Promoting Reactive Oxygen Species Accumulation Research Articles

NpCIPK6–NpSnRK1 module facilitates inter-subgeneric hybridization barriers in water lily (nymphaea) by reducing abscisic acid content

Abstract Pre-fertilization hybridization barriers are the main causes of inter-subgeneric hybridization challenges in water lily. However, the mechanism underlying low compatibility between pollen and stigma of water lily remains unclear. This study demonstrates that CBL-interacting protein kinase 6 (CIPK6) responded to the signaling exchange between incompatible pollen and stigma through interactions with SNF1-related kinase 1 (SnRK1) and promotes the accumulation of SnRK1 protein. Activated SnRK1 interacted with 9-cis-epoxycarotenoid dioxygenase 2 (NCED2) to promote its degradation, thereby inhibiting abscisic acid (ABA) synthesis. A decrease in ABA content in the stigma impaired the ABA-mediated removal of reactive oxygen species (ROS), ultimately resulting in the rejection of the incompatible pollen by the stigma. Our results highlight the essential role of the NpCIPK6–NpSnRK1–NpNCED2 module in conferring inter-subgeneric hybridization barriers in water lily by interfering with ABA synthesis and promoting ROS accumulation. This study offers valuable mechanistic insights into cellular signaling and reproductive barriers in water lily as well as across other biological contexts.

Tobacco Transcription Factor NtWRKY70b Facilitates Leaf Senescence via Inducing ROS Accumulation and Impairing Hydrogen Sulfide Biosynthesis.

Leaf senescence is the terminal stage of leaf development, and its initiation and progression are closely controlled by the integration of a myriad of endogenous signals and environmental stimuli. It has been documented that WRKY transcription factors (TFs) play essential roles in regulating leaf senescence, yet the molecular mechanism of WRKY-mediated leaf senescence still lacks detailed elucidation in crop plants. In this study, we cloned and identified a tobacco WRKY TF gene, designated NtWRKY70b, acting as a positive regulator of natural leaf senescence. The expression profile analysis showed that NtWRKY70b transcript levels were induced by aging and hydrogen peroxide (H2O2) and downregulated upon hydrogen sulfide (H2S) treatment. The physiological and biochemical assays revealed that overexpression of NtWRKY70b (OE) clearly promoted leaf senescence, triggering increased levels of reactive oxygen species (ROS) and decreased H2S content, while disruption of NtWRKY70b by chimeric repressor silencing technology (SRDX) significantly delayed the onset of leaf senescence, leading to a decreased accumulation of ROS and elevated concentration of H2S. The quantitative real-time PCR analysis showed that the expression levels of various senescence-associated genes and ROS biosynthesis-related genes (NtRbohD and NtRbohE) were upregulated in OE lines, while the expression of H2S biosynthesis-related genes (NtDCD and NtCYSC1) were inhibited in OE lines. Furthermore, the Yeast one-hybrid analysis (Y1H) and dual luciferase assays showed that NtWRKY70b could directly upregulate the expression of an ROS biosynthesis-related gene (NtRbohD) and a chlorophyll degradation-related gene (NtPPH) by binding to their promoter sequences. Accordingly, these results indicated that NtWYKY70b directly activated the transcript levels of NtRbohD and NtPPH and repressed the expression of NtDCD and NtCYCS1, thereby promoting ROS accumulation and impairing the endogenous H2S production, and subsequently accelerating leaf aging. These observations improve our knowledge of the regulatory mechanisms of WRKY TFs controlling leaf senescence and provide a novel method for ensuring high agricultural crop productivity via genetic manipulation of leaf senescence in crops.

Dihydroartemisinin eliminates senescent cells by promoting autophagy-dependent ferroptosis via AMPK/mTOR signaling pathway.

