GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules.

Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. Here we show that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. We propose a model in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs.

Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules.

Neurons in the central nervous system (CNS) fail to regenerate axons after injuries due to the diminished intrinsic axon growth capacity of mature neurons and the hostile extrinsic environment composed of a milieu of inhibitory factors. Recent studies revealed that targeting a particular group of extracellular inhibitory factors is insufficient to trigger long-distance axon regeneration. Instead of antagonizing the growing list of impediments, tackling a common target that mediates axon growth inhibition offers an alternative strategy to promote axon regeneration. Neuronal growth cone, the machinery that derives axon extension, is the final converging target of most, if not all, growth impediments in the CNS. In this study, we aim to promote axon growth by directly targeting the growth cone. Here we report that pharmacological inhibition or genetic silencing of nonmuscle myosin II (NMII) markedly accelerates axon growth over permissive and nonpermissive substrates, including major CNS inhibitors such as chondroitin sulfate proteoglycans and myelin-associated inhibitors. We find that NMII inhibition leads to the reorganization of both actin and microtubules (MTs) in the growth cone, resulting in MT reorganization that allows rapid axon extension over inhibitory substrates. In addition to enhancing axon extension, we show that local blockade of NMII activity in axons is sufficient to trigger axons to grow across the permissive-inhibitory border. Together, our study proposes NMII and growth cone cytoskeletal components as effective targets for promoting axon regeneration.

Impact of Acupuncture on Sleep and Comorbid Symptoms for Chronic Insomnia: A Randomized Clinical Trial.

STUDY OBJECTIVES: To evaluate the efficacy and safety of acupuncture at HT 7 (Shenmen) and KI 7 (Fuliu) on sleep and comorbid symptoms for chronic insomnia. METHODS AND DESIGN: A randomized, single-blind, parallel and sham-controlled trial consisted of an acupuncture group (n = 41) and a sham acupuncture group (n = 41). Setting: a tertiary hospital of integrated Chinese and Western medicine. Participants: 82 subjects with chronic insomnia based on the International Classification of Sleep Disorders, Third Edition (ICSD-3). Interventions: a 10-session acupuncture treatment at bilateral HT 7 and KI 7 or sham acupoints with shallow needling was performed over 3 weeks. Measurements: the Pittsburgh sleep quality index (PSQI) and insomnia severity index (ISI) were evaluated at baseline, posttreatment, and at two follow-ups as the primary outcome measures. Polysomnography (PSG) on two consecutive nights, the Beck anxiety inventory (BAI), the Beck depression inventory (BDI) fatigue severity scale (FSS) and the Epworth sleepiness scale (ESS) were evaluated at baseline and posttreatment as the secondary outcome measures. RESULTS: After the treatments, PSQI scores decreased by 5.04 in the acupuncture group and 2.92 in the sham acupuncture group. ISI scores decreased by 7.65 in the acupuncture group and 5.05 in the sham acupuncture group. The between-group differences in the primary outcome measures posttreatment were statistically significant. However, no differences were found between the two groups during the two follow-ups. Regarding the PSG data, there were significantly lower levels of sleep onset latency (SOL), a lower percentage of sleep stage N1 and a higher percentage of sleep stage N3 in the acupuncture group than in the sham acupuncture group. After treatment, there were lower levels of comorbid symptoms (BAI, BDI, FSS and ESS) in both groups. However, no significant differences were observed between the groups. CONCLUSION: Acupuncture at HT 7 and KI 7 is an effective and safe nonpharmacologic intervention option for chronic insomnia. CLINICAL TRIAL REGISTRATION: The study was registered at the Chinese Clinical Trial Registry, registration ID: ChiCTR1900023787, China.

[CD44 variant increases the invasive ability of human breast cancer cell line MCF-7 cells].

