Influence of proteasome activator γ on iodine uptake capability of anaplastic thyroid carcinoma / 中华核医学与分子影像杂志

Objective To investigate the changes of iodine uptake capability (IUC) and mRNA expression of iodine uptake-related proteins in ARO and WRO thyroid carcinoma cell lines after silence of proteasome activator γ (REGγ),and observe the relation between REGγ and IUC of thyroid carcinoma.Methods The AROshN,AROshR,WROshN and WROshR (shN =blank plasmid,shR =plasmid with silence of REGγ) thyroid carcinoma cell lines were routinely cultured.Low dosage (3.7 kBq) of Na125I was added and then IUC was determined at different time points (5,10,15,20,40 and 70 min).The mRNA expressions of sodium/iodine symporter (NIS),thyroid stimulating hormone receptor (TSHR),thyroid peroxidase (TPO) and thyroglobulin (Tg) were examined by real-time PCR.Paired t test was used to analyze the data.Results After the silence of REGγ,the peak values of IUC in AROshR and WROshR cells were increased from (1 974±12) to (4 502±23) counts/min,and from (2 988±25) to (5 001±16) counts/min,respectively.The increase rates were 128.1％ in AROshR cells and 67.4％ in WROshR cells (t values:17.30,13.20,both P＜0.05).The mRNA expressions of NIS,TSHR,TPO,Tg in AROshR cells were 2.82,1.98,2.65 and 2.31 times higher than those in AROshN cells,and the expressions in WROshR cells were 2.21,1.78,2.51 and 1.78 times higher than those in WROshN cells (t values:13.80-21.93,all P＜0.05).Conclusion Silence of REGγcan increase the gene expressions of the iodine uptake-related proteins and elevate the IUC of thyroid carcinoma cells.

Genes related to maintenance of autophagy and successful aging / Genes relacionados à manutenção da autofagia e envelhecimento bem-sucedido

ABSTRACT Considering aging as a phenomenon in which there is a decline in essential processes for cell survival, we investigated the autophagic and proteasome pathways in three different groups: young, older and oldest old male adults. The expression profile of autophagic pathway-related genes was carried out in peripheral blood, and the proteasome quantification was performed in plasma. No significant changes were found in plasma proteasome concentrations or in correlations between proteasome concentrations and ages. However, some autophagy- and/or apoptosis-related genes were differentially expressed. In addition, the network and enrichment analysis showed an interaction between four of the five differentially expressed genes and an association of these genes with the transcriptional process. Considering that the oldest old individuals maintained both the expression of genes linked to the autophagic machinery, and the proteasome levels, when compared with the older group, we concluded that these factors could be considered crucial for successful aging.

RESUMO Considerando o envelhecimento como um fenômeno em que há um declínio nos processos essenciais a sobrevivência celular, investigamos as vias autofágica e proteassômica em três grupos: jovens, idosos e longevos. O perfil de expressão dos genes relacionados à via autofágica foi analisado em sangue periférico, e a quantificação do proteassoma realizada em plasma. Não foram encontradas alterações significativas nas concentrações plasmáticas de proteassoma ou na correlação entre as concentrações de proteassoma e as idades. No entanto, alguns genes relacionados a autofagia e / ou apoptose foram expressos diferencialmente. Além disso, as análises de rede e de enriquecimento mostraram uma interação entre quatro dos cinco genes diferencialmente expressos e a associação desses ao processo transcricional. Considerando que os indivíduos longevos mantiveram tanto a expressão de genes ligados à maquinaria autofágica, quanto os níveis de proteassoma quando comparados aos idosos, concluímos que esses fatores poderiam ser considerados cruciais para o envelhecimento bem-sucedido.

