[Correlation of miR-155 Expression with Drug Sensitivity of FLT3-ITD+ Acute Myeloid Leukemia Cell Line and Its Mechanism].

OBJECTIVE: To investigate the correlation of miR-155 expression with drug sensitivity of FLT3-ITD+ acute myeloid leukemia (AML) cell line and its potential regulatory mechanism. METHODS: By knocking out miR-155 gene in FLT3-ITD+ AML cell line MV411 through CRISPR/Cas9 gene-editing technology, monoclonal cells were screened. The genotype of these monoclonal cells was validated by PCR and Sanger sequencing. The expression of mature miRNA was measured by RT-qPCR. The treatment response of doxorubicin, quizartinib and midostaurin were measured by MTT assay and IC50 of these drugs were calculated to identify the sensitivity. Transcriptome sequencing was used to analyze change of mRNA level in MV411 cells after miR-155 knockout, gene set enrichment analysis to analyze change of signaling pathway, and Western blot to verify expressions of key molecules in signaling pathway. RESULTS: Four heterozygotes with gene knockout and one heterozygote with gene insertion were obtained through PCR screening and Sanger sequencing. RT-qPCR results showed that the expression of mature miR-155 in the monoclonal cells was significantly lower than wild-type clones. MTT results showed that the sensitivity of MV411 cells to various anti FLT3-ITD+ AML drugs increased significantly after miR-155 knockout compared with wild-type clones. RNA sequencing showed that the mTOR signaling pathway and Wnt signaling pathway were inhibited after miR-155 knockout. Western blot showed that the expressions of key molecules p-mTOR, Wnt5α and ß-catenin in signaling pathway were down-regulated. CONCLUSION: Drug sensitivity of MV411 cells to doxorubicin, quizartinib and midostaurin can be enhanced significantly after miR-155 knockout, which is related to the inhibition of multiple signaling pathways including mTOR and Wnt signaling pathways.

Trpm2 deficiency in microglia attenuates neuroinflammation during epileptogenesis by upregulating autophagy via the AMPK/mTOR pathway.

Epilepsy is one of the most common neurological disorders. Neuroinflammation involving the activation of microglia and astrocytes constitutes an important and common mechanism in epileptogenesis. Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that plays pathological roles in various inflammation-related diseases. Our previous study demonstrated that Trpm2 knockout exhibits therapeutic effects on pilocarpine-induced glial activation and neuroinflammation. However, whether TRPM2 in microglia and astrocytes plays a common pathogenic role in this process and the underlying molecular mechanisms remained undetermined. Here, we demonstrate a previously unknown role for microglial TRPM2 in epileptogenesis. Trpm2 knockout in microglia attenuated kainic acid (KA)-induced glial activation, inflammatory cytokines production and hippocampal paroxysmal discharges, whereas Trpm2 knockout in astrocytes exhibited no significant effects. Furthermore, we discovered that these therapeutic effects were mediated by upregulated autophagy via the adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in microglia. Thus, our findings highlight an important deleterious role of microglial TRPM2 in temporal lobe epilepsy.

Targeting nucleolin improves sensitivity to chemotherapy in acute lymphoblastic leukemia.

PURPOSE: Most patients with acute lymphoblastic leukemia (ALL) are treated with chemotherapy as primary care. Although the treatment response is usually positive, resistance and relapse often occur via unknown mechanisms. The purpose of this study was to identify factors associated with chemotherapy resistance in ALL. Here, we present clinical and experimental evidence that overexpression of nucleolin (NCL), a multifunctional nucleolar protein, is linked to drug resistance in ALL. METHODS: NCL mRNA and protein levels were compared between cell lines and patient samples using qRT-PCR and immunoblotting. NCL mRNA levels were compared between patients of different disease stages from our clinic patients' specimens and publicly available ALL patient datasets. Cells and patient-derived xenograft mouse experiments were performed to assess the effect of NCL inhibition on ALL chemotherapy effectiveness. RESULTS: Analysis of patient specimens, and publicly available RNA-sequencing datasets revealed a strong correlation between the abundance of NCL and disease relapse or poor survival in B-ALL. Altering NCL expression results in changes in drug sensitivity in ALL cell lines. High levels of NCL upregulated components of the ATP-binding cassette transporters via activation of the ERK pathway, resulting in a decrease in drug accumulation inside the cells. Targeting NCL with AS1411, an NCL-binding oligonucleotide aptamer, significantly increased the sensitivity of ALL cell lines and cells/patient-derived ALL xenograft mice to chemotherapeutic drugs and prolonged mouse survival. CONCLUSION: Our results highlight NCL as a prognostic marker in B-ALL and a potential therapeutic target to combat chemotherapy resistance in ALL.