OBJECTIVE: To evaluate the impact of a new CD44 variant on invasion of human breast cancer cell line MCF-7, and its possible mechanisms. METHODS: The full length cDNA encoding CD44v17 was obtained from the total RNA isolated from the MCF-7/ADR cells by reverse transcript-polymerase chain reaction (RT-PCR) and subcloned into pMD19-T vector. The CD44v17 gene sequence and reading frame were confirmed by two restriction enzymes and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44v17 was transfected into MCF-7 cells by Lipofectamine. The changes of MMP-2 and MMP-9 expression at gene and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells through the artificial matrix membrane in every group was counted to compare the change of the invasive ability regulated by CD44 variant. The ERK and p-ERK were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by CD44 variant. RESULTS: The new gene sequence was successfully cloned into recombinant vector pcDNA3.1 and identified by the two restriction enzymes. It was confirmed that the reconstructed plasmid contained the sequence of CD44 gene variant which was composed of 1 to 4 exons, 16 to 17 exons, and 1 to 205 bases of 18 exons. The new gene sequence was sent to NCBI for publication and obtained the registered number FJ216964. The up-regulated levels of the CD44 gene mRNA and protein were respectively detected by RT-PCR and flow cytometry in MCF-7 cells transfected with pcDNA3.1-CD44v17. The invasiveness of the cells and the activity of MMP-2 and MMP-9 were clearly activated by hyaluronic acid (HA) treatment and blocked by CD44 neutralizing antibody. Pretreated MCF-7/CD44v17 cells with the neutralizing antibody against CD44 and the inhibitor of MAPKs signaling pathway strongly block the expression of p-ERK. CONCLUSION: A new CD44 gene variant has been found in adriamycin-resistant human breast cancer MCF-7/ADR cells. The expression vector pcDNA3.1-CD44v17 has been cloned and constructed successfully. HA can be integrated with CD44 variant and then regulates the expression of MMP-2 and MMP-9, which increases the invasion ability of MCF-7 cells through the Ras/MAPK signaling pathway.

Effects of imatinib and 5-bromotetrandrine on the reversal of multidrug resistance of the K562/A02 cell line.

BACKGROUND AND OBJECTIVE: Research has shown that 5-bromotetrandrine (BrTet) can effectively reverse multidrug resistance (MDR). Imatinib plays an important role in cell proliferation. This study explored the efficacy of the combination of imatinib and BrTet on reversing MDR of tumor cells and its mechanism. METHODS: Cytoxicity was assessed by MTT assay. Apoptosis of K562/A02 cells was analyzed by flow cytometry. The expressions of mdr1 mRNA and P-glycoprotein (P-gp) were detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. RESULTS: After 48 h of treatment with 0.0625 micromol/L imatinib, 0.5 micromol/L BrTet, or both, the 50% inhibition concentration (IC50) of daunorubicin (DNR) for the K562/A02 cells were 5.69 mg/L, 5.41 mg/L, and 2.19 mg/L, respectively. The gray-scale values of mdr1 mRNA expression in the K562/A02 cells were 0.65+/-0.02, 0.64+/-0.01, and 0.25+/-0.03, respectively. The expression levels of P-gp were 0.74+/-0.02, 0.52+/-0.02, and 0.29+/-0.02, respectively. All decreased significantly in the K562/A02 cells treated with both imatinib and BrTet compared to cells treated with imatinib and BrTet alone (P<0.05). The apoptosis rates of the K562/A02 cells increased without a significant difference after treatment with DNR, imatinib, or BrTet (P>0.05), while increased significantly after treatment with DNR combined with imatinib, BrTet, or both (P<0.05). CONCLUSIONS: The MDR of K562/A02 cells may be partially reversed by imatinib or BrTet, and the mechanism may be related to the downregulation of mdr1 mRNA and P-gp expression and the upregulation of the rate of apoptosis in K562/A02 cells. Imatinib combined with BrTet showed a synergistic effect on K562/A02 cells.

Acupuncture for insomnia with short sleep duration: protocol for a randomised controlled trial.

