Exogenous Hsp70 delays senescence and improves cognitive function in aging mice

Molecular chaperone Heat Shock Protein 70 (Hsp70) plays an important protective role in various neurodegenerative disorders often associated with aging, but its activity and availability in neuronal tissue decrease with age. Here we explored the effects of intranasal administration of exogenous recombinant human Hsp70 (eHsp70) on lifespan and neurological parameters in middle-aged and old mice. Long-term administration of eHsp70 significantly enhanced the lifespan of animals of different age groups. Behavioral assessment after 5 and 9 mo of chronic eHsp70 administration demonstrated improved learning and memory in old mice. Likewise, the investigation of locomotor and exploratory activities after eHsp70 treatment demonstrated a significant therapeutic effect of this chaperone. Measurements of synaptophysin show that eHsp70 treatment in old mice resulted in larger synaptophysin-immunopositive areas and higher neuron density compared with control animals. Furthermore, eHsp70 treatment decreased accumulation of lipofuscin, an aging-related marker, in the brain and enhanced proteasome activity. The potential of eHsp70 intranasal treatment to protect synaptic machinery in old animals offers a unique pharmacological approach for various neurodegenerative disorders associated with human aging.Heat shock proteins (HSPs) serve to maintain intracellular protein homeostasis and have been shown to prevent protein damage during aging in different animal models (1). HSPs are required for longevity (2, 3), and a number of studies suggest that longer-lived species have higher constitutive expression of HSPs (4–7). Consistent with this finding, overexpression of HSP genes increased longevity in Drosophila, Caenorhabditis elegans, and vertebrates (1, 8, 9). Hsp70 is the major cytoprotective molecular chaperone with many different functions in the cell (10–12). Observations suggest that genetic variants of the Hsp70 family contribute to longevity in a wide range of organisms (9, 13, 14). Its defensive role in multiple neurodegenerative disorders (15, 16) can be explained by the multifaceted action of this protein. Indeed, the induction of Hsp70 has been shown to diminish oxidative stress damage (17, 18), suppress apoptosis (19), support proteasomal and lysosomal functioning (20), suppress toxic protein aggregation such as Aβ (21), inhibit proinflammatory signaling (22), and increase survival of endogenous neural progenitor cells (21). Notwithstanding Hsp70’s importance, its chaperone activity, as well as the rate of its synthesis and induction in response to stimuli, decreases in neurons with age (3, 6, 21, 22), suggesting that a pharmacological approach aiming to recover this chaperone in the aging brain may counter neurodegeneration.To our knowledge, the effect of exogenous HSPs on longevity has not yet been investigated. We previously showed that intranasally injected Hsp70 rapidly entered the brain of wild-type mice and was transported within neurons (23, 24). Furthermore, chronic Hsp70 treatment ameliorated multiple behavioral and molecular disturbances in two models of Alzheimer’s disease (AD)-type neurodegeneration (23). In this study, we explored the geroprotection potential of recombinant exogenous Hsp70 (eHsp70) in healthy mice. For all of the described experiments, we used highly pure LPS-free human eHsp70 (25), which rules out a possibility of confounding inflammatory responses associated with contaminated Hsp70. Our results demonstrate that long-term intranasal administration of human eHsp70 improves longevity and ameliorates aging-related behavioral deficits and molecular alterations to synaptic structure in the brains of aging mice.     

