancer-binding protein homologous protein (CHOP) (2D), and the upregulation of TIMP4 (2D and 3D), MMP2, 9 and 14 (2D), respectively, were observed. The findings presented herein suggest that 2D and 3D cell cultures can be useful in screening for the drug-induced effects of PGF2α and OMD toward DEX-treated HTM cells. Full article