A Systematic Review and Meta-Analysis of the R778L Mutation in ATP7B With Wilson Disease in China.

BACKGROUND: Wilson disease (WD) is a hereditary disorder of copper metabolism, caused by mutations in the ATP7B gene. There are more than 1000 pathogenic variants identified in ATP7B. R778L is the most common ATP7B mutation in China. METHODS: To estimate whether R778L is associated with the onset age of WD and other clinical variables. Genotyping results of ATP7B gene were collected in our 22 patients with WD. We then conducted a systematic review and meta-analysis in databases, using the keywords Wilson disease and R778L mutation. RESULTS: After the screening, a total of 23 studies were included, including 3007 patients with WD. Patients with R778L mutation presented at an earlier age (standardized mean difference [SMD] = -0.18 [95% confidence interval, -0.28 to 0.08], P = 0.0004) and had lower ceruloplasmin concentration (SMD = -0.21 [95% confidence interval, -0.40 to -0.02], P = 0.03) than the patients without the R778L mutation. However, sex (odds ratio [OR] = 1.07 [95% confidence interval, 0.89 to 1.29], P = 0.32) and first presentation were not associated with R778L mutation in WD (hepatic: OR = 1.37 [95% confidence interval, 0.87 to 2.16, P = 0.17; neurological: OR = 0.79 [95% confidence interval, 0.48 to 1.30, P = 0.35; mix: OR = 1.04 [95% confidence interval, 0.42 to 2.53, P = 0.87; asymptomatic/others: OR = 1.98 [95% confidence interval, 0.49 to 7.96, P = 0.34). CONCLUSIONS: Our results indicated that the R778L mutation is associated with an earlier presentation and lower ceruloplasmin concentration in China.

Case report: Pediatric anti-gamma aminobutyric acid-B receptor encephalitis with benign prognosis.

Objective: To explore the clinical characteristics of pediatric anti-gamma-aminobutyric acid-B receptor (GABABR) encephalitis to enhance the understanding and improve the diagnostic and therapeutic strategies for this disease. Methods: We report a rare case of a female pediatric patient with anti-GABABR encephalitis who was treated at the Children's Hospital of Zhejiang University School of Medicine. Literature search was performed to explore the clinical characteristics of pediatric anti-GABABR encephalitis. Results: The patient exhibited recurrent epileptic seizure, status epilepticus, and psychiatric symptoms at the age of 11 years and 10 months. Anti-GABABR antibodies were positive in cerebrospinal fluid and serum. Brain magnetic resonance imaging (MRI) exhibited abnormal signals in the left hippocampus. Symptoms and abnormality of brain MRI were improved after administration of immunosuppressants, anti-seizure and antipsychotic drugs. Two of pediatric anti-GABABR encephalitis with clinical data were identified through literature search. Analysis of these three cases suggested that the pediatric patients primarily experienced limbic encephalitis, with no tumor incidence. A favorable immunotherapy response was demonstrated with a superior prognosis in all the cases. Conclusions: We reported a pediatric anti-GABABR encephalitis case with early age of onset. Promt autoimmune antibody testing and tumor screening, as well as immunomodulatory treatment immediately after a definitive diagnosis are warranted to improve prognosis.

Loss of MiR-155 Sensitizes FLT3-ITD+AML to Chemotherapy and FLT3 Inhibitors via Glycolysis Blocking by Targeting PIK3R1.

