DNA Damage-inducible Gene Research Articles

Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2.

Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆lhr cells by mutant lhr alleles depended on Lhr's ATPase/helicase activities and its tetrameric quaternary structure. The lhr-nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen-UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr-nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen-UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen-UVA.

The DNA damage response of Escherichia coli, revisited: Differential gene expression after replication inhibition

In 1967, in this journal, Evelyn Witkin proposed the existence of a coordinated DNA damage response in Escherichia coli, which later came to be called the "SOS response." We revisited this response using the replication inhibitor azidothymidine (AZT) and RNA-Seq analysis and identified several features. We confirm the induction of classic Save our ship (SOS) loci and identify several genes, including many of the pyrimidine pathway, that have not been previously demonstrated to be DNA damage-inducible. Despite a strong dependence on LexA, these genes lack LexA boxes and their regulation by LexA is likely to be indirect via unknown factors. We show that the transcription factor "stringent starvation protein" SspA is as important as LexA in the regulation of AZT-induced genes and that the genes activated by SspA change dramatically after AZT exposure. Our experiments identify additional LexA-independent DNA damage inducible genes, including 22 small RNA genes, some of which appear to activated by SspA. Motility and chemotaxis genes are strongly down-regulated by AZT, possibly as a result of one of more of the small RNAs or other transcription factors such as AppY and GadE, whose expression is elevated by AZT. Genes controlling the iron siderophore, enterobactin, and iron homeostasis are also strongly induced, independent of LexA. We confirm that IraD antiadaptor protein is induced independent of LexA and that a second antiadaptor, IraM is likewise strongly AZT-inducible, independent of LexA, suggesting that RpoS stabilization via these antiadaptor proteins is an integral part of replication stress tolerance.

#245 Deficiency of GADD45α expression mediated tubulointerstitial injury in diabetic nephropathy

Abstract Background and Aims The tubulointerstitial injury plays crucial roles in the development of diabetic nephropathy (DN). Recently, some studies have reported that disorder of mitochondrial fatty acid β-oxidation in proximal tubules is involved in tubulointerstitial injury of DN. The growth arrest and DNA damage-inducible gene 45 (GADD45) family proteins, especially, GADD45α, plays a crucial role in various cellular processes, such as mitochondrial biogenesis and lipid metabolism. Recent studies have demonstrated that GADD45α facilitates gene activation by inducing DNA demethylation. Therefore, this study aimed to investigate the role of GADD45α in tubulointerstitial injury of DN and its underlying mechanisms. Methods Bioinformatics and machine learning techniques were employed to identify differentially expressed genes between healthy individuals and DN patients. Experiments were conducted by using high glucose-treated HK-2 cells and streptozotocin (STZ)-induced mice. Lentivirus carrying the GADD45α gene was used to induce GADD45α overexpression in HK-2 cells. Adeno-Associated Virus vectors carrying LoxP-flanked GADD45α were intravenously injected into γGT-Cre transgenic mice expressing Cre recombinase in renal proximal tubular cells, specifically leading to GADD45α overexpression in proximal tubules. Results Through bioinformatics and machine learning techniques, GADD45α was identified and found to exhibit a strong association with tubulointerstitial injury in DN. Further analysis revealed that the expression of GADD45α was decreased in the kidneys of DN patients, diabetic mice, and high glucose-treated HK-2 cells. Notably, GADD45α overexpression ameliorated kidney damage and mitochondrial injury, leading to improved fatty acid β-oxidation, restored ATP production, and reduced ROS production. Transcriptomics analyses further demonstrated that GADD45α overexpression enhanced the expression of genes related to mitochondrial biogenesis, dynamics, and electron transport chain, with six transmembrane epithelial antigen of prostate 4 (STEAP4) being a key protein in this process. Knocking down STEAP4 using siRNA in HK-2 cells exacerbated the damage induced by high glucose. In addition, our further results indicated that high glucose increased methylation of the STEAP4 promoter, while GADD45α overexpression decreased methylation and increased STEAP4 expression. Conclusions Deficiency of GADD45α expression mediated tubulointerstitial injury in DN, which was mainly through the decreased STEAP4 expression by demethylating CpG motifs in the STEAP4 promoter region, thereby resulting in reduced fatty acid oxidation, energy synthesis, and increased ROS production, and finally accelerated tubular injuries. These findings suggested that targeting the GADD45α-STEAP4 pathway could be a potential therapeutic strategy for DN.