Changes in 20S proteasome acitivities in brain and spinal cord of acute and chronic morphine-dependent mice / 中华麻醉学杂志

Objective To investigate the changes in 20S proteasome activities in the brain and spinal cord of acute and chronic morphine-dependent mice.Metbods Male ICR mice,weighing 25-30 g,were used in the study.The experiment was performed in 2 parts.In experiment Ⅰ,16 mice were randomly divided into 5 groups (n =8 each) using a random number table:control group (group C) and acute morphine dependence group (AMD group).In experiment Ⅱ,16 mice were randomly divided into 5 groups (n =8 each) using a random number table:control group (group C) and chronic morphine dependence group (CMD group).Acute morphine dependence was induced with morphine 100 mg/kg injected subcutaneously,and the mice were sacrificed 3 h later.Chronic morphine dependence was induced by increasing doses of morphine for 4 days,the initial dose of morphine was 20 mg/kg injected subcutaneously twice a day and was increased by 10 mg/kg every day,the dose of morphine was 10 mg/kg injected subcutaneously on 5th day,and then the mice were sacrificed 1 h later.In group C,the equal volume of normal saline was given instead,and the other treatments were similar to those previously described in morphine dependence groups.After the mice were sacrificed,the hippocampus,prefrontal cortex,striatum and spinalcord were isolated for determination of 20S proteasome activity,measured as chymotrypsin-like (ChT-L),trypsin-like (T-L) and peptidylglutamyl-like hydrolyzing (PGLH) activities.Results Experiment Ⅰ Compared with C group,PGLH activity in the spinal cord and T-L activity in the striatum or prefrontal cortex were significantly weakened in group AMD.There was no significant difference in 20S proteasome activity in the hippocampus between the two groups.Experiment Ⅱ Compared with C group,ChT-L and T-L activities in the spinal cord were significantly weakened,and PGLH activity in the striatum was enhanced in CMD group.There was no significant difference in 20S proteasome activity in the prefrontal cortex and hippocampus between the two groups.Conclusion 20S proteasome activity in the spinal cord and brain is weakened in acute morphine-dependent mice,20S proteasome activity in the spinal cord is weakened,20S proteasome activity in the striatum is enhanced in chronic morphine-dependent mice,these changes have specificity in terms of position and type of activity,and the changes mentioned above may be related to development of morphine dependence in mice.

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

The effect of proteasome inhibitor on NF-κB signal path in a rat model of knee osteoarthritis / 中华骨科杂志

Objective To observe the effect of MG-132 on NF-κB signal path of cartilage and synovium in a rat model of knee osteoarthritis.Methods The rat models of knee osteoarthritis were established by performing anterior cruciate ligament amputation and partial medial meniscectomy.Totally 144 adult SD rats were randomly divided into 4 groups:MG-132 group,100 ml 0.007 g/L MG-132 solution was injected in to the knee joints of rat model 24 h after surgery; DMSO group,100 ml 0.1％ DMSO solution was injected 24 h after surgery; sham surgery group,merely the knee capsulotomy was performed and no solution was injected;control group,100 ml 0.007 g/L MG-132 solution was injected into the knee joints.The cartilage and synovium specimens were obtained at 2,4,12 weeks postoperatively.Pathomorphological observation was taken.The levels of NF-κB p65,I-κB,TNF-α and IL-1β at mRNA were detected by real-time PCR,and the activityof 20S proteasome was measured by fluorospectrophotometry.Resnlts The Mankin score of MG-132 groupwas lower than that of DMSO group.The Mankin scores of sham surgery and control groups were lower thanthose of MG-132 and DMSO groups with significant difference.The mRNA levels of NF-κB p65,IL-1 β,TNF-α of cartilage and synovium in MG-132 group were lower than those of DMSO group with significant differenceexcept for NF-κB p65 of synovium at 2 weeks and IL-1β of cartilage at 12 weeks.The mRNA levels of I-κB of cartilage at 2 weeks and I-κB of synovium at 4 weeks in MG-132 group were higher than those in DMSO group with statistical significance.Conclusion MG-132,the proteasome inhibitor,could postpone the progress of osteoarthritis through alleviating synovial inflammation and defending the articular cartilage.

