Sirt3 protects dopaminergic neurons from mitochondrial oxidative stress.

Age-dependent elevation in mitochondrial oxidative stress is widely posited to be a major factor underlying the loss of substantia nigra pars compacta (SNc) dopaminergic neurons in Parkinson's disease (PD). However, mechanistic links between aging and oxidative stress are not well understood. Sirtuin-3 (Sirt3) is a mitochondrial deacetylase that could mediate this connection. Indeed, genetic deletion of Sirt3 increased oxidative stress and decreased the membrane potential of mitochondria in SNc dopaminergic neurons. This change was attributable to increased acetylation and decreased activity of manganese superoxide dismutase (MnSOD). Site directed mutagenesis of lysine 68 to glutamine (K68Q), mimicking acetylation, decreased MnSOD activity in SNc dopaminergic neurons, whereas mutagenesis of lysine 68 to arginine (K68R), mimicking deacetylation, increased activity. Introduction of K68R MnSOD rescued mitochondrial redox status and membrane potential of SNc dopaminergic neurons from Sirt3 knockouts. Moreover, deletion of DJ-1, which helps orchestrate nuclear oxidant defenses and Sirt3 in mice led to a clear age-related loss of SNc dopaminergic neurons. Lastly, K68 acetylation of MnSOD was significantly increased in the SNc of PD patients. Taken together, our studies suggest that an age-related decline in Sirt3 protective function is a major factor underlying increasing mitochondrial oxidative stress and loss of SNc dopaminergic neurons in PD.

Motor neuron mitochondrial dysfunction in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, predominantly affects high metabolic tissues including motor neurons, skeletal muscles and the heart. Although the genetic cause of SMA has been identified, mechanisms underlying tissue-specific vulnerability are not well understood. To study these mechanisms, we carried out a deep sequencing analysis of the transcriptome of spinal motor neurons in an SMA mouse model, in which we unexpectedly found changes in many genes associated with mitochondrial bioenergetics. Importantly, functional measurement of mitochondrial activities showed decreased basal and maximal mitochondrial respiration in motor neurons from SMA mice. Using a reduction-oxidation sensitive GFP and fluorescence sensors specifically targeted to mitochondria, we found increased oxidative stress level and impaired mitochondrial membrane potential in motor neurons affected by SMA. In addition, mitochondrial mobility was impaired in SMA disease conditions, with decreased retrograde transport but no effect on anterograde transport. We also found significantly increased fragmentation of the mitochondrial network in primary motor neurons from SMA mice, with no change in mitochondria density. Electron microscopy study of SMA mouse spinal cord revealed mitochondria fragmentation, edema and concentric lamellar inclusions in motor neurons affected by the disease. Intriguingly, these functional and structural deficiencies in the SMA mouse model occur during the presymptomatic stage of disease, suggesting a role in initiating SMA. Altogether, our findings reveal a critical role for mitochondrial defects in SMA pathogenesis and suggest a novel target for improving tissue health in the disease.

Numb directs the subcellular localization of EAAT3 through binding the YxNxxF motif.

Excitatory amino acid transporter type 3 (EAAT3, also known as SLC1A1) is a high-affinity, Na(+)-dependent glutamate carrier that localizes primarily within the cell and at the apical plasma membrane. Although previous studies have reported proteins and sequence regions involved in EAAT3 trafficking, the detailed molecular mechanism by which EAAT3 is distributed to the correct location still remains elusive. Here, we identify that the YVNGGF sequence in the C-terminus of EAAT3 is responsible for its intracellular localization and apical sorting in rat hepatoma cells CRL1601 and Madin-Darby canine kidney (MDCK) cells, respectively. We further demonstrate that Numb, a clathrin adaptor protein, directly binds the YVNGGF motif and regulates the localization of EAAT3. Mutation of Y503, N505 and F508 within the YVNGGF motif to alanine residues or silencing Numb by use of small interfering RNA (siRNA) results in the aberrant localization of EAAT3. Moreover, both Numb and the YVNGGF motif mediate EAAT3 endocytosis in CRL1601 cells. In summary, our study suggests that Numb is a pivotal adaptor protein that mediates the subcellular localization of EAAT3 through binding the YxNxxF (where x stands for any amino acid) motif.

Non-aggregating tau phosphorylation by cyclin-dependent kinase 5 contributes to motor neuron degeneration in spinal muscular atrophy.

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35(-/-) compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration.