Impact of Complex Apoptotic Signaling Pathways on Cancer Cell Sensitivity to Therapy.

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.

Identification of potential crucial genes and therapeutic targets for epilepsy

BackgroundEpilepsy, a central neurological disorder, has a complex genetic architecture. There is some evidence suggesting that genetic factors play a role in both the occurrence of epilepsy and its treatment. However, the genetic determinants of epilepsy are largely unknown. This study aimed to identify potential therapeutic targets for epilepsy.MethodsDifferentially expressed genes (DEGs) were extracted from the expression profiles of GSE44031 and GSE1834. Gene co-expression analysis was used to confirm the regulatory relationship between newly discovered epilepsy candidate genes and known epilepsy genes. Expression quantitative trait loci analysis was conducted to determine if epilepsy risk single-nucleotide polymorphisms regulate DEGs’ expression in human brain tissue. Finally, protein–protein interaction analysis and drug–gene interaction analysis were performed to assess the role of DEGs in epilepsy treatment.ResultsThe study found that the protein tyrosine phosphatase receptor-type O gene (PTPRO) and the growth arrest and DNA damage inducible alpha gene (GADD45A) were significantly upregulated in epileptic rats compared to controls in both datasets. Gene co-expression analysis revealed that PTPRO was co-expressed with RBP4, NDN, PAK3, FOXG1, IDS, and IDS, and GADD45A was co-expressed with LRRK2 in human brain tissue. Expression quantitative trait loci analysis suggested that epilepsy risk single-nucleotide polymorphisms could be responsible for the altered PTPRO and GADD45A expression in human brain tissue. Moreover, the protein encoded by GADD45A had a direct interaction with approved antiepileptic drug targets, and GADD45A interacts with genistein and cisplatin.ConclusionsThe results of this study highlight PTPRO and GADD45A as potential genes for the diagnosis and treatment of epilepsy.

Identify GADD45G as a potential target of 4-methoxydalbergione in treatment of liver cancer: bioinformatics analysis and in vivo experiment

BackgroundThe growth arrest and DNA damage-inducible gene gamma (GADD45G), an important member of GADD45 family, has been connected to the development of certain human cancers. Our previous studies have confirmed that GADD45G expression could be upregulated by 4-methoxydalbergione (4MOD) in liver cancer cells, but its potential pathological role in hepatocellular carcinoma (HCC) has not been fully understood. This study aimed to determine potential role of GADD45G in HCC, and the effects of 4-methoxydalbergione (4MOD) on the regulation of GADD45G expression in vivo were also analyzed.MethodsPublicly available data and in-house immunohistochemistry (IHC) experiments were utilized to explore the expression profiles and clinical significance of GADD45G in HCC samples. Functional enrichment analysis based on GADD45G co-expression genes was used to excavate the molecular mechanism of GADD45G in HCC. We also conducted in vivo experiment on BALB/c nude mice to excavate the inhibitory effect of 4MOD on HCC and to evaluate the differences in the expression of GADD45G in xenograft tissues between the 4MOD-treated and untreated groups.ResultsGADD45G displayed significant low expression in HCC tissues. Downregulated expression of GADD45G was positively correlated with some high risk factors in HCC patients and predicted worse prognosis of HCC patients. There was a close association of GADD45G mRNA expression and immune cells, including neutrophils, NK cells, CD8 T cells, and macrophages. Co-expressed genes of GADD45G were involved in several pathways including cell cycle, carbon metabolism, and peroxisome. 4MOD could significantly suppress the growth of HCC in vivo, and this inhibitory effect was dependent on the upregulation of GADD45G expression.ConclusionGADD45G expression can be used as a new clinical biomarker for HCC and GADD45G may be a potential target for the anti-cancer effect of 4MOD in liver cancer.

