Lactacystin

The Streptomyces sp.

Lactacystin is a specific and an irreversible inhibitor of proteasome with IC50 value of 4.8 μM [1].

Lactacystin binds to the catalytic subunits of the 20 S proteasome and inhibits all the three peptidase activities of the proteasome, chymotrypsin-like, trypsin-like and caspase-like. With this feature, lactacystin is used to study the role of the proteasome. In Neuro-2a cells, lactacystin treatment resulted in the induction of neurite outgrowth. It caused a transient increase in cAMP levels and the bipolar morphology of the cells. Lactacystin also inhibited cell cycle progression in MG-63 human osteosarcoma cells. Moreover, lactacystin was reported to have therapeutic effects on glioma cells. In rat C6 glioma cells, lactacystin significantly inhibited cell growth with IC50 value of about 10 μM. Besides that, in a mouse gliomaxenograft model, administration of lactacystin resulted in tumor size reduction at a dose of 1-5 μg / 20 g body weight [2, 3].

References:
1. Csizmadia V, Csizmadia E, Silverman L, et al. Effect of proteasome inhibitors with different chemical structures on the ubiquitin–proteasome system in vitro. Veterinary Pathology Online, 2010, 47(2): 358-367.
2. Fenteany G, Schreiber S L. Lactacystin, proteasome function, and cell fate. Journal of Biological Chemistry, 1998, 273(15): 8545-8548.
3. Wang H, Zhang S, Zhong J, et al. The proteasome inhibitor lactacystin exerts its therapeutic effects on glioma via apoptosis: An in vitro and in vivo study. Journal of International Medical Research, 2013, 41(1): 72-81.

Chronic L-DOPA treatment attenuates behavioral and biochemical deficits induced by unilateral lactacystin administration into the rat substantia nigra.[Pubmed:24361083]

Behav Brain Res. 2014 Mar 15;261:79-88.

The aim of the study was to determine whether the dopamine (DA) precursor l-DOPA attenuates parkinsonian-like symptoms produced by the ubiquitin-proteasome system inhibitor Lactacystin. Wistar rats were injected unilaterally with Lactacystin (2.5 mug/2 mul) or 6-OHDA (8 mug/2 mul) into the substantia nigra (SN) pars compacta. Four weeks after the lesion, the animals were treated chronically with l-DOPA (25 or 50 mg/kg) for two weeks. During l-DOPA treatment, the Lactacystin-treated rats were tested for catalepsy and forelimb asymmetry. Rotational behavior was evaluated after apomorphine (0.25 mg/kg) and l-DOPA in both PD models. After completion of experiments, the animals were killed and the levels of DA and its metabolites in the striatum and SN were assayed. We found that acute l-DOPA administration effectively decreased catalepsy and increased the use of the compromised forelimb in the cylinder test. However, the Lactacystin group did not respond to apomorphine or acute l-DOPA administration in the rotational test. Repeated l-DOPA treatment produced contralateral rotations in both PD models, but the number of rotations was much greater in the 6-OHDA-lesioned rats. Both toxins markedly (>90%) reduced the levels of DA and its metabolites in the striatum and SN, while l-DOPA diminished these decreases, especially in the SN. By demonstrating the efficacy of l-DOPA in several behavioral tests, our study confirms the usefulness of the Lactacystin lesion as a model of PD. However, marked differences in the rotational response to apomorphine and l-DOPA suggest different mechanisms of neurodegeneration evoked by Lactacystin and 6-OHDA.

Decreased behavioral response to intranigrally administered GABAA agonist muscimol in the lactacystin model of Parkinson's disease may result from partial lesion of nigral non-dopamine neurons: comparison to the classical neurotoxin 6-OHDA.[Pubmed:25655509]

Behav Brain Res. 2015 Apr 15;283:203-14.