Cellular senescence is an irreversible cell-cycle arrest in response to a variety of cellular stresses, which contribute to the pathogenesis of a variety of age-related degenerative diseases. However, effective antisenescence strategies are still lacking. Drugs that selectively target senescent cells represent an intriguing therapeutic strategy to delay aging and age-related diseases. Thus, we thought to investigate the effects of dihydroartemisinin (DHA) on senescent cells and elucidated its mechanisms underlying aging. Stress-induced premature senescence (SIPS) model was built in NIH3T3 cells using H2O2 and evaluated by β-galactosidasestaining. Cells were exposed to DHA and subjected to cellular activity assays including viability, ferroptosis, and autophagy. The number of microtubule-associated protein light-chain 3puncta was detected by immunofluorescence staining. The iron content was assessed by spectrophotometer and intracellular reactive oxygen species (ROS) was measured by fluorescent probe dichlorodihydrofluorescein diacetate. We found that DHA triggered senescent cell death via ferroptosis. DHA accelerated ferritin degradation via promoting autophagy, increasing the iron contents, promoting ROS accumulation, thus leading to ferroptotic cell death in SIPS cells. In addition, autophagy inhibitor BafA1 preconditioning inhibited ferroptosis induced by DHA. Moreover, Atg5 silencing and autophagy inhibitor BafA1 preconditioning inhibited ferroptosis induced by DHA. We also revealed that the expression of p-AMP-activated protein kinase (AMPK)and p-mammalian target of rapamycin (mTOR) in senescent cells was downregulated. These results suggested that DHA may be a promising drug candidate for clearing senescent cells by inducing autophagy-dependent ferroptosis via AMPK/mTOR signaling pathway.

A novel quinoline derivative, DFIQ, sensitizes NSCLC cells to ferroptosis by promoting oxidative stress accompanied by autophagic dysfunction and mitochondrial damage

BackgroundThe development of nonapoptotic programmed cell death inducers as anticancer agents has emerged as a cancer therapy field. Ferroptosis, ferrous ion-driven programmed cell death that is induced by redox imbalance and dysfunctional reactive oxygen species (ROS) clearance, is triggered during sorafenib and PD-1/PD-L1 immunotherapy. DFIQ, a quinoline derivative, promotes apoptosis by disrupting autophagic flux and promoting ROS accumulation. Our pilot experiments suggest that DFIQ participates in ferroptosis sensitization. Thus, in this study, we aimed to reveal the mechanisms of DFIQ in ferroptosis sensitization and evaluate the clinical potential of DFIQ.MethodsWe treated the non-small cell lung cancer (NSCLC) cell lines H1299, A549, and H460 with the ferroptosis inducer (FI) DFIQ and analyzed viability, protein expression, ROS generation, and fluorescence staining at different time points. Colocalization analysis was performed with ImageJ.ResultsDFIQ sensitized cells to FIs such as erastin and RSL3, resulting in a decrease in IC50 of at least 0.5-fold. Measurement of ROS accumulation to explore the underlying mechanism indicated that DFIQ and FIs treatment promoted ROS accumulation and SOD1/SOD2 switching. Mitochondria, known ROS sources, produced high ROS levels during DFIQ/FI treatment. RSL3 treatment promoted mitochondrial damage and mitophagy, an autophagy-associated mitochondrial recycling system, and cotreatment with DFIQ induced accumulation of mitochondrial proteins, which indicated disruption of mitophagic flux. Thus, autophagic flux was measured in cells cotreated with DFIQ. DFIQ treatment was found to disrupt autophagic flux, leading to accumulation of damaged mitochondria and eventually inducing ferroptosis. Furthermore, the influence of DFIQ on the effects of clinical FIs, such as sorafenib, was evaluated, and DFIQ was discovered to sensitize NSCLC cells to sorafenib and promote ferroptosis.ConclusionsThis study indicates that DFIQ not only promotes NSCLC apoptosis but also sensitizes cells to ferroptosis by disrupting autophagic flux, leading to accumulation of dysfunctional mitochondria and thus to ferroptosis. Ferroptosis is a novel therapeutic target in cancer therapy. DFIQ shows the potential to enhance the effects of FIs in NSCLC and act as a potential therapeutic adjuvant in ferroptosis-mediated therapy.

