M4 and M5 acute myeloid leukaemias display a high sensitivity to Bortezomib-mediated apoptosis

The present study explored the sensitivity of leukaemic blasts derived from 30 acute myeloid leukaemia (AML) patients to Bortezomib. Bortezomib induced apoptosis of primary AML blasts: 18/30 AMLs were clearly sensitive to the proapoptotic effects of Bortezomib, while the remaining cases were moderately sensitive to this molecule. The addition of tumour necrosis factor-related-apoptosis-inducing ligand, when used alone, did not induce apoptosis of AML blasts and further potentiated the cytotoxic effects of Bortezomib. The majority of AMLs sensitive to Bortezomib showed immunophenotypic features of the M4 and M5 French-American-British classification subtypes and displayed myelomonocytic features. All AMLs with mutated FLT3 were in the Bortezomib-sensitive group. Biochemical studies showed that: (i) Bortezomib activated caspase-8 and caspase-3 and decreased cellular FLICE [Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme]-inhibitory protein (c-FLIP) levels in AML blasts; (ii) high c-FLIP levels in AML blasts were associated with low Bortezomib sensitivity. Finally, analysis of the effects of Bortezomib on leukaemic cells displaying high aldehyde dehydrogenase activity suggested that this drug induced in vitro killing of leukaemic stem cells. The findings of the present study, further support the development of Bortezomib as an anti-leukaemic drug and provide simple tools to predict the sensitivity of AML cells to this drug.     

NSAIDs enhance proteasomic degradation of survivin, a mechanism of gastric epithelial cell injury and apoptosis

NSAIDs cause severe gastrointestinal injury, in part by suppressing survivin, an inhibitor of apoptosis protein, both in cultured gastric epithelial cells and in human and rat gastric mucosa. The mechanism(s) of survivin down-regulation by NSAIDs is unclear. In this study, we examined whether NSAID treatment decreases survivin mRNA expression and/or enhances degradation of survivin protein via ubiquitin proteasome system in rat gastric mucosal, RGM-1 cells, and whether survivin overexpression prevents indomethacin-induced cell injury and apoptosis. Effects of indomethacin on survivin mRNA expression, survivin protein half-life and ubiquitination were examined in RGM-1 cells. Proteasome inhibitors were utilized to prevent indomethacin-induced survivin protein degradation in RGM-1 cells. The effects of stable overexpression of survivin on indomethacin-induced RGM-1 cell injury and apoptosis were examined. Results showed: (1) Indomethacin treatment did not alter survivin mRNA expression, but significantly reduced survivin protein half-life from 1.5h to approximately 1h and increased survivin ubiquitination. (2) Inhibition of ubiquitin proteasome prolonged survivin protein half-life to over 2h and inhibited indomethacin-induced survivin degradation. (3) Overexpression of survivin significantly reduced indomethacin-induced cell injury and apoptosis. In conclusion, indomethacin treatment enhances degradation of survivin via the ubiquitin proteasome machinery in RGM-1 cells, and maintenance of survivin levels is important for prevention of gastric epithelial cell injury and apoptosis.     

The pattern of neuronal loss and survival may reflect differential expression of proteasome activators in Parkinson's disease

The basis of neuronal vulnerability, degeneration, and sparing in PD are unknown, but there is increasing evidence to suggest that the ubiquitin‐proteasome system (UPS) plays an important role in the pathogenesis of the disorder. In this study, we employed an immunocytochemical approach to determine if the differential expression of key UPS components in various brain regions and cells might underlie the pattern of neuronal degeneration and survival seen in PD. We showed that the ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and 26/20S proteasome α‐ and β‐subunits, are abundantly expressed in the substantia nigra pars compacta (SNc) and in cultured dopaminergic neurons. Although the proteasome activator PA700 is expressed in the medial SNc, levels are low in the lateral region, and expression of the other proteasome activator, PA28, is near absent in the entire SNc. PA28 (but not PA700) was found to be poorly expressed in noradrenergic neurons in the locus coeruleus (LC) compared with adjacent cells in the mesencephalic nucleus. PA700 and PA28 are also poorly expressed in dopaminergic neurons compared with other cell types in culture. Inhibition of proteasomal function, generation of misfolded proteins, induction of oxidative stress or impairment of mitochondrial complex I activity, caused a compensatory upregulation in PA700 and PA28 in a variety of cells but not in dopaminergic neurons in culture. These findings are consistent with the demonstration that, in sporadic PD, proteasomal activity and levels of PA700/PA28 are reduced in the SNc butare markedly upregulated in regions/cells that are spared from the neurodegenerative process. Thus, the differential distribution and activity of proteasome activations could play a significant role in the pathogenesis of PD. Synapse 64:241–250, 2010. © 2009 Wiley‐Liss, Inc.     

The proteasome inhibitor bortezomib stimulates osteoblastic differentiation of human osteoblast precursors via upregulation of vitamin D receptor signalling

Interactions of myeloma cells with the bone marrow microenvironment lead to enhanced osteoclast recruitment and impaired osteoblast activity. Recent evidence revealed that the proteasome inhibitor bortezomib stimulates osteoblast differentiation, but the mechanisms are not fully elucidated. We hypothesised that bortezomib could influence osteoblastic differentiation via alteration of vitamin D signalling by blocking the proteasomal degradation of the vitamin D receptor (VDR). This is of clinical importance, as a high rate of vitamin D deficiency was reported in patients with myeloma. We performed cocultures of primary human mesenchymal stem cells (hMSCs) and human osteoblasts (hOBs) with myeloma cells, which resulted in an inhibition of the vitamin D‐dependent differentiation of osteoblast precursors. Treatment with bortezomib led to a moderate increase in osteoblastic differentiation markers in hMSCs and hOBs. Importantly, this effect could be strikingly increased when vitamin D was added. Bortezomib led to enhanced nuclear VDR protein levels in hMSCs. Primary hMSCs transfected with a VDR luciferase reporter construct showed a strong increase in VDR signalling with bortezomib. In summary, stimulation of VDR signalling is a mechanism for the bortezomib‐induced stimulation of osteoblastic differentiation. The data suggest that supplementation of vitamin D in patients with myeloma treated with bortezomib is crucial for optimal bone formation.     