DNA-Damage Inducible 1 is a Property of Human Non-Small Cell Lung Cancer / 결핵및호흡기질환

BACKGROUND: DNA damage-inducible 1 (Ddi1), one of the ubiquitin-like and ubiquitin-associated family of proteins, may function in the regulation of the ubiquitin-proteasome pathway, which has been validated as a target for antineoplastic therapy. We investigated Ddi1 expression in human lung cancer tissues and evaluated the relationship of this expression pattern with clinicopathological factors in patients with non-small-cell lung cancer (NSCLC). METHODS: Ddi1 expression was examined by immunohistochemistry in tumor tissues from 97 patients with stage I NSCLC, who had undergone curative surgical resection at two tertiary referral hospitals from 1993~2004. None of the patients received preoperative chemotherapy and/or radiation therapy. RESULTS: Thirty-nine (40.2%) of the 97 cases were positive for Ddi1. Ddi1 expression was dominantly seen in cytoplasm rather than in the nuclei of cancer cells in all histological types, whereas adjacent nontumoral lung tissue showed negative Ddi1 staining in most cases. Ddi1 expression tended to increase in well-differentiated tumors but without statistical significance. Positive Ddi1 expression was associated with a tendency for better disease-free survival and disease-specific survival, although the difference was not significant. CONCLUSION: Ddi1 expression is a property of NSCLC. Because Ddi1 could be a potential target for cancer therapy, more research is needed to evaluate its role in NSCLC.

Transient exposure to hydrogen peroxide inhibits the ubiquitination of phosphorylated IkappaBalpha in TNFalpha-stimulated HEK293 cells

During ischemia-reperfusion injury, brief pre-exposure to oxidative stress renders organs resistant to subsequent severe damage. NF-kappaB is a transcription factor that is involved in reperfusion-induced inflammatory and immune responses. The activity of NF-kappaB has been shown to be modulated by oxidative stress in various cell types through different pathways. We studied the effect of pre-exposure to oxidative stress on subsequent NF-kappaB activation in TNFalpha-stimulated HEK293 cells. The cells were transiently exposed to 0.5 mM H2O2 for 20 min, prior to stimulation with TNFalpha, and the subsequent expression of NF-kappaB-dependent genes and the levels of NF-kappaB signaling molecules were measured. Pre-exposure to H2O2 significantly delayed the TNFalpha-induced expression of an NF-kappaB reporter gene and inflammatory proteins (intercellular adhesion molecule-1 and IL-1beta). The degradation of inhibitor of NF-kappaB alpha (IkappaBalpha) and the nuclear translocation of NF-kappaB were also delayed by H2O2 treatment, whereas IkappaBalpha phosphorylation and IkappaB kinase activity were not changed. When we examined the ubiquitin/proteosome pathway in H2O2-treated cells, we could not detect significant changes in proteosomal peptidase activities, but we were able to detect a delay of IkappaBalpha poly-ubiquitination. Our results suggest that transient exposure to oxidative stress temporally inhibits NF-kappaB-dependent gene expression by suppressing the poly-ubiquitination of phosphorylated IkappaBalpha in HEK293 cells.

Proteolytic system is dysfunctional in diabetic nephropathy model rats / 中华肾脏病杂志

Objective To investigate autophagy and proteasome system alteration in vivo and in vitro of diabetic nephropathy (DN) model rats.Methods Rat glomerular mesangial cells were primaryly cultured,and cell proliferation was tested by MTT assay.The mesangial cells were cultured under different concentrations of glucose (5.4 mmol/L for normal control and 30 mmol/L for high glucose) for 0,8,16,72 hours.The expression of autophagy (LC3) and proteasome (PSMAs) proteins was examined by Western blotting analysis.Spontaneous type 2 diabetes model OLETF and its normal control LETO rats were observed for 36 weeks.The levels of blood glucose and 24 hours urinary protein were evaluated in every 4 weeks.All the rats were sacrificed at the 36th week,and renal pathological changes were semi-quantitively analyzed.The expression of PSMAs and LC3 proteins was also examined in kidney cortex by Western blotting.Results Under high glucose concentrations,the abundance of PSMAs and LC3 proteins significantly reducedin the mesangial cells at 8 hours.There was no significant difference at other time points.The levels of blood glucose and 24 h urinary protein in OLETT rats exhibited progressive increase compared to those in LETO rats (all P＜0.01).And glomerular sclerosis index and tubulointerstitial injury index were significantly higher than those in LETO rats (all P＜0.01).The abundance of PSMAs proteins was significantly reduced in renal cortex of OLETF rats compared with LETO rats,while the abundance of LC3 proteins had no significant difference between two groups.Conclusion Proteolytic system dysfunction may play a role in pathogenesis of DN.

Role of spinal cord proteasome in chronic morphine tolerance in rats / 中华麻醉学杂志

ObjectiveTo evaluate the role of the spinal cord proteasome in chronic morphine tolerance in rats.MethodsTwenty-four healthy male SD rats in which intrathecal catheters were successfully placed without complications were randomized into 4 groups ( n =6 each):normal saline group ( group NS),chronic morphine tolerance group (group M),chronic morphine tolerance + proteasome inhibitor MG-132 group (group M + MG) and MG-132 group (group MG).Normal saline 10 μl,morphine 10 μg,morphine 10 μg+ MG-132 2.5 μg and MG-132 2.5 μg were injected intrathecally twice a day for 7 consecutive days in groups NS,M,M + MG and MG respectively.Tail flick latency was measured on day 1 before intrathecal injection and on day 1,3,5 and 7 of intrathecal injection to calculate the percentage of maximum possible antinociceptive effect (MPAE).After the last intrathecal injection,5 rats were sacrificed,and L3-5 spinal cord was removed for determination of the expression of glutamate-aspartate transporter (GLAST) and excitatory amino acids carrier 1 (EAAC1)by Western blot.Results MPAE was gradually decreased during the intrathecal injection in groups M and M + MG( P ＜ 0.05).Compared with group NS,MPAE was gradually incresed during the intrathecal injection in groups M and M + MG,and the expression of GLAST and EAAC1 in the spinal cord was significantly down-regulated in group M (P ＜ 0.05).There was no significant difference in the parameters mentioned above between group MG and group NS ( P ＞0.05 ).Compared with group M,MPAE was significantly increased and the expression of GLAST and EAAC1 in the spinal cord was significantlyup-regulatedingroupM+MG(P＜0.05or0.01).ConclusionSpinal cord proteasome is involved in the development of chronic morphine tolerance in rats.

Inhibitory effect of proteasome inhibitor on the proliferation of human lens epithelial cells / 中国医师杂志

Objective To investigate the effect of proteasome inhibitor MG132 on the proliferation of human lens epithelial cells SRA01/04. Methods The SRA01/04 cells were treated with MG132 by different concentrations (0, 0. 1, 0. 5, 1. 0, 2. 5, 5.0, 10. 0μmol/L) for 36 hours. The cell viability in all groups was determined using methylthiazoltetrazolium (MTT) test. The effect of MG132 on the apoptosis and regulation of cell cycle about SRA01/04 cells were detected by flow cytometry (FCM). The SRA01/04 cells treated with MG132 were observed after Annexin V/FITC-PI staining by fluorescence microscope. Results The inhibitory effect of MG132 on SRA01/04 cells proliferation was enhanced with the increase of MG132 concentration. The 50% inhibiting concentration ( IC50 ) of MG132 was 2. 50μmol/L after SRA01/04 cells were treated with MG132 for 36 hours. The apoptosis index of the cells treated by MG132 at 2. 5μmol/L and 5 μmol/L for 36 hours was 6. 55 ± 0. 35% and 13.75 ± 3.18%, and 0. 75 ± 0. 21% for 5.0μmol/L for 36 hours in control group. After cells were treated with MG132 for 48h, the percentages of cells at G0/G1 phase were (42. 57 ± 0. 64) %, (73.42 ± 3.10) %, ( 80. 95 ± 3.83 ) % 0, 2. 5,5.0 μmol/Lgroups respectively, and those at S phase were (49. 44±1.36)%, ( 17. 40 ± 1.50)%, ( 19. 57 ± 1.29)%.Annexin V/FITC-PI staining was used, and MG132 was found to result to apoptosis. Conclusions MG132 could inhibit the proliferation of SRA01/04 cells by the effect of inducing apoptosis and regulation of cell cycle. The proteasome inhibitor-might play a key role in the prevention of posterior capsular opacification.

Role of autophagy and proteasome degradation pathways in apoptosis of PC12 cells transfected with A53T α-synuclein / 中华神经科杂志

Objective To explore the specific role of autophagy and ubiquitin-proteasome pathway in apoptosis, specific protease inhibitor and (or) macroautophagy inhibitors.Methods The stimulators were selected to work on the pheochromocytoma (PC12) cell lines transfected with human mutant α-synuclein (A53T).Cell activity and apeptosis rate were detected by MTT law and flow cytometry.NO energy, heat shock protein 70 (Hsp70) and Caspase-3 expression were determined in cell culture.Results A53T cell survival rate significantly decreased 24 hours after handling with the protease inhibitor (100 nmol/L) and (or) autophagy inhibitors 3-MA (10 mmol/L, A =0.23±0.01,0.19±0.01 and 0.17±0.01 respectively; P <0.05) compared with the control group (A =0.32±0.06).Cell survival rate was significantly higher than the other drug group after 24 hours handling with autophagy stimulators (A =0.44±0.08).Compared with the control group or autophagy stimulator of rapamycin (0.2 μg/ml) group (1.55%±1.15%), A53T cells apeptosis percentage rate was significantly higher after treated with proteasome inhibitor and macroautophagy inhibitors 24 hours (4.74%±0.91%, 4.59%±1.18% and 5.40%±1.75%respectively, P <0.05); and a slight decrease with stimulators.Protein Hsp70 and NO were significantly higher in proteasome inhibitor groups than the control group.But in antophagy inhibitor and stimulator group, NO and Hsp70 protein was similar to the control group.Conclusion The inhibition of macroautophagy and proteasome can promote apoptosis.Inhibiting or stimulating autophagy has less impact on Hsp70 and NO than proteasome pathway.

A Pilot Study of Bortezomib in Korean Patients with Relapsed or Refractory Myeloma

Recent clinical trials showed that bortezomib, a novel proteasome inhibitor, had therapeutic activity in multiple myeloma. However, there was no data about the feasibility of bortezomib in Korean patients. We performed a pilot study of bortezomib in patients with relapsed or refractory myeloma (1.3 mg/m2 twice weekly for 2 week in a 3-week cycle). Seven patients were enrolled. The median age of patients was 59 yr. All patients previously received VAD (vincristine, doxorubicin and dexamethasone) and thalidomide chemotherapy. Three patients previously received alkylator-containing chemotherapy and 4 patients, autologous stem cell transplantation. Bortezomib monotherapy resulted in 3 partial remissions (43%), 3 no changes (43%) and 1 progressive disease (14%). One patient who had no response to bortezomib monotherapy experienced partial remission after addition of dexamethasone to bortezomib. The most common serious toxicity was thrombocytopenia (grade 3/4, 10 of 20 cycles (50%)) and grade 3 peripheral neuropathy was developed in 2 of 20 cycles (10%). Drug-related adverse event led to discontinuation of bortezomib in 1 patient. There was no treatment related mortality. Overall, bortezomib seems to be effective and feasible. Conduction of larger clinical studies on Korean patients is necessary to characterize clinical efficacy and safety of bortezomib more precisely.