Changing trends in prognostic factors for patients with multiple myeloma after autologous stem cell transplantation during the immunomodulator drug/proteasome inhibitor era

We evaluated the clinical significance of prognostic factors including the International Staging System (ISS) and modified European Group for Blood and Marrow Transplantation response criteria in 1650 Japanese patients with multiple myeloma (MM) who underwent upfront single autologous stem cell transplantation (ASCT). We categorized patients into two treatment cohorts: pre‐novel agent era (1995–2006) and novel agent era (2008–2011). The combined percentage of pre‐ASCT complete response and very good partial response cases (463 of 988, 47%) significantly increased during the novel agent era compared with the pre‐novel agent era (164 of 527, 31%; P < 0.0001). The 2‐year overall survival (OS) rate of 87% during the novel agent era was a significant improvement relative to that of 82% during the pre‐novel agent era (P = 0.019). Although significant differences in OS were found among ISS stages during the pre‐novel agent era, no significant difference was observed between ISS I and II (P = 0.107) during the novel agent era. The factors independently associated with a superior OS were female gender (P = 0.002), a good performance status (P = 0.024), lower ISS (P < 0.001), pre‐ASCT response at least partial response (P < 0.001) and ASCT during the novel agent era (P = 0.017). These results indicate that the response rate and OS were significantly improved, and the ISS could not clearly stratify the prognoses of Japanese patients with MM who underwent upfront single ASCT during the novel agent era.     

Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug-resistant cancer cells

Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.     

PS-341对小鼠重症急性胰腺炎核因子-κB活化的影响

目的观察PS-341对重症急性胰腺炎(SAP)小鼠胰腺组织核因子-κB(NF-κB)活化的影响,阐明PS-341对SAP的作用机制。方法采用连续7次腹内注射雨蛙素(每次间隔1h)及脂多糖制作小鼠SAP模型,将60只ICR雌性小鼠随机分为PS-341治疗组(注射脂多糖前0.5h腹内注射0.5mg/kgPS-341),模型对照组(注射脂多糖前0.5h腹内注射50%DMSO),空白对照组(生理盐水制模)。最后一次注射雨蛙素后2h麻醉小鼠,取血检测血淀粉酶,光镜下观察小鼠胰腺和肺脏的病理形态,测定胰腺组织中髓过氧化物酶水平,实时荧光定量PCR测定胰腺组织中黏附分子(ICAM-1)的含量,免疫印迹法(Westernblotting)检测胰腺组织中IκBα的表达,电泳迁移率实验(EMSA)测定NF-κB活性。结果 PS-341治疗组和模型对照组比较,胰腺组织中I-κB降解减少,NF-κB活性显著下降,血清淀粉酶、MPO、胰腺组织中细胞黏附分子ICAM-1的表达都明显降低(P<0.05)。胰腺和肺脏组织病理学有明显的改善(P<0.05)。结论 PS-341通过抑制胰腺内NF-κB活化而抑制炎症反应。PS-341可能是治疗SAP的一个新措施。     

人肝癌细胞氧化应激模型中热休克蛋白90α对20S蛋白酶体功能的影响

目的观察人肝癌细胞氧化应激模型中细胞内Hsp90α和20S蛋白酶体的表达的变化以及Hsp90α表达下调后对20S蛋白酶体功能的影响;明确热休克蛋白90α对20s蛋白酶体功能的影响。方法以500μMH2O2建立氧化应激HepG2细胞模型;通过ROS探针法观察细胞在不同氧化应激条件下ROS的分布和含量,通过MTT比色法观察氧化应激对细胞存活的影响;以免疫印迹方法检测氧化应激对细胞内Hsp90α及20S蛋白酶体的合成的影响;以pSilencer2.1-U6neo载体,用电穿孔法建立稳定的Hsp90α抑制表达细胞株,通过检测糜蛋白酶活性观察氧化应激和降低Hsp90α表达对蛋白酶体活性的影响。结果氧化应激后细胞内ROS的含量增多,且分布从胞浆向胞核转移;随着作用时间延长,H2O2对HepG2细胞增殖的抑制程度增强;氧化应激导致胞内Hsp90α表达量先增加后降低,20S蛋白酶体表达量明显降低;siRNA干扰组蛋白酶体活性显著下降。结论氧化应激可影响细胞内Hsp90α和20S蛋白酶体的表达并改变其细胞内分布;氧化应激及降低Hsp90α的表达均可影响蛋白酶体的活性;Hsp90α对蛋白酶体功能维持起保护性的作用。     

