Involvement of Hydrogen Peroxide in Safingol-Induced Endonuclease G-Mediated Apoptosis of Squamous Cell Carcinoma Cells

Safingol, a L-threo-dihydrosphingosine, induced the nuclear translocation of a mitochondrial apoptogenic mediator—endonuclease G (endo G)—and apoptosis of human oral squamous cell carcinoma (SCC) cells. Upstream mediators remain largely unknown. The levels of hydrogen peroxide (H₂O₂) in cultured oral SCC cells were measured. Treatment with safingol increased intracellular H₂O₂ levels but not extracellular H₂O₂ levels, indicating the production of H₂O₂. The cell killing effect of safingol and H₂O₂ was diminished in the presence of reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC). Dual staining of cells with annexin V and propidium iodide (PI) revealed that apoptotic cell death occurred by treatment with H₂O₂ and safingol. The number of apoptotic cells was reduced in the presence of NAC. In untreated cells, endo G distributed in the cytoplasm and an association of endo G with mitochondria was observed. After treatment with H₂O₂ and safingol, endo G was distributed to the nucleus and cytoplasm, indicating the nuclear translocation of the mitochondrial factor. NAC prevented the increase of apoptotic cells and the translocation of endo G. Knock down of endo G diminished the cell killing effect of H₂O₂ and safingol. These results suggest that H₂O₂ is involved in the endo G-mediated apoptosis of oral SCC cells by safingol.     

Kansuinine A Ameliorates Atherosclerosis and Human Aortic Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Production and Suppressing IKKβ/IκBα/NF-κB Signaling

Reactive oxygen species (ROS)-induced vascular endothelial cell apoptosis is strongly associated with atherosclerosis progression. Herein, we aimed to examine whether Kansuinine A (KA), extracted from Euphorbia kansui L., prevents atherosclerosis development in a mouse model and inhibits cell apoptosis through oxidative stress reduction. Atherosclerosis development was analyzed in apolipoprotein E-deficient (ApoE^−/−) mice fed a high-fat diet (HFD) using Oil Red O staining and H&E staining. Human aortic endothelial cells (HAECs) were treated with KA, followed by hydrogen peroxide (H₂O₂), to investigate the KA-mediated inhibition of ROS-induced oxidative stress and cell apoptosis. Oil Red O staining and H&E staining showed that atherosclerotic lesion size was significantly smaller in the aortic arch of ApoE^−/− mice in the HFD+KA group than that in the aortic arch of those in the HFD group. Further, KA (0.1–1.0 μM) blocked the H₂O₂-induced death of HAECs and ROS generation. The H₂O₂-mediated upregulation of phosphorylated IKKβ, phosphorylated IκBα, and phosphorylated NF-κB was suppressed by KA. KA also reduced the Bax/Bcl-2 ratio and cleaved caspase-3 expression, preventing H₂O₂-induced vascular endothelial cell apoptosis. Our results indicate that KA may protect against ROS-induced endothelial cell apoptosis and has considerable clinical potential in the prevention of atherosclerosis and cardiovascular diseases.     

Complexity of NAC Action as an Antidiabetic Agent: Opposing Effects of Oxidative and Reductive Stress on Insulin Secretion and Insulin Signaling

Dysregulated redox balance is involved in the pathogenesis of type 2 diabetes. While the benefit of antioxidants in neutralizing oxidative stress is well characterized, the potential harm of antioxidant-induced reductive stress is unclear. The aim of this study was to investigate the dose-dependent effects of the antioxidant N-acetylcysteine (NAC) on various tissues involved in the regulation of blood glucose and the mechanisms underlying its functions. H₂O₂ was used as an oxidizing agent in order to compare the outcomes of oxidative and reductive stress on cellular function. Cellular death in pancreatic islets and diminished insulin secretion were facilitated by H₂O₂-induced oxidative stress but not by NAC. On the other hand, myotubes and adipocytes were negatively affected by NAC-induced reductive stress, as demonstrated by the impaired transmission of insulin signaling and glucose transport, as opposed to H₂O₂-stimulatory action. This was accompanied by redox balance alteration and thiol modifications of proteins. The NAC-induced deterioration of insulin signaling was also observed in healthy mice, while both insulin secretion and insulin signaling were improved in diabetic mice. This study establishes the tissue-specific effects of NAC and the importance of the delicate maintenance of redox balance, emphasizing the challenge of implementing antioxidant therapy in the clinic.     

