Effect Of Proteasome Inhibitor MG-132 Research Articles

The anti-tumor effect of proteasome inhibitor MG132 for human adenoid cystic carcinoma: correlate with the emerging role of Nrf2/Keap1 signaling pathway.

BackgroundAdenoid cystic carcinoma (ACC) is one of the most common malignant salivary gland tumors. Moreover, the unique biological characteristics and complex structures of ACC contribute to its poor survival rates. Recently, proteasome inhibitors have been shown to elicit satisfactory therapeutic effects in the treatment of certain solid tumors, but few studies have been implemented to investigate the effects of proteasome inhibitor therapy for ACC.MethodsIn this present study, cell counting kit-8 assay and flow cytometry assay were performed to examine the effects of proteasome inhibitor (MG132) on cell viability and apoptosis. We applied western blot and immunofluorescence staining to explore the expression of the Nrf2/Keap1 signaling pathway and P62, additionally Nrf2 inhibitor (ML385) was utilized to evaluate the role of Nrf2/Keap1 signaling pathway in MG132-induced cell apoptosis.ResultsOur data indicated that MG132 significantly suppressed the growth of ACC-83 cells(MG132 10µM P = 0.0046; 40µM P = 0.0033; 70µM P = 0.0007 versus control) and induced apoptosis (MG132 10µM P = 0.0458; 40µM P = 0.0018; 70µM P = 0.0087 versus control). The application of MG132 induced the up-regulation of Nrf2/Keap1 signaling pathway. Furthermore, inhibition of Nrf2 attenuated the therapeutic effects of MG132 for ACC (both ML385 + MG132 10µM P = 0.0013; 40µM P = 0.0057; 70µM P = 0.0003 versus MG132). P < 0.05 was considered statistically significant.ConclusionsOur results revealed that proteasome inhibitors MG132 could inhibit the cell viability and induce the apoptosis of ACC through Nrf2/Keap1 signaling pathway.

Effect of proteasome inhibitor MG132 on the expression of oxidative metabolism related genes in tomato

Expression analysis of antioxidant related genes may provide key information to develop plants more tolerant to abiotic and biotic stress. For this purpose, in this study, proteosome was inhibited by using MG132 proteosome inhibitor in Lycopersicon esculentum (tomato) tissues with high antioxidant content. We aimed to explain the response of gene isoforms associated with oxidative stress metabolism as a result of proteasome inhibition induced by MG132 in tomato. Gene expression levels of SLGR1, SLPhGPX, SLCAT1, SLFe-SOD, SLGPX, SLCu/Zn-SOD, SLcAPX and SLGST genes which are known to be associated with antioxidant mechanisms, were determined by real-time PCR in tomato. There was no significant change in SLPhGPX, SLCAT1, SL-Fe-SOD and SLcAPX genes in root, stem and leaf tissues. In addition, expression of SLGR1 and SLGST genes in root tissue was increased. In leaf tissue, expression of SLGPX, SLCu/Zn-SOD and SLGST genes increased significantly. There was no significant change in the expression of the genes studied in the shoot tissue. These results obtained from tomato root, stem and leaf tissues by creating proteasome inhibition will help to understand the relationship between antioxidant system and proteasome system in plants.

The inhibitive effects of proteasome inhibitor MG-132 on pterygium fibroblasts in vitro and the potential key regulators involved