FLT3 tyrosine kinase inhibitors in combination with chemotherapy have shown some success in patients with FLT3 mutations. But a variety of mechanisms have led to the rapid resistance to the treatment. One of the most prominent is the metabolic alteration on aerobic glycolysis. We aim to explore the role of a high expressing microRNA, miR-155, in mediating resistance to chemotherapy and FLT3 inhibitor treatment. The deep sequencing data mining revealed the connection between glycolysis and drug resistance. MV411 cells with miR-155 knockout (KO) not only had increased sensitivity to FLT3 inhibitors but also Adriamycin (ADM) treatment. When combined with glycolysis inhibition the treatment response in MV411 cells further increased. Whereas in miR-155 KO cells, a lower glucose consumption level and lactic acid level were observed, and western blotting showed a decreased expression of key enzymes in glycolysis pathways. A negative correlation between PIK3R1 and miR-155 level can be observed in the sequencing data from FLT3-ITD+ AML patients. Moreover, luciferase reporter assay revealed that the 3'UTR of PIK3R1 mRNA can interact with the seed sequence of miR-155-5p. In conclusion, the loss of miR-155 increased treatment sensitivity to both chemotherapy and FLT3 inhibitors in FLT3-ITD+ AML cells via glycolysis blocking by targeting PIK3R1.

Optical tweezers and Raman spectroscopy for single-cell classification of drug resistance in acute lymphoblastic leukemia.

Laser Tweezers Raman Spectroscopy (LTRS) is a combination of laser tweezers and Raman spectroscopy. It is a physical tool based on the mechanical effects of the laser, which can be used to study single living cells in suspension in a fast and non-destructive way. Our work aims to establish a methodology system based on LTRS to rapidly and non-destructively detect the resistance of acute lymphoblastic leukemia (ALL) cells and to provide a new idea for the evaluation of the resistance of ALL cells. Two specific adriamycin-resistant and parental ALL cells BALL-1 and Nalm6 were included in this study. Adriamycin resistant cells can induce the spectral differences, which can be detected by LTRS initially. To ensure the accuracy of the results, we use the principal components analysis (PCA) as well as the classification and regression trees (CRT) algorithms, which show that the specificity and sensitivity of LTRS are above 90%. In addition, to further clarify the chemoresistance status of ALL cells, we used the CRT models and receiver operating characteristic (ROC) curves which are based on the band data to look for some important bands and band intensity ratios that have strong pointing significance. Our work proves that LTRS analysis combined with multivariate statistical analyses have great potential to be a novel analytical strategy at the single-cell level for rapidly evaluating the chemoresistance status of ALL cells.

[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].

OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes. METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate. CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].

OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes. METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate. CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

Research progress on transient receptor potential melastatin 2 channel in nervous system diseases.

Transient receptor potential M2 (TRPM2) ion channel is a non-selective cationic channel that can permeate calcium ions, and plays an important role in neuroinflammation, ischemic reperfusion brain injury, neurodegenerative disease, neuropathic pain, epilepsy and other neurological diseases. In ischemic reperfusion brain injury, TRPM2 mediates neuronal death by modulating the different subunits of glutamate N-methyl-D-aspartic acid receptor in response to calcium/zinc signal. In Alzheimer's disease, TRPM2 is activated by reactive oxygen species generated by ß-amyloid peptide to form a malignant positive feedback loop that induces neuronal death and is involved in the pathological process of glial cells by promoting inflammatory response and oxidative stress. In epilepsy, the TRPM2-knockout alleviates epilepsy induced neuronal degeneration by inhibiting autophagy and apoptosis related proteins. The roles of TRPM2 channel in the pathogenesis of various central nervous system diseases and its potential drug development and clinical application prospects are summarized in this review.

Identification BCL6 and miR-30 family associating with Ibrutinib resistance in activated B-cell-like diffuse large B-cell lymphoma.

