补骨脂素通过炎症和Ras/Raf1介导对类风湿关节炎大鼠的保护作用

目的：研究补骨脂素减少炎症和抑制Ras/Raf1表达保护类风湿关节炎大鼠的机制。方法：将SD大鼠分成对照组、模型组（类风湿关节炎大鼠模型）、补骨脂素低剂量组（22 mg/kg补骨脂素处理）、补骨脂素中剂量组（44 mg/kg补骨脂素处理）、补骨脂素高剂量组（88 mg/kg补骨脂素处理）、补骨脂素高剂量+NC组（88 mg/kg补骨脂素+阴性对照慢病毒载体处理）、补骨脂素高剂量+Ras组（88 mg/kg补骨脂素+Ras过表达慢病毒载体处理）;采用Western Blotting检测大鼠的肾素-血管紧张素系统（RAS）、原癌基因丝苏氨酸蛋白激酶-1（Raf1）、成骨细胞特异性转录因子（Osx）、Ⅰ型胶原蛋白α1链（COL1A1）、骨钙素（OC）、基质金属蛋白酶-3（MMP-3）、基质金属蛋白酶-13（MMP-13）蛋白表达;检测关节肿胀度,HE染色对关节组织病理评分,ELISA法检测炎症介质白细胞介素-2（IL-2)、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6)水平。结果：与对照组比较,模型组大鼠关节组织中Ras、Raf1、MMP-3、MMP-13蛋白表达以及关节肿胀度、关节组织病理评分和IL-2、TNF-α、IL-6水平均升高,Osx、COL1A1、OC蛋白表达均降低（均P<0.05）;与模型组比较,补骨脂素低、中、高剂量组大鼠关节组织中除Osx、COL1A1、OC蛋白表达均升高外,其他指标均显著降低（均P<0.05）;与补骨脂素高剂量+NC组比较,补骨脂素高剂量+Ras组大鼠关节组织中Osx、COL1A1、OC蛋白表达均降低,而其他指标均显著升高（均P<0.05）。结论：补骨脂素通过减少炎症和抑制Ras/Raf1表达减轻类风湿关节炎大鼠关节损伤,促进软骨修复。     

Development of farsenyl transferase inhibitors as anticancer agents

Ras proteins belong to the monomeric GTPases familly. They control cell growth, differentiation, proliferation, and survival. Ras mutations are frequently found in human cancers and play a fundamental role in tumorigenesis. Ras requires localization to the plasma membrane to exert its oncogenic effects. This subcelllular localization is dependent of protein farnesylation which is a post translational modification catalysed by the farnesyl transferase enzyme. Farnesyl transferase Inhibitors (FTI) were then designed ten to twelve years ago to inhibit ras processing and consequently the growth of ras mutated tumor. Preclinical data show that FTIs inhibit cell proliferation and survival in vitro and in vivo of a wide range of cancer cell lines, many of which contain wild type ras suggesting that mutated Ras is not the only target of the FTIs effects. Four FTIs went then through clinical trials and three of then are still developed in the clinic. Phase I et II clinical trials confirmed a relevant antitumor activity and a low toxicity. Phase III clinical trials are currently undergoing for both solid and hematologic tumors. The expected results should allow to define the position of FTIs as anticancer drugs, particularly in combination with conventional chemotherapy, hormone therapy, radiotherapy or any other new targeted compound.     

Simvastatin induces a central hypotensive effect via Ras-mediated signalling to cause eNOS up-regulation

BACKGROUND AND PURPOSE

Clinical studies indicate that statins have a BP-lowering effect in hypercholesterolemic individuals with hypertension. Specifically, statins modulate BP through the up-regulation of endothelial NOS (eNOS) activation in the brain. However, the signalling mechanisms through which statins enhance eNOS activation remain unclear. Therefore, we examined the possible signalling pathways involved in statin-mediated BP regulation in the nucleus tractus solitarii (NTS).

EXPERIMENTAL APPROACH

To investigate the involvement of Ras and other signalling pathways in simvastatin-induced effects on BP, BP and renal sympathetic nerve activity (RSNA) were determined in spontaneously hypertensive rats (SHRs) before and after i.c.v. administration of simvastatin in the absence and presence of a Ras-specific inhibitor (farnesyl thiosalicylic acid, FTS), a geranylgeranyltransferase inhibitor (GGTI-2133), a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or a MAPK-ERK kinase (MEK) inhibitor (PD98059).

KEY RESULTS

FTS significantly attenuated the decrease in BP and increased NO evoked by simvastatin and reversed the decrease in basal RSNA induced by simvastatin. Immunoblotting and pharmacological studies showed that inhibition of Ras activity by FTS significantly abolished simvastatin-induced phosphorylation of ERK1/2, ribosomal protein S6 kinase (RSK), Akt and decreased eNOS phosphorylation. Likewise, administration of Akt and ERK1/2 signalling inhibitors, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98059, attenuated the reduction in BP evoked by simvastatin. Furthermore, i.c.v. simvastatin decreased Rac1 activation and the number of ROS-positive cells in the NTS.