This study aimed to determine whether MG-132 as a proteasome inhibitor can effectively hinder pterygium progression, and to screen out potential regulators involved in MG-132 mediated process. Human pterygium fibroblasts (HPFs) were derived from pterygium tissues from 5 patients. Cell proliferation was examined by MTT, cell cycle and apoptosis were detected by flow cytometry. The overgrowth pterygium tissues were characterized by H&E staining and IHC compared with normal tissues. Differential mRNA expression with MG-132 treatment was determined by RNA sequencing and analyzed by GO and KEGG pathways. The expression levels of Nrf2, MCPIP1, CDKN1B and XBP1, four genes closely associated with pterygium, were detected by RT-qPCR and western blotting. MG-132 dose-dependently inhibited the growth of HPFs, induced G2/M phase arrest of cell cycle at a certain dose, and also caused cell apoptosis, with the levels of cleaved caspase3, cleaved PARP, Bax and p21 increased. Ki-67 and Bcl-2 were highly expressed while Bax was decreased in pterygium tissues. Total 7199 differentially expressed genes (DEGs) were identified, including HSPA family most significantly increased, and AL590428.1, AL122125.1 and lincRNAs such as FGF14-AS2 decreased. The up-regulated DEGs were mainly enriched in RNA degradation pathway, while down-regulated DEGs were related to the regulation of cell cycle. The expressions of Nrf2 and MCPIP1 were significantly increased, while XBP1 and CDKN1B were decreased. In conclusion, MG-132 inhibited the proliferation and induced apoptosis of HPFs in vitro with 7199 DEGs participated in, which may provide a useful reference for the exploitation of MG-132 in treating pterygium.

Proteasome inhibitor MG132 enhances TRAIL-induced apoptosis and inhibits invasion of human osteosarcoma OS732 cells

MG132 as a proteasome inhibitor could induce apoptosis in various cancer cells. This study aimed to discuss the effect of proteasome inhibitor MG132 on the TRAIL-induced apoptosis of human osteosarcoma OS732 cells. MG132 and TRAIL were applied on OS732 cells respectively or jointly. Cell survival rates, changes of cellular shape, cell apoptosis and cell invasion were analyzed, respectively, by 3-(4,5)-dimethylthiahiazo(-z-y1)-2,5-di-phenytetrazoliumromide (MTT) assay, inverted phase contrast microscope, flow cytometry, and transwell invasion chamber methods. The protein levels of DR5, caspase-3, caspase-8, p27kip1 and MMP-9 were measured by Western blot analysis. The results indicated that combination of MG132 and TRAIL had the effect of up-regulating expression of DR5, caspase-3, caspase-8 and p27kip1, down-regulating expression of MMP-9 and inducing apoptosis as well as suppressing the ability of invasion of OS732 cells. The survival rate of combined application of 10μM MG132 and 100ng/ml TRAIL on OS732 cells was significantly lower than that of the individual application (p<0.01). Changes of cellular shape and apoptotic rates also indicated the apoptosis-inducing effect of combined application was much stronger than that of individual application. Cell cycle analysis showed combination of MG132 and TRAIL mostly caused OS732 cells arrested at G2–M-phase. The invasion ability of OS732 cells was restrained significantly in the combined group compared with the individual group and control group.

Effects of proteasome inhibitor MG132 on memory acquisition and retrieval in one-trial passive avoidance in mice

Objective To investigate the effects of proteasome inhibitor MG132 on memory acquisition and memory retrieval in one-trail passive avoidance for mice. Methods MG132 (25 mg/kg) was administrated at different time points to analyze the time-dependent effects of MG132 on hippocampus proteasome activity. MG132 (25 mg/kg) was administrated 3 hours before-training or 1.5 hours pre-test to investigate the effect on memory acquisition and retrieval. The interaction of low dose (12 mg/kg) of MG132 and novelty interference on memory retrieval to studied. Results Hippocampus proteasome activity was significantly blocked when MG132 (25 mg/kg) was administrated 0.5-1.5 hours before analysis. The inhibition recovered as time passing and backed to normal state 24 hours later.A significant difference was showed between vehicle and pre-training, pre-test group(pre-training vehicle (296.03±3.97)s, pre-training (173.30±47.51)s; pre-test vehicle (199.23±43.01)s, pre-test (44.10±11.16)s). Proteasome inhibitor did not produce a significant effect on memory retrieval (vehicle (174.19±30.54)s, lower dose (174.19±30.54)s), then only together with a novelty task the retrieval was significantly altered(vehicle (164.02±35.26)s, MG132+ novelty group (84.70±23.92)s). Conclusion Proteasome inhibitor MG132 can inhibit memory acquisition and memory retrieval. Thereby the effect on memory retrieval is enhanced by novelty. Thus, a physiological proteasome activity is essential for learning abilities. Key words: Memory acquisition; Memory retrieval; Ubquitin-proteasome system; MG132; One-trial passive avoidance

Inhibition of hepatitis B virus production in vitro by proteasome inhibitor MG132.

