非编码RNA在索拉非尼治疗肝细胞癌耐药中的作用机制

索拉非尼作为晚期肝细胞癌的一线治疗药物,能够有效改善肝癌患者预后。但索拉非尼耐药已经成为影响肝细胞癌治疗效果的主要障碍。近年来研究发现,非编码RNA在肝细胞癌索拉非尼耐药中起着关键作用。总结了非编码RNA通过诱导肝癌细胞自噬、促进肝癌干细胞增殖、促进肝癌细胞上皮-间质转化过程、抑制肝癌细胞凋亡以及调节肝癌组织微环境来调节肝癌细胞对索拉非尼的敏感性。指出了非编码RNA调控肝细胞癌索拉非尼耐药的分子机制在克服耐药方面的潜在临床意义。     

射频消融联合索拉非尼治疗进展期肝细胞癌的临床观察

原发性肝癌在全球发病率已超过62.6万/年,居于恶性肿瘤发病率的第5位;病死率也接近60万/年,位居肿瘤相关死亡的第3位[1]。我国发病人数约占全球的55%,其中85%～90%为肝细胞癌,     

耐索拉非尼肝癌细胞株的构建及耐药机制研究

［摘要］　目的　构建耐索拉非尼肝癌细胞株并探讨其耐药机制。方法　应用HepG2、Huh7细胞通过浓度递增法建立耐索拉非尼细胞模型。通过CCK8法检测细胞对不同浓度索拉非尼的敏感性。通过划痕实验检测细胞的迁移能力。通过流式细胞术分析细胞凋亡情况。通过荧光实时定量RT-PCR和Western blot实验检测凋亡相关因子caspase-3和自噬标志因子P62、LC3的表达水平。结果　获得耐索拉非尼肝癌细胞株HepG2-SR、Huh7-SR。随着索拉非尼药物干预浓度的升高,耐药细胞株及野生型细胞株的存活率均逐渐降低。HepG2-SR细胞的半数抑制浓度（IC₅₀）显著高于HepG2细胞［（15.74±0.38）μM vs （7.27±0.44）μM ;t=5.450,P<0.001］;Huh7-SR细胞的IC₅₀显著高于Huh7细胞［（11.73±0.27）μM vs （4.92±0.31）μM;t=4.807,P<0.001］。相较于野生型细胞株,耐药细胞株具有更高的抗凋亡和迁徙能力。耐药细胞株的caspase-3、P62表达水平显著低于野生型细胞株（P<0.05）,而LC3表达水平显著高于野生型细胞株（P<0.05）。结论　耐索拉非尼肝癌细胞株HepG2-SR、Huh7-SR具有更强的抗凋亡、抗生殖抑制和迁移能力,且自噬水平更高,为研究肝细胞癌对索拉非尼的耐药机制提供了基础。     

索拉非尼治疗原发性肝细胞癌导致心脑血管不良事件2例报道

目的 分析应用索拉非尼治疗肝细胞癌（HCC）发生心脑血管不良事件2例患者的资料,为临床诊治提供参考。方法 选择2例晚期HCC患者,治疗前均无心脑血管病变证据,均在应用靶向抗肿瘤药物索拉非尼治疗过程中发生心脑血管不良事件,对二者的发病过程及诊治情况进行分析。结果 2例HCC患者经索拉非尼与肝动脉化疗栓塞术（TACE）联合治疗后病情完全缓解。其中1例患者出现右冠状动脉完全闭塞,并发生致死性恶性心律失常,经治疗后好转,已停用索拉非尼。另外1例患者发生急性脑梗死,经早期识别、干预后症状好转并继续给予索拉非尼治疗,但患者最终因突发心肌梗死死亡。结论 临床应用索拉非尼治疗HCC时应早期识别心脑血管不良事件,并进行干预。     

