蛋白酶体抑制剂MG132对PC12细胞的增殖抑制作用

目的:了解蛋白酶体抑制剂MG132对多巴胺能细胞PC12增殖和凋亡的影响. 方法:用不同浓度的MG132处理PC12细胞3 d,通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)微量比色法检测MG132对PC12细胞增殖的影响,通过姬姆萨染色法和流式细胞术检测MG132对PC12细胞凋亡的影响. 结果:在作用时间相同(3 d)的条件下,随着MG132浓度的增高(0, 1, 2.5, 5, 10, 20 μmol/L),对PC12细胞增殖的抑制作用逐渐增强. 当MG132浓度达到2.5 μmol/L时,对PC12细胞的抑制率超过40%, IC50＝3.78 μmol/L. 同时,MG132能够明显诱导PC12细胞凋亡:姬姆萨染色显示,浓度为2.5 μmol/L的MG132作用3 d,细胞凋亡率为24.7%,而对照为1.3%;流式细胞术分析结果表明,浓度为2.5 μmol/L的MG132作用3 d,细胞凋亡率为25.6%,而对照为0%. 结论:蛋白酶体抑制剂MG132能够抑制多巴胺能细胞PC12的增殖并诱导细胞凋亡,蛋白酶体活性的丧失可能影响到多巴胺能细胞的生存.     

LMP/TAP基因多态性的研究进展

低分子量蛋白酶体和抗原处理相关转运体与内源性抗原处理、呈递有关,被认为与肿瘤发生、病毒清除密切相关。有研究表明,LMP/TAP基因具有多态性。目前LMP/TAP基因与疾病的相关性,以及其多态性与疾病的遗传易感关联日益受到广泛的关注,认为它们可能为独立于HLA基因之处的另一自身免疫疾病的易感/保护基因。     

过氧化氢对人视网膜色素上皮细胞ARPE-19中20S蛋白酶体表达的上调作用

目的:探讨过氧化氢诱导的氧化应激对人视网膜色素上皮细胞ARPE-19中20S蛋白酶体表达的影响。方法:分别选择0,100,300μmol/L过氧化氢作用ARPE-19细胞4h后收集样本,Western blot方法检测细胞中20S蛋白酶体表达水平,细胞免疫荧光方法检测20S蛋白酶体在细胞中的分布。结果:100和300μmol/L过氧化氢溶液处理的细胞中20S蛋白酶体的表达和对照组相比明显增加(100μmol/L过氧化氢处理组vs0和300μmol/L过氧化氢处理组,P<0.05),并呈现剂量依赖性。正常培养情况下,细胞内20S蛋白酶体在核周微量表达,100和300μmol/L过氧化氢溶液处理的细胞内20S蛋白酶体和对照组相比明显增加,其分布集中在核内及核膜周围。结论:过氧化氢对ARPE-19的20S蛋白酶体表达有上调作用。     

20S proteasome inhibitory activity of flavonoids isolated from Spatholobus suberectus

The promising biological role of the ubiquitin proteasome pathway (UPP) in cancer therapy has recently emerged. Inhibition of proteasome has been proposed as a new therapeutic target for treatment of cancer. Bioassay‐guided fractionation of a MeOH extract of the stems of Spatholobus suberectus resulted in seven compounds, liquiritigenin (1), isoliquiritigenin (2), genistein (3), daidzein (4), medicarpin (5), 7‐hydroxyflavanone (6) and formononetin (7), which were evaluated for the first time for their inhibitory effect on 20S proteasome. Among the isolated compounds, 2, 3 and 6 exhibited inhibitory activities on human 20S proteasome with IC₅₀ values of 4.88 ± 1.5, 9.26 ± 1.2 and 5.21 ± 1.5 µm , respectively. Copyright © 2010 John Wiley & Sons, Ltd.     

Threonine peptidases as drug targets against malaria

Introduction: Malaria is caused by the intracellular parasite Plasmodium falciparum. Although numerous therapies are available to fight the disease, the number of pharmacophores is small, and constant development of novel therapies, especially with new targets, is desirable to fight developing resistance against presently prescribed drugs.

Areas covered: This review discusses research on plasmodial threonine peptidases along with recent advances in proteasome inhibitor development.

Expert opinion: While PfHslV is an attractive drug target in malaria, more investigation is required to clarify its functional role in the parasite. More efforts should also be invested in assessing the plasmodial proteasome as a drug target. The few papers investigating the effect of proteasome inhibitors on different stages of the life cycle point towards important roles not only during asexual, but also in hepatic and sexual stages, in humans and the mosquito. If this holds true, this is a key argument to further develop proteasome inhibitors for use against malaria.     

Mechanisms of MHC class I-restricted antigen presentation

The vertebrate immune system monitors whether an organism is invaded by pathogens. Therefore, each cell has to prove itself as healthy. This is achieved by presenting fragments of intracellular protein degradation products on the surface, i.e., each cell displays peptides on specialised proteins known as major histocompatibility complex (MHC) class I proteins. A displayed peptide has to pass certain constraints before its presentation: It has to be excised out of a protein, translocated into the endoplasmic reticulum (ER) and fit into the binding groove of a MHC molecule. In theory, alteration of the cellular protein profile by mutation or infection should force pathogen-specific T-cells to take action via recognition of foreign peptide bound to MHC class I molecules on the cell surface. Unfortunately, pathogens and tumours have evolved many ways to affect antigen presentation and to escape from immune response. Understanding the exact mechanisms of antigen presentation, i.e., protein cleavage and peptide binding by MHC molecules, would allow their manipulation by drugs and lead to the re-establishment of the correct antigen presentation pathway. This review will summarise current knowledge of the mechanisms of antigen presentation and discuss putative targets for therapeutic treatment as well as for vaccination strategies.     