CONCLUSIONS AND IMPLICATIONS

Simvastatin modulates central BP control in the NTS of SHRs by increasing Ras-mediated activation of the PI3K-Akt and ERK1/2-RSK signalling pathways, which then up-regulates eNOS activation.     

Brief overview of selected approaches in targeting pancreatic adenocarcinoma

Introduction: Pancreatic adenocarcinoma (PDAC) has the worst prognosis of any major malignancy, with 5-year survival painfully inadequate at under 5%. Investigators have struggled to target and exploit PDAC unique biology, failing to bring meaningful results from bench to bedside. Nonetheless, in recent years, several promising targets have emerged.

Areas covered: This review will discuss novel drug approaches in development for use in PDAC. The authors examine the continued efforts to target Kirsten rat sarcoma viral oncogene homolog (KRas), which have recently been successfully abated using novel small interfering RNA (siRNA) eluting devices. The authors also discuss other targets relevant to PDAC including those downstream of mutated KRas, such as MAPK kinase and phosphatidylinositol 3-kinase.

Expert opinion: Although studies into novel biomarkers and advanced imaging have highlighted the potential new avenues toward discovering localized tumors earlier, the current therapeutic options highlight the fact that PDAC is a highly metastatic and chemoresistant cancer that often must be fought with virulent, systemic therapies. Several newer approaches, including siRNA targeting of mutated KRas and enzymatic depletion of hyaluronan with PEGylated hyaluronidase are particularly exciting given their early stage results. Further research should help in elucidating their potential impact as therapeutic options.     

Approaches to Ras signaling modulation and treatment of Ras-dependent disorders: a patent review (2007 – present)

Introduction: Ras proteins are small GTPases molecular switches that cycle through two alternative conformational states, a GDP-bound inactive state and a GTP-bound active state. In the active state, Ras proteins interact with and modulate the activity of several downstream effectors regulating key cellular processes including proliferation, differentiation, survival, senescence, migration and metabolism. Activating mutations of RAS genes and of genes encoding Ras signaling members have a great incidence in proliferative disorders, such as cancer, immune and inflammatory diseases and developmental syndromes. Therefore, Ras and Ras signaling represent important clinical targets for the design and development of pharmaceutically active agents, including anticancer agents.

Areas covered: The authors summarize methods available to down-regulate the Ras pathway and review recent patents covering Ras signaling modulators, as well as methods designed to kill specifically cancer cells bearing activated RAS oncogene.

Expert opinion: Targeted therapy approach based on direct targeting of molecules specifically altered in Ras-dependent diseases is pursued with molecules that down-regulate expression or inhibit the biological function of mutant Ras or Ras signaling members. The low success rate in a clinical setting of molecules targeting activated members of the Ras pathway may require development of novel approaches, including combined and synthetic lethal therapies.     

Therapeutic potential of thiazolidinediones as anticancer agents

There has been great interest in the development of oncolytic viruses – viruses that selectively destroy tumour cells – as cancer therapeutics. Reovirus holds great promise as an anticancer therapy, not just because it is a wild type virus that inherently displays selective tumour cytotoxicity in cancers with active Ras signalling pathways but also because it results only in relatively benign infections with few minor symptoms. As many tumours have an activated Ras pathway, the potential for utilizing reovirus as an effective anticancer agent is substantial. The several challenges that need to be overcome in the development of oncolytic viruses as anticancer agents, including issues of systemic toxicity, tumour selectivity and immune response, are addressed in this review. Clinical studies with the objective of developing Reolysin (human reovirus serotype 3 Dearing) as a human cancer therapeutic are currently underway. The first human Phase I study with intravenous Reolysin has now been completed and further studies, including Phase I and II clinical trials using Reolysin alone and in combination with radiation or chemotherapy, delivered via local or systemic intravenous administration, have commenced.     