The mycobacterial ImuA'-ImuB-DnaE2 mutasome: composition and recruitment in live cells.

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

Impact of GADD45A on Radiation Biodosimetry Using Mouse Peripheral Blood.

High-dose-radiation exposure in a short period of time leads to radiation syndromes characterized by severe acute and delayed organ-specific injury accompanied by elevated organismal morbidity and mortality. Radiation biodosimetry based on gene expression analysis of peripheral blood is a valuable tool to detect exposure to radiation after a radiological/nuclear incident and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including chronic inflammation, can potentially obscure the predictive power of the method. GADD45A (Growth arrest and DNA damage-inducible gene a) plays important roles in cell growth control, differentiation, DNA repair, and apoptosis. GADD45A-deficient mice develop an autoimmune disease, similar to human systemic lupus erythematosus, characterized by severe hematological disorders, kidney disease, and premature death. The goal of this study was to elucidate how pre-existing inflammation in mice, induced by GADD45A ablation, can affect radiation biodosimetry. We exposed wild-type and GADD45A knockout male C57BL/6J mice to 7 Gy of X rays and 24 h later RNA was isolated from whole blood and subjected to whole genome microarray and gene ontology analyses. Dose reconstruction analysis using a gene signature trained on gene expression data from irradiated wild-type male mice showed accurate reconstruction of either a 0 Gy or 7 Gy dose with root mean square error of ± 1.05 Gy (R^2 = 1.00) in GADD45A knockout mice. Gene ontology analysis revealed that irradiation of both wild-type and GADD45A-null mice led to a significant overrepresentation of pathways associated with morbidity and mortality, as well as organismal cell death. However, based on their z-score, these pathways were predicted to be more significantly overrepresented in GADD45A-null mice, implying that GADD45A deletion may exacerbate the deleterious effects of radiation on blood cells. Numerous immune cell functions and quantities were predicted to be underrepresented in both genotypes; however, differentially expressed genes from irradiated GADD45A knockout mice predicted an increased deterioration in the numbers of T lymphocytes, as well as myeloid cells, compared with wild-type mice. Furthermore, an overrepresentation of genes associated with radiation-induced hematological malignancies was associated with GADD45A knockout mice, whereas hematopoietic and progenitor cell functions were predicted to be downregulated in irradiated GADD45A knockout mice. In conclusion, despite the significant differences in gene expression between wild-type and GADD45A knockout mice, it is still feasible to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of pre-existing inflammation status.

Pharmacological inhibition of LSD1 suppresses growth of hepatocellular carcinoma by inducing GADD45B.

Lysine-specific histone demethylase 1 (LSD1) is an attractive target for malignancies therapy. Nevertheless, its role in hepatocellular carcinoma (HCC) progression and the potential of its inhibitor in HCC therapy remains unclear. Here, we show that LSD1 overexpression in human HCC tissues is associated with HCC progression and poor patient survival. ZY0511, a highly selective and potent inhibitor of LSD1, suppressed human HCC cell proliferation in vitro and tumor growth in cell-derived and patient-derived HCC xenograft models in vivo. Mechanistically, ZY0511 induced mRNA expression of growth arrest and DNA damage-inducible gene 45beta (GADD45B) by inducing histone H3 at lysine 4 (H3K4) methylation at the promoter of GADD45B, a novel target gene of LSD1. In human HCC tissues, LSD1 level was correlated with a decreased level of GADD45B, which was associated with HCC progression and predicted poor patient survival. Moreover, co-administration of ZY0511 and DTP3, which specifically enhanced the pro-apoptotic effect of GADD45B, effectively inhibited HCC cell proliferation both in vitro and in vivo. Collectively, our study revealed the potential value of LSD1 as a promising target of HCC therapy. ZY0511 is a promising candidate for HCC therapy through upregulating GADD45B, thereby providing a novel combinatorial strategy for treating HCC.