INTRODUCTION: Insomnia with short sleep duration has a more serious negative impact on patient health. The existing literature suggests that medication therapy is more effective for this phenotype of insomnia compared with cognitive-behavioural therapy. However, the potential side effects of hypnotic medications hinder their clinical application. Acupuncture has been widely used in the treatment of insomnia, but it remains unclear whether it has therapeutic efficacy for insomnia with short sleep duration. The purpose of this trial is to evaluate the efficacy and safety of acupuncture for insomnia with short sleep duration. METHODS AND ANALYSIS: This study is designed as a randomised, single-centre, single-blinded, placebo acupuncture controlled trial involving 152 participants. Eligible patients will be divided into two groups according to the objective total sleep time: insomnia with normal sleep duration group and insomnia with short sleep duration group. Then, patients in each group will be randomly assigned to two subgroups, the treatment group (acupuncture) and the control group (placebo acupuncture), in a 1:1 ratio with 38 subjects in each subgroup. The primary outcome is the Pittsburgh Sleep Quality Index and the Insomnia Severity Index. Secondary outcomes are actigraphy, the Beck Anxiety Inventory, the Beck Depression Inventory and the Fatigue Severity Scale. All adverse effects will be assessed by the Treatment Emergent Symptom Scale. Outcomes will be evaluated at baseline, post treatment, as well as at 1-week and 1-month follow-up. ETHICS AND DISSEMINATION: This protocol has been approved by the ethics committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine (no. 2019-17). Written informed consent will be obtained from all participants. The results will be disseminated through peer-reviewed journals for publications. TRIAL REGISTRATION NUMBER: ChiCTR1900023473; Pre-results.

Electro-catalytic degradation of phenol on several metal-oxide anodes.

Three kinds of Ti-based multilayer metal-oxide electrode, including Ti/SnO(2)+Sb(2)O(3)/PbO(2), Ti/SnO(2)+Sb(2)O(3)/MnO(x) and Ti/SnO(2)+Sb(2)O(3)/RuO(2)+PbO(2) electrodes, were prepared by thermal decomposition, and SnO(2)+Sb(2)O(3) coatings were produced with a polymeric precursor method (PPM). The conversion of phenol was carried out with these electrodes as anodes under galvanostatic control. Samples during the electrolyses were characterized with UV-vis spectra and chromatography, and chemical oxygen demand (COD) and instantaneous current efficiency (ICE) for phenol degradation were also determined. The results show that phenol can be oxidized and degraded for all of the three anodes, and the oxidation reactions of phenol follow first-order kinetics, but there are considerable differences in the effectiveness and performance of electro-catalytic degradation. Phenol can be degraded relatively fast on the Ti/SnO(2)+Sb(2)O(3)/PbO(2) anode and the degradation rate of phenol is slower with the Ti/SnO(2)+Sb(2)O(3)/MnO(x) electrode, and the slowest with the Ti/SnO(2)+Sb(2)O(3)/RuO(2)+PbO(2) electrode, whose apparent rate constants are 2.49 x 10(-2), 1.42 x 10(-2) and 9.76 x 10(-3) min(-1), respectively. The rates of electro-catalytic degradation relate to oxygen evolution potential, and the higher the oxygen evolution potential, the better the performance of electro-catalytic degradation. The potential for oxygen evolution at the Ti/SnO(2)+Sb(2)O(3)/PbO(2) anode is highest, then Ti/SnO(2)+Sb(2)O(3)/MnO(x), following Ti/SnO(2)+Sb(2)O(3)/RuO(2)+PbO(2). The accelerated life tests at 60 degrees C and in 1.0 mol L(-1) aqueous H(2)SO(4) with an anodic current density of 4.0 Acm(-2) show that the service life is prolonged when the SnO(2)+Sb(2)O(3) interlayer coating are inserted between Ti substrate and active layers.

[Effect of YB-1 gene knockdown on human leukemia cell line K562/A02].

OBJECTIVE: To investigate the potential effects of YB-1 gene knockdown on gene expression profile, cell growth and apoptosis in leukemia cell line K562/A02. METHODS: The recombinant eukaryotic expression plasmid containing YB-1 short hairpin RNA (shRNA) or random-sequence (HK) were transfected into K562/A02 cells by lipofectamine mediation. cDNA microarray was performed to explore the alteration of gene expression profile when YB-1 gene expression was decreased. Expression of CARD8 and RHOC genes were verified by semi-quantitative reverse transcription-PCR (RT-PCR). The proliferative ability of the cells was determined by methyl thiazolyltetrazolium (MTT) assay and cell cycle analysis. Cell apoptosis was assayed by Annexin V-FITC/PI double labeled flow cytometry. RESULTS: The levels of YB-1 mRNA and protein decreased dramatically in three positively transfected cells when compared with untransfected K562/A02 cells or K562/A02-HK thansfected cells. Gene expression profile was altered by transfection of YB-1 shRNA into K562/A02 cells. Among 47,000 genes on the microarray, 252 genes were detected to have changes, with 143 down-regulated and 109 up-regulated. They were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, etc. An increase in CARD8 gene expression and a decrease in RHOC gene expression have been confirmed by RT-PCR in K562/A02-YBX13 cells. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased proliferation, higher G1, lower G2 and S ratio in cell cycle distribution in comparison with the control groups. Annexin V/PI detection indicated higher Annexin V+ ratio in the three positively transfected cells 24 hours after cells were treated with 0.5 micromol/L of As2O3. CONCLUSION: Down-regulation of YB-1 gene by shRNA-YB-1 can alter the gene expression profile in K562/A02 cells, leading to change of cell proliferation and apoptosis.