甲胎蛋白应答评价索拉非尼联合卡瑞利珠单抗治疗晚期肝细胞癌的效果分析

目的 探讨甲胎蛋白(AFP)应答在索拉非尼联合卡瑞利珠单抗治疗晚期肝细胞癌中的临床疗效及安全性。方法 选取2020年9月—2022年2月于新疆医科大学第一附属医院接受索拉非尼联合卡瑞利珠单抗治疗的48例晚期肝细胞癌患者的临床资料,按照治疗后AFP应答水平进行分组：应答组(治疗6～8个月时,与基础AFP比较,AFP下降率>20%,n=32);无应答组(治疗6～8个月时,与基础AFP比较,AFP下降率≤20%,n=16)。计量资料组间比较采用Mann-Whitney U检验;计数资料组间比较采用χ²检验。绘制生存曲线,行Cox单因素/多因素回归分析得到与OS相关的独立危险因素。比较两组患者无进展生存时间、总生存期与治疗效果。结果 两组患者中均未观察到达到临床缓解的病例。AFP应答组的客观有效率(21.88%vs 0)和疾病控制率(84.38%vs 43.75%)均高于无应答组(χ²值分别为2.530、6.668,P值分别为0.112、0.010)。应答组无进展生存期(9.9个月)、总生存期(13.8个月)均高于无应答组(6.8个月、11.1...     

索拉非尼联合奥曲肽对HepG2细胞的抑制作用

目的 观察索拉非尼联合奥曲肽在体外对人肝癌细胞株HepG2的抑制作用,并探讨其机制. 方法 采用不同浓度梯度的索拉非尼、奥曲肽单独和联合作用于人肝癌细胞株HepG2,分别于24、48、72 h用CCK-8试剂盒测定各组细胞的杀伤效应,流式细胞术检测HepG2细胞的凋亡率,荧光显微镜观察细胞的凋亡情况;并用酶联免疫吸附法检测细胞培养上清液中血管内皮生长因子(VEGF)的水平,Western blot法测定细胞中髓样细胞白血病-1(Mcl-1)、细胞外信号调节激酶(ERK) 1/2、磷酸化ERK1/2的表达.数据采用均数±标准差((x)±s)表示,用SPSS17.0软件对数据进行单因素方差分析. 结果 索拉非尼、奥曲肽单独使用和联合使用均对HepG2细胞具有抑制作用,并诱导其凋亡,联合用药组抑制效果优于单独用药组F=200.398,(P＜0.05).荧光显微镜观察显示,联合用药组和单独用药组细胞膜上均出现代表细胞早期凋亡的标志物磷脂酰丝氨酸.联合用药组VEGF表达水平为(10.31±4.69) pg/ml,低于索拉非尼组[(22.73±5.88)pg/ml]、奥曲肽组[(25.46±3.45) pg/ml]及正常对照组[(57.15±6.32) pg/ml],F=1019.725,P值均＜0.05.Western blot 法结果显示,各组ERK1/2表达水平差异并无统计学意义(P＞0.05);与正常对照相组比,索拉非尼、奥曲肽及联合用药组的磷酸化ERK1/2表达均减少(F=2.401,P＜0.05),联合用药组表达水平低于单独用药组(P值均＜0.05);奥曲肽组Mcl-1的表达水平与对照组差异无统计学意义,索拉非尼组和联合用药组的表达水平低于正常对照组(P值均＜ 0.05),联合用药组的Mcl-1表达水平低于各单独用药组(P值均＜ 0.05).结论 索拉非尼、奥曲肽均可抑制人肝癌细胞HepG2增殖,并诱导其凋亡,联合应用效果更为显著;其机制可能与二者协同抑制VEGF的表达,并下调抗凋亡蛋白Mcl-1及磷酸化ERK1/2的表达有关.     