Targeting apoptosis in solid tumors: the role of bortezomib from preclinical to clinical evidence

The ubiquitin–proteasome pathway is the main proteolytic system present in the nucleus and cytoplasm of all eukaryotic cells. Apoptosis activation induced by ubiquitin–proteasome pathway inhibition makes the proteasome a new target of anticancer therapy. Bortezomib is the first proteasome inhibitor to be approved by the US FDA; in 2003 as a third line and in 2005 as a second line therapy for the treatment of multiple myeloma only. This review focuses on the use of bortezomib, not only in its therapeutic role but also, more specifically, in its biologic role and discusses the most recent applications of the drug in solid tumors, both at a preclinical and clinical level.     

泛素羧基末端水解酶-1抑制剂对多巴胺能神经元的神经毒性作用

Objective To investigate the neurotoxic effects ofLDN-57444, a specific ubiquitin C-termiual hydrolase L1 (UCH-L1) inhibitor, on dopaminergic neurons and the possible mechanism. Methods The viability of SK-N-SH cells exposed to 5, 10, 25, 50, 75 or 100 μmol/L LDN-57444 for 24 h was assessed using MTT assay, and the cell apoptosis was detected with Hoechst staining. Western blot was performed to identify the expressions of UCH-L1 protein, ubiquitin monomer and polyubiquitinated proteins, and the activity of the ubiquitin-proteasome system (UPS) was evaluated with fluorometry. Results After exposure to UCH-LI inhibitor for 24 h, the cell process-like structures of SK-N-SH cells diminished, and the cell body shrank and became spherical. Exposure to LDN-57444 resulted in concentration-dependent reduction of the cell viability, and the reduction became statistically significant following the exposure to 50 μmol/L LDN-57444, as compared with that in the control group (P<0.05). The exposure also resulted in obvious cell apoptosis as shown by nuclear fragmentation and presence of the apoptotie bodies. Western blot detected no obvious changes in UCH-L1 protein expression but identified reduced ubiquitin monomer and increased polyubiquitinated protein expression in the cells. Fluorometry showed reduced activity of UPS in the exposed cells. Conclusion UCH-L1 inhibitor produces neurotoxicity to dopaminergie neurons and induces cell apoptosis possibly as the result of impaired UPS activity and intracellular accumulation of polyubiquitinated proteins following the exposure.     

Prolonged toxicokinetics and toxicodynamics of paraquat in mouse brain

BACKGROUND: Paraquat (PQ) has been implicated as a risk factor for the Parkinson disease phenotype (PDP) in humans and mice using epidemiologic or experimental approaches. The toxicokinetics (TK) and toxicodynamics (TD) of PQ in the brain are not well understood. OBJECTIVES: The TK and TD of PQ in brain were measured after single or repeated doses. METHODS: Brain regions were analyzed for PQ levels, amount of lipid peroxidation, and functional activity of the 20S proteasome. RESULTS: Paraquat (10 mg/kg, ip) was found to be persistent in mouse ventral midbrain (VM) with an apparent half-life of approximately 28 days and was cumulative with a linear pattern between one and five doses. PQ was also absorbed orally with a concentration in brain rising linearly after single doses between 10 and 50 mg/kg. The level of tissue lipid peroxides (LPO) was differentially elevated in three regions, being highest in VM, lower in striatum (STR), and least in frontal cortex (FCtx), with the earliest significant elevation detected at 1 day. An elevated level of LPO was still present in VM after 28 days. Despite the cumulative tissue levels of PQ after one, three, and five doses, the level of LPO was not further increased. The activity of the 20S proteasome in the striatum was altered after a single dose and reduced after five doses. CONCLUSIONS: These data have implications for PQ as a risk factor in humans and in rodent models of the PDP.     

Keratin inclusions alter cytosolic protein localization in hepatocytes

Aim:  Mallory bodies have been observed in various liver diseases, however, the precise mechanism and significance of these structures have yet to be determined.
Methods:  Previously we reported on the redistribution of cytosolic proteins to keratin inclusions in mutant keratin 18-transfected cells. In this study, we treated green fluorescent protein-tagged wild-type keratin 18-transfected cells with several proteasome inhibitors and performed immunofluorescent analyses.
Results:  Proteasome inhibitors induced intracellular keratin inclusions, and desmoplakin, zonula occludens-1 and β-catenin were relocated to keratin inclusions, while theintegral membrane proteins were intact. The cytosolic proteins, 14-3-3 ζ protein and glucose-6-phosphate dehydrogenase were also relocated to inclusions. Moreover, E-cadherin, a basolateral membrane protein, was present on both the apical and basolateral domains in inclusion-containing cells.
Conclusion:  These data are identical to those in the mutant keratin 18 transfection study and suggest that keratin inclusions induced by different treatments affect localization of various cytosolic components, which may influence cellular functions performed by these proteins.