Linalool Induces Cell Cycle Arrest and Apoptosis in Leukemia Cells and Cervical Cancer Cells through CDKIs

Plantaginaceae, a popular traditional Chinese medicine, has long been used for treating various diseases from common cold to cancer. Linalool is one of the biologically active compounds that can be isolated from Plantaginaceae. Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible tumor cells. However, the signaling pathway for apoptosis remains undefined. In this study, the cytotoxic effect of linalool on human cancer cell lines was investigated. Water-soluble tetrazolium salts (WST-1) based colorimetric cellular cytotoxicity assay, was used to test the cytotoxic ability of linalool against U937 and HeLa cells, and flow cytometry (FCM) and genechip analysis were used to investigate the possible mechanism of apoptosis. These results demonstrated that linalool exhibited a good cytotoxic effect on U937 and HeLa cells, with the IC₅₀ value of 2.59 and 11.02 μM, respectively, compared with 5-FU with values of 4.86 and 12.31 μM, respectively. After treating U937 cells with linalool for 6 h, we found an increased sub-G1 peak and a dose-dependent phenomenon, whereby these cells were arrested at the G0/G1 phase. Furthermore, by using genechip analysis, we observed that linalool can promote p53, p21, p27, p16, and p18 gene expression. Therefore, this study verified that linalool can arrest the cell cycle of U937 cells at the G0/G1 phase and can arrest the cell cycle of HeLa cells at the G2/M phase. Its mechanism facilitates the expression of the cyclin-dependent kinases inhibitors (CDKIs) p53, p21, p27, p16, and p18, as well as the non-expression of cyclin-dependent kinases (CDKs) activity.     

Sod1 Loss Induces Intrinsic Superoxide Accumulation Leading to p53-Mediated Growth Arrest and Apoptosis

Oxidative damages induced by a redox imbalance cause age-related changes in cells and tissues. Superoxide dismutase (SOD) enzymes play a major role in the antioxidant system and they also catalyze superoxide radicals (O₂^•−). Since the loss of cytoplasmic SOD (SOD1) resulted in aging-like phenotypes in several types of mouse tissue, SOD1 is essential for the maintenance of tissue homeostasis. To clarify the cellular function of SOD1, we investigated the cellular phenotypes of Sod1-deficient fibroblasts. We demonstrated that Sod1 deficiency impaired proliferation and induced apoptosis associated with O₂^•− accumulation in the cytoplasm and mitochondria in fibroblasts. Sod1 loss also decreased the mitochondrial membrane potential and led to DNA damage-mediated p53 activation. Antioxidant treatments effectively improved the cellular phenotypes through suppression of both intracellular O₂^•− accumulation and p53 activation in Sod1-deficient fibroblasts. In vivo experiments revealed that transdermal treatment with a vitamin C derivative significantly reversed the skin thinning commonly associated with the upregulated p53 action in the skin. Our findings revealed that intrinsic O₂^•− accumulation promoted p53-mediated growth arrest and apoptosis as well as mitochondrial disfunction in the fibroblasts.     