To investigate the suppressive effects of proteasome inhibitor MG132 on hepatitis B virus production. HepG2 2.2.15 hepatoblastoma cells, which constitutively produce HBV particles, were used in the present study. MTT assay was used to evaluate the cytotoxicity of MG132. A Proteasome-Glo chymotrypsin-like cell-based assay was used to access the proteasome activity. Quantitative PCR were performed to analyze HBV-DNA. Secreted HBV antigens in the culture medium were measured by ELISA. Western blot and immunofluorescent staining of HBV antigen were also performed. After 6 days of MG132 treatment, proteasome activity was greatly decreased to 64.3 ± 7.8% and 36.4 ± 7.7% of untreated cells by 0.1μM and 0.3μM of MG132, respectively. The levels of HBsAg and HBeAg, and the copy number of extracellular HBV-DNA, were decreased to nearly half of the control group by 0.1μM MG132. The HBV replicative intermediates were also suppressed by MG132. Western blot and immunofluorescent staining clearly showed the lower levels of the expression of HBV proteins induced by MG132. MG132 could effectively inhibit the HBV replication in vitro. Ubiquitin-proteasome pathway plays an important role in HBV life cycle and could be a promising therapeutic target for anti-HBV drugs.

MG132-mediated inhibition of the ubiquitin–proteasome pathway ameliorates cancer cachexia

PurposeTo evaluate the effect of proteasome inhibitor MG132 in cancer cachexia and to delineate the molecular mechanism underlying.MethodsWe established an experimental cancer cachexia model by subcutaneously implanting colon 26 cells into the armpits of BALB/c mice. Following administration of MG132 at various time points, body weight, food intake, gastrocnemius muscle weight, spontaneous activity and survival of tumor-bearing mice were examined along with tumor growth. Moreover, cachectic markers including glucose, triglyceride, albumin and total proteins as well as levels of the proinflammatory cytokines TNF-α and IL-6 in serum and gastrocnemius tissue were measured. Finally, mRNA and protein levels of p65, IκBα, and ubiquitin E3 ligases MuRF1 and MAFbx in gastrocnemius muscle were assessed.ResultsMG132 treatment significantly alleviated cancer cachexia as demonstrated by attenuated weight loss, altered carbohydrate metabolism and muscle atrophy and increased spontaneous activity and survival time of tumor-bearing mice. MG132 reduced tumor growth and the levels of TNF-α and IL-6 in serum and gastrocnemius tissue. NF-κB, MuRF1 and MAFbx were also inhibited by MG132. Unexpectedly, MG132 was more efficient when administrated during the early stages of cachexia. MG132 had no effect on food intake of tumor-bearing mice.ConclusionOur results demonstrate that MG132-induced inhibition of the ubiquitin–proteasome pathway in cancer cachexia decreased the activity of NF-κB and the degradation of IκBα, and reduced the levels of TNF-α and IL-6 in serum and gastrocnemius tissue, accompanied by downregulation of MuRF1 and MAFbx. These data suggest that MG132 is a potential therapeutic and preventive agent for cancer cachexia.

The effect of proteasome inhibitor MG132 on experimental inflammatory bowel disease

Immunoproteasome up-regulation enhances the processing of nuclear factor-kappaB (NF-kappaB) and degradation of IkappaBalpha, which correlates with increased amounts of NF-kappaB in the various cells. Aberrant activation of NF-kappaB is involved in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to elucidate the effect of proteasome inhibitor MG132 on experimental IBD. We investigated the effects of MG132 on intestinal inflammation and epithelial regeneration in both interleukin-10-deficient (IL-10(-/-)) mice and mice with dextran sulphate sodium (DSS)-induced colitis. Body weight, histological findings and tumour necrosis factor (TNF)-alpha mRNA expression, epithelial cell proliferation and NF-kappaB p65 activity in colonic tissues were examined. The effects of MG132 on cell proliferation, migration and multiple drug resistance 1 (MDR1) gene expression were determined in vitro. MG132 ameliorated intestinal inflammation of IL-10(-/-) mice by decreasing TNF-alpha mRNA expression in the colonic tissues, which was associated with suppression of NF-kappaB activation, and reduced significantly the number of Ki-67-positive intestinal epithelial cells. On the other hand, MG132 did not reduce intestinal inflammation in mice with DSS-induced colitis, and delayed significantly the recovery of body weight and epithelial regeneration. MG132 also suppressed significantly epithelial cell proliferation, cell migration and MDR1 gene expression in vitro. Proteasome inhibition reduces T cell-mediated intestinal inflammation, but may interrupt both epithelial regeneration and barrier function of colonic mucosa. Optimal use of proteasome inhibitor should be kept in mind when we consider its clinical application for patients with IBD.

