Cytoplasmic APE1 promotes resistance response in osteosarcoma patients with cisplatin treatment

Chemotherapy resistance has become a hold back and major clinical challenge in osteosarcoma cancer. The alteration and subcellular distribution of apurinic/apyrimidinic endonuclease 1 (APE1) has been reported to be involved in chemotherapy resistance in many cancers. Here, we report that the cytoplasmic distribution of APE1 plays a key role in the sensitivity of combination platinum chemotherapy in osteosarcoma. Interestingly, the prevalence of cisplatin-induced DNA damage and apoptosis in low cytoplasmic APE1 osteosarcoma cell lines was higher than in high expression of cytoplasmic APE1 cell lines. Overexpression of cytoplasmic APE1 protected the osteosarcoma cells from CDDP-induced apoptosis. In addition, clinical data also show that the level of cytoplasmic APE1 was negatively associated with sensitivity to combination chemotherapy of cisplatin in osteosarcoma patients. Our findings suggest that cytoplasmic APE1 plays a significant role in chemotherapy resistance. This role is a supplement to the extranuclear function of APE1, and cytoplasmic APE1 expression level could be a promising predictor of platinum treatment prognosis for osteosarcoma patients.     

Genistein induces apoptosis by stabilizing intracellular p53 protein through an APE1-mediated pathway

Genistein (GEN) has been previously shown to have a proapoptotic effect on cancer cells through a p53-dependent pathway, the mechanism of which remains unclear. One of its intracellular targets, APE1, protects against apoptosis under genotoxic stress and interacts with p53. In this current study, we explored the mechanism of the proapoptotic effect of GEN by examining the APE1–p53 protein–protein interaction. We initially showed that the p53 protein level was elevated in GEN-treated human non-small lung cancer A549 cells and cervical cancer HeLa cells. By examining both protein synthesis and degradation, we found that GEN enhances p53 intracellular stability by interfering with the interaction of APE1 and p53, which provided a plausible explanation for how GEN initiates apoptosis. Furthermore, we found that the interaction between APE1 and p53 is important for the degradation of p53 and is dependent on the redox domain of APE1 by utilizing the redox domain mutant APE1 C65A. Our data suggest that the degradation of wild-type p53 is blocked when the redox domain of APE1 is masked or interrupted. Based on this evidence, we hereby report a novel mechanism of p53 degradation through an APE1-mediated, redox-dependent pathway.     

MAD2B promotes podocyte injury through regulating Numb-dependent Notch 1 pathway in diabetic nephropathy

Rationale: Recent studies have demonstrated that the loss of podocyte is a critical event in diabetic nephropathy (DN). Previously, our group have found that the mitotic arrest deficient protein MAD2B was involved in high glucose (HG)-induced podocyte injury by regulating APC/C activity. However, the exact mechanism of MAD2B implicated in podocyte injury is still lacking.Methods: The experiments were conducted by using kidney tissues from streptozotocin (STZ) induced diabetic mice with or without podocyte-specific deletion of MAD2B and the cultured podocytes exposed to different treatments. Glomerular pathological injury was evaluated by periodic acid-Schiff staining and transmission electron microscopy. The endogenous interaction between MAD2B and Numb was discovered by yeast two-hybrid analysis and co-immunoprecipitation assay. The expressions of MAD2B, Numb and related pathway were detected by western blot, immunochemistry and immunofluorescence.Results: The present study revealed that MAD2B was upregulated in diabetic glomeruli and cultured podocytes under hyperglycemic conditions. Podocyte-specific deletion of MAD2B alleviated podocyte injury and renal function deterioration in mice of diabetic nephropathy. Afterwards, MAD2B was found to interact with Numb, which was downregulated in diabetic glomeruli and HG-stimulated cultured podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes exposed to HG and in diabetic mice, and the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased accordingly. In addition, overexpression of Numb ameliorated HG-induced podocyte injury.Conclusions: The present findings suggest that upregulated MAD2B expression contributes to Numb depletion and activation of Notch 1 signaling pathway, which ultimately leads to podocyte injury during DN progression.     