Characterization of an HL-60 cell variant resistant to the antineoplastic ether lipid 1-O-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine

A resistant cell line (HL-60R) was selected by incubating HL-60 cells with increasing concentrations of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) and used to examine the mechanism of resistance to the antineoplastic ether-linked lipid. The HL-60R cells exhibited a>10-fold increase in resistance when measured by [³H]-thymidine incorporation in comparison to the HL-60 cell line. ET-18-OCH₃ binding occurred at 4°C and was not saturable at the concentrations tested (1–100 μM), indicating that the binding was receptor-independent. At 4°C, association of ET-18-OCH₃ was low for each cell line. At 37°C, uptake in the HL-60 cells was approximately 5-fold greater in comparison to HL-60R cells at each concentration tested. However, when the cellular content of ET-18-OCH₃ was equal, both cell lines experienced similar declines in cell growth. Cellular incorporation of ether lipid was determined using serum-free media and in the presence of serum albumin or lipoproteins. Reduced uptake by the resistant cell line was observed only in the presence of albumin. A greater proportion of ether lipid could be removed from prelabeled HL-60R cells than from HL-60 cells, by an albumin wash procedure, indicating an increased rate of internalization and retention by the sensitive cell line. ET-18-OCH₃ uptake in the HL-60 cell line was also more sensitive to treatment with endocytic (chloroquine, monensin) or metabolic (NaF, KCN) inhibitors. These results suggest that uptake is the principal determinant influencing sensitivity of the resistant cell line and consists of receptor-independent binding followed by internalization. Differential uptake requires the presence of serum albumin and is dependent on the energy-dependent endocytosis of the ether lipid.     

Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro

Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1), 3β-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13)-en-12,6α-olide (2), iso-seco-tanapartholide 3-O-methyl ether (3) and 4β,15-dihydro-3-dehydrozaluzanin C (4), were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica). When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC₅₀ values in the range 3.6–13.5 μM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1–3 elicited increases in the hypodiploid (subG1) population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258–propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents.     

Chemical Compounds Released from Specific Osteoinductive Bioactive Materials Stimulate Human Bone Marrow Mesenchymal Stem Cell Migration

In this work, a poly(L-lactide-co-glycolide) (PLGA)-based composite was enriched with one of the following sol-gel bioactive glasses (SBG) at 50 wt.%: A1—40 mol% SiO₂, 60 mol% CaO, CaO/SiO₂ ratio of 1.50; S1—80 mol% SiO₂, 20 mol% CaO, CaO/SiO₂ ratio of 0.25; A2—40 mol% SiO₂, 54 mol% CaO, 6 mol% P₂O₅, CaO/SiO₂ ratio of 1.35; S2—80 mol% SiO₂,16 mol% CaO, 4 mol% P₂O₅, CaO/SiO₂ ratio of 0.20. The composites and PLGA control sheets were then soaked for 24 h in culture media, and the obtained condition media (CM) were used to treat human bone marrow stromal cells (hBMSCs) for 72 h. All CMs from the composites increased ERK 1/2 activity vs. the control PLGA CM. However, expressions of cell migration-related c-Fos, osteopontin, matrix metalloproteinase-2, C-X-C chemokine receptor type 4, vascular endothelial growth factor, and bone morphogenetic protein 2 were significantly increased only in cells treated with the CM from the A1/PLGA composite. This CM also significantly increased the rate of human BMSC migration but did not affect cell metabolic activity. These results indicate important biological markers that are upregulated by products released from the bioactive composites of a specific chemical composition, which may eventually prompt osteoprogenitor cells to colonize the bioactive material and accelerate the process of tissue regeneration.     

Xanthine Oxidoreductase-Mediated Superoxide Production Is Not Involved in the Age-Related Pathologies in Sod1-Deficient Mice