索拉非尼联合肝动脉化疗栓塞术治疗中晚期肝细胞癌疗效观察

目的观察经导管肝动脉化疗栓塞术(TACE)与靶向治疗药相结合治疗中晚期肝细胞癌的疗效和安全性。方法 2008年1月至2012年12月我院收治的中晚期肝细胞癌患者60例,均采用TACE治疗,其中37例术后口服索拉非尼(400 mg,2次/d),根据不良反应调整用量,定期复查腹部CT。根据m RECIST标准进行疗效评价,观察患者的肿瘤进展时间和总生存期,并记录索拉非尼不良反应和TACE前后的肝功能变化。结果 37例应用TACE联合索拉非尼治疗患者的中位生存期为(13±0.98)m,显著长于23例单纯TACE治疗组[(7.3±1.20)m,P=0.001],肿瘤进展时间为(7.5±1.21)m,显著长于单纯TACE治疗组[(5±0.62)m,P=0.001];在随访结束时,联合治疗组疾病控制率为48.6%,显著高于单纯TACE组的17.4%(P0.01);多因素分析显示有无联合应用索拉非尼、有无门脉癌栓、是否抗病毒治疗是显著影响生存时间的风险因素;索拉非尼治疗主要的相关不良反应为手足皮肤反应。结论 TACE联合索拉非尼治疗可延长中晚期肝细胞癌患者的疾病进展时间及总生存期,安全性好。     

经肝动脉化疗栓塞术联合索拉非尼治疗中晚期肝细胞癌研究进展

经肝动脉化疗栓塞术(TACE)联合索拉非尼是目前治疗失去手术机会的中晚期肝细胞癌(HCC)最常用的策略,但具体治疗模式还未形成共识。分析了TACE联合索拉非尼治疗HCC的治疗模式及其有效性和安全性。认为不同的治疗模式均具有其利弊,还需要进行更多的临床研究,为中晚期肝癌患者的治疗提供一定参考。     

索拉非尼联合国产PD-1抑制剂治疗不可手术切除的肝细胞癌的疗效及不良反应

目的研究索拉非尼联合国产程序性细胞死亡受体-1(programmed cell death receptor-1,PD-1)抑制剂在不可手术切除的肝细胞癌(hepatocellular carcinoma, HCC)中的疗效及不良反应。方法回顾性分析2019年6月至2021年1月于北京地坛医院肿瘤内科使用国产PD-1抑制剂联合索拉非尼治疗的不可手术切除的HCC患者的临床资料,资料完整且符合入组条件者共22例,其中卡瑞利珠单抗联合索拉非尼组9例,信迪利单抗联合索拉非尼组13例,随访患者,主要研究终点为统计客观缓解率(objective response rate, ORR)、疾病控制率(disease control rate, DCR)和无进展生存期(progression-free survival, PFS),次要研究终点为总生存期(overall survival, OS)和安全性。结果在可评价疗效的22例患者中,7例患者疗效评价为部分缓解(partial remission, PR),10例患者疗效评价为疾病稳定(stable disease, SD),5例患者疗效评价为疾病进展(progressive disease, PD),ORR为31.8%,DCR为77.3%。中位无进展生存期(median progression-free survival, mPFS)为8.0个月(5.4~10.6个月)。不良反应发生率为77.3%,最常见的不良反应为腹泻(27.3%)、手足综合征(22.7%)、转氨酶升高(22.7%)、疲乏(18.2%)。结论索拉非尼联合国产PD-1抑制剂治疗不可手术切除的HCC临床效果显著,不良反应可控,是一种安全、有效的治疗方案。     

TACE联合索拉非尼对不能手术切除肝细胞肝癌的疗效分析

目的分析经导管肝动脉化疗栓塞(transcatheter arterial chemoembolization,TACE)联合索拉非尼对不能手术切除肝细胞肝癌(hepatocellular carcinoma,HCC)的疗效。方法选取海军总医院2008年10月-2010年10月收治的不能手术切除的HCC患者,共162例,按照随机数字表分为联合组及TACE组,各81例,分别接受TACE联合索拉非尼治疗及单纯TACE治疗,比较两组患者的疗效。结果联合组部分缓解11例,稳定65例,有效率93.8%,TACE组部分缓解7例,稳定42例,有效率60.5%,联合组治疗效果显著优于TACE组(P0.05);联合组生存质量改善36例,稳定26例,稳定及改善率76.5%,TACE组改善19例,稳定23例,稳定及改善率51.9%,联合组生存质量改善程度显著优于对照组(P0.05);两组患者治疗后丙氨酸氨基转移酶(ALT)、总胆红素(TBIL)均显著升高,血清白蛋白(ALB)均无明显变化,两组患者治疗后ALT、TBIL及ALB水平差异无统计学意义(P0.05);两组不良反应发生率分别为80.2%、81.5%,差异无统计学意义(P0.05);联合组6个月、12个月生存率分别为77.8%、69.1%,TACE组分别为59.3%、50.6%,联合组生存率显著高于TACE组(P0.05)。结论 TACE联合索拉非尼较单纯TACE治疗具有更好的疗效,能够有效延长患者生存期、保证其生活质量,且不良反应耐受性好,为不能手术切除HCC的治疗提供了新的方法。     