Dikkopf-1 promotes matrix mineralization of osteoblasts by regulating Ca+-CAMK2A-CREB1 pathway

Dickkopf-1 (DKK1) is a secreted protein that acts as an antagonist of the canonical WNT/β-catenin pathway, which regulates osteoblast differentiation. However, the role of DKK1 on osteoblast differentiation has not yet been fully clarified. Here, we investigate the functional role of DKK1 on osteoblast differentiation. Primary osteoprogenitor cells were isolated from human spinal bone tissues. To examine the role of DKK1 in osteoblast differentiation, we manipulated the expression of DKK1, and the cells were differentiated into mature osteoblasts. DKK1 overexpression in osteoprogenitor cells promoted matrix mineralization of osteoblast differentiation but did not promote matrix maturation. DKK1 increased Ca⁺ influx and activation of the Ca⁺/calmodulin-dependent protein kinase II Alpha (CAMK2A)-cAMP response element-binding protein 1 (CREB1) and increased translocation of p-CREB1 into the nucleus. In contrast, stable DKK1 knockdown in human osteosarcoma cell line SaOS2 exhibited reduced nuclear translocation of p-CREB1 and matrix mineralization. Overall, we suggest that manipulating DKK1 regulates the matrix mineralization of osteoblasts by Ca⁺-CAMK2A-CREB1, and DKK1 is a crucial gene for bone mineralization of osteoblasts.     

Nrf2抗氧化的分子调控机制

Nrf2是调控细胞氧化应激反应的重要转录因子,同时也是维持细胞内氧化还原稳态的中枢调节者。Nrf2通过诱导调控一系列抗氧化蛋白的组成型和诱导型表达,可以减轻活性氧和亲电体引起的细胞损伤,使细胞处于稳定状态,维持机体氧化还原动态平衡。本研究为了从分子层面深入探讨剖析Nrf2发挥抗氧化功能的作用机制,通过查找阅读大量相关文献并进行整理归纳,最终从Nrf2的结构与激活、Nrf2抗氧化功能以及Nrf2抗氧化的分子调控机制三个方面进行了概述分析。其中在对Nrf2抗氧化的分子调控机制的探讨部分,既探析了对Nrf2起激活作用的相关调节因子的作用机制,又分析了Nrf2被激活后对其下游多种抗氧化因子及谷胱甘肽氧化还原系统的诱导调控机制,以期较深入了解Nrf2抵抗机体氧化应激损伤作用及其抗氧化分子调控机制。     

The role of Asn-212 in the catalytic mechanism of human endonuclease APE1: Stopped-flow kinetic study of incision activity on a natural AP site and a tetrahydrofuran analogue

Mammalian AP endonuclease 1 is a pivotal enzyme of the base excision repair pathway acting on apurinic/apyrimidinic sites. Previous structural and biochemical studies showed that the conserved Asn-212 residue is important for the enzymatic activity of APE1. Here, we report a comprehensive pre-steady-state kinetic analysis of two APE1 mutants, each containing amino acid substitutions at position 212, to ascertain the role of Asn-212 in individual steps of the APE1 catalytic mechanism. We applied the stopped-flow technique for detection of conformational transitions in the mutant proteins and DNA substrates during the catalytic cycle, using fluorophores that are sensitive to the micro-environment. Our data indicate that Asn-212 substitution by Asp reduces the rate of the incision step by ∼550-fold, while Ala substitution results in ∼70,000-fold decrease. Analysis of the binding steps revealed that both mutants continued to rapidly and efficiently bind to abasic DNA containing the natural AP site or its tetrahydrofuran analogue (F). Moreover, transient kinetic analysis showed that N212A APE1 possessed a higher binding rate and a higher affinity for specific substrates compared to N212D APE1. Molecular dynamics (MD) simulation revealed a significant dislocation of the key catalytic residues of both mutant proteins relative to wild-type APE1. The analysis of the model structure of N212D APE1 provides evidence for alternate hydrogen bonding between Asn-212 and Asp-210 residues, whereas N212A possesses an extended active site pocket due to Asn removal. Taken together, these biochemical and MD simulation results indicate that Asn-212 is essential for abasic DNA incision, but is not crucial for effective recognition/binding.     

Wheat gliadin promotes the interleukin-4-induced IgE production by normal human peripheral mononuclear cells through a redox-dependent mechanism