LtGAPR1 Is a Novel Secreted Effector from Lasiodiplodia theobromae That Interacts with NbPsQ2 to Negatively Regulate Infection

The effector proteins secreted by a pathogen not only promote the virulence and infection of the pathogen but also trigger plant defense response. Lasiodiplodia theobromae secretes many effectors that modulate and hijack grape processes to colonize host cells, but the underlying mechanisms remain unclear. Herein, we report LtGAPR1, which has been proven to be a secreted protein. In our study, LtGAPR1 played a negative role in virulence. By co-immunoprecipitation, 23 kDa oxygen-evolving enhancer 2 (NbPsbQ2) was identified as a host target of LtGAPR1. The overexpression of NbPsbQ2 in Nicotiana benthamiana reduced susceptibility to L. theobromae, and the silencing of NbPsbQ2 enhanced L. theobromae infection. LtGAPR1 and NbPsbQ2 were confirmed to interact with each other. Transiently, expressed LtGAPR1 activated reactive oxygen species (ROS) production in N. benthamiana leaves. However, in NbPsbQ2-silenced leaves, ROS production was impaired. Overall, our report revealed that LtGAPR1 promotes ROS accumulation by interacting with NbPsbQ2, thereby triggering plant defenses that negatively regulate infection.

The MAPK-Alfin-like 7 module negatively regulates ROS scavenging genes to promote NLR-mediated immunity

Nucleotide-binding leucine-rich repeat (NLR) receptor-mediated immunity includes rapid production of reactive oxygen species (ROS) and transcriptional reprogramming, which is controlled by transcription factors (TFs). Although some TFs have been reported to participate in NLR-mediated immune response, most TFs are transcriptional activators, and whether and how transcriptional repressors regulate NLR-mediated plant defenses remains largely unknown. Here, we show that the Alfin-like 7 (AL7) interacts with N NLR and functions as a transcriptional repressor. Knockdown and knockout of AL7 compromise N NLR-mediated resistance against tobacco mosaic virus, whereas AL7 overexpression enhances defense, indicating a positive regulatory role for AL7 in immunity. AL7 binds to the promoters of ROS scavenging genes to inhibit their transcription during immune responses. Mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK) directly interact with and phosphorylate AL7, which impairs the AL7-N interaction and enhances its DNA binding activity, which promotes ROS accumulation and enables immune activation. In addition to N, AL7 is also required for the function of other Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeats (TNLs) including Roq1 and RRS1-R/RPS4. Our findings reveal a hitherto unknown MAPK-AL7 module that negatively regulates ROS scavenging genes to promote NLR-mediated immunity.

PDC1 is activated by ABF4 and inhibits seed germination by promoting ROS accumulation in Arabidopsis

Seed germination is tightly regulated by multiple endogenous signaling molecules and metabolites, such as abscisic acid (ABA), the products of fatty acid β-oxidation for energy production and reactive oxygen species (ROS). In Arabidopsis the expression of pyruvate decarboxylase 1 (PDC1), which is involved in fermentation metabolism, was strongly induced by ABA during seed germination, suggesting that PDC1 might regulate seed germination in response to ABA. To prove this hypothesis, we characterized the role of PDC1 in ABA-mediated inhibition of seed germination. The percentages of seed germination and cotyledon greening of PDC1-deficiency pdc1 mutants were higher, whereas those of PDC1-overexpression lines were lower, than those of wild-type (WT) plants in the presence of ABA. Chromatin immunoprecipitation and electrophoretic mobility shift assays indicated that the ABA-responsive element BINDING FACTOR 4 (ABF4) transcription factor could bind to the PDC1 promoter and activate gene expression. ABA-induction of PDC1 expression was impaired in abf4–1 mutants, and overexpression of ABF4 in pdc1–1 plants can partially rescue their ABA-insensitive phenotypes. In addition, ABA-induced ROS accumulation was impaired in pdc1–1 seedlings, whereas it was enhanced in PDC1-overexpression lines, as compared with WT plants. Furthermore, the levels of free fatty acids were lower in pdc1–1 mutants, but higher in the PDC1-overexpression lines, than WT plants. This is consistent with the altered expression of fatty acid β-oxidation-related genes in these plants. Taken together, our results indicated that ABF4 activates PDC1 expression, thereby promoting fatty acid β-oxidation and enhancing ROS accumulation, leading to inhibition of seed germination in Arabidopsis.

