Inhititory effect of proliferation and promotion effect of apoptosis of lactacystin combined with carboplatin on ovarian cancer SKOV3 cells / 吉林大学学报(医学版)

Objective:To study the influence of proteasome inhibitor lactacystin (LAC) and carboplatin in proliferation and apoptosis of the human ovarian cancer SKOV3 cells in vitro ,and to clarify the mechanisms. Methods:The SKOV3 ovarian cancer cells were cultured in vitro ;0,2.5,5.0,10.0 and 20.0 μmol· L-1 LAC were used to intervent the SKOV3 cells for 48 h;5 μmol·L-1 LAC was used to intervent the SKOV3 cells for 0, 24,48,and 72 h;the SKOV3 cells were divided into control group (treated without medical intervention),LAC group (treated with 5 μmol · L-1 LAC), carboplatin group (treated with 10, 20, 40 and 80 μmol · L-1 carboplatin),LAC and carboplatin group (treated with 5 μmol· L-1 LAC and 10,20,40,and 80 μmol· L-1 carboplatin,respectively).MTT method and FCM were used to detect the inhibitory rates of proliferation and apoptotic rates of the SKOV3 cells in various groups.Results:The MTT test results showed that the proliferation of the SKOV3 cells were inhibited with the prolongation of time and increasing of LAC concentration;the half inhibitory concentration (IC50 )of LAC at 48 h was 5.36 μmol · L-1 ;compared with carboplatin group,the inhibitory rates of proliferation of SKOV3 cells in LAC and carboplatin groups were significantly increased (P <0.05).The IC50 of carboplatin was dropped from 58.08 μmol·L-1 to 18.37 μmol·L-1 .The FCM results showed that with the prolongation of treated time of LAC,the apoptotic rates of SKOV3 cells were increased;compared with carboplatin group and LAC group,the apoptotic rate of cells in LAC and carboplatin group was increased (P <0.05).Conclusion:LAC can inhibit the proliferation of the ovarian cancer SKOV3 cells and induce the apoptosis, and LAC can enhance the inhibitory effect of proliferation of carboplatin on the ovarian cancer SKOV3 cells.

Effect of HDAC-6 on PD cell induced by lactacystin

Objective: To explore the effects of histone deacetylase 6(HDAC-6) on the PD cell model induced by proteasome inhibitor lactacystin. Methods: Human neuroblastoma SK-N-SH cells were cultured. The wild type pcDNA3.1-alpha-synuclein eukaryotic expression plasmid was transferred into the cells which then were divided into control group, group L, group T and group T+L. The cells of group L were added with 5 μmol/L lactacystin dissolved indimethylsulfoxide (DMSO) to induce PD cell model with abnormal protein aggregation, the cells of control group were treated with 5 μmol/L DMSO, the cells of group T were treated with 5 μmol/L selective HDAC-6 inhibitor tubacin dissolved in DMSO, and the cells of group T+L were treated with 5 μmol/L lactacystin and 10 μmol/L tubacin dissolved in DMSO. The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 were detected by Western blot and the cell survival rate of all the groups was detected by MTT colorimetric assay, and compared 24 h after the cells were treated. Results: The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 of the cells of group L were significantly higher than the control group, and the cell survival rate was significantly lower (P 0.05). Conclusions: The expression level of alpha-synuclein oligomers can be improved and the cell survival rate can be reduced by the PD cell model induced by lactacystin and treated with selective HDAC-6 inhibitor tubacin, which means that alpha-synuclein oligomers of the PD cell model induced by lactacystin can be inhibited and the cell survival rate can be improved by HDAC-6, and the mechanism may be related to the increased of HSP-27 and HSP-70.