The apoptosis of bovine lens epithelial cells induced by proteasome inhibitor MG132

To investigate the effect of proteasome inhibitor MG132 on the apoptosis of bovine lens epithelial cells (BLECs), the cells were treated with MG132 at different concentrations for 12, 24 and 36 h. The cell viability was analyzed by MTT assay and the effect of MG132 on the apoptosis of BLECs was assessed by flow cytometry (FCM). The results showed that after treatment for the same period, the inhibitory effect of MG132 on BLECs proliferation was enhanced with the increment of the concentration of MG132 (0, 2, 5, 10, mumol/L) (P<0.05). The 50% inhibiting concentration (IC(50)) was 2.03 mumol/L when the BLECs were treated with MG132 for 36 h. MG132 also induced the apoptosis of BLECs obviously. FCM showed that the apoptosis index of the cells treated by MG132 at 2 mumol/L for 12 h was (20.24+/-1.51)%, and that of the control was (0.98+/-0.20)% respectively (P<0.01, n=3). It was concluded that MG132 could lead to apoptosis of BLECs. The decrease of proteasome activity may play an important role in the formation and development of cataract.

Proteasome inhibitor MG-132 modifies coxsackie and adenovirus receptor expression in colon cancer cell line lovo

The efficacy of adenovirus vector-based cancer gene therapy is controversial. Its uptake by cells in many cases requires the major receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR). Low transduction is believed to be one of the main barriers as the expression of CAR on tumor cells is frequently reduced. Increasing CAR expression on tumor cells thus offers a promising opportunity for more effective adenovirus based treatment. Expression of CAR in 62 cases of colon tumor specimens were examined with immunohistochemistry. To modify the CAR expression, the effects of proteasome inhibitor MG132 on CAR expression of colon cancer cell lines were determined by flow cytometry, RT-PCR, and western blot. To evaluate adenovirus transfer, we further used rAd.EGFP, rAd.p53, and oncolytic adenovirus to infect target cells. The CAR expression was significantly decreased in colon carcinomas, both in primary tumors and lymphonode metastasis. Though the deregulation of CAR occurred in early disease and showed no relationship with TNM stage, when primary tumors are more than 5 cm in diameter, this deregulation becomes more frequent. More importantly, proteasome inhibitor MG-132 could enhance CAR expression in colon carcinoma cell line lovo, accompanied with enhanced adenovirus transfer, target gene expression, and oncolysis. These data provide a rational basis for evaluation of CAR expression in tumors and pretreatment with CAR conditioner prior to adenovirus vector-based gene therapy.

Proteasome inhibitor ameliorates severe acute pancreatitis and associated lung injury of rats

To observe the effect of proteasome inhibitor MG-132 on severe acute pancreatitis (SAP) and associated lung injury of rats. Male adult SD rats were randomly divided into SAP group, sham-operation group, and MG-132 treatment group. A model of SAP was established by injection of 5% sodium taurocholate into the biliary-pancreatic duct of rats. The MG-132 group was pretreated with 10 mg/kg MG-132 intraperitoneally (ip) 30 min before the induction of pancreatitis. The changes in serum amylase, myeloperoxidase (MPO) activity of pancreatic and pulmonary tissue were measured. The TNF-alpha level in pancreatic cytosolic fractions was assayed with an enzyme-linked immunosorbent assay (ELISA) kit. Meanwhile, the pathological changes in both pancreatic and pulmonary tissues were also observed. MG-132 significantly decreased serum amylase, pancreatic weight/body ratio, pancreatic TNF-alpha level, pancreatic and pulmonary MPO activity (P < 0.05). Histopathological examinations revealed that pancreatic and pulmonary samples from rats pretreated with MG-132 demonstrated milder edema, cellular damage, and inflammatory activity (P < 0.05). The proteasome inhibitor MG-132 shows a protective effect on severe acute pancreatitis and associated lung injury of rats.