Mutation of Leaf Senescence 1 Encoding a C2H2 Zinc Finger Protein Induces ROS Accumulation and Accelerates Leaf Senescence in Rice.

Premature senescence of leaves causes a reduced yield and quality of rice by affecting plant growth and development. The regulatory mechanisms underlying early leaf senescence are still unclear. The Leaf senescence 1 (LS1) gene encodes a C2H2-type zinc finger protein that is localized to both the nucleus and cytoplasm. In this study, we constructed a rice mutant named leaf senescence 1 (ls1) with a premature leaf senescence phenotype using CRISPR/Cas9-mediated editing of the LS1 gene. The ls1 mutants exhibited premature leaf senescence and reduced chlorophyll content. The expression levels of LS1 were higher in mature or senescent leaves than that in young leaves. The contents of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were significantly increased and catalase (CAT) activity was remarkably reduced in the ls1 plants. Furthermore, a faster decrease in pigment content was detected in mutants than that in WT upon induction of complete darkness. TUNEL and staining experiments indicated severe DNA degradation and programmed cell death in the ls1 mutants, which suggested that excessive ROS may lead to leaf senescence and cell death in ls1 plants. Additionally, an RT-qPCR analysis revealed that most senescence-associated and ROS-scavenging genes were upregulated in the ls1 mutants compared with the WT. Collectively, our findings revealed that LS1 might regulate leaf development and function, and that disruption of LS1 function promotes ROS accumulation and accelerates leaf senescence and cell death in rice.

New Insights into the Role of Alternating Temperatures and Cyanide in the ROS-Mediated Cardoon Seed Dormancy Termination

Physiological dormancy in wild cardoon (Cynara cardunculus var. sylvestris) can be terminated by achenes exposure to alternating temperatures, likely with the participation of reactive oxygen species (ROS). Cyanide is a natural compound that mediates seed dormancy removal in some plant species in association with oxidative signalling exerted by ROS. To date, no study has been conducted on the cyanide effect on ROS homeostasis during the germination of cardoon. Here, we showed that the addition of cyanide at low concentrations in dormant cardoon achenes promotes dormancy breakage at a constant temperature, speeds up germination to alternating temperatures and promotes ROS accumulation in embryonic axes of dormant achenes. The in-silico transcriptome analysis showed that the expression levels of transcripts of genes associated with ROS signalling and production, calcium signalling, gibberellins biosynthesis and cell wall loosening were significantly up-regulated at the alternating temperatures imbibition condition. In contrast, the expression of gene transcripts associated with the inhibition of germination, ABA biosynthesis and signalling were up-regulated at the constant temperature imbibition. However, no significant difference in lipid peroxidation or protein carbonylation levels was observed when achenes were imbibed at constant or alternating temperature conditions. These results suggest that dormancy termination triggered by alternating temperatures or cyanide could be mediated by ROS production and signalling in the cardoon embryonic axis, but this does not determine extensive protein carbonylation.

Effect of high glucose supplementation on pulmonary fibrosis involving reactive oxygen species and TGF-β