Ibrutinib has clear efficacy for activated B-cell-like diffuse large B cell lymphoma (ABC-DLBCL) in previous clinical researches. However, the resistance of Ibrutinib has limited its therapeutic benefit and the potential mechanism remains unclear. This study was aimed to identify potential candidate genes and miRNA targets to overcome Ibrutinib resistance in ABC-DLBCL. First, two expression profiles were downloaded from the GEO database, which used to identify the DEGs related to Ibrutinib resistance in ABC-DLBCL cell lines by GEO2R analysis separately. And the common DEGs were obtained though Venn diagram. Then Gene ontology (GO) and pathway enrichment analysis were conducted by DAVID database. From STRING database, BCL6, IL10, IL2RB, IRF4, CD80, PRDM1and GZMB were determined to be the hub genes by protein-protein interaction (PPI) network. Through miRNA-mRNA targeting network, we found that BCL6, IRF4, CD80, and PRDM1 were common target genes of miR-30 family. The cBioPortal database showed that BCL6 had the highest level of genetic alterations among DLBCL. In addition, another expression profile from GEO database showed that BCL6 was significantly high expression in no responsive patients after Ibrutinib treatment, and the receiver operating characteristic (ROC) curve which was used to evaluate the relationship between BCL6 expression and its effect was 0.67. MTT assay showed that treatment with FX1 (a BCL6 inhibitor) can enhance the sensitivity of Ibrutinib in C481S BTK HBL-1 cells. The results suggested that BCL6 and miR-30 family maybe associate with Ibrutinib resistance in ABC-DLBCL.

Identification of Early Recurrence Factors in Childhood and Adolescent B-Cell Acute Lymphoblastic Leukemia Based on Integrated Bioinformatics Analysis.

Over the past 50 years, great progress has been made in the diagnosis and treatment of acute lymphoblastic leukemia (ALL), especially in pediatric patients. However, early recurrence is still an important threat to the survival of patients. In this study, we used integrated bioinformatics analysis to look for biomarkers of early recurrence of B-cell ALL (B-ALL) in childhood and adolescent patients. Firstly, we obtained gene expression profiles from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database and the Gene Expression Omnibus (GEO) database. Then, we identified differentially expressed genes (DEGs) based on whether the disease relapsed early. LASSO and Cox regression analysis were applied to identify a subset of four genes: HOXA7, S100A11, S100A10, and IFI44L. A genetic risk score model was constructed based on these four optimal prognostic genes. Time-dependent receiver operating characteristic (ROC) curves were used to evaluate the predictive value of this prognostic model (3-, 5-, and 10-year AUC values >0.7). The risk model was significantly associated with overall survival (OS) and event-free survival in B-ALL (all p < 0.0001). In addition, a high risk score was an independent poor prognostic risk factor for OS (p < 0.001; HR = 3.396; 95% CI: 2.387-4.832). Finally, the genetic risk model was successfully tested in B-ALL using an external validation set. The results suggested that this model could be a novel predictive tool for early recurrence and prognosis of B-ALL.

Iron overload as a risk factor for poor graft function following allogeneic hematopoietic stem cell transplantation.

Hematological malignancies are increasingly treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, iron overload is a frequent adverse effect of allo-HSCT and is associated with poor prognosis. In the present study, we investigated hematopoiesis in iron-overloaded mice and elucidated the effects of iron overload on the bone marrow (BM) microenvironment. Iron-overloaded BALB/C mice were generated by injecting 20 mg/mL saccharated iron oxide intraperitoneally. Hematoxylin-eosin staining was performed to evaluate the effects of an iron overload in mice. BM cells obtained from C57BL/6 mice were transplanted into irradiated BALB/C mice (whole-body irradiation of 4 Gy, twice with a 4-hours interval) by tail vein injection. Two weeks after allo-HSCT, the hematopoietic reconstitution capacity was evaluated in recipients by colony-forming assays. Histopathological examinations showed brown-stained granular deposits, irregularly arranged lymphocytes in the liver tissues, and blue-stained blocks in the BM collected from mice received injections of high-dose saccharated iron oxide (20 mg/mL). Iron-overloaded mice showed more platelets, higher-hemoglobin (HGB) concentration, fewer granulocyte-macrophage colony-forming units (CFU-GM), erythrocyte colony-forming units (CFU-E), and mixed granulocyte/erythrocyte/monocyte/megakaryocyte colony-forming units (CFU-mix) than healthy mice. Iron-overloaded recipients presented with reduced erythrocytes and HGB concentration in peripheral blood, along with decreased marrow stroma cells, CFU-GM, CFU-E, and CFU-mix relative to healthy recipients. Taken together, our findings demonstrate that iron overload might alter the number of red blood cells after transplantation in mice by destroying the BM microenvironment, thereby affecting the recovery of BM hematopoietic function.