Nano‑zinc enhances gene regulation of non‑specific immunity and antioxidative status to mitigate multiple stresses in fish

The toxicity of ammonia surged with arsenic pollution and high temperature (34 °C). As climate change enhances the pollution in water bodies, however, the aquatic animals are drastically affected and extinct from nature. The present investigation aims to mitigate arsenic and ammonia toxicity and high-temperature stress (As + NH3 + T) using zinc nanoparticles (Zn-NPs) in Pangasianodon hypophthalmus. Zn-NPs were synthesized using fisheries waste to developing Zn-NPs diets. The four isonitrogenous and isocaloric diets were formulated and prepared. The diets containing Zn-NPs at 0 (control), 2, 4 and 6 mg kg−1 diets were included. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-s-transferase (GST) were noticeably improved using Zn-NPs diets in fish reared under with or without stressors. Interestingly, lipid peroxidation was significantly reduced, whereas vitamin C and acetylcholine esterase were enhanced with supplementation of Zn-NPs diets. Immune-related attributes such as total protein, globulin, albumin, myeloperoxidase (MPO), A:G ratio, and NBT were also improved with Zn-NPs at 4 mg kg−1 diet. The immune-related genes such as immunoglobulin (Ig), tumor necrosis factor (TNFα), and interleukin (IL1b) were strengthening in the fish using Zn-NPs diets. Indeed, the gene regulations of growth hormone (GH), growth hormone regulator (GHR1), myostatin (MYST) and somatostatin (SMT) were significantly improved with Zn-NPs diets. Blood glucose, cortisol and HSP 70 gene expressions were significantly upregulated by stressors, whereas the dietary Zn-NPs downregulated the gene expression. Blood profiling (RBC, WBC and Hb) was reduced considerably with stressors (As + NH3 + T), whereas Zn-NPs enhanced the RBC, WBC, and Hb count in fish reread in control or stress conditions. DNA damage-inducible protein gene and DNA damage were significantly reduced using Zn-NPs at 4 mg kg−1 diet. Moreover, the Zn-NPs also enhanced the arsenic detoxification in different fish tissues. The present investigation revealed that Zn-NPs diets mitigate ammonia and arsenic toxicity, and high-temperature stress in P. hypophthalmus.

GADD34 suppresses eIF2α phosphorylation and improves cognitive function in Alzheimer's disease-model mice

Alzheimer's disease (AD) causes neurodegeneration, leading to cognitive impairment and memory loss. Our previous studies have demonstrated that the induction of growth arrest and DNA damage-inducible gene 34 (GADD34) by quercetin can affect eukaryotic translation initiation factor 2α (eIF2α) phosphorylation-activated transcription factor 4 (ATF4) signaling. However, the relationship between GADD34 expression and cognitive function has not been clarified. In this study, we determined the direct effect of GADD34 on memory. To achieve this, truncated GADD34 (GADD34.5) was injected into the mouse brain to suppress eIF2α phosphorylation and evaluate memory. The injection of GADD34.5 into the hippocampus in AD-model mice did not improve novel object recognition but improved novel object location. The injection of GADD34.5 into the amygdala also resulted in the maintenance of contextual fear memory based on the fear condition test. These results suggest that GADD34 is effective in improving memory for spatial cognition and contextual fear conditioning in AD by inhibiting eIF2α phosphorylation. In summary, GADD34 suppresses eIF2α phosphorylation in the brain and prevents memory loss. As quercetin feeding increases GADD34 expression, it might be used in preventative applications for AD.

Semaglutide (GLP-1 receptor agonist) stimulates browning on subcutaneous fat adipocytes and mitigates inflammation and endoplasmic reticulum stress in visceral fat adipocytes of obese mice.