Lactacystin is a selective UPS inhibitor recently used to destroy dopamine (DA) neurons in animal models of Parkinson's disease (PD). However, both in vitro and in vivo studies show discrepancies in terms of the sensitivity of non-DA neurons to its toxicity. Therefore, our study was aimed to examine the toxic effect of intranigral administration of Lactacystin on DA and non-DA neurons in the rat substantia nigra (SN), compared to the classic neurotoxin 6-OHDA. Tissue DA levels in the striatum and SN and GABA levels in the SN were also examined. Moreover, behavioral response of nigral GABAA receptors to locally administered muscimol was evaluated in these two PD models. We found that both Lactacystin and 6-OHDA induced a strong decrease in DA level in the lesioned striatum and SN but only Lactacystin slightly reduced GABA levels in the SN. A stereological analysis showed that both neurotoxins highly decreased the number of DA neurons in the SN, while only Lactacystin moderately reduced the number of non-DA ones. Finally, in the Lactacystin group, the number of contralateral rotations after intranigrally administrated muscimol was decreased in contrast to the increased response in the 6-OHDA model. Our study proves that, although Lactacystin is not a fully selective to DA neurons, these neurons are much more vulnerable to its toxicity. Partial lesion of nigral non-DA neurons in this model may explain the decreased behavioral response to the GABAA agonist muscimol.

Investigation the mechanism of the apoptosis induced by lactacystin in gastric cancer cells.[Pubmed:25541208]

Tumour Biol. 2015 May;36(5):3465-70.

The study aims to investigate the relationship between nuclear factor (nuclear factor kappa B (NF-kappaB)) viability and Lactacystin-mediated cell apoptosis in gastric cancer cells. Two gastric cancer cell lines (MKN28 and SGC7901) were treated with Lactacystin-a proteasome inhibitor for 24 h. The cell viability, toxicity, and death were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. DNA binding viability of NF-kappaB and caspase-3 viability were analyzed by ELISA; the expression of p65 NF-kappaB nuclear protein was detected by immunocytochemistry and Western blot. Lactacystin reduced DNA binding viability of NF-kappaB (t = 3.0,P = 0.013) and the NF-kappaB viability (compared to the 5, 10 mumol/L MKN28 cell (p53 mutant) line, P < 0.001) and the expression of p65 NF-kappaB nuclear protein decreased parallelled to concentrations of Lactacystin in MKN28 cell line, while without obvious effects on NF-kappaB viability in SGC7901 cell line (P = 0.381), while the viability of caspase-3 increased also along with the raising of Lactacystin concentrations (compared to control, 5 mumol/L: SGC7901 cell line P = 0.029, MKN28 cell line P < 0.001; 10 mumol/L: SGC7901 cell line, P < 0.001, MKN28 cell line, P < 0.001). It was concluded that Lactacystin had diversified killing effects on gastric cancer cells. The mechanism may be related to induce the apoptosis by downregulation of nuclear factor kappa B viability. There may be additional cell survival/death pathway in SGC7901 gastric cancer cells.

Differential protein profile of PC12 cells exposed to proteasomal inhibitor lactacystin.[Pubmed:24858133]

Neurosci Lett. 2014 Jul 11;575:25-30.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide and recent studies implicate a central role for ubiquitin-proteasome system (UPS) impairment in the etiopathogenesis of PD. To explore the possible role of UPS dysfunction in PD and the proteins involved, PC12 cells were treated with 10muM Lactacystin, a 20S proteasome inhibitor, for 24h. Lactacystin induced cell death and alpha-synuclein-positive inclusions in cytoplasm. Following two-dimensional difference in-gel electrophoresis (2-D DIGE) which was used to separate the cellular proteins, the proteins that were significantly altered were analyzed and identified. Proteomic study identified 6 differentially expressed proteins between Lactacystin-treated and control cells in this study. Four proteins (heat shock 70kDa protein 8, 78kDa glucose-regulated protein, serine proteinase inhibitor clade B member 6 and aldehyde reductase) were increased and 2 proteins (peripherin and tyrosine hydroxylase) were decreased following proteasomal inhibition. The results revealed that PC12 cells treated with 10muM Lactacystin for 24h could be used as a cellular model of PD. The proteins identified in the present indicate not only the damage of proteasomal inhibition to the cells but also the possible responses of the cells. These data show that proteomic study may provide information relevant to biological basis for PD and potential new treatment targets.

Neurorestoration induced by the HDAC inhibitor sodium valproate in the lactacystin model of Parkinson's is associated with histone acetylation and up-regulation of neurotrophic factors.[Pubmed:26040297]

Br J Pharmacol. 2015 Aug;172(16):4200-15.