Effect of HDAC-6 on PD cell induced by lactacystin

OBJECTIVE@#To explore the effects of histone deacetylase 6(HDAC-6) on the PD cell model induced by proteasome inhibitor lactacystin.@*METHODS@#Human neuroblastoma SK-N-SH cells were cultured. The wild type pcDNA3.1-alpha-synuclein eukaryotic expression plasmid was transferred into the cells which then were divided into control group, group L, group T and group T+L. The cells of group L were added with 5 μmol/L lactacystin dissolved indimethylsulfoxide (DMSO) to induce PD cell model with abnormal protein aggregation, the cells of control group were treated with 5 μmol/L DMSO, the cells of group T were treated with 5 μmol/L selective HDAC-6 inhibitor tubacin dissolved in DMSO, and the cells of group T+L were treated with 5 μmol/L lactacystin and 10 μmol/L tubacin dissolved in DMSO. The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 were detected by Western blot and the cell survival rate of all the groups was detected by MTT colorimetric assay, and compared 24 h after the cells were treated.@*RESULTS@#The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 of the cells of group L were significantly higher than the control group, and the cell survival rate was significantly lower (P  0.05).@*CONCLUSIONS@#The expression level of alpha-synuclein oligomers can be improved and the cell survival rate can be reduced by the PD cell model induced by lactacystin and treated with selective HDAC-6 inhibitor tubacin, which means that alpha-synuclein oligomers of the PD cell model induced by lactacystin can be inhibited and the cell survival rate can be improved by HDAC-6, and the mechanism may be related to the increased of HSP-27 and HSP-70.

Transcriptional Upregulation of Plasminogen Activator Inhibitor-1 in Rat Primary Astrocytes by a Proteasomal Inhibitor MG132

Plasminogen activator inhibitor-1 (PAI-1) is a member of serine protease inhibitor family, which regulates the activity of tissue plasminogen activator (tPA). In CNS, tPA/PAI-1 activity is involved in the regulation of a variety of cellular processes such as neuronal development, synaptic plasticity and cell survival. To gain a more insights into the regulatory mechanism modulating tPA/PAI-1 activity in brain, we investigated the effects of proteasome inhibitors on tPA/PAI-1 expression and activity in rat primary astrocytes, the major cell type expressing both tPA and PAI-1. We found that submicromolar concentration of MG132, a cell permeable peptide-aldehyde inhibitor of ubiquitin proteasome pathway selectively upregulates PAI-1 expression. Upregulation of PAI-1 mRNA as well as increased PAI-1 promoter reporter activity suggested that MG132 transcriptionally increased PAI-1 expression. The induction of PAI-1 downregulated tPA activity in rat primary astrocytes. Another proteasome inhibitor lactacystin similarly increased the expression of PAI-1 in rat primary astrocytes. MG132 activated MAPK pathways as well as PI3K/Akt pathways. Inhibitors of these signaling pathways reduced MG132-mediated upregulation of PAI-1 in varying degrees and most prominent effects were observed with SB203580, a p38 MAPK pathway inhibitor. The regulation of tPA/PAI-1 activity by proteasome inhibitor in rat primary astrocytes may underlie the observed CNS effects of MG132 such as neuroprotection.

Investigation of the apoptosis mechanism induced by lactacystin in prostate cancer cell / 中国医师进修杂志

Objective To investigate the relationship between nuclear factor(NF)-κB activity and lactacystin induced prostate cancer cell apoptosis.Methods Two prostate cancer cell were divided into two groups:blank control group treated with culture solution,lactacystin group treated with different concentration of lactacystin(0.5,1.0,2.0,4.0 μ mol/L),the action time were 8,16 and 24 hours.The cell survival rate was measured by MTT assay.NF-κB DNA binding activity was measured by enzyme-linked immunosorbent assay,the expression of NF-κB P65 nuclear protein was detected by Western blot assay,and caspase-3 activity was analyzed by enzyme analysis assay.Results On basal condition,the NF-κ B DNA binding activity was much higher in DU145 cell than that in LNCaP cell(t=4.728,P=0.001).Compared with blank control group,different concentration of lactacystin groups'NF-κ B DNA binding activity in both the LNCaP and DU145 cell were reduced.The expression of NF-κB p65 nuclear protein decreased along with raising of lactacystin concentration in LNCaP cell,but it did not change in DU145 cell.On basal condition,caspase-3activity in DU145 cell was higher than that in LNCaP cell(t=4.519,P=0.001).After lactacystin acting of 24 hours,caspase-3 activity increased along with raising of lactacystin concentration in both the LNCaP and DU145 cell(2.0 μmol/L lactacystin group compared with 1.0 μmol/L lactacystin group,DU145 cell P=0.000,LNCaP cell P=0.000).Conclusions Lactacystin has different killing effects on prostate cancer cell.The mechanism may be related to inducing the apoptosis by down-regulation of NF-κB activity.There may be additional cell survival/death pathway in androgen-independent prostate cancer cell.