[Determination of the conversion of styrene with UV spectroscopy in microemulsion polymerization induced by ultrasound].

The determination of the conversion of styrene with UV spectroscopy was studied in microemulsion polymerization induced by ultrasound. The effects of SDS, pentrol and polystyrene on the styrene spectra were discussed. The experimental results show that the concentration of styrene in microemulsion polymerization system can be determined quantitatively at the wavelength of 247nm by UV spectroscopy, and the relationship between the styrene concentration and the absorbence is linear in the range of 9.7 x 10(-6) mol x L(-1)-6.95 x 10(-5) mol x L(-1), meanwhile the molar absorptivity is 1.384 x 10(4) L x mol(-1) x cm(-1). The SDS and pentrol presented in microemulsion polymerization system have no effects on the determination of the conversion of styrene. Polystyrene, a resultant in the process of microemulsion polymerization, could be removed by adding 95% alcohol to the microemulsion polymerization system and making polystyrene to be precipitated. Residual polystyrene does not affect the determination of the concentration of styrene. Comparing the results of UV spectroscopy with those of the chemical analysis and gravimetric analysis, the determination of the conversion of styrene by UV spectroscopy is feasible, and the process is simple and easy.

Combination of tetrandrine as a potential-reversing agent with daunorubicin, etoposide and cytarabine for the treatment of refractory and relapsed acute myelogenous leukemia.

The potential mechanism of the chemotherapy resistance in acute myeloid leukemia (AML) is the multidrug resistance (MDR-1) gene product P-glycoprotein (P-gp), which is often overexpressed in myeloblasts from acute myeloid leukemia. In a multicenter clinical trial, 38 patients with poor risk forms of AML were treated with tetrandrine (TET), a potent inhibitor of the MDR-1 efflux pump, combined with daunorubicin (DNR), etoposide and cytarabine (TET-DEC). Overall, post-chemotherapy marrow hypoplasia was achieved in 36 patients. Sixteen patients (42%) achieved complete remission or restored chronic phase, 9 achieved partial remission (PR) and 13 failed therapy. Toxicities included infection, myelosuppression, stomatitis, mucositis, cerebellar toxicity and reversible cardiotoxicity. There was no significant difference in response for P-gp-positive and -negative patients. P-gp function was assessed in 26 patients by flow cytometric analysis, TET-contained plasma-augmented DNR accumulation relative to pretreatment plasma in K562/A02 cells by a median value of 88+/-101% (range, 11-501%). However, there was no difference in DNR uptake between responding and non-responding patients. Our data showed that TET-DEC was relatively well tolerated in these patients with poor risk AML, and had encouraging antileukemic effects.

[The down regulation of VEGF gene expression may link to change the expression profile of genes in leukemia cell line K562].

OBJECTIVE: To explore the potential effects of anti-VEGF hairpin ribozyme gene to gene expression profiles in leukemia cell line K562. METHODS: The lipofectamine mediation was used to transfect the recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the non-recombinant vector as control into K562 cells. And the positive clones were screened by G418. Ribozyme gene in K562 cells was confirmed by PCR. Fluorescent real time reverse transcription-PCR(RT-PCR) and Western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. cDNA microarray was used to explore the alteration of gene expression profiles when decreasing VEGF gene expression in leukemia cells. Expression of PCNA and GSN genes were verified by semi-quantitative RT-PCR. RESULTS: The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418. Stable expression of the ribozyme gene in K562 cells was confirmed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC (K562 cell transfected with empty vector) cells. The gene expression profiles were changed by transfection of anti-VEGF hairpin ribozyme gene into K562 cells. Among 4096 gene clones on the microarray, 191 (4.86%) genes were detected to have the marked changes with 104 down-regulated and 87 up-regulated, that were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, and oncogenes etc. An increased expression of GSN gene and a decreased expression of PCNA gene in K562/RZ cells have been detected by RT-PCR. CONCLUSION: Down-regulation of VEGF gene by introducing anti-VEGF hairpin ribozyme gene can alter the gene expression profiles in K562 cells, leading to change of cell growth, differentiation and apoptosis in K562/RZ cells.