Targeting the proteasome pathway

Background: The ubiquitin–proteasome pathway functions as a main pathway in intracellular protein degradation and plays a vital role in almost all cellular events. Various inhibitors of this pathway have been developed for research purposes. The recent approval of bortezomib (PS-341, Velcade^®), a proteasome inhibitor, for the treatment of multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and other components of the ubiquitin–proteasome pathway. Objectives: We review the current understanding of the ubiquitin–proteasome pathway and inhibitors targeting this pathway, including proteasome inhibitors, as candidate drugs for chemical therapy. Methods: Preclinical and clinical data for inhibitors of the proteasome and the ubiquitin–proteasome pathway are discussed. Conclusions: The proteasome and other members in the ubiquitin–proteasome pathway have emerged as novel therapeutic targets.     

Targeting protein aggregation in neurodegeneration – lessons from polyglutamine disorders

Polyglutamine diseases, such as Huntington’s disease, are among the most common inherited neurodegenerative disorders. They share salient clinical and pathological features with major sporadic neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and amyotropic lateral sclerosis. Over the last decade, protein aggregation has emerged as a common pathological hallmark in neurodegenerative diseases and has, therefore, attracted considerable attention as a likely shared therapeutic target. Because of their clearly defined molecular genetic basis, polyglutamine diseases have allowed researchers to dissect the relationship between neurodegeneration and protein aggregation. In this review, the authors discuss recent progress in understanding polyglutamine-mediated neurotoxicity, and discuss the most promising therapeutic strategies being developed in the polyglutamine diseases and related neurodegenerative disorders.     

Investigational drugs for head and neck cancer

Introduction: In the treatment of advanced/metastatic head and neck cancer (HNC), resistance to chemotherapy and to anti-EGFR agents remains a major issue, and new molecular drugs are eagerly awaited. Over the last decade, knowledge of the genetic landscape of HNC has rapidly grown. However, no tailored therapeutic intervention targeting HNC molecular abnormalities is currently available outside from clinical trials.

Areas covered: In this review, the authors analyze new drugs in the HNC setting which have been investigated in recently published trials or are currently being investigated. The article excludes strategies directed towards the EGFR pathway and antivascular agents.

Expert opinion: Agents acting on the PI3K axis have a strong biological rationale and show the preliminary signs of activity, in particular when combined with other agents. There is limited clinical data of the other discussed pathways; the CMET/HGF pathway as a possible modulator of anti-EGFR drug sensitivity and agents directed towards MEK, WEE-1, NOTCH represent new interesting approaches to HNC. It is of the utmost importance to try and incorporate the molecular dissection of the tumor profiles in clinical trials with such agents. Moreover, the mutational status of other cross-talking pathways should be assessed, since potential resistance mechanisms can be recognized and possibly overcome by a careful selection of patients and combination regimens. Immunotherapy represents a growing field in HNC and its wider application will impact on future therapeutic strategies, including the association with chemotherapy, targeted agents and radiation.     

Proteasome inhibitor inhibits proliferation and induces apoptosis in renal interstitial fibroblasts

BackgroundThe ubiquitin proteasome pathway plays a pivotal role in controlling cell proliferation, apoptosis and differentiation in a variety of normal and tumor cells. This study aimed to investigate the role of a proteasome inhibitor on proliferation, apoptosis and related proteins in renal interstitial fibroblasts (NRK-49F).MethodsNRK-49F cells were induced using transforming growth factor-β1 (TGF-β1) and pretreated with the proteasome inhibitor MG-132. Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The cell cycle and apoptosis were analyzed using flow cytometry. Apoptosis was also analyzed using a DNAladder. The protein expression of p53, p27, p21, caspase-3, Bcl-2 and Bax was examined using western blots.ResultsThe results showed that TGF-β1 (5 ng/ml) can stimulate the proliferation of NRK-49F cells.MG-132 (0.25–5 μM) inhibited TGF-β1-induced proliferation in a dose-dependent manner through G1-arrest; TGF-β1 alone did not induce apoptosis (3.8 ± 0.4% vs. 4.7 ± 1.6%). However, pretreatment with MG-132 significantly induced apoptosis in TGF-β1-stimulated NRK-49F cells in a dosedependentmanner. AtypicalDNAladderwas also confirmed in these two groups.Western blot analysis showed thatMG-132 activated p53, p21, caspase-3 and Bax, and inhibited Bcl-2 in a dose-dependent manner, while p27 expression remained unchanged.ConclusionsA proteasome inhibitor inhibited proliferation and induced apoptosis in renal interstitial fibroblasts stimulated by TGF-β1. The mechanism may relate to the p53, p21, caspase-3, Bcl-2 and Bax pathways. Our results suggest that a proteasome inhibitor could be a new strategy to treat renal interstitial fibrosis.