Reactive oxygen species (ROS) metabolism is regulated by the oxygen-mediated enzyme reaction and antioxidant mechanism within cells under physiological conditions. Xanthine oxidoreductase (XOR) exhibits two inter-convertible forms (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)), depending on the substrates. XO uses oxygen as a substrate and generates superoxide (O₂^•−) in the catalytic pathway of hypoxanthine. We previously showed that superoxide dismutase 1 (SOD1) loss induced various aging-like pathologies via oxidative damage due to the accumulation of O₂^•− in mice. However, the pathological contribution of XO-derived O₂^•− production to aging-like tissue damage induced by SOD1 loss remains unclear. To investigate the pathological significance of O₂^•− derived from XOR in Sod1^−/− mice, we generated Sod1-null and XO-type- or XDH-type-knock-in (KI) double-mutant mice. Neither XO-type- nor XDH-type KI mutants altered aging-like phenotypes, such as anemia, fatty liver, muscle atrophy, and bone loss, in Sod1^−/− mice. Furthermore, allopurinol, an XO inhibitor, or apocynin, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, failed to improve aging-like tissue degeneration and ROS accumulation in Sod1^−/− mice. These results showed that XOR-mediated O₂^•− production is relatively uninvolved in the age-related pathologies in Sod1^−/− mice.     

Catalase (CAT) Gene Family in Rapeseed (Brassica napus L.): Genome-Wide Analysis,Identification, and Expression Pattern in Response to Multiple Hormones and Abiotic Stress Conditions

Catalase (CAT) is an antioxidant enzyme expressed by the CAT gene family and exists in almost all aerobic organisms. Environmental stresses induce the generation of reactive oxygen species (ROS) that eventually hinder plant growth and development. The CAT enzyme translates the hydrogen peroxide (H₂O₂) to water (H₂O) and reduce the ROS levels to shelter the cells’ death. So far, the CAT gene family has not been reported in rapeseed (Brassica napus L.). Therefore, a genome-wide comprehensive analysis was conducted to classify the CAT genes in the rapeseed genome. The current study identified 14 BnCAT genes in the rapeseed genome. Based on phylogenetic and synteny analysis, the BnCATs belong to four groups (Groups I–IV). A gene structure and conserved motif analysis showed that Group I, Group II, and Group IV possess almost the same intron/exon pattern, and an equal number of motifs, while Group III contains diverse structures and contain 15 motifs. By analyzing the cis-elements in the promoters, we identified five hormone-correlated responsive elements and four stress-related responsive elements. Further, six putative bna-miRNAs were also identified, targeting three genes (BnCAT4, BnCAT6, and BnCAT8). Gene ontology (GO) enrichment analysis showed that the BnCAT genes were largely related to cellular organelles, ROS response, stimulus response, stress response, and antioxidant enzymes. Almost 10 BnCAT genes showed higher expression levels in different tissues, i.e., root, leaf, stem, and silique. The expression analysis showed that BnCAT1–BnCAT3 and BnCAT11–BnCAT13 were significantly upregulated by cold, salinity, abscisic acid (ABA), and gibberellic acid (GA) treatment, but not by drought and methyl jasmonate (MeJA). Notably, most of the genes were upregulated by waterlogging stress, except BnCAT6, BnCAT9, and BnCAT10. Our results opened new windows for future investigations and provided insights into the CAT family genes in rapeseed.     

ROS/TNF-α Crosstalk Triggers the Expression of IL-8 and MCP-1 in Human Monocytic THP-1 Cells via the NF-κB and ERK1/2 Mediated Signaling

IL-8/MCP-1 act as neutrophil/monocyte chemoattractants, respectively. Oxidative stress emerges as a key player in the pathophysiology of obesity. However, it remains unclear whether the TNF-α/oxidative stress interplay can trigger IL-8/MCP-1 expression and, if so, by which mechanism(s). IL-8/MCP-1 adipose expression was detected in lean, overweight, and obese individuals, 15 each, using immunohistochemistry. To detect the role of reactive oxygen species (ROS)/TNF-α synergy as a chemokine driver, THP-1 cells were stimulated with TNF-α, with/without H₂O₂ or hypoxia. Target gene expression was measured by qRT-PCR, proteins by flow cytometry/confocal microscopy, ROS by DCFH-DA assay, and signaling pathways by immunoblotting. IL-8/MCP-1 adipose expression was significantly higher in obese/overweight. Furthermore, IL-8/MCP-1 mRNA/protein was amplified in monocytic cells following stimulation with TNF-α in the presence of H₂O₂ or hypoxia (p ˂ 0.0001). Synergistic chemokine upregulation was related to the ROS levels, while pre-treatments with NAC suppressed this chemokine elevation (p ≤ 0.01). The ROS/TNF-α crosstalk involved upregulation of CHOP, ERN1, HIF1A, and NF-κB/ERK-1,2 mediated signaling. In conclusion, IL-8/MCP-1 adipose expression is elevated in obesity. Mechanistically, ROS/TNF-α crosstalk may drive expression of these chemokines in monocytic cells by inducing ER stress, HIF1A stabilization, and signaling via NF-κB/ERK-1,2. NAC had inhibitory effect on oxidative stress-driven IL-8/MCP-1 expression, which may have therapeutic significance regarding meta-inflammation.     

Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

Helicobacter pylori neutrophil-activating protein (HP-NAP)-induced production of reactive oxygen species (ROS) by neutrophils and monocytes is regulated by pertussis toxin (PTX)-sensitive G proteins, whereas HP-NAP-induced cytokine secretion by monocytes is mediated by Toll-like receptor 2 (TLR2). However, it is unclear whether TLR2 participates in HP-NAP-induced cytokine secretion by neutrophils. Here, all-trans retinoic acid (ATRA)-induced differentiated HL-60 cells were first employed as a neutrophil model to investigate the molecular mechanisms underlying neutrophil responses to HP-NAP. HP-NAP-induced ROS production in ATRA-induced differentiated HL-60 cells is mediated by the PTX-sensitive heterotrimeric G protein-dependent activation of extracellular signal-regulated kinase 1/2 and p38-mitogen-activated protein kinase, which is consistent with the findings reported for human neutrophils. Next, whether TLR2 participated in HP-NAP-induced secretion of interleukin-8 (IL-8) was investigated in neutrophils and ATRA-induced differentiated HL-60 cells. In both cells, TLR2 participated in HP-NAP-induced IL-8 secretion but not HP-NAP-induced ROS production. Interestingly, PTX-sensitive G proteins also contributed to the HP-NAP-induced secretion of IL-8 from neutrophils and the differentiated HL-60 cells. Our ELISA-based binding assay further revealed the competitive binding of Pam₃CSK₄, a TLR2 agonist, and HP-NAP to TLR2, which suggests the presence of specific and direct interactions between HP-NAP and TLR2. Thus, HP-NAP directly interacts with and activates TLR2 to induce IL-8 secretion in neutrophils and ATRA-induced differentiated HL-60 cells.     

Morin,a Flavonoid from Moraceae,Induces Apoptosis by Induction of BAD Protein in Human Leukemic Cells

Evidence suggests that phytochemicals can safely modulate cancer cell biology and induce apoptosis. Here, we investigated the anti-cancer activity of morin, a flavone originally isolated from members of the Moraceae family in human leukemic cells, focusing on apoptosis. An anti-cancer effect of morin was screened with several human leukemic cell lines. U937 cells were most sensitive to morin, where it induced caspase-dependent apoptosis in a dose-dependent manner. It also induced loss of MMP (ΔΨ_m) along with cytochrome c release, down-regulated Bcl-2 protein, and up-regulated BAX proteins. The apoptotic activity of morin was significantly attenuated by Bcl-2 augmentation. In conclusion, morin induced caspase-dependent apoptosis through an intrinsic pathway by upregulating BAD proteins. In addition, Bcl-2 protein expression is also important in morin-induced apoptosis of U937 cells. This study provides evidence that morin might have anticancer properties in human leukemic cells.     