Semaglutide (GLP-1 agonist) was approved for treating obesity. Although the effects on weight loss and metabolism are known, the responses of adipocytes to semaglutide are yet limited. C57BL/6 male mice (n = 20/group) were fed a control diet (C) or a high-fat(HF)diet for 16 weeks and then separated into four groups (n = 10/group) for an additional four weeks: C, C diet and semaglutide, HF, and HF diet and semaglutide. Epididymal white adipose tissue(eWAT) and subcutaneous white adipose tissue(sWAT) fat pads were studied with biochemistry, immunohistochemistry/fluorescence, stereology, and reverse transcription-quantitative polymerase chain reaction. In obese mice, semaglutide reduced the fat pad masses (eWAT, -55%; sWAT, -40%), plasmatic cytokines, and proinflammatory gene expressions: tumor necrosis factor-alpha (-60%); interleukin (IL)-6 (-55%); IL-1beta (-40%); monocyte chemoattractant protein-1 (-90%); andleptin (-80%). Semaglutide also lessened endoplasmic reticulum (ER) stress genes of activating transcription factor-4 (-85%), CCAAT enhancer-binding protein homologous protein (-55%), and growth arrest and DNA damage-inducible gene 45 (-45%). The obese mice's adipocyte hypertrophy and macrophage infiltration were equally reduced by semaglutide. Semaglutide enhanced multiloculation and uncoupled protein 1 (UCP1) labeling in obese mice: peroxisome proliferator-activated receptor-alpha (+560%) and gamma (+150%), fibronectin type III domain-containing protein 5 (+215%), peroxisome proliferator-activated receptor-alpha coactivator (+110%), nuclear respiratory factor 1 (+260%), and mitochondrial transcription factor A (+120%). Semaglutide also increased thermogenetic gene expressions for the browning phenotype maintenance: beta-3 adrenergic receptor (+520%), PR domain containing 16 (+90%), and Ucp1 (+110%). In conclusion, semaglutide showed significant beneficial effects beyond weight loss, directly on fat pads and adipocytes of obese mice, remarkably anti-inflammatory, and reduced adipocyte size and ER stress. Besides, semaglutide activated adipocyte browning, improving UCP1, mitochondrial biogenesis, and thermogenic marker expressions help weight loss.

A DNA-Damage Inducible Gene Promotes the Formation of Antibiotic Persisters in Response to the Quorum Sensing Signaling Peptide in Streptococcus mutans.

Bacteria use quorum sensing (QS) to communicate with each other via secreted small autoinducers produced by individuals. QS allows bacteria to display a unified response that benefits the species during adaptation to environment, colonization, and defense against competitors. In oral streptococci, the CSP-ComDE QS is an inducible DNA damage repair system that is pivotal for bacterial survival. In the oral pathogen Streptococcus mutans, the QS system positively influences the formation of antibiotic persisters, cells that can survive antibiotic attack by entering a non-proliferative state. We recently identified a novel gene, pep299, that is activated in the persister cell fraction induced by QS. In this study, we focused our investigation on the role of pep299, a gene encoding a bacteriocin-like peptide, in the formation of antibiotic persisters. Mutant Δ299, unable to produce Pep299, showed a dramatic reduction in the number of stress-induced persisters. Using a co-culture assay, we showed that cells overproducing pep299 induced the formation of persisters in the mutant, suggesting that Pep299 was actively secreted and detected by neighboring cells. Cells exposed to DNA damage conditions activated the gene expression of pep299. Interestingly, our results suggested that the pep299 gene was also involved in the regulation of a QS-inducible toxin–antitoxin system. Our study suggests that the pep299 gene is at the core of the triggered persistence phenotype in S. mutans, allowing cells to transition into a state of reduced metabolic activity and antibiotic tolerance.

Synthesis and anti-melanoma effect of 3-O-prenyl glycyrrhetinic acid against B16F10 cells via induction of endoplasmic reticulum stress-mediated autophagy through ERK/AKT signaling pathway.