This study explored the profibrotic impact of high glucose in the lung and potential mechanisms using latent TGF-β1-induced human epithelial cell pulmonary fibrosis and bleomycin (BLM)-induced pulmonary fibrosis models. Results demonstrated that high glucose administration induced epithelial–mesenchymal transition (EMT) in human epithelial cells in a dose-dependent manner via activating latent TGF-β1, followed by increased expression of mesenchymal-related proteins and decreased expression of epithelial marker protein E-cadherin. Further mechanism analysis showed that administration of high glucose dose-dependently promoted total and mitochondrial reactive oxygen species (ROS) accumulation in human epithelial cells, which promoted latent TGF-β1 activation. However, N-acetyl-L-cysteine, a ROS eliminator, inhibited such effects. An in vivo feed study found that mice given a high-glucose diet had more seriously pathological characteristics of pulmonary fibrosis in BLM-treated mice, including increasing infiltrated inflammatory cells, collagen I deposition, and the expression of mesenchymal-related proteins while decreasing the expression of the epithelial marker E-cadherin. In addition, high glucose intake further increased TGF-β1 concentration and upregulated p-Smad2/3 and snail in lung tissues from BLM-treated mice when compared to BLM-treated mice. Finally, supplementation with high glucose further increased the production of lipid peroxidation metabolite malondialdehyde and decreased superoxide dismutase activity in BLM-treated mice. Collectively, these findings illustrate that high glucose supplementation activates a form of latent TGF-β1 by promoting ROS accumulation and ultimately exacerbates the development of pulmonary fibrosis.

Plant Defense Responses to a Novel Plant Elicitor Candidate LY5-24-2.

Plant elicitors enhance plant defense against pathogen attacks by inducing systemic acquired resistance (SAR) with no or low direct fungicidal activity. Here we report the synthesis of a novel plant elicitor candidate LY5-24-2 [3,4-dichloro-N-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)isothiazole-5-carboxamide] and evaluation of its SAR inducing activity. Bioassays indicated that LY5-24-2 did not show significant anti-fungal activity but provided long-lasting resistance in Arabidopsis thaliana (A. thaliana) through promoting the accumulation of lignin, cellulose and pectin by 60.1%, 82.4% and 305.6%, respectively, at a concentration of 100 µM. LY5-24-2 also facilitated the closure of leaf stomata and increased the intracellular free Ca2+ by 47.8%, induced reactive oxygen species (ROS) accumulation, and inhibited the activity of ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6) by 38.9% and 34.0%, respectively, as compared with the control at a concentration of 100 µM. LY5-24-2 induced SAR in plants and was dependent on the NPR1-mediated SA pathway by up-regulating expression of 2273 genes in A. thaliana. Meanwhile, LY5-24-2 also improved cucumber (Cucumis sativus L.) defense against Pseudoperonospora cubensis (P. cubensis) through promoting ROS accumulation and inhibiting activity of APX and CAT by 30.7% and 23.1%, respectively. Its expression of SA signaling genes CsNPR1, CsPR4 and CsPR5 was enhanced by 10.8, 5.8 and 6.6 times, respectively. These results demonstrated that LY5-24-2 is a novel elicitor candidate for plant protection via inducing SAR.

Grafting resulting in alleviating tomato plant oxidative damage caused by high levels of ofloxacin

Antibiotic pollution has become a global problem threatening human health. Ofloxacin is one of the more widely used antibiotics, but reports on the reaction of plant to ofloxacin pollution are limited. In this study, using adversity-resistant (R), adversity-sensitive (S) and grafted plant S/R as models, we investigated the biological response of tomato to exogenous ofloxacin residues. The results showed that lower levels of ofloxacin treatment (5 mg L−1 and 10 mg L−1) promoted tomato growth, and 10 mg L−1 ofloxacin was the critical dose to stimulate growth among the different treatments. In addition, the photosynthetic and fluorescence parameters, antioxidant enzyme activities and transcription-level expression of the enzymes were stimulated by low ofloxacin treatment. However, high ofloxacin treatment (20 mg L−1 and 40 mg L−1) exhibited a significantly negative effect on plant growth, photosynthesis, fluorescence parameters, antioxidant enzyme activities and transcript levels expression. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels increased with increasing ofloxacin concentrations, indicating that the oxidative damage of plants was severe with increasing doses. In contrast, the role of antioxidant enzymes in the antibiotic response was limited at high ofloxacin concentrations. The grafting experiment demonstrated that grafted plants had the ability to alleviate ofloxacin stress. In conclusion, ofloxacin can damage the photosynthetic machinery by promoting ROS accumulation, which results in the etiolation of tomato leaves and inhibits plant growth, but grafting can reduce its.