The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice

Blood–brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti–ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Stroke continues to be a leading cause of death and the most frequent cause of disability in adults. Despite significant advances in decoding the pathophysiology of cerebral ischemia, therapeutic options for stroke are still limited. Ischemic injury to the brain rapidly triggers adhesion molecule expression on the activated endothelium (1), resulting in rolling, adhesion, and extravasation of blood-derived inflammatory cells (2). Infiltrating inflammatory cells, including neutrophils, result in irreversible impairment of blood–brain barrier (BBB) function and tissue damage through the release of reactive oxygen species (ROS), proteolytic enzymes, and proinflammatory mediators (3). However, our understanding of the links between BBB breakdown and peripheral neutrophils infiltrating the ischemic brain is still incomplete.Neddylation is the process of posttranslational protein modification by conjugating the ubiquitin-like protein, neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8), to target proteins (4). This process is catalyzed by NEDD8-activating enzyme E1 (NAE1 and UBA3), NEDD8-conjugating enzyme E2 (UBC12), and NEDD8 E3 ligase (5). The best characterized substrates of NEDD8 are cullins (6), which are scaffold proteins for the cullin-RING E3 ligase (CRL) (7). The conjugation of NEDD8 to cullins leads to the activation of CRL (8), which ubiquitinates a multitude of different proteins for targeted degradation (9). Recently, the neddylation pathway was reported to contribute to growth of a variety of cancer cells and inflammatory responses (10). In contrast, inhibition of neddylation by MLN4924, which is a small molecule inhibitor of NAE, suppressed tumor growth, reduced inflammation, and prevented atherogenesis (11–13). However, to the best of our knowledge, the role of neddylation in ischemic stroke has not been addressed so far.Using a mouse model of focal transient cerebral ischemia, we show that neddylation was up-regulated in the peri-infarct cortex after stroke and was expressed in neutrophils. Treatment with the neddylation inhibitor MLN4924 reduced brain infarction and improved neurological functions. We also demonstrated that MLN4924-afforded neuroprotection was mediated via anti-inflammatory and BBB-protective effects involving the accumulation of CRL substrate neurofibromatosis 1 (NF1).     

Blockage of neddylation modification stimulates tumor sphere formation in vitro and stem cell differentiation and wound healing in vivo