Proteasome inhibitor MG-132 enhances whole-body protein turnover in rat

Proteasome inhibitors are novel therapeutic agents which may be used in treatment of cancer and other severe disorders. We studied the effect of proteasome inhibitor MG-132 on protein and amino acid metabolism. In MG-132-treated rats we observed a significant decrease in proteasome-dependent proteolysis in skeletal muscle and an increase in whole-body protein turnover (i.e., increase in whole-body proteolysis and protein synthesis). Proteasome-dependent proteolysis was activated in the liver and kidney, protein synthesis increased in skeletal muscle, liver, and kidney. Insignificant changes were found in jejunum and colon. MG-132 administration induced a significant increase in concentration of several amino acids in blood plasma and their decrease in jejunum and colon. We conclude that administration of MG-132 affects both protein anabolic and protein catabolic pathways via the direct effect on proteasome-dependent proteolysis and indirect effect on proteolysis and protein synthesis via unidentified mediators.

PKCα is involved in phorbol ester TPA-mediated stabilization of p14 ARF

We generated A21-13 cells expressing p14 ARF in the presence of doxycycline in order to examine the stability of p14 ARF protein. The effects of proteasome inhibitor MG132 on p14 ARF protein stabilization were detectable using our experimental procedure. Introduction of mutant p53 did not affect MG132-mediated p14 ARF protein stabilization. We found that phorbol ester TPA (12- o-tetradecanoyl-phorbol 13-acetate) stabilized p14 ARF protein and that p53 status had no effect on TPA-mediated stabilization. TPA-mediated stabilization was abolished by staurosporine but not by lovastatin or U0126. We further investigated which isoforms of PKC were involved in TPA-mediated p14 ARF stabilization using short-interference RNA. Knockdown of PKCα, but not PKCδ, attenuated TPA-mediated p14 ARF stabilization. These findings suggest that PKCα is involved in TPA-mediated stabilization of p14 ARF protein, and this effect of TPA was not affected by the Ras/MAPK pathway or p53 status. Our results are indicative of a novel role of PKC in p14 ARF protein stability.

Effect of proteasome inhibitor MG132 on in vitro maturation of pig oocytes.

The present study aimed to demonstrate the dependence of meiotic maturation in pig oocytes on the activity of the protease complex proteasome. The proteasome inhibitor MG132 blocked the exit of maturing pig oocytes from metaphase I stage. Seventy-five per cent of the oocytes were blocked at metaphase I when they were cultured with 10 microM MG132. The blocking effect of MG132 was expressed only when the oocytes were exposed to an inhibitor before the 18th hour of in vitro culture. The effects of MG132 are fully reversible. However, a significant proportion of oocytes (46%) cultured for 48 h in MG132-supplemented medium and then for 24 h in MG132-free medium did not block meiosis at the stage of metaphase II and underwent spontaneous parthenogenetic activation. On the basis of our data we can conclude that exit from the metaphase I stage of meiosis is proteasome-dependent in pig oocytes matured in vitro. On the other hand, our data also indicate that other proteasome-independent events are involved in regulating the exit from metaphase I.

Anti-neoplastic effects of proteasome inhibitor MG-132

Anti-neoplastic effects of proteasome inhibitor MG-132

Dyclonine and alverine citrate enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells

Proteasome is an important target in cancer therapy. To enhance the efficacy of proteasome inhibitors is a challenging task due to the paucity of understanding the functional interactions between proteasome and other cellular pathways in mammalian cells. Taking advantage of the knowledge gained from Saccharomyces cerevisiae, we show that dyclonine and alverine citrate, the major components of two over-the-counter medicines, can substantially enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells. This study also highlights an important yeast genetic approach to identification of potential therapeutics that can be used for combination therapy with proteasome inhibitors.