The ABI4-RbohD/VTC2 regulatory module promotes reactive oxygen species (ROS) accumulation to decrease seed germination under salinity stress.

Salinity stress enhances reactive oxygen species (ROS) accumulation by activating the transcription of NADPH oxidase genes such as RbohD, thus mediating plant developmental processes, including seed germination. However, how salinity triggers the expression of ROS-metabolism-related genes and represses seed germination has not yet been fully addressed. In this study, we show that Abscisic Acid-Insensitive 4 (ABI4), a key component in abscisic acid (ABA) signaling, directly combines with RbohD and Vitamin C Defective 2 (VTC2), the key genes involved in ROS production and scavenging, to modulate ROS metabolism during seed germination under salinity stress. Salinity-induced ABI4 enhances RbohD expression by physically interacting with its promoter, and subsequently promotes ROS accumulation, thus resulting in cell membrane damage and a decrease in seed vigor. Additional genetic evidence indicated that the rbohd mutant largely rescues the salt-hypersensitive phenotype of ABI4 overexpression seeds. Consistently, the abi4/vtc2 double mutant showed the salt-sensitive phenotype, similar to the vtc2 mutant, suggesting that both RbohD and VTC2 are epistatic to ABI4 genetically. Altogether, these results suggest that the salt-induced RbohD transcription and ROS accumulation is dependent on ABI4, and that the ABI4-RbohD/VTC2 regulatory module integrates both ROS metabolism and cell membrane integrity, ultimately repressing seed germination under salinity stress.

Sucralose promotes accumulation of reactive oxygen species (ROS) and adipogenesis in mesenchymal stromal cells

Consumption of non-nutritive sweeteners (NNS) has been consistently associated with obesity and cardiometabolic disease in epidemiologic studies. Herein, we investigated effects of sucralose, a widely used NNS, at a cellular level. We wanted to investigate effect of sucralose on reactive oxygen species accumulation and adipogenesis in a human adipocyte tissue-derived mesenchymal stromal cells (MSCs) in a controlled fashion.MethodsIn vitro experiments were conducted on commercially available MSCs obtained from human adipose tissue. hMSCs were exposed with sucralose at 0.2 mM (a concentration which could plausibly be observed in the circulatory system of high NNS consumers) up to 1.0 mM (supra-physiologic concentration) in the presence of both normal and high glucose media to detect a dose response based on the outcome measures. Reactive oxygen species (ROS) were detected using Mitosox Red staining and further analyzed by ImageJ and gene expression analysis. Effect of sucralose on adipogenic differentiation was observed in different concentrations of sucralose followed by gene expression analysis and Oil Red O staining.ResultsIncreased ROS accumulation was observed within 72 h of exposure. Increased adipogenesis was also noted when exposed to higher dose of sucralose.ConclusionSucralose promotes ROS accumulation and adipogenesis in human adipose tissue derived mesenchymal stromal cells.

Inhibition of NR4A1 Promotes ROS Accumulation and IL24-Dependent Growth Arrest in Rhabdomyosarcoma.

Nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in rhabdomyosarcoma (RMS), and inactivation of NR4A1 (siNR4A1) or treatment with the NR4A1 antagonist 1,1-bis(3'-indoly)-1-(p-hydroxy-phenyl)methane (DIM-C-pPhOH) has antiproliferative and proapoptotic effects on RMS cells. However, the mechanism by which NR4A1 inhibition exerts these effects is poorly defined. Here, we report that NR4A1 silencing or inhibition resulted in accumulation of reactive oxygen species (ROS) and ROS-dependent induction of the tumor suppressor-like cytokine IL24 in RMS cells. Mechanistically, NR4A1 was found to regulate the expression of the proreductant genes thioredoxin domain-containing 5 (TXNDC5) and isocitrate dehydrogenase 1 (IDH1), which are downregulated in RMS cells following NR4A1 knockdown or inhibition. Silencing TXNDC5 and IDH1 also induced ROS accumulation and IL24 expression in RMS cells, suggesting that NR4A1 antagonists mediate their antiproliferative and apoptotic effects through modulation of proreductant gene expression. Finally, cotreatment with the antioxidant glutathione or IL24-blocking antibody reversed the effects of NR4A1 inhibition, demonstrating the importance of both ROS and IL24 in mediating the cellular responses. IMPLICATIONS: Overall, these data elucidate the mechanism by which NR4A1 inhibition functions to inhibit the proliferation, survival, and migration of RMS cells.