Melanoma is an aggressive form of cancer with poor prognosis and survival rates and limited therapeutic options. Here, we report the anti-melanoma effect of 3-O-prenyl glycyrrhetinic acid (NPC-402), a derivative of glycyrrhtinic acid, from a reputed medicinal plant Glycyrrhiza glabra against B16F10 cells. We studied the cytotoxic effect of NPC-402 on melanoma cells and investigated the role of mitogen-activated protein (MAP) kinase, AKT axis, and endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated autophagy as the involved signaling cascade by studying specific marker proteins. In this study, 4-phenylbutyric acid (4PBA, a chemical chaperone) and small interference RNA (siRNA) knockdown of C/EBP Homologous Protein (CHOP)/growth arrest- and DNA damage-inducible gene 153(GAD153) blocked NPC-402-mediated autophagy induction, thus confirming the role of ER stress and autophagy in melanoma cell death. NPC-402 induced oxidative stress and apoptosis in melanoma cells, which were effectively mitigated by treatment with N-acetylcysteine (NAC). In vivo studies showed that intraperitoneal (i.p.) injection of NPC-402 at 10 mg/kg (5 days in 1 week) significantly retarded angiogenesis in the Matrigel plug assay and reduced the tumor size and tumor weight without causing any significant toxic manifestation in C57BL/6J mice. We conclude that NPC-402 has a high potential to be developed as a chemotherapeutic drug against melanoma.

Starch-Based Hydrogel Nanoparticles Loaded with Polyphenolic Compounds of Moringa Oleifera Leaf Extract Have Hepatoprotective Activity in Bisphenol A-Induced Animal Models.

Bisphenol A (BPA) is an xenoestrogenic chemical used extensively in the fabrication of baby bottles, reusable plastic water bottles and polycarbonate plastic containers. The current study aims to investigate the hepatoprotective activity of Moringa oleifera Lam leaf extract (MOLE) and hydrogel NPs made of starch-MOLE-Bovine Serum Albumin (BSA) against Bisphenol A-induced liver toxicity in male rats. Fabrication and characterization of hydrogel NPs formed of starch-MOLE-BSA were investigated using FTIR, TEM, zeta potential, UV-visible spectroscopy and fluorescence spectrophotometer. The potential efficacy of hydrogel NPs was studied. Compared to the results of control, the level of liver function, oxidative stress markers and lipid profile status were remodulated in the groups treated with MOLE and hydrogel NPs (Encap. MOLE). Meanwhile, the administration of MOLE and Encap MOLE significantly increased antioxidant activity and decreased the level of apoptotic pathways. Heme oxygenase (HO)-1 and growth arrest -DNA damage-inducible gene 45b (Gadd45b) were also regulated in the groups treated with MOLE and Encap. MOLE compared to the group which received BPA alone. In the present study, MOLE and hydrogel NPs led to remarkable alterations in histological changes during BPA administration. Overall, MOLE has a potential antioxidant activity which can be used in the treatment of liver disorders.

Chloroquine attenuates chronic hypoxia-induced testicular damage via suppressing endoplasmic reticulum stress and apoptosis in experimental rat model.

Chronic hypoxia negatively affects male fertility by causing pathological changes in male reproductive system. However, underlying mechanisms of this damage are unknown. Chloroquine (CLQ) is an anti-inflammatory agent that is widely used in the treatment of inflammation-related diseases such as malaria and rheumatoid arthritis. This study aimed to investigate the therapeutic effects of CLQ in the hypoxia-induced testicular damage via assessment of hypoxic response, endoplasmic reticulum stress and apoptosis. For this purpose, 32 Wistar albino rats were divided into 4 groups as control (given 20%-21% O2 , no treatment), CLQ (given 50 mg/kg and 20%-21% O2 for 28 days), hypoxia (HX) (given 10% O2 for 28 days) and HX + CLQ (given 50 mg/kg and 10% O2 for 28 days). After the experiment, blood samples and testicular tissues were taken. Histopathological evaluation was performed on testicular tissues and hypoxia-inducible factor 1-α (HIF1-α), heat shock proteins (HSPs) HSP70, HSP90 and growth arrest and DNA damage-inducible gene 153 (GADD153) expression levels were detected via immunohistochemistry. Moreover, apoptotic cells were detected via terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining and serum testosterone levels were determined by enzyme-linked immunosorbent assay (ELISA) assay. Histopathological changes, apoptotic cell numbers and HIF1-α, HSP70, HSP90 and GADD153 expressions significantly increased in HX group (P < .05). Moreover, serum testosterone levels decreased in this group (P > .05). However, CLQ exerted a strong ameliorative effect on all parameters in HX + CLQ group. According to our results, we suggested that CLQ can be considered as an alternative protective agent for eliminating the negative effects of hypoxic conditions on male fertility.