Increased levels of serum IgE have been described in gliadin-intolerant patients; however, biological mechanisms implicated in this immunoglobulin production remained unknown. In this study, we demonstrated that in vitro crude gliadins and gliadin lysates (Glilys) promoted the IL-4-induced IgE production by human peripheral blood mononuclear cells (PBMC), indicating that the biological process related to gliadin intolerance and/or allergy may lead to IgE production in vivo. It was found that crude gliadin and Glilys potentiated, after 13 days of culture in a dose-dependent manner, IL-4-induced IgE production and, to a lesser extent, the IgG production, while they did not affect IgA or IgM productions. This promoting effect of gliadin and Glilys on the IL-4-induced activation of normal human PBMC was also observed on the early release (2 days) of the soluble fraction of CD23, suggesting its possible involvement in IgE potentiation. The promoting effect of crude gliadin and Glilys appeared to be indirect because they did not modify purified B-lymphocytes IgE production after IL-4 and anti-CD40 monoclonal antibody stimulation.In addition, as revealed by luminol-dependent chemiluminescence, we demonstrated that crude gliadin and Glilys promoted a substantial production of free radicals by normal human PBMC, treated or not with IL-4. This redox imbalance associated with an increased IgE production led us to evaluate the effect of pharmacological antioxidants (N-acetyl-cysteine (NAC) and Cu/Zn-superoxide dismutase (SOD1)) on IgE production by human PBMC. The NAC and the intracellularly delivered SOD1 were found to suppress the IL-4+/-crude gliadin or Glilys-induced IgE production by normal human PBMC. Taken together, these data indicated that gliadin specifically enhanced IL-4-induced IgE production by normal human PBMC, probably by the regulation of redox pathways, and that this 'pro-allergenic' effect could be counteracted by natural antioxidants: thiols and/or vectorized SOD1.     

The APE2 nuclease is essential for DNA double-strand break repair by microhomology-mediated end joining

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Retracted: Receptor for advanced glycation end products reveals a mechanism regulating thyroid hormone secretion through the SIRT1/Nrf2 pathway

Advanced glycation end products (AGEs) play a causative role in the complications involved with diabetes mellitus (DM). Nowadays, DM with hypothyroidism (DM-hypothyroidism) is indicative of an ascended tendency in the combined morbidity. In this study, we examine the role of the receptor (RAGE) played for AGEs in thyroid hormone (TH) secretion via the silent information regulator 1 (SIRT1)/nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) pathway. Blood samples were collected from patients with type 2 DM (T2DM)-hypothyroidism and from patients with T2DM, followed by detection of serum AGEs level. The underlying regulatory mechanisms of RAGE were analyzed in association with the treatment of high glucose, siRNA against RAGE, AGE, SIRT1, or Nrf2 vector in normal immortalized thyroid Nthy-ori 3-1 cells. Serum of patients with T2DM-hypothyroidism indicated promoted levels of AGEs vs those with just T2DM. Both AGEs and high glucose triggered cellular damage, increased oxidative stress, as well as displayed a decreased survival rate along with TH secretion in the Nthy-ori 3-1 cells. Moreover, AGEs and high glucose also led to RAGE upregulation, both SIRT1 and NRF2 downregulation, and the decreased expression of TH secretion–related proteins in Nthy-ori 3-1 cells. Notably, these alternations induced by the AGEs can be reserved by silencing RAGE or upregulating either SIRT1 or Nrf2, indicating a mechanism of regulating TH secretion through the SIRT1/Nrf2 pathway. Collectively, our data proposed that AGEs and high glucose exerted a potent effect on cellular damage and TH deficiency in Nthy-ori 3-1 cells through the RAGE upregulation as well as SIRT1/Nrf2 pathway inactivation. This mechanism may underlie the occurrence of DM-hypothyroidism.     

IGF1 regulates PKM2 function through Akt phosphorylation

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.     

Redox regulation of neutral sphingomyelinase-1 activity in HEK293 cells through a GSH-dependent mechanism

Phospholipases are essential enzymes in cellular signalling processes such as cellular differentiation, proliferation and apoptosis. Based on its high degree of homology with sequences of prokaryote SMases, a type of Mg(2+)-dependent PLC (nSMase-1) was recently discovered which displayed strong redox dependence for activity in vitro [F. Rodrigues-Lima, A.C. Fensome, M. Josephs, J. Evans, R.J. Veldman, M. Katan (2000), J. Biol. Chem. 275 (36) 28316-28325]. The aim of this work was to test the hypothesis that glutathione could be a natural regulator of nSMase-1 activity ex vivo. We studied how altering glutathione levels and redox ratio modulate nSMase-1 activity in a HEK293 cell line that ectopically overexpressed the nSMase-1 gene. Diminishing total glutathione with BSO without altering significantly the GSH/GSSG ratio did not affect nSMase-1 activity. Treatment of cells with diamide produced a transient decrease of total glutathione and a sharp, but also transient, decrease of the GSH/GSSG ratio. Under these conditions, nSMase-1 activity was temporarily activated and then returned to normal levels. Simultaneous treatment with BSO and diamide that resulted in permanent decreases of total glutathione and GSH/GSSG redox ratio produced a sustained activation of nSMase-1 activity. Taken together, these data indicate that altering the GSH/GSSG ratio by increasing GSSG or decreasing GSH levels, but not the total concentration of glutathione, modulates nSMase-1 activity. Our findings are the first evidence supporting the ex vivo regulation of nSMase-1 through a redox glutathione-dependent mechanism.