[Identification of interaction and interaction domains between neuroglobin and Na(+), K(+)-ATPase beta2 subunit].

The pre-transformed human fetal brain cDNA library was used to screen the protein interacting with neuroglobin by using yeast two hybrid system III from ClonTech Inc. The protein encoded by one of the clones interacting with neuroglobin (NGB) was confirmed to be the C terminus of the Na(+), K(+)-ATPase beta2 subunit (NKA1b2) based on amino acid sequences. Then the full-length coding region cDNA sequence of NKA1b2 was obtained from human fetal brain cDNA library by PCR. A set of experiments were designed to test the interaction between NGB and NKA1b2. Interaction between NGB and NKA1b2 was confirmed by binding assay in vitro. Furthermore, the interaction was also proved by co-immunoprecipitation test in vivo. Moreover, the structure integrity of neuroglobin was found to be essential for the interaction between NGB and NKA1b2 by yeast two hybrid method with a series of neuroglobin truncated mutants.

Relationship between p53 status and response to chemotherapy in patients with gastric cancer: a meta-analysis.

BACKGROUND: Previous studies have yielded conflicting results regarding the relationship between p53 status and response to chemotherapy in patients with gastric cancer. We therefore performed a meta-analysis to expound the relationship between p53 status and response to chemotherapy. METHODS/FINDINGS: Thirteen previously published eligible studies, including 564 cases, were identified and included in this meta-analysis. p53 positive status (high expression of p53 protein and/or a mutant p53 gene) was associated with improved response in gastric cancer patients who received chemotherapy (good response: risk ratio [RR] = 0.704; 95% confidence intervals [CI] = 0.550-0.903; P = 0.006). In further stratified analyses, association with a good response remained in the East Asian population (RR = 0.657; 95% CI = 0.488-0.884; P = 0.005), while in the European subgroup, patients with p53 positive status tended to have a good response to chemotherapy, although this did not reach statistical significance (RR = 0.828, 95% CI = 0.525-1.305; P = 0.417). As five studies used neoadjuvant chemotherapy (NCT) and one used neoadjuvant chemoradiotherapy (NCRT), we also analyzed these data, and found that p53 positive status was associated with a good response in gastric cancer patients who received chemotherapy-based neoadjuvant treatment (RR = 0.675, 95% CI = 0.463-0.985; P = 0.042). CONCLUSION: This meta-analysis indicated that p53 status may be a useful predictive biomarker for response to chemotherapy in gastric cancer. Further prospective studies with larger sample sizes and better study designs are required to confirm our findings.

PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1.

In contrast to neurons in the central nervous system, mature neurons in the mammalian peripheral nervous system (PNS) can regenerate axons after injury, in part, by enhancing intrinsic growth competence. However, the signalling pathways that enhance the growth potential and induce spontaneous axon regeneration remain poorly understood. Here we reveal that phosphatidylinositol 3-kinase (PI3K) signalling is activated in response to peripheral axotomy and that PI3K pathway is required for sensory axon regeneration. Moreover, we show that glycogen synthase kinase 3 (GSK3), rather than mammalian target of rapamycin, mediates PI3K-dependent augmentation of the growth potential in the PNS. Furthermore, we show that PI3K-GSK3 signal is conveyed by the induction of a transcription factor Smad1 and that acute depletion of Smad1 in adult mice prevents axon regeneration in vivo. Together, these results suggest PI3K-GSK3-Smad1 signalling as a central module for promoting sensory axon regeneration in the mammalian nervous system.

[Effect of triptolide on sensitivity of K562/A02 cell line to adriamycin].