Integrated bioinformatics analysis of the crucial candidate genes and pathways associated with glucocorticoid resistance in acute lymphoblastic leukemia.

Glucocorticoids (GC) are the foundation of the chemotherapy regimen in acute lymphoblastic leukemia (ALL). However, resistance to GC is observed more frequently than resistance to other chemotherapy agents in patients with ALL relapse. Moreover, the mechanism underlying the development of GC resistance in ALL has not yet been fully uncovered. In this study, we used bioinformatic analysis methods to integrate the candidate genes and pathways participating in GC resistance in ALL and subsequently verified the bioinformatics findings with in vitro cell experiments. Ninety-nine significant common differentially expressed genes (DEGs) associated with GC resistance were determined by integrating two gene profile datasets, including GC-sensitive and -resistant samples. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) and REACTOME pathways analysis, the signaling pathways in which DEGs were significantly enriched were clustered. The GC resistance-related biologically functional interactions were visualized as DEG-associated Protein-Protein Interaction (PPI) network complexes, with 98 nodes and 127 edges. MYC, a node which displayed the highest connectivity in all edges, was highlighted as the core gene in the PPI network. Increased C-MYC expression was observed in adriamycin-resistant BALL-1/ADR cells, which we demonstrated was also resistant to dexamethasone. These results outlined a panorama in which the solitary and scattered experimental results were integrated and expanded. The potential promising target of the candidate pathways and genes involved in GC resistance of ALL was concomitantly revealed.

ß-Catenin Controls the Electrophysiologic Properties of Skeletal Muscle Cells by Regulating the α2 Isoform of Na+/K+-ATPase.

ß-Catenin is a key component of the canonical Wnt signaling pathway. It has been shown to have an important role in formation of the neuromuscular junction. Our previous studies showed that in the absence of ß-catenin, the resting membrane potential (RMP) is depolarized in muscle cells and expression of the α2 subunit of sodium/potassium adenosine triphosphatase (α2NKA) is reduced. To understand the underlying mechanisms, we investigated the electrophysiologic properties of a primary cell line derived from mouse myoblasts (C2C12 cells) that were transfected with small-interfering RNAs and over-expressed plasmids targeting ß-catenin. We found that the RMP was depolarized in ß-catenin knocked-down C2C12 cells and was unchanged in ß-catenin over-expressed muscle cells. An action potential (AP) was not released by knockdown or over-expression of ß-catenin. α2NKA expression was reduced by ß-catenin knockdown, and increased by ß-catenin over-expression. We showed that ß-catenin could interact physically with α2NKA (but not with α1NKA) in muscle cells. NKA activity and α2NKA content in the cell membranes of skeletal muscle cells were modulated positively by ß-catenin. These results suggested that ß-catenin (at least in part) regulates the RMP and AP in muscle cells, and does so by regulating α2NKA.

TRPM2 channel regulates cytokines production in astrocytes and aggravates brain disorder during lipopolysaccharide-induced endotoxin sepsis.