Pathological Relationship between Intracellular Superoxide Metabolism and p53 Signaling in Mice

Intracellular superoxide dismutases (SODs) maintain tissue homeostasis via superoxide metabolism. We previously reported that intracellular reactive oxygen species (ROS), including superoxide accumulation caused by cytoplasmic SOD (SOD1) or mitochondrial SOD (SOD2) insufficiency, induced p53 activation in cells. SOD1 loss also induced several age-related pathological changes associated with increased oxidative molecules in mice. To evaluate the contribution of p53 activation for SOD1 knockout (KO) (Sod1^−/−) mice, we generated SOD1 and p53 KO (double-knockout (DKO)) mice. DKO fibroblasts showed increased cell viability with decreased apoptosis compared with Sod1^−/− fibroblasts. In vivo experiments revealed that p53 insufficiency was not a great contributor to aging-like tissue changes but accelerated tumorigenesis in Sod1^−/− mice. Furthermore, p53 loss failed to improve dilated cardiomyopathy or the survival in heart-specific SOD2 conditional KO mice. These data indicated that p53 regulated ROS-mediated apoptotic cell death and tumorigenesis but not ROS-mediated tissue degeneration in SOD-deficient models.     

Asymmetric Synthesis and Evaluation of Danshensu-Cysteine Conjugates as Novel Potential Anti-Apoptotic Drug Candidates

We have previously reported that the danshensu-cysteine conjugate N-((R)-3-benzylthio-1-methoxy-1-oxo-2-propanyl)-2-acetoxy-3-(3,4-diacetoxyphenyl) propanamide (DSC) is a potent anti-oxidative and anti-apoptotic agent. Herein, we further design and asymmetrically synthesize two diastereoisomers of DSC and explore their potential bioactivities. Our results show that DSC and its two diastereoisomers exert similar protective effects in hydrogen peroxide (H₂O₂)-induced cellular injury in SH-SY5Y cells, as evidenced by the increase of cell viability, superoxide dismutase (SOD), and reduced glutathione (GSH) activity, and glutathione peroxidase (GPx) expression, and the decrease of cellular morphological changes and nuclear condensation, lactate dehydrogenase (LDH) release, and malondialdehyde (MDA) production. In H₂O₂-stimulated human umbilical vein endothelial cells (HUVEC), DSC concentration-dependently attenuates H₂O₂-induced cell death, LDH release, mitochondrial membrane potential collapse, and modulates the expression of apoptosis-related proteins (Bcl-2, Bax, caspase-3, and caspase-9). Our results provide strong evidence that DSC and its two diastereoisomers have similar anti-oxidative activity and that DSC exerts significant vascular-protective effects, at least in part, through inhibition of apoptosis and modulation of endogenous antioxidant enzymes.     

Why Is the Invasive Plant Sphagneticola trilobata More Resistant to High Temperature Than Its Native Congener?

Climate change and invasive alien species threaten biodiversity. High temperature is a worrying ecological factor. Most responses of invasive plants aimed at coping with adversity are focused on the physiological level. To explore the molecular mechanisms underlying the response of an invasive plant (Sphagneticola trilobata L.) to high temperature, using a native species (Sphagneticola calendulacea L.) as the control, relevant indicators, including photosynthetic pigments, gas exchange, chlorophyll fluorescence, the antioxidant system, and related enzyme-coding genes were measured. The results showed that the leaves of S. calendulacea turned yellow, photosynthetic pigment content (Chl a, Chl b, Car, Chl) decreased, gas exchange (P_n) and chlorophyll fluorescence parameters (F_v/F_m, Φ_PSII) decreased under high temperature. It was also found that high temperature caused photoinhibition and a large amount of ROS accumulated, resulting in an increase in MDA and relative conductivity. Antioxidant enzymes (including SOD, POD, CAT, and APX) and antioxidants (including flavonoids, total phenols, and carotenoids) were decreased. The qPCR results further showed that the expression of the PsbP, PsbA, and RubiscoL, SOD, POD, CAT, and APX genes was downregulated, which was consistent with the results of physiological data. Otherwise, the resistance of S. trilobata to high temperature was better than that of S. calendulacea, which made it a superior plant in the invasion area. These results further indicated that the gradual warming of global temperature will greatly accelerate the invasion area of S. trilobata.