BACKGROUND AND PURPOSE: Histone hypoacetylation is associated with Parkinson's disease (PD), due possibly to an imbalance in the activities of enzymes responsible for histone (de)acetylation; correction of which may be neuroprotective/neurorestorative. This hypothesis was tested using the anti-epileptic drug sodium valproate, a known histone deacetylase inhibitor (HDACI), utilizing a delayed-start study design in the Lactacystin rat model of PD. EXPERIMENTAL APPROACH: The irreversible proteasome inhibitor Lactacystin was unilaterally injected into the substantia nigra of Sprague-Dawley rats that subsequently received valproate for 28 days starting 7 days after Lactacystin lesioning. Longitudinal motor behavioural testing, structural MRI and post-mortem assessment of nigrostriatal integrity were used to track changes in this model of PD and quantify neuroprotection/restoration. Subsequent cellular and molecular analyses were performed to elucidate the mechanisms underlying valproate's effects. KEY RESULTS: Despite producing a distinct pattern of structural re-modelling in the healthy and Lactacystin-lesioned brain, delayed-start valproate administration induced dose-dependent neuroprotection/restoration against Lactacystin neurotoxicity, characterized by motor deficit alleviation, attenuation of morphological brain changes and restoration of dopaminergic neurons in the substantia nigra. Molecular analyses revealed that valproate alleviated Lactacystin-induced histone hypoacetylation and induced up-regulation of brain neurotrophic/neuroprotective factors. CONCLUSIONS AND IMPLICATIONS: The histone acetylation and up-regulation of neurotrophic/neuroprotective factors associated with valproate treatment culminate in a neuroprotective and neurorestorative phenotype in this animal model of PD. As valproate induced structural re-modelling of the brain, further research is required to determine whether valproate represents a viable candidate for disease treatment; however, the results suggest that HDACIs could hold potential as disease-modifying agents in PD.

Inhibition of NF-kappa B activity induces apoptosis in murine hepatocytes.[Pubmed:9327720]

Am J Pathol. 1997 Oct;151(4):891-6.

Recently we have demonstrated that inhibition of the nuclear factor (NF)-kappa B/Rel family of transcription factors induces apoptosis of B cells. Interestingly, mice lacking the relA gene encoding the p65 subunit of NF-kappa B exhibit embryonic lethality at days 15 to 16 of gestation, accompanied by massive destruction of liver via apoptosis. To determine whether p65 protein plays a direct role in hepatocyte survival, we employed a nontransformed murine hepatocyte (NMH) cell line, which maintains to a high degree the differentiated hepatocyte phenotype. Exponentially growing NMH cells were found to possess a constitutive level of functional classical (p50/p65) NF-kappa B as assayed by electrophoretic mobility shift analysis, antibody supershift, and transient transfection assays. Treatment of NMH cells with the proteasome inhibitor Lactacystin, which prevents degradation of the NF-kappa B inhibitor proteins I kappa B, induced apoptosis. Direct inhibition of the endogenous NF-kappa B activity by microinjection of NMH cells with purified specific inhibitor I kappa B-alpha-glutathione-S-transferase fusion protein or an antibody against p65 protein induced apoptosis. These findings suggest that expression of NF-kappa B/Rel activity in murine hepatocytes acts directly to promote survival of these cells and suggest that apoptosis observed in hepatocytes of mice lacking relA is a direct effect of p65 deficiency.

Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin.[Pubmed:7732382]

Science. 1995 May 5;268(5211):726-31.

Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces neurite outgrowth in a murine neuroblastoma cell line. Tritium-labeled Lactacystin was used to identify the 20S proteasome as its specific cellular target. Three distinct peptidase activities of this enzyme complex (trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activities) were inhibited by Lactacystin, the first two irreversibly and all at different rates. None of five other proteases were inhibited, and the ability of Lactacystin analogs to inhibit cell cycle progression and induce neurite outgrowth correlated with their ability to inhibit the proteasome. Lactacystin appears to modify covalently the highly conserved amino-terminal threonine of the mammalian proteasome subunit X (also called MB1), a close homolog of the LMP7 proteasome subunit encoded by the major histocompatibility complex. This threonine residue may therefore have a catalytic role, and subunit X/MB1 may be a core component of an amino-terminal-threonine protease activity of the proteasome.

Lactacystin, an antibiotic Streptomyces spp. metabolite, is a potent and selective proteasome inhibitor with an IC50 of 4.8 μM for 20S proteasome. Lactacystin also inhibits the lysosomal enzyme cathepsin A. Lactacystin inhibits cell growth and induces neurite outgrowth.

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