This study was aimed to evaluate the effect of triptolide (TPL) on the reversal of multidrug resistance in K562/A02 cell line. The sensitivity of K562 and K562/A02 to adriamycin (ADM) and reversal of drug resistance were determined with MTT method. The concentration of intracellular ADM and P-glycoprotein expression were detected by flow cytometry. Luciferase reporter gene assay was used to detect the transcriptional activity of MDR1 promoter. The results showed that TPL significantly decreased the resistance degree of K562/A02 cells, inhibited P-glycoprotein expression (mean fluorescent intensity decreased from 123 ± 13 to 39 ± 13) and increased the intracellular concentration of ADM (mean fluorescent intensity increased from 18 ± 5 to 34 ± 6) in K562/A02 cells. Luciferase reporter gene assay demonstrated that TPL inhibited the transcriptional activity of MDR1 promoter by 75%. It is concluded that TPL may effectively reverse the multidrug resistance in K562/A02 cells via modulating P-glycoprotein expression and increasing intracellular ADM accumulation.

Intravascular infusion of lidocaine: a novel way to relieve sudden internal carotid artery occlusion in embolization of intracranial aneurysms.

BACKGROUND: Sudden internal carotid artery (ICA) occlusive vasospasm is a serious complication of intracranial aneurysm embolization. Conventional spasmolysis with papaverine yields a generally poor outcome. We believe that arterial infusion of lidocaine may offer a better outcome. MATERIALS AND METHODS: We retrospectively reviewed the outcome of patients treated with either papaverine or lidocaine infusion for vasospasm during embolization. RESULTS: 14 patients undergoing intracranial aneurysm embolization had a ICA occlusive vasospasm. Among the 8 patients who received conventional treatment with papaverine the vasospasm improved partially in 5. In 3 cases, treatment was ineffective. 6 of the patients died within 3 days. 2 patients developed hemispheric infarction and underwent a decompressive craniectomy and subtotal resection of the infarct; 1 of these 2 patients died after 4 months and the other was severely disabled. In the 6 patients treated with lidocaine, spasmolysis and subsequent aneurysm treatment was successful in 5. In 1 patient who had preoperative stenosis of the carotid artery proximal to the aneurysm spasmolysis failed. CONCLUSIONS: ICA occlusive spasm is an extremely serious and often lethal complication in embolization of intracranial aneurysms. Conventional treatment with papaverine has a poor outcome, whereas arterial infusion of lidocaine may achieve better results.

Preventing chemoresistance of human breast cancer cell line, MCF-7 with celecoxib.

PURPOSE: To investigate the preventive effect of celecoxib, a specific cyclooxygenase-2 (Cox-2) inhibitor, on the development of chemoresistance in breast cancer cell line, MCF-7, and explore the mechanism of the action. METHODS: Chemoresistance phenotype was established by treating MCF-7 cells with 0.05 µg/ml doxorubicin for 7 days, and then the effect of preventive chemoresistance was investigated by the combination of same dose of doxorubicin with 10 µM celecoxib. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to assess cytostatic efficacy of doxorubicin and 50% inhibiting concentration (IC(50)) of MCF-7 cells. RT-PCR was performed to examine mRNA expression of multidrug resistance gene 1 (MDR1) and its transcription factors c-Jun and NF-κB. Western blotting analysis was performed to detect the expression of protein. Flow cytometry (FCM) was applied to analyze the expression and function of P-glycoprotein (P-gp). Electrophoretic gel mobility shift assay (EMSA) was performed to determine the DNA-binding activity of nuclear transcription factors AP-1 and NF-κB. RESULTS: Compared with sensitive MCF-7 cells, MDR1 and its transcription factors c-Jun and NF-κB were up-regulated at both mRNA level (P < 0.01) and protein level (P < 0.01) by treatment with 0.05 µg/ml doxorubicin for 7 days. After co-incubation with both the same dose of doxorubicin and 10 µM celecoxib for 7 days, both mRNA level and protein level of MDR1, c-Jun and NF-κB up-regulated by doxorubicin were partly reversed (P < 0.01); DNA-binding activity of nuclear transcription factors AP-1 and NF-κB were inhibited; and the function of P-gp was decreased (P < 0.01). When MCF-7 cells were treated with increasing doses of doxorubicin in the presence of 10 µM celecoxib, IC50 value of doxorubicin and doxorubicin plus 10 µM celecoxib was 0.67 ± 0.03 and 0.38 ± 0.04 µg/ml, respectively (P < 0.01). CONCLUSION: Celecoxib effectively prevents the development of chemoresistance in breast cancer cell line MCF-7 induced by doxorubicin, which was partly involved in inhibiting the expression and DNA-binding activity of nuclear transcription factors AP-1 and NF-κB and downstream expression and function of P-gp. Furthermore, cytostatic efficacy of celecoxib and doxorubicin on MCF-7 cell was synergistic.