Increased prosomal proteins in breast cancer cells and in neighboring normal cells in Parsi and non-Parsi populations

Monoclonal antibodies were raised against the prosomal proteins p27K, p29K and the prosome-like protein p21K (PLP) from normal breast glandular cells and from benign and malignant tumors. They were used to clarify the involvement of prosomes in tumorigenesis of human breast cells. Immunostaining showed the distribution of prosomes in the cytoplasm and nuclei of cells from European normal women (EN) and Parsi (P) and non-Parsi (NP) benign (B) and malignant (M) tissues. The flow-cytometry studies showed an increased mean percentage of labeled cells, particularly with anti-p27K prosomal protein mAb, in malignant tissue from NP compared to EN. The p21K data indicated an increase in the number of cells labeled by flow-cytometry studies in all groups compared to EN, while p29K-expressing cells were more abundand in NPN, PB, PM and NPM. Intergroup comparison showed that the mean percentage of cells labeled with anti-p27K and anti-p29K was significantly higher in PB than in NPB, as seen by flow cytometry, whereas there was a higher production or accumulation of the p21K (PLP) prosomal protein in NPM than in PM, as seen by immunostaining. By comparison with EN, there were also significantly more normal cells containing the three antigens in the apparently normal tissue in the neighborhood of the tumor in NPM, and more cells containing p21K in PM patients than in EN. As prosomes are involved in the cell differentiation and in the cell cycle control, the changes observed in breast tissues may be related to oncogenic processes. Furthermore, the modified subunit pattern of prosomes in cancer and, possibly, pre-cancer tissue may be of interest for diagnosis purposes. Received: 12 June 1997 / Accepted: 21 October 1997     

去泛素化酶与肿瘤相关性的研究进展

泛素-蛋白酶体途径是大多数细胞内蛋白质降解的主要方式,去泛素化酶（Deubiquitinating enzymes,DUBs）可以逆转这一过程,对其进行反向调节.此外,去泛素化酶可以稳定抑癌基因,从而抑制了肿瘤的发生.对去泛素化酶及其抑制剂的深入研究,为抗肿瘤药物的靶向治疗提供理论依据.本文将重点对去泛素化酶及其在肿瘤中的作用进行综述.     

Apolipoprotein B100 quality control and the regulation of hepatic very low density lipoprotein secretion

Apolipoprotein B （apoB） is the main protein component of very low density lipoprotein （VLDL） and is necessary for the assembly and secretion of these triglyceride （TG）-rich particles. Following release from the liver, VLDL is converted to low density lipoprotein （LDL） in the plasma and increased production of VLDL can therefore play a detrimental role in cardiovascular disease. Increasing evidence has helped to establish VLDL assembly as a target for the treatment of dyslipidemias. Multiple factors are involved in the folding of the apoB protein and the formation of a secretion-competent VLDL particle. Failed VLDL assembly can initiate quality control mechanisms in the hepatocyte that target apoB for degradation. ApoB is a substrate for endoplasmic reticulum associated degradation （ERAD） by the ubiquitin proteasome system and for autophagy. Efficient targeting and disposal of apoB is a regu- lated process that modulates VLDL secretion and partitioning of TG. Emerging evidence suggests that significant overlap exists between these degradative pathways. For example, the insulin-mediated targeting of apoB to autop- hagy and postprandial activation of the unfolded protein response （UPR） may employ the same cellular machinery and regulatory cues. Changes in the quality control mechanisms for apoB impact hepatic physiology and pathology states, including insulin resistance and fatty liver. Insulin signaling, lipid metabolism and the hepatic UPR may impact VLDL production, particularly during the postprandial state. In this review we summarize our current understanding of VLDL assembly, apoB degradation, quality control mechanisms and the role of these processes in liver physiology and in pathologic states.