Molecular mechanisms and animal models of spinal muscular atrophy.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the degeneration of spinal motor neurons and muscle atrophy. Although the genetic cause of SMA has been mapped to the Survival Motor Neuron1 (SMN1) gene, mechanisms underlying selective motor neuron degeneration in SMA remain largely unknown. Here we review the latest developments and our current understanding of the molecular mechanisms underlying SMA pathogenesis, focusing on the animal model systems that have been developed, as well as new diagnostic and treatment strategies that have been identified using these model systems. This article is part of a special issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

Excessive Wnt/beta-catenin signaling promotes midbrain floor plate neurogenesis, but results in vacillating dopamine progenitors.

The floor plate (FP), a ventral midline structure of the developing neural tube, has differential neurogenic capabilities along the anterior-posterior axis. The midbrain FP, unlike the hindbrain and spinal cord floor plate, is highly neurogenic and produces midbrain dopaminergic (mDA) neurons. Canonical Wnt/beta-catenin signaling, at least in part, is thought to account for the difference in neurogenic capability. Removal of beta-catenin results in mDA progenitor specification defects as well as a profound reduction of neurogenesis. To examine the effects of excessive Wnt/beta-catenin signaling on mDA specification and neurogenesis, we have analyzed a model wherein beta-catenin is conditionally stabilized in the Shh+domain. Here, we show that the Foxa2+/Lmx1a+ domain is extended rostrally in mutant embryos, suggesting that canonical Wnt/beta-catenin signaling can drive FP expansion along the rostrocaudal axis. Although excess canonical Wnt/beta-catenin signaling generally promotes neurogenesis at midbrain levels, less tyrosine hydroxylase (Th)+, mDA neurons are generated, particularly impacting the Substantia Nigra pars compacta. This is likely because of improper progenitor specification. Excess canonical Wnt/beta-catenin signaling causes downregulation of net Lmx1b, Shh and Foxa2 levels in mDA progenitors. Moreover, these progenitors assume a mixed identity to that of Lmx1a+/Lmx1b+/Nkx6-1+/Neurog1+ progenitors. We also show by lineage tracing analysis that normally, Neurog1+ progenitors predominantly give rise to Pou4f1+ neurons, but not Th+ neurons. Accordingly, in the mutant embryos, Neurog1+ progenitors at the midline generate ectopic Pou4f1+ neurons at the expense of Th+ mDA neurons. Our study suggests that an optimal dose of Wnt/beta-catenin signaling is critical for proper establishment of the mDA progenitor character. Our findings will impact embryonic stem cell protocols that utilize Wnt pathway reagents to derive mDA neuron models and therapeutics for Parkinson's disease.

HAP1 gene expression is associated with radiosensitivity in breast cancer cells.

OBJECTIVES: The purpose of this study was to investigate the relationship between huntingtin-associated protein1 (HAP1) gene and radiation therapy of breast cancer cells. METHODS: HAP1 gene was transfected into breast cancer MCF-7 cells, which was confirmed by quantitative reverse transcription-polymerase chain reaction analysis (qRT-PCR) and Western blot in vitro. The changes of cell radiosensitivity were assessed by colony formation assay. Apoptosis were examined by flow cytometry. The expressions of two radiation-induced genes were evaluated by Western blot. Tumor growth was investigated in nude mice xenograft models in vivo. RESULTS: Our data showed that HAP1 gene expression was significantly increased in HAP1-transfected MCF-7 cells in comparison with the parental cells or negative control cells. The survival rate in MCF-7/HAP1 cells was significantly decreased after irradiation (0, 2, 4, 6, 8Gy), compared to cells in MCF-7 and MCF-7/Pb groups in vitro. HAP1 gene increased apoptosis in MCF-7 cells after irradiation. Additionally, the tumor volume and weight in MCF-7/HAP1+RT group were observably lower than in MCF-7/HAP1 group and MCF-7/Pb+RT group. CONCLUSION: The present study indicated that HAP1 gene expression was related to the radiosensitivity of breast cancer cells and may play an important role in the regulation of cellular radiosensitivity.

Effect of Furin inhibitor on lung adenocarcinoma cell growth and metastasis.

BACKGROUND: To investigate the mechanisms of lung adenocarcinoma cell metastasis and provide a theoretical basis for the in-depth study of lung adenocarcinoma. METHODS: A549 cells are incubated with different concentrations of Furin inhibitor for indicated times. The proliferation and migration were confirmed with MTT, colony formation, wound Healing and Transwell assayes. Hochest 33342 / PI double staining was used to detect apoptosis. Cell migration and apoptosis associated proteins were analysed by enzyme-linked immunosorbent assay (ELISA) and western blot. RESULTS: We have found that Furin inhibitor play a significant role in inhibition A549 cell growth. And we also found cell migration was inhibited significantly upon Furin inhibitor treatment. CONCLUSION: The proliferration and migration of A549 cell were inhibited by Furin inbitor through down-regulation the expression of migration and apoptosis related proteins.