[Influence of YB-1 protein on the biological behaviour in K562/A02 cells].

The aim of this study was to investigate whether the growth, apoptosis and sensitivity to anticancer agent could be altered after introduction of YB-1 shRNA eukaryotic expression vector into the K562/A02 cells, and its possible molecular mechanisms. The recombinant eukaryotic expression plasmids including YB-1 shRNA and the vector-random-sequence were introduced into K562/A02 cells by lipofectamine mediation, and the positive clones were screened by G418. RT-PCR and Western blot were employed to detect the expression of mRNA and protein of YB-1 in leukemia cells, respectively. The proliferative ability of the cells was determined by MTT assay and cell cycle analysis. Apoptosis of K562/A02 cells was assayed by AnnexinV-FITC/PI double labeled flow cytometry. The drug sensitivity to anticancer agent was determined by MTT assay. The expressions of MDR1 gene and P-gp were detected by RT-PCR and flow cytometry respectively. The results indicated that the levels of mRNA and protein of YB-1 decreased dramatically in three groups of positively transfected cells when compared with control cells. The inhibitory rates of 3 different shRNA sequences targeting YB-1 gene were (65.1 ± 2.1)%, (27.4 ± 1.3)% and (67.4 ± 1.6)% respectively. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased levels of the proliferative ability in K562/A02 cells, and displayed higher at G(1), lower at G(2) and S phase in cell cycle distribution in comparison with the control groups. AnnexinV/PI detection indicated higher AnnexinV(+) ratio in 3 groups of positively transfected cells after being treated with As(2)O(3) of 0.5 µmol/L for 24 hours. The IC(50) values of doxorubicin in 3 groups of positively transfected cells were significantly lower than that in control group. The level of MDR1 gene and P-gp decreased significantly in 3 groups of positively transfected cells. It is concluded that the transfection with YB-1 shRNA gene can inhibit the proliferation of leukemia cells and induce cell apoptosis. The expression of MDR1 mRNA and P-gp decrease after transfection of YB-1 shRNA into K562/A02 cells.

[Mechanisms of preventive effect of tetrandrine on acquired multidrug resistance in K562 cells].

This study was purposed to explore the mechanisms of preventive effect of tetrandrine (TTD) on doxorubicin (ADM)-induced multidrug resistance (MDR) in human leukemia cell line K562 from two aspects of the transcription control of MDR1 gene and cell apoptosis. The experiment was divided into 3 groups: group I-blank control; group II-ADM-induced drug-resistance; group III-ADM-induced drug-resistance after pretreatment with TTD. Reverse transcription-PCR (RT-PCR) was used to detect the mRNA expression levels of c-Jun, YB-1 and Survivin genes. Western blot was used to determine the nuclear protein expression levels of c-Jun and YB-1. Flow cytometry was used to assay the apoptosis of cells. The results showed that as compared with group I, the expression levels of c-Jun mRNA and nuclear protein decreased (p < 0.05), as well as the expression levels of YB-1 mRNA and nuclear protein increased in group II (p < 0.05). However, the expression of Survivin mRNA had no change (p > 0.05); the apoptosis rate of cells was 8.31%. As compared with group II, the expression levels of c-Jun mRNA and nuclear protein increased (p < 0.05), expression levels of YB-1 mRNA and nuclear protein as well as Survivin mRNA decreased in group III (p < 0.05). The apoptosis of cells was 97.2%. It is concluded that TTD can inhibit the expression of YB-1 and up-regulate the expression of c-Jun, thus inhibit the expression of MDR1 gene. TTD can also inhibit the expression of Survivin and increase the apoptosis of cells induced by ADM.