Phenotypic Changes Produced by Endogenous DNA Damage in Yeast Mutants Deficient in Recombination and Base Excision Repair

DNA is under constant attack by exogenous and endogenous factors. Endogenous sources of DNA lesions include free radicals such as reactive oxygen and nitrogen species (RONS), as well as many naturally occurring enzymatic and chemical processes. To prevent mutations and possibly cell death these lesions must be repaired prior to DNA replication. There are five major pathways that are utilized for repair depending on the type of DNA damage. Eukaryotes utilize two pathways, homologous recombination (HR) and nonhomologous end‐joining (NHEJ), for repair of DNA double‐strand breaks (DSBs). In addition, the cells utilize base excision repair (BER) for repair of damaged or missing bases. Previous work in the lab has shown that HR‐deficient yeast mutant cell cultures exhibit high levels of G2 phase cells during normal growth, without exposure to exogenous DNA damaging agents. Inactivation of DNA damage checkpoint genes, but not spindle checkpoint genes, abolished the high G2 cell phenotype, indicating that the damage checkpoint is persistently activated in the cells. In a screen of yeast mutant library strains deficient in each of the major repair pathways, the high G2 phenotype was observed in HR‐ and BER‐deficient cells, but not in mutants specifically defective in nucleotide excision repair (NER), mismatch repair (MMR) or NHEJ.Experiments performed for the current project have assessed the consequences of inactivation of the HR and BER pathways on yeast cells in more detail. The high G2 cell phenotype was heightened in both diploid HR (rad52) and BER (apn1, ogg1) mutants relative to their haploid counterparts. Expression of DIN1 (RNR3), a DNA damage‐inducible gene, was found to be constitutively induced in the HR mutant cells. Preliminary tests of the role of free radicals in generation of endogenous signaling were performed by overexpression of Yap1p, a transcriptional activator of antioxidant enzyme genes. The high G2 cell phenotype was abolished in ogg1 mutants and strongly reduced in rad52 cells when Yap1p was overexpressed. Current work is focused on (a) analysis of DIN1 promoter activation in BER mutants, (b) assessment of the impact of neutralization of free radicals using chemical antioxidants, and (c) measurement of phosphorylated Rad53 checkpoint protein levels using Western blots to characterize the DNA damage checkpoint signaling response.

Folate ameliorates homocysteine-induced osteoblast dysfunction by reducing endoplasmic reticulum stress-activated PERK/ATF-4/CHOP pathway in MC3T3-E1 cells.

Homocysteine (Hcy) is considered a newly identified risk factor for osteoporosis. Nevertheless, the underlying mechanism of folate (FA), a key factor in the metabolism of Hcy, in protection against osteoblast dysfunction remains unclear. The purpose of this study was to investigate the mechanism by which FA attenuates Hcy-induced osteoblast damage. The Hcy-induced MC3T3-E1 cells were treated with different concentrations of FA. Cell morphology, cell density, cell proliferation ability, alkaline phosphatase (ALP) activity and mineralization capacity were observed and determined; the gene expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX) and ERS-associated factors, including glucose-regulated protein 78 (GRP-78), activating transcription factor 4 (ATF-4) and growth arrest and DNA damage inducible gene 153 (CHOP/GADD153), were assessed by RT-PCR; and protein levels of GRP-78 and ATF-4 were analyzed by western blotting. Hcy suppressed the proliferation, differentiation and mineralization ability of MC3T3-E1 cells in a concentration-dependent manner and activated the ERS signaling pathway. After intervention with different concentrations of FA, the cell viability and density, ALP activity, number of mineralized nodules, calcium content and Bcl-2 gene expression were all significantly increased, whereas the gene expression of GRP-78, CHOP/GADD153, ATF-4 and Bax was markedly downregulated, and protein levels of GRP-78 and ATF-4 were also markedly decreased. The adverse effects of Hcy on osteoblast differentiation are dose dependent. FA not only protects against osteoblasts apoptosis but also has a direct osteogenic effect on Hcy-induced osteoblasts, which could be partially mediated by inhibition of the PERK-activated ERS pathway.