The survival motor neuron gene smn-1 interacts with the U2AF large subunit gene uaf-1 to regulate Caenorhabditis elegans lifespan and motor functions.

Spinal muscular atrophy (SMA), the most frequent human congenital motor neuron degenerative disease, is caused by loss-of-function mutations in the highly conserved survival motor neuron gene SMN1. Mutations in SMN could affect several molecular processes, among which aberrant pre-mRNA splicing caused by defective snRNP biogenesis is hypothesized as a major cause of SMA. To date little is known about the interactions of SMN with other splicing factor genes and how SMN affects splicing in vivo. The nematode Caenorhabditis elegans carries a single ortholog of SMN, smn-1, and has been used as a model for studying the molecular functions of SMN. We analyzed RNA splicing of reporter genes in an smn-1 deletion mutant and found that smn-1 is required for efficient splicing at weak 3' splice sites. Genetic studies indicate that the defective lifespan and motor functions of the smn-1 deletion mutants could be significantly improved by mutations of the splicing factor U2AF large subunit gene uaf-1. In smn-1 mutants we detected a reduced expression of U1 and U5 snRNAs and an increased expression of U2, U4 and U6 snRNAs. Our study verifies an essential role of smn-1 for RNA splicing in vivo, identifies the uaf-1 gene as a potential genetic modifier of smn-1 mutants, and suggests that SMN-1 has multifaceted effects on the expression of spliceosomal snRNAs.

GSK3 temporally regulates neurogenin 2 proneural activity in the neocortex.

The neocortex is comprised of six neuronal layers that are generated in a defined temporal sequence. While extrinsic and intrinsic cues are known to regulate the sequential production of neocortical neurons, how these factors interact and function in a coordinated manner is poorly understood. The proneural gene Neurog2 is expressed in progenitors throughout corticogenesis, but is only required to specify early-born, deep-layer neuronal identities. Here, we examined how neuronal differentiation in general and Neurog2 function in particular are temporally controlled during murine neocortical development. We found that Neurog2 proneural activity declines in late corticogenesis, correlating with its phosphorylation by GSK3 kinase. Accordingly, GSK3 activity, which is negatively regulated by canonical Wnt signaling, increases over developmental time, while Wnt signaling correspondingly decreases. When ectopically activated, GSK3 inhibits Neurog2-mediated transcription in cultured cells and Neurog2 proneural activities in vivo. Conversely, a reduction in GSK3 activity promotes the precocious differentiation of later stage cortical progenitors without influencing laminar fate specification. Mechanistically, we show that GSK3 suppresses Neurog2 activity by influencing its choice of dimerization partner, promoting heterodimeric interactions with E47 (Tcfe2a), as opposed to Neurog2-Neurog2 homodimer formation, which occurs when GSK3 activity levels are low. At the functional level, Neurog2-E47 heterodimers have a reduced ability to transactivate neuronal differentiation genes compared with Neurog2-Neurog2 homodimers, both in vitro and in vivo. We thus conclude that the temporal regulation of Neurog2-E47 heterodimerization by GSK3 is a central component of the neuronal differentiation "clock" that coordinates the timing and tempo of neocortical neurogenesis in mouse.

The expression of squalene epoxidase in human gastric cancer and its clinical significance.

Context: Squalene epoxidase (SQLE) is overexpressed in a variety of tumors, which may play an important role in their tumorigenesis, development, and prognosis. Aims: The aim of this study is to investigate the expression of SQLE and explore its clinicopathological significance in gastric cancer. Settings and Design: The correlation between its positive expression and the pathological characteristics of patients (such as sex, age, tumor size, survival, tumor differentiation, TNM staging, and lymph node metastasis) was analyzed. Materials and Methods: Immunohistochemical method was used to detect its expression in 107 cases of gastric carcinoma and 34 cases of tumor-adjacent tissues. Statistical Analysis Used: Counting data were analyzed by Chi-square test. Its overall survival was analyzed by Kaplan-Meier method and log-rank test. Its hazard factors were analyzed by Cox multivariate analysis. Results: The positive rate of SQLE in gastric cancer is 67.3%, which is higher than that in tumor-adjacent tissues (17.6%), <0.001. Expression of SQLE is closely related to tumor differentiation, TNM staging and lymph node metastasis (P = 0.030, P = 0.009, and P = 0.011, respectively). Furthermore, compared with those low expression of SQLE, the patients of overexpression had worse overall survival by Kaplan-Meier analysis (P = 0.025). Cox multivariate analysis shows that lymph node metastasis, tumor differentiation, SQLE, and TNM staging are independent factors for prognosis of gastric cancer (P = 0.003, 0.020, 0.018, and P = 0.001 respectively). Conclusions: SQLE is overexpressed in gastric cancer. It could be used for the diagnosis and prognosis of the gastric cancer patients.