MLN4924, also known as pevonedistat, is the first-in-class inhibitor of NEDD8-activating enzyme, which blocks the entire neddylation modification of proteins. Previous preclinical studies and current clinical trials have been exclusively focused on its anticancer property. Unexpectedly, we show here, to our knowledge for the first time, that MLN4924, when applied at nanomolar concentrations, significantly stimulates in vitro tumor sphere formation and in vivo tumorigenesis and differentiation of human cancer cells and mouse embryonic stem cells. These stimulatory effects are attributable to (i) c-MYC accumulation via blocking its degradation and (ii) continued activation of EGFR (epidermal growth factor receptor) and its downstream pathways, including PI3K/AKT/mammalian target of rapamycin and RAS/RAF/MEK/ERK, via inducing EGFR dimerization. Finally, MLN4924 accelerates EGF-mediated skin wound healing in mouse and stimulates cell migration in an in vitro culture setting. Taking these data together, our study reveals that neddylation modification could regulate stem cell proliferation and differentiation and that a low dose of MLN4924 might have a therapeutic value for stem cell therapy and tissue regeneration.In cells, homeostasis is reached by a fine balance between the rates of protein synthesis and degradation. The process of protein degradation by the ubiquitin-proteasome system (UPS) involves two sequential steps: (i) ubiquitylation catalyzed by the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) and (ii) degradation catalyzed by 26S proteasome (1). Abnormal UPS activity abrogates the homeostasis and is associated with many human diseases, including cancer (2, 3). Bortezomib, the first Food and Drug Administration-approved proteasome inhibitor for the treatment of multiple myeloma and mantle cell lymphoma (4), possesses significant cytotoxicity against normal cells due to its universal inhibition of the UPS (5). This stimulated an intensive search for inhibitors targeting the enzymes upstream of the proteasome for improved specificity.Cullin-RING ligases (CRLs), consisting of scaffold cullins, adaptor proteins, substrate-recognizing receptors, and RING finger proteins, are the largest family of E3 ubiquitin ligases and are responsible for the ubiquitylation of about 20% of all cellular proteins (6, 7). The activity of CRLs requires neddylation of the cullin proteins (8, 9), a ubiquitylation-like process, which is sequentially catalyzed by three enzymes: a NEDD8-activating enzyme (NAE or E1), NEDD8-conjugating enzyme (E2), and NEDD8 ligase (E3). Abnormal elevation of CRL activity has been found in multiple human cancers, which makes them favorable targets for cancer treatment (10, 11).MLN4924 was descried in 2009 as the first-in-class NAE inhibitor exclusively in the application for cancer treatment by inactivation of CRLs via blockage of cullin neddylation (7). Since the initial report, more than 150 papers (by PubMed search) were published to show its preclinical anticancer activity, either acting alone or in combination with current chemotherapy and/or radiation (6, 11). One of the seven clinical trials of MLN4924 ({"type":"clinical-trial","attrs":{"text":"NCT00911066","term_id":"NCT00911066"}}NCT00911066) was published recently, concluding a modest effect of MLN4924 against acute myeloid leukemia (AML) (12).To further elucidate the role of blocking neddylation in cancer treatment, we thought to study the effect of MLN4924 on cancer stem cells (CSCs) or tumor-initiating cells (TICs), a small group of tumor cells with stem cell properties that have been claimed to be responsible for cancer initiation and relapse (13). Meanwhile, a recent study showed that pharmacological inactivation of SKP2 SCF (SKP1–Cullins–F-box proteins) E3 ubiquitin ligase (also known as CRL1) by a small molecule inhibitor, compound 25, could restrict CSC traits and cancer progression in prostate cancer (14).To our utter surprise, we found that at low drug concentrations (30–100 nM), MLN4924 stimulates in vitro tumor sphere (TS) formation and in vivo tumorigenesis of both cancer cells and embryonic stem cells. Mechanistic studies revealed the involvement of accumulation of c-MYC, and dimerization and activation of EGFR (epidermal growth factor receptor). We also found that MLN4924 could accelerate the process of EGF-induced skin wound healing in mouse and cell migration in an in vitro culture setting. Thus, MLN4924, when applied at low concentrations, may have potential utility in the field of stem cell therapy and tissue regeneration.     

Cullin neddylation inhibitor attenuates hyperglycemia by enhancing hepatic insulin signaling through insulin receptor substrate stabilization

Hepatic insulin resistance is a hallmark feature of nonalcoholic fatty liver disease and type-2 diabetes and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress-sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin-RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover, but the pathophysiological roles and therapeutic implications of this cellular signaling regulation is largely unknown. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a cullin scaffold. Here, we report that pharmacological inhibition of cullin neddylation by MLN4924 (Pevonedistat) rapidly decreases hepatic glucose production and attenuates hyperglycemia in mice. Mechanistically, neddylation inhibition delays CRL-mediated IRS protein turnover to prolong insulin action in hepatocytes. In vitro knockdown of either cullin 1 or cullin 3, but not other cullin members, attenuates insulin-induced IRS protein degradation and enhances cellular insulin signaling activation. In contrast, in vivo knockdown of liver cullin 3, but not cullin 1, stabilizes hepatic IRS and decreases blood glucose, which recapitulates the effect of MLN4924 treatment. In summary, these findings suggest that pharmacological inhibition of cullin neddylation represents a therapeutic approach for improving hepatic insulin signaling and lowering blood glucose.