The Novel Autophagy Inhibitor Alpha-Hederin Promoted Paclitaxel Cytotoxicity by Increasing Reactive Oxygen Species Accumulation in Non-Small Cell Lung Cancer Cells.

Chemoresistance is a major limiting factor that impairs the outcome of non-small cell lung cancer (NSCLC) chemotherapy. Paclitaxel (Tax) induces protective autophagy in NSCLC cells, leading to the development of drug resistance. We recently identified a new autophagy inhibitor (alpha-hederin) and hypothesized that it may promote the killing effect of Tax on NSCLC cells. We found that alpha-hederin (α-Hed) could block late autophagic flux in NSCLC cells by altering lysosomal pH and inhibiting lysosomal cathepsin D maturation. Combination treatment of α-Hed and Tax synergistically reduced NSCLC cell proliferation and increased NSCLC cell apoptosis compared with treatment with α-Hed or Tax alone. Furthermore, α-Hed plus Tax enhanced the accumulation of intracellular reactive oxygen species (ROS) in NSCLC cells, while the ROS inhibitor N-acetylcysteine reversed the inhibitory effect of the combination treatment. Our findings suggest that α-Hed can increase the killing effect of Tax on NSCLC cells by promoting ROS accumulation, and that combining α-Hed with classical Tax represents a novel strategy for treating NSCLC.

Effects of 5-hydroxy-4'-nitro-7-propionyloxy-genistein on inhibiting proliferation and invasion via activating reactive oxygen species in human ovarian cancer A2780/DDP cells.

5-hydroxy-4'-nitro-7-propionyloxy-genistein (HNPG), a novel synthetic isoflavone derivative, was demonstrated to possess antitumor activity in gastric cancer and breast cancer in vitro, but its antitumor effect and mechanism in ovarian cancer has not been characterized. The aim of the present study was to investigate the effects of HNPG on inhibiting the proliferation and invasion in human ovarian cancer A2780 cell lines of cisplatin resistance (A2780/DDP) and elucidate its underlying molecular mechanism. The results indicated that HNPG presented with marked antitumor activity against A2780/DDP cells in vitro, significantly inhibited the rates of proliferation, clone formation, invasion and metastasis, and markedly induced apoptosis in dose- and time-dependent manner. Simultaneously, levels of reactive oxygen species (ROS) were increased and mitochondrial membrane potential was decreased. In addition, Bcl-2 expression was downregulated, Bax expression was upregulated, and the ratio of Bcl-2/Bax was decreased. Concurrently, levels of Cyt-C were markedly enhanced and the caspase cascade was triggered. Taken together, the results suggested that HNPG exerted anticancer effects through promoting ROS accumulation in cells, triggering mitochondrial apoptotic pathways and ultimately resulting in cells apoptosis. Therefore, HNPG serves as a potential candidate in the chemoprevention and/or treatment of cisplatin-resistant human ovarian cancer.

ABNORMAL INFLORESCENCE MERISTEM1 Functions in Salicylic Acid Biosynthesis to Maintain Proper Reactive Oxygen Species Levels for Root Meristem Activity in Rice.