[Clinical Characteristics and Prognosis of Patients with Hematological Malignancies Superimposed with Solid Tumors].

OBJECTIVE: To investigate the clinical characteristics and prognosis of hematological malignancies superimposed patients with solid tumors. METHODS: The clinical data of 30 patients with more than two kinds of malignancy (the second is hematological malignancy) from October 2011 to October 2020 in Department of Hematology, Jiangning Hospital Affiliated to Nanjing Medical University were collected and analyzed retrospectively. The overall survival time was used as the prognostic evaluation standard, and the survival of patients were analyzed by KaplanMeier method. Logrank test and Cox regression model were used to carry out univariate and multivariate retrospective analysis on clinical and laboratory parameters of 30 patients. RESULTS: Among 30 cases, 20 were male, 10 were female, the median age of onset of the second tumor was 70 years old. The common types of the secondary hematological malignancies to solid tumors are myelodysplastic syndrome, acute myeloid leukemia, multiple myeloma. Univariate analysis showed that patients' gender, age, type of solid tumors, the onset of interval between two kinds of tumor, chromosome karyotype were not related to do with the patients' overall survival time. Type of hematologic disease, ECOG score were associated with patients' overall survival time, and the multivariate analysis showed that the type of hematologic disease and ECOG score were independent risk factors for patients with poor prognosis. CONCLUSION: Patients superimposed with solid tumors complicated with myelodysplastic syndrome or acute leukemia and ECOG score ≥3 have poor prognosis and shorter overall survival time, which are independent risk factors influencing the prognosis. Bone marrow injury, immune dysfunction and genetic susceptibility after chemoradiotherapy may be the main causes of these diseases.

Allicin enhances the radiosensitivity of colorectal cancer cells via inhibition of NF-κB signaling pathway.

Radioresistance is an important factor affecting the radiotherapy effect of colorectal cancer (CRC). Allicin is a versatile sulfur-containing organic compound extracted from garlic (Allium sativum L.), which has many pharmacological effects. However, the effect of allicin on the sensitivity of CRC radiotherapy has not been confirmed. The present study is to observe the radiosensitivity effects of allicin and to explore its mechanism in CRC radiotherapy. The proliferation inhibition effects of allicin combined with X-ray radiotherapy in HCT116 cells were measured by growth curve of cell and colony formation assays. The cell apoptosis was detected by Hoechst 33258 nucleus staining assay. The migration ability of cells was detected by Transwell chamber migration assay. The animal model of CRC was established in BALB/c mice via transplantation of CT26 cell, and the radiosensitization effect of allicin on CRC was detected in vivo. The mRNA expressions of NF-κB, IKKß, and IκBα were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The protein expressions of NF-κB, p-NF-κB, IKKß, p-IKKß, IκBα, and p-IκBα were detected by western blotting. Our results showed that allicin improves the sensitivity of X-ray radiotherapy in CRC, and its mechanism may be associated with inhibition of NF-κB signaling pathway. These findings suggest that allicin may be used as a potential sensitizer for tumor radiotherapy in the clinic.

Csk, a critical link of g protein signals to actin cytoskeletal reorganization.

Heterotrimeric G proteins can signal to reorganize the actin cytoskeleton, but the mechanism is unclear. Here we report that, in tyrosine kinase Csk-deficient mouse embryonic fibroblast cells, G protein (Gbetagamma, Galpha(12), Galpha(13), and Galpha(q))-induced, and G protein-coupled receptor-induced, actin stress fiber formation was completely blocked. Reintroduction of Csk into Csk-deficent cells restored the G protein-induced actin stress fiber formation. Chemical rescue experiments with catalytic mutants of Csk demonstrated that the catalytic activity of Csk was required for this process. Furthermore, we uncovered that Gbetagamma can both translocate Csk to the plasma membrane and directly increase Csk kinase activity. Our genetic and biochemical studies demonstrate that Csk plays a critical role in mediating G protein signals to actin cytoskeletal reorganization.