Sepsis is one of the most significant challenges in intensive care units, which is associated with increased morbidity and mortality. Sepsis-associated encephalopathy (SAE) is a severe complication which can cause death and serious disabilities. Calcium signaling in astrocyte is essential for cellular activation and the potential resolution of infection or inflammation in SAE patients. The transient receptor potential melastatin 2 (TRPM2) channel has been identified as a unique fusion of a Ca2+-permeable nonselective cation channel, which plays an important role in inflammation and immune response. Because of its role as an oxidative stress sensor in astrocytes, we investigated the function of TRPM2 in inflammation mediators (interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α) release, Bcl-2/E1B-19 K-interacting protein 3 (BNIP3), apoptosis inducing factor (AIF) and Endonuclease G (Endo G) expression. We showed that TRPM2-KO mice, when intraperitoneally (i.p) injected with LPS, exhibited better neurologic assessment scores and decreased inflammatory injury in hippocampal neurons compared with wild-type (WT) mice. The absence of TRPM2 triggered less production of inflammatory mediators (IL-1ß, IL-6, TNF-α) and decreased apoptosis related proteins (BNIP3, AIF, Endo G) expressions in response to LPS induced sepsis. Furthermore, TRPM2-deficient astrocytes (transfected with TRPM2 siRNA) upon LPS stimulation also induced decreased IL-1ß, IL-6 and TNF-α level. Our data suggested that decreased production of inflammatory cytokines and apoptosis related proteins with TRPM2 deletion could regulate inflammatory stress and decrease inflammatory injury in hippocampal neurons, and consequently, ameliorate brain disorder.

[Inhibitory Effect of NPM Gene Knockdown on Proliferation of Chronic Myeloid Leukemia Cell Line K562 and Its Mechanism].

OBJECTIVE: To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology. METHODS: shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins. RESULTS: The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G0/G1 phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex. CONCLUSION: Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.

Low concentrations of chloroquine and 3-methyladenine suppress the viability of retinoblastoma cells synergistically with vincristine independent of autophagy inhibition.

BACKGROUND: To study the inhibition of retinoblastoma cell viability by two commonly used autophagy inhibitors, chloroquine (CQ) and 3-methyladenine (3-MA), alone or in combination with the conventional chemotherapeutic drug vincristine (VCR), and to investigate whether the mechanisms of these drugs are related to inhibition of autophagy. METHODS: On retinoblastoma cell line HXO-Rb44, VCR, CQ and 3-MA were used individually or combined. The cell viability was determined by CCK8 method, and the cellular autophagic activity was determined by Western blotting of LC3 and p62. Caspase 3 fragmentation and Akt activation was also determined by Western blotting. RESULTS: VCR induced cell cycle arrest and apoptosis in HXO-Rb44 cells, but only inhibited autophagy at relatively high doses. Both CQ and 3-MA were synergistic with VCR to inhibit the growth of retinoblastoma cells and the combinational use significantly reduced the dosage of each drug. The lowest effective dose of CQ and 3-MA was most efficient to add on VCR; however, such dose was not sufficient to suppress autophagy in these cells. CQ could directly induce caspase activation, while 3-MA significantly inhibited Akt phosphorylation. CONCLUSIONS: CQ and 3-MA were synergistic with VCR to inhibit retinoblastoma cells. Our result suggested a novel strategy to combine CQ or 3-MA with VCR to reduce the side effects of each drug. However, lack of change in the autophagic activity when using the two drugs at lower doses suggests multiple mechanisms of action of the same drug at different doses. At higher doses, the drugs could inhibit autophagy, while at lower doses, they suppress tumor growth via autophagy-independent mechanisms.

The role of an Ommaya reservoir in the management of children with cryptococcal meningitis.

Cryptococcal meningitis is the most common life-threatening fungal infection and is associated with high mortality in children. Amphotericin B plus flucytosine and fluconazole is the optimal current therapy. Implantation of an Ommaya reservoir for intraventricular infusion of medication and aspiration of cerebrospinal fluid (CSF) for the treatment of increased intracranial pressure (ICP) has been reported. Intraventricular injection of amphotericin B through an Ommaya reservoir in children with cryptococcal meningitis has not been reported previously. We report two children who had cryptococcal meningitis and associated increased intracranial pressure, and were treated with an Ommaya reservoir. Both patients experienced rapid reversal of symptoms. At the time of discharge both patients had recovered and have remained asymptomatic.