MAGEH1 interacts with GADD45G and induces renal tubular cell apoptosis.

BackgroundMelanoma-associated antigen H1 (MAGEH1) is a protein that belongs to melanoma-associated antigen (MAGE) superfamily. Growth arrest and DNA damage 45G (GADD45G) is a member of the DNA damage-inducible gene family which responds to environmental stresses. We have previously shown that GADD45G is a protein that promotes apoptosis of renal tubular cells in response to a nephrotoxic injury. In this study, we show evidence that MAGEH1 interacts with GADD45G and is involved in the induction of nephrotoxin-induced apoptosis of renal tubular cells.MethodsPrimary human renal tubular epithelial (HRE) cells and human kidney 2 (HK-2) cells were used in this study. To produce stable cell lines in which MAGEH1 expression was silenced, HRE cells were transduced with a lentiviral vector encoding a single guide RNA construct targeting the MAGEH1 gene. To knockdown GADD45G expression in HRE cells, a vector containing short hairpin RNA (shRNA) was used. We used short interfering RNAs (siRNA) to achieve transient silencing of genes in HK-2 cells. Recombinant adenoviruses were synthesized to overexpress MAGEH1 and GADD45G proteins. Human protein microarray was used to identify proteins that binds to GADD45G. Co-immunoprecipitation assays were then performed to confirm microarray results. Cell death was induced by cyclosporine A (CsA). Real-time quantitative PCR assay was used to evaluate gene expression levels. The degree of apoptosis and necrosis of cultured cells was evaluated by flow cytometry. Expression levels of caspases were examined using western blot analysis.ResultsWe found that GADD45G bound to one protein spotted in the protein microarray, which was subsequently identified as MAGEH1. We confirmed the interaction between GADD45G and MAGEH1 protein using the co-immunoprecipitation assay. MAGEH1 gene expression was not altered by CsA-induced cytotoxic injury, whereas GADD45G gene expression was increased significantly upon CsA treatment. MAGEH1 expression was significantly downregulated in GADD45G knockdown HRE stable cells suggesting that MAGEH1 expression may be dependent on GADD45G expression. CsA-induced apoptosis was significantly reduced in MAGEH1 knockdown HRE stable cells which led to an increased survival of these cells. Similar results were observed in GADD45G knockdown HRE stable cells. Accordingly, CsA-induced apoptosis was significantly decreased in MAGEH1 siRNA and GADD45G siRNA transfected HK-2 cells. CsA-induced activation of caspase-7 and caspase-9 was inhibited in MAGEH1 knockdown HRE stable cells, and similarly in GADD45G knockdown HRE stable cells.ConclusionsTo the best of our knowledge, this is the first study to show that MAGEH1 interacts with GADD45G and that MAGEH1 is involved in caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.

The molecular mechanism of GADD153 in apoptosis of keloid fibroblasts exposed to botulinum toxin type A

Apoptosis plays a key role in keloids. Growth arrest and DNA damage‐inducible gene 153 (GADD153) is regulated by apoptosis. Botulinum toxin type A (BTXA) can induce apoptosis in keloid fibroblasts. This research aimed to explore the hypothesis that GADD153 mediates apoptosis in keloid fibroblasts exposed to BTXA. BTXA significantly induced GADD153 protein and mRNA expression in keloid fibroblasts. Treatment with c‐Jun N‐terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA) and tumour necrosis factor‐alpha (TNF‐α) antibodies reversed the BTXA‐induced GADD153 expression. BTXA enhanced the transcriptional activity of GADD153, whereas the GADD153 mutant plasmid, JNK siRNA and anti‐TNF‐α antibody treatment abolished the BTXA‐induced transcriptional activity of GADD153. The addition of TNF‐α to keloid fibroblasts markedly increased GADD153 protein expression. The addition of GADD153 siRNA, SP600125 and anti‐TNF‐α antibodies reversed cell death and caspase 3 and 9 activity induced by BTXA.