Survival factor-mediated BAD phosphorylation raises the mitochondrial threshold for apoptosis.

Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, we generated mice with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. We show that BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo.

Disodium Guanylate Alleviates Acute Hepatic Injury Induced by Carbon Tetrachloride Via Antioxidative Stress and Antiapoptosis In Vivo and In Vitro.

Hepatic injury is one of the most common digestive system diseases worldwide in clinic. Guanylic acid or guanosine monophosphate (GMP) was an important component of nucleotides, which is mainly in the form of sodium salt (disodium guanylate, GMP-Na2 ). However, its effect on hepatic injury has not yet been investigated. This study is to investigate the protective effects of GMP-Na2 on acute hepatic injury induced by carbon tetrachloride (CCl4 ), and to explore its mechanism. The hepatic injury models of mice and HL-7702 cells were induced by CCl4 . The alanine transaminase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), total antioxidant capacity (T-AOC) were determined by biochemical method. Hematoxylin-eosin staining were used to determine the morphological changes on liver tissue in mice. The mRNA and protein expressions of caspase-3, Bax, and Bcl-2 were detected by RT-PCR and Western blot analysis. Our results show that GMP-Na2 treatment significantly decreased the activities of ALT and AST, and the levels of MDA as well as increased the levels of SOD, GSH-Px, and T-AOC. Importantly, GMP-Na2 effectively enhanced the antiapoptosis function by upregulating Bcl-2 expression and downregulating caspase-3 and Bax expressions in vivo and in vitro. Moreover, the histopathological changes of liver tissue were obviously improved after GMP-Na2 treatment. These findings suggest that GMP-Na2 has protective effects on hepatic injury, and its mechanisms may be associated with antioxidative stress and antiapoptosis.

Prognostic and clinicopathologic significance of AEG-1/MTDH and E-cadherin expression in human gallbladder carcinoma.

Astrocyte elevated gene-1 (AEG-1) and E-cadherin are associated with tumorigenesis and progression. The aim of this study is to investigate the expression of AEG-1 and E-cadherin in human gallbladder cancer (GBC) and explore their clinical and pathological significance. The expression of AEG-1 and E-cadherin protein were detected in 71 cases of human GBC and 22 cases of tumor-adjacent tissue by the immunohistochemical method. Our results demonstrate that the positive expression (high expression) rate of AEG-1 was 62.0% in human GBC which was higher than that in tumor-adjacent tissues (13.6%), P<0.001. The positive expression of AEG-1 protein was correlated with tumor TNM classification, histologic grade, and lymph node metastasis (P=0.037, P=0.033 and P=0.020, respectively). The positive expression rate of E-cadherin was 40.8% in GBC, which was lower than that in tumor-adjacent tissues (77.3%), P=0.003. Negative expression (Low expression) of E-Cadherin was significantly related with tumor TNM classification, histologic grade and lymphatic metastasis (P=0.028, P=0.003 and P=0.040, respectively). The expression of AEG-1 was negatively correlated with the expression of E-Cadherin (r=0.530, P<0.001). The log-rank test statistical analysis suggested that patients with positive expression of AEG-1 or negative expression of E-Cadherin protein had shorter overall survival time. Cox multivariate analysis showed that tumor TNM classification, histologic grade and lymphatic metastasis, AEG-1 and E-cadherin expression were independent factors for prognosis of GBC (P=0.013, P=0.019, P=0.001, P=0.011 and P=0.025 respectively). In conclusion, positive expression of AEG-1 and negative expression of E-Cadherin are markedly correlated with tumor TNM classification, histologic grade and lymphatic metastasis. The expression of AEG-1 was negatively correlated with the expression of E-Cadherin. Cox multivariate analysis showed that tumor TNM classification, histologic grade and lymphatic metastasis, positive expression of AEG-1 and negative expression of E-Cadherin were risk factors for prognosis of GBC. Detection of AEG-1 and E-Cadherin may be helpful to evaluate prognosis and infiltrative capability of gallbladder carcinoma.

Impaired Autophagy and Defective Mitochondrial Function: Converging Paths on the Road to Motor Neuron Degeneration.