A Cullin-RING E3 ligase (CRL) is a multiprotein complex generally consisting of a cullin protein, a RING E3 ligase, and a substrate receptor that recognizes specific substrates for ubiquitination and proteasomal degradation (1). Each of the cullin member proteins serves as the scaffold of a functionally distinct CRL complex. Mammalian cells express a large number of substrate receptors in a tissue-specific manner, which further determines the substrate specificity of a unique CRL complex. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a conserved lysine on a cullin protein (1). Neddylation is mediated by a set of specialized Nedd8 E1, E2, and E3 enzymes that sequentially transfer Nedd8 to a cullin protein. Unlike protein ubiquitination, current knowledge supports that cullin proteins are the predominant neddylation targets in mammalian cells (2). Recently, CRLs have emerged as attractive targets for drug development (3). In the last 10 y, CRLs have drawn major attention in cancer research owing to CRL regulation of oncogenes and tumor suppressors (4). The Nedd8-activating E1 enzyme (NAE1) is the only known Nedd8 E1 enzyme (1). MLN4924 (Pevonedistat) is the first in-class small molecule inhibitor of NAE1 and has entered Phase-I/II clinical trials for various cancer treatments (5). In contrast, the translational potential of targeting cullin neddylation for treating other diseases is still largely unknown.Hepatic insulin resistance is a hallmark pathogenic feature of fatty liver disease and type-2 diabetes, and decreased hepatocellular insulin receptor substrate (IRS) is a key underlying cause (6, 7). Following insulin activation, IRS signaling is feedback inactivated by mechanistic target of rapamycin (mTOR)-mediated serine/threonine phosphorylation and subsequent IRS proteosome degradation (8, 9). Although this feedback mechanism prevents sustained insulin signaling activation in cells, overactivation of this desensitization mechanism by mTOR and other stress/nutrient kinases significantly contributes to hepatic insulin resistance in fatty livers (8, 10). However, the broad cellular functions of these kinases present a major obstacle in targeting them to prevent insulin resistance (11). Serine/threonine-phosphorylated IRS proteins are destined for ubiquitylation and proteasome degradation (9, 12), but targeting the proteasome pathway is unlikely a viable approach due to the perceived broad biological impact. Interestingly, studies of mouse embryonic fibroblasts have revealed that CRLs are involved in mediating IRS1 protein turnover (13, 14). These findings prompted us to ask two intriguing questions: First, are CRLs involved in regulating hepatic insulin signaling, and if yes, which CRL may mediate such an effect in hepatocytes? Second, can CRLs serve as potentially new therapeutic targets for modulating insulin signaling and glucose homeostasis in fatty liver disease in vivo? Here, we report findings that neddylation inhibition effectively enhances hepatic insulin signaling by delaying CRL-mediated IRS turnover, resulting in attenuated hyperglycemia in obese mice.     

Pevonedistat (MLN4924), a First‐in‐Class NEDD8‐activating enzyme inhibitor,in patients with acute myeloid leukaemia and myelodysplastic syndromes: a phase 1 study

This trial was conducted to determine the dose‐limiting toxicities (DLTs) and maximum tolerated dose (MTD) of the first in class NEDD8‐activating enzyme (NAE) inhibitor, pevonedistat, and to investigate pevonedistat pharmacokinetics and pharmacodynamics in patients with acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). Pevonedistat was administered via a 60‐min intravenous infusion on days 1, 3 and 5 (schedule A, n = 27), or days 1, 4, 8 and 11 (schedule B, n = 26) every 21‐days. Dose escalation proceeded using a standard ‘3 + 3’ design. Responses were assessed according to published guidelines. The MTD for schedules A and B were 59 and 83 mg/m², respectively. On schedule A, hepatotoxicity was