Root meristem activity determines root growth and root architecture and consequently affects water and nutrient uptake in plants. However, our knowledge about the regulation of root meristem activity in crop plants is very limited. Here, we report the isolation and characterization of a short root mutant in rice (Oryza sativa) with reduced root meristem activity. This root growth defect is caused by a mutation in ABNORMAL INFLORESCENCE MERISTEM1 (AIM1), which encodes a 3-hydroxyacyl-CoA dehydrogenase, an enzyme involved in β-oxidation. The reduced root meristem activity of aim1 results from reduced salicylic acid (SA) levels and can be rescued by SA application. Furthermore, reduced SA levels are associated with reduced levels of reactive oxygen species (ROS) in aim1, likely due to increased expression of redox and ROS-scavenging-related genes, whose increased expression is (at least in part) caused by reduced expression of the SA-inducible transcriptional repressors WRKY62 and WRKY76. Like SA, ROS application substantially increased root length and root meristem activity in aim1 These results suggest that AIM1 is required for root growth in rice due to its critical role in SA biosynthesis: SA maintains root meristem activity through promoting ROS accumulation by inducing the activity of WRKY transcriptional repressors, which repress the expression of redox and ROS-scavenging genes.

Berry anthocyanins reduce proliferation of human colorectal carcinoma cells by inducing caspase-3 activation and p21 upregulation.

Colorectal cancer is the fourth most common type of cancer worldwide, and adenocarcinoma cells that form the majority of colorectal tumors are markedly resistant to antineoplastic agents. Epidemiological studies have demonstrated that consumption of fruits and vegetables that are rich in polyphenols, is linked to reduced risk of colorectal cancer. In the present study, the effect of a standardized anthocyanin (ACN)‑rich extract on proliferation, apoptosis and cell cycle in the Caco-2 human colorectal cancer cell line was evaluated by trypan blue and clonogenic assays and western blot analysis of cleaved caspase‑3 and p21Waf/Cif1. The results of the current study demonstrated that the ACN extract markedly decreased Caco‑2 cell proliferation, induced apoptosis by activating caspase‑3 cleavage, and upregulated cyclin‑dependent kinase inhibitor1 (p21Waf/Cif1) expression in a dose dependent manner. Furthermore, ACN extract was able to produce a dose‑dependent increase of intracellular reactive oxygen species (ROS) in Caco‑2 cells, together with a light increase of the cell total antioxidant status. In conclusion, the present study demonstrated that a standardized berry anthocyanin rich extract inhibited proliferation of Caco‑2 cells by promoting ROS accumulation, inducing caspase‑3 activation, and upregulating the expression of p21Waf/Cif1.

Dioscin, a natural steroid saponin, induces apoptosis and DNA damage through reactive oxygen species: A potential new drug for treatment of glioblastoma multiforme

Dioscin, a natural product obtained from medicinal plants shows lipid-lowering, anti-cancer and hepatoprotective effects. However, the effect of it on glioblastoma is unclear. In this study, dioscin significantly inhibited proliferation of C6 glioma cells and caused reactive oxygen species (ROS) generation and Ca²⁺ release. ROS accumulation affected levels of malondialdehyde, nitric oxide, glutathione disulfide and glutathione, and caused cell apoptosis. In addition, ROS generation caused mitochondrial damage including structural changes, increased mitochondrial permeability transition and decreased mitochondria membrane potential, which led to the release of cytochrome C, nuclear translation of programmed cell death-5 and increased activities of caspase-3,9. Simultaneously, dioscin down-regulated protein expression of Bcl-2, Bcl-xl, up-regulated expression of Bak, Bax, Bid and cleaved poly (ADP-ribose) polymerase. Also, oxygen stress induced S-phase arrest of cancer cells by way of regulating expression of DNA Topo I, p53, CDK2 and Cyclin A and caused DNA damage. In a rat allograft model, dioscin significantly inhibited tumor size and extended the life cycle of the rats. In conclusion, dioscin shows noteworthy anti-cancer activity on glioblastoma cells by promoting ROS accumulation, inducing DNA damage and activating mitochondrial signal pathways. Ultimately, we believe dioscin has promise as a new therapy for the treatment of glioblastoma.