Selective motor neuron degeneration is a hallmark of amyotrophic lateral sclerosis (ALS). Around 10% of all cases present as familial ALS (FALS), while sporadic ALS (SALS) accounts for the remaining 90%. Diverse genetic mutations leading to FALS have been identified, but the underlying causes of SALS remain largely unknown. Despite the heterogeneous and incompletely understood etiology, different types of ALS exhibit overlapping pathology and common phenotypes, including protein aggregation and mitochondrial deficiencies. Here, we review the current understanding of mechanisms leading to motor neuron degeneration in ALS as they pertain to disrupted cellular clearance pathways, ATP biogenesis, calcium buffering and mitochondrial dynamics. Through focusing on impaired autophagic and mitochondrial functions, we highlight how the convergence of diverse cellular processes and pathways contributes to common pathology in motor neuron degeneration.

Role of PHLPP1 in inflammation response: Its loss contributes to gliomas development and progression.

PH domain leucine-rich repeats protein phosphatase 1(PHLPP1) belongs to a novel family of Ser/Thr protein phosphatases: PHLPP serves as tumor suppressor in several cancers. However, little knowledge about the expression of PHLPP1 in human glioma tumor tissue and its role in inflammation response in glioma cells was known. Glioma samples were obtained from a total of 37 patients including 16 males and 21 females with surgical removal of the brain tumor. PHLPP1 protein and inflammatory cytokines were measured by Western blot analysis and immunohistochemistry while mRNA was determined by RT-PCR. The levels of inflammatory cytokines including TNF-α, IL-17, IL-1ß in U251 glioma cells were evaluated by siRNA PHLPP1 and PHLPP1 addition. The loss of PHLPP1 expression occurs at high frequency in human gliomas. The highest mean values of PHLPP1 mRNA and protein were found in non-glioma brain tissues whereas the lowest mean values were found in those in glioblastoma with an increase of TNF-α, IL-17, IL-1ß (p<0.05). PHLPP1 expression in human glioma was associated negatively with the severity of the tumor and inflammatory cytokines. siRNA PHLPP1 could increase the levels of inflammatory cytokines in U251 glioma cells while PHLPP1 addition could inhibit significantly inflammatory cytokines. We concluded that PHLPP1 played a suppression role in inflammatory response of glioma. The present study indicated that PHLPP1 could be used as a predictor for the prediction of the patients or as a therapeutic target for the treatment of human glioma.

Dual Inhibition of Topoisomerase II and Tyrosine Kinases by the Novel Bis-Fluoroquinolone Chalcone-Like Derivative HMNE3 in Human Pancreatic Cancer Cells.

Both tyrosine kinase and topoisomerase II (TopII) are important anticancer targets, and their respective inhibitors are widely used in cancer therapy. However, some combinations of anticancer drugs could exhibit mutually antagonistic actions and drug resistance, which further limit their therapeutic efficacy. Here, we report that HMNE3, a novel bis-fluoroquinolone chalcone-like derivative that targets both tyrosine kinase and TopII, induces tumor cell proliferation and growth inhibition. The viabilities of 6 different cancer cell lines treated with a range of HMNE3 doses were detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was determined using Hoechst 33258 fluorescence staining and the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The expression of activated Caspase-3 was examined by immunocytochemistry. The tyrosine kinase activity was measured with a human receptor tyrosine kinase (RTK) detection kit using a horseradish peroxidase (HRP)-conjugated phosphotyrosine (pY20) antibody as the substrate. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. The expression levels of the P53, Bax, Bcl-2, Caspase-3, -8, -9, p-cSrc, c-Src and topoisomerase II proteins were detected by western blot analysis. The proliferation of five of the six cancer cell lines was significantly inhibited by HMNE3 at 0.312 to 10 µmol/L in a time- and dose-dependent manner. Treatment of the Capan-1 and Panc-1 cells with 1.6 to 3.2 µM HMNE3 for 48 h significantly increased the percentage of apoptotic cells (P<0.05), and this effect was accompanied by a decrease in tyrosine kinase activity. HMNE3 potentially inhibited tyrosine kinase activity in vitro with an IC50 value of 0.64±0.34 µmol/L in Capan-1 cells and 3.1±0.86 µmol/L in Panc-1 cells. The activity of c-Src was significantly inhibited by HMNE3 in a dose- and time-dependent manner in different cellular contexts. Compared with the control group, HMNE3 induced increased expression of cellular apoptosis-related proteins. Consistent with cellular apoptosis data, a significant decrease in topoisomerase IIß activity was noted following treatment with HMNE3 for 24 h. Our data suggest that HMNE3 induced apoptosis in Capan-1 and Panc-1 cells by inhibiting the activity of both tyrosine kinases and topoisomerase II.