Ras as a therapeutic target in hematologic malignancies

The RAS gene product is normally a membrane-localized G protein (N-Ras, K-Ras and H-Ras) of 21 kDa classically described as a molecular off/on switch. It is inactive when bound to guanosine diphosphate and active when bound to GTP. When mutated, the gene produces an abnormal protein resistant to GTP hydrolysis by GTPase, resulting in a constitutively active GTP-bound protein that stimulates a critical network of signal transduction pathways that lead to cellular proliferation, survival and differentiation. At least three downstream effector pathways have been described, including Raf/MEK/ERK, PI3K/AKT and RalGDS, but they are not completely understood. Ras pathways are also important downstream effectors of several receptor tyrosine kinases localized in the cell membrane, most notably the BCR-ABL fusion protein seen in patients with Philadelphia chromosome positive chronic myelogenous leukemia. An important consideration in designing strategies to block Ras stimulatory effect is that Ras proteins are synthesized in the cytosol, but require post-translational modifications and attachment to anchor proteins or membrane binding sites in the cell membrane to be biologically active. Farnesyl transferase inhibitors (FTIs) are probably the best-studied class of Ras inhibitors in hematologic malignancies. They block the enzyme farnesyl-transferase (FTase), which is essential for post-translational modification. However, it has been observed that the Ras proteins also can be geranylgeranylated in the presence of FTIs, thus allowing membrane localization and activation, which limits their effectiveness. It is now hypothesized that their mechanism of action may be through FTase inhibition involving other signal transduction pathways. S-trans, trans-farnesylthiosalicylic acid, which was first designed as a prenylated protein methyltransferase inhibitor, has shown in vitro activity against all activated Ras proteins by dislodging them from their membrane-anchoring sites. Here, Ras biology, its signaling pathways and its implications as a therapeutic target in hematologic malignancies are reviewed.     

The effects of chronic aluminum exposure on learning and memory of rats by observing the changes of Ras/Raf/ERK signal transduction pathway

Objective

To investigate the effects of chronic aluminum (Al) exposure on learning and memory function of rats by observing the changes of Ras/Raf/ERK (Ras/ERK) signaling pathway.

Methods

Eighty weaned Wistar rats were divided into four groups ad libitum, 20 rats in each group. The four groups were fed with drinking water containing 0% (control), 0.2%, 0.4% and 0.6% (Al exposure) AlCl₃ for 3 months individually to set up aluminum exposure models. The laboratory was maintained at 18–23 °C and 45–55% relative humidity. Graphite furnace atomic absorption spectrometry was used to detect the content of Al in brain and blood. Western blot and real-time PCR (RT-PCR) were used to determine the protein and mRNA expression levels for Ras, Raf1, ERK2 and CREB.

Results

Chronic Al exposure increased the content of Al in rats’ blood and brain. It increased expression of Ras in the hippocampi compared with the control but the expression decreased along the Al exposure groups (p < 0.05). Similarly, Raf1, ERK2 and CREB expressions decreased compared to the control in a dose-dependent manner (p < 0.05).

Conclusion

Chronic Al exposure may affect learning and memory through impact on Ras/ERK signal pathway.     

ERK1/2通路及其介导多发性硬化发病的研究进展

丝裂原活化蛋白激酶1/2（ERK1/2）通路是第一个被发现的细胞信号转导通路,由Ras、Raf、MEK1/2和ERK1/2组成。ERK1/2通路激活后可以将细胞外信号从细胞膜转到细胞核,参与了细胞的多种生理病理功能,如细胞的生长、增殖、分化和凋亡等,并且与多种疾病的发病有关,包括多发性硬化（MS）和实验性自身免疫性脑脊髓炎（EAE）。ERK1/2通路的激活可以引起星形胶质细胞、MG、T细胞、巨噬细胞等活化,释放多种炎性因子,引起髓鞘损伤,导致MS/EAE的发病。多项研究表明,通过抑制ERK1/2通路可以减少炎性因子的释放,减轻髓鞘损伤,改善MS/EAE的病情,为治疗MS药物的开发提供了重要的靶点。     

Naringin-induced p21WAF1-mediated G1-phase cell cycle arrest via activation of the Ras/Raf/ERK signaling pathway in vascular smooth muscle cells

The flavonoid naringin has been shown to play a role in preventing the development of cardiovascular disease. However, the exact molecular mechanisms underlying the roles of integrated cell cycle regulation and MAPK signaling pathways in the regulation of naringin-induced inhibition of cell proliferation in vascular smooth muscle cells (VSMCs) remain to be identified. Naringin treatment resulted in significant growth inhibition and G₁-phase cell cycle arrest mediated by induction of p53-independent p21WAF1 expression; expression of cyclins and CDKs in VSMCs was also down-regulated. In addition, among the pathways examined, blockade of ERK function inhibited naringin-dependent p21WAF1 expression, reversed naringin-mediated inhibition of cell proliferation and decreased cell cycle proteins. Moreover, naringin treatment increased both Ras and Raf activations. Transfection of cells with dominant negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed naringin-induced ERK activity and p21WAF1 expression. Finally, naringin-induced reduction in cell proliferation and cell cycle protein was abolished in the presence of RasN17 and RafS621A mutant genes. The Ras/Raf/ERK pathway participates in p21WAF1 induction, leading to a decrease in cyclin D1/CDK4 and cyclin E/CDK2 complexes and in naringin-dependent inhibition of cell growth. These novel and unexpected findings provide a theoretical basis for preventive use of flavonoids to the atherosclerosis disease.