Apoptotic Effect of Co-Treatment with a Natural Product, Chios Gum Mastic, and a Proteasome Inhibitor, Lactacystin, on Human Osteosarcoma Cells / 대한해부학회지

Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. Recently, it was reported that CGM induced apoptosis in a few cancer cells in vitro. Since recent studies indicated the synergistic interactions between the apoptotic stimulus and a proteasome inhibitor, the ubiquintin-proteasome pathway has become an attractive target in cancer therapy. And to date, there has been no report of the synergistic apoptotic effect between CGM and a proteasome inhibitor to become an attractive target in cancer therapy. Therefore, this study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM, and a proteasome inhibitor, lactacystin, on human osteosarcoma (HOS) cells. To investigate whether the co-treatment of CGM and lactacystin compared with each single treatment efficiently induced apoptosis on HOS cells, MTT assay, DNA electrophoresis, Hoechst staining, DNA hypoploidy assay, Westen blot analysis, immunofluorescent staining, proteasome activity and mitochondrial membrane potential (MMP) change were performed. In this study, HOS cells co-treated with CGM and lactacystin showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-7, caspase-3, PARP and DFF45 (ICAD) whereas each single treated HOS cells hardly showed. We presented data indicating that the co-treatment of CGM and lactacystin induced potentially apoptosis whereas each single treatment did slightly. Moreover, the co-treatment of CGM and lactacystin potentiated the inhibition of proteasome activity. Therefore, our data provide the possibility that combination therapy of CGM and lactacystin could be considered as a novel therapeutic strategy for human osteosarcoma.

RPE Cell Apoptosis by the Combination Treatment of SAHA and Lactacystin

PURPOSE: To establish a new therapeutic strategy for proliferative vitreoretinopathhy (PVR), we examined the effect of combined treatment with HDAC inhibitor SAHA and proteasome inhibitor lactacystin in human retinal pigment epithelial (RPE) cells, ARPE-19. METHODS: Viability was determined by trypan blue exclusion assay. Mitochondrial membrane potential (MMP) was measured by flow cytometry. Proteasome activity was measured by fluorophotometry. The expression and degradation of apoptosis-related proteins were assesssed by Western blotting. Subcellular location of apoptosis-related factors was monitored by confocal miscroscopy. RESULTS: A single treatment with 5 micro M SAHA or 10 micro M lactacystin did not reduce cell viability. However, combination treatment with 5 micro M SAHA and 10 micro M lactacystin substantially reduced the viability, because the mixture induced the reduction of MMP and nuclear condensation or fragmentation. Moreover, the combination treatment triggered the activation of caspase-3 and the production of PARP cleavage products. These data indicate that the combination treatment efficiently induces apoptosis in ARPE-19 cells. However, co-treatment of SAHA did not augment the proteasome inhibitory activity of lactacystin, nor did co-treatment of lactacystin augment acetylation of histones. It is notable that while p53 and CAD were observed in the mitochondria of cells treated with SAHA, they were translocated into the nucleus after the combination treatment. CONCLUSIONS: These results suggest that the combination treatment of SAHA and lactacystin effectively induced apoptosis in ARPE-19 cells. Further work is warranted to develop this combination therapy as a novel therapeutic strategy for PVR.

Apoptotic Effect of Combination Treatment with a Proteasome Inhibitor, Lactacystin, and a Histone Deacetylase Inhibitor, Trichostatin A, on MCF-7 Cells / 대한해부학회지

Although much information has been accumulated about the synergistic interaction of proteasome inhibitors and HDAC inhibitors to induce apoptosis in a certain type of cells, much less is known currently about the underlying mechanism. This study was undertaken to explore the combination effect of a histone deacetylase inhibitor, TSA, and a proteasome inhibitor, lactacystin, on the induction of apoptosis. Pretreatment of TSA and subsequent treatment of lactacystin showed the strong antitumor activity and nuclear condensation. Western blot assay showed that combination treatment of TSA and lactacystin increased Bax/Bcl-2 ratio and decreased level of XIAP. Activation of caspase-7 and cleavage of PARP were demonstrated after the combination treatment. In combination treatment group, cell cycle arrest was induced at G2/M phase and abolished increase in proteasome activity. This study is elucidating the mechanims whereby targeting apoptotic machineries may help in directing therapeutic strategies.

Effect of Lactacystin on the Sulindac-Induced Apoptosis Mechanisms in HT-29 Cells

PURPOSE: One of possible mechanisms of the antineoplastic effect by nonsteroidal anti-inflammatory drugs (NSAIDs) is an induction of apoptosis. The NSAIDs-induced apoptosis appears to be caspase- and mitochondria-dependent. The ubiquitin-proteasome system, which is a fundamental non- lysosomal tool that cells use to process or degrade a variety of short-lived proteins, is known to be involved in apoptosis and to be located upstream of mitochondrial changes and caspase activation. The present study was conducted to explore the potential role of proteasome pathway in NSAIDs-induced apoptosis. METHODS: We employed sulindac as a NSAID, and the lactacystin as a proteasome inhibitor to investigate the extent of the apoptosis in colon cancer cell line, HT-29 cells. The proteasome activity and the amount of apoptosis were quantified after cells were treated with 1 mM sulindac, 1micrometer lactacystin or both. RESULTS: Sulindac treatment caused apoptosis of the HT-29 cells in a time-dependent manner with resultant changes in nuclear morphology. Western blots also showed caspase-3 activation and PARP cleavage after sulindac treatment. Not only single treatment with lactacystin decreased proteasome activity, but co-treatment with sulindac enhanced decrease in proteasome activity further (P<0.01). Treatment with lactacystin only did not induce apoptosis. However, lactacystin augmented the induction of sulindac-induced apoptosis (P<0.01). This synergistic effect was also proven by Western blot analyses, where co-treatment augmented the caspase-3 activation and PARP degradation. CONCLUSIONS: The combination treatment of sulindac with a proteasome inhibitor lactacystin is suggested to be a very effective strategy for the induction of cancer cell apoptosis. Elucidation of the mechanism underlying the regression of colon cancers by combination of sulindac and lactacystin seems to be an immediate challenge in the near future.

Applying Lactacystin,a selective proteasome inhibitor,to set up the rat model of Parkinson's disease with Lewy body / 临床神经病学杂志

Objective To set up the Parkinson's disease(PD)rat model with pathologic characteristic of Lewy body in nigral cells.Methods SD rats were injected respectively with 8 mg Lactacystin(Lactacystin group),sodium saline(NS group)and 12 mg 6-OHDA(6-OHDA group)by stereotaxic unilateral injection into the pars compacta of substantia nigral.The spontaneous and apomorphine-induced contralateral behaviors of rats were observed.The changes of midbrain histology were viewed by microscope;expression of ?-synuclein and tyrosine hydroxylase(TH)positive cells were investigated by immunohistochemistry.The contents of dopamine and homovanillic acid in striatum were determined.Results Rats of NS group did not display abnormal behavior.The animals treated with Lactacystin developed progressively bradykinesia,hypokinesia,tremor,contralateral head tilting,and displayed apomorphine-induced contralateral rotation behavior;3 weeks later the number of TH positive cells were decreased by 83.29% compared with NS group(P