ABSTRACT
Objective@#To investigate the inhibitory effect of bortezomib (PS-341) on enterovirus replication.@*Methods@#The methyl thiazolyl tetrazolium (MTT) assay was used to value cell viability in response to PS-341 treatment. The protein and viral gene mRNAs were measured by real-time quantitative PCR (qRT-PCR).@*Results@#Our result show that after enterovirus (EV)-D68 or coxsackievirus B3 (CV-B3) infected cells were treated with PS-341, compared with the control group, the inhibition rate of the intracellular viral RNA reached 50%~70% or 60%~90%. PS-341 was added after RD cells were infectd with EV-D68, the intracellular virus titer was down-regulated by 90.23% and 83.40% in the supernatant, the intracellular virus titer was down-regulated by 93% and 90% in the supernatant and in RD cells. PS-341 had no effect on virus adsorption and importing. The cells were treated with PS-341 and apoptosis-inhibiting agent Ac-YVAD-CHO, the viral RNA replication inhibition rate reached 10%-30%, and the expression of viral protein was increased, which indicated that the inhibitory effect of PS-341 on viral replication was attenuated.@*Conclusions@#According to the result of the study, PS-341 could reduce apoptosis by regulating the proteasome pathway, inhibiting the gene replication and assemble, without effect on virus adsorption, entry and release. In addition, PS-341 also inhibited the replication of CV-B3 in cells, which suggest that PS-341 has a broad spectrum anti-EVs effects.
ABSTRACT
Objective To compare the proteome difference between multiple myeloma cell line U266 cells treated and untreated with PS-341, to investigate the potential drug targets, and to provide theoretical evidence for clinical therapy of multiple myeloma. Methods Two-dimensional gel electrophoresis (2-DE) was performed to separate proteins from treated and untreated U266 cells with proteasome inhibitor PS-341. ImageMaster 2D Platinum software was used to analyze 2-DE image, and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to identify the differentially expressed proteins. The expression levels of differential protein BAG-2 in the 2 groups of U266 cells lines were detected by Western blot. Results The 2-DE reference pattern of treated and untreated U266 cells with PS-341 was established. A total of 31 differential proteins were identified by MALDI-TOF-MS, 27 of which were down-regulated after PS-341 treatment. The differential expression level of BAG-2 in the 2 groups of U266 cells was confirmed by Western blot. Conclusion Some down-regulated proteins may be the potential drug targets of proteasome inhibitor PS-341.
ABSTRACT
Objective To explore the effect and molecular mechanism of proteasome inhibitor in TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis resistance on malignant lymphoma cells.Methods Raji cells were treated with TRAIL and proteasome inhibitor (PS-341) in vitro and the cell growth index was evaluated by MTT assay; cell cycle was analysed by flow cytometry; the protein and mRNA level of Bax were measured by Western blotting and real time RT-PCR. Results TRAIL inhibited proliferation of Raji cells at the concentration of 500 μg/L, but the inhibition rate was lower than that of the control cell:Hmy2.ciR.TRAIL arrested cell in G0/G1 phase. The Bax protein in Raji is degraded, but the Bax mRNA expression level does not change significantly .The effects of TRAIL was enhanced significantly 10 nmol/L PS-341 was added. Conclusion Raji cells are resistant in TRAIL-induced apoptosis. This effect may be related to the decrease of Bax protein. The Ubiquitin-proteasome pathway is involved in the degradation of Bax in TRAIL-treated Raji cells.
ABSTRACT
BACKGROUND: PS-341 is a novel, highly selective and potent proteasome inhibitor, which showed cytotoxicity against some tumor cells. Its anti-tumor activity has been suggested to be associated with modulation of the expression of apoptosis-associated proteins, such as p53, p21(WAF/CIP1), p27(KIP1), NF-kappa, Bax and Bcl-2. c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-3beta(GSK-3beta are important modulators of apoptosis. However, their role in PS-341-induced apoptosis is unclear. This study was undertaken to elucidate the role of JNK and GSK-3beta in the PS-341-induced apoptosis in lung cancer cells. METHOD: NCI-H157 and A549 cells were used in the experiments. The cell viability was assayed using the MTT assay and apoptosis was evaluated by proteolysis of PARP. The JNK activity was measured by an in vitro immuno complex kinase assay and by phosphorylation of endogenous c-Jun. The protein expression was evaluated by Western blot analysis. Dominant negative JNK1 (DN-JNK1) and GSK-3betawere overexpressed using plasmid and adenovirus vectors, respectively. RESULT: PS-341 reduced the cell viability via apoptosis, activated JNK and increased the c-Jun expression. Blocking of the JNK activation by overexpression of DN-JNK1, or pretreatment with SP600125, suppressed the apoptosis induced by PS-341. The activation of caspase 3 was mediated by JNK activation. Blocking of the caspase 3 activation suppressed PS-341-induced apoptosis. PS-341 activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but its blockade enhanced the PS-341-induced cell death via apoptosis. GSK-3betawas inactivated by PS-341 via the PI3K/Akt pathway. Overexpression of constitutively active GSK-3beta enhanced PS-341-induced apoptosis; in contrast, this was suppressed by dominant negative GSK-3beta(DN-GSK-3beta. Inactivation of GSK-3beta by pretreatment with lithium chloride or the overexpression of DN-GSK-3beta suppressed both the JNK activation and c-Jun up-regulation induced by PS-341. CONCLUSION: The JNK/caspase pathway is involved in PS-341-induced apoptosis, which is negatively regulated by the PI3K/Akt-mediated inactivation of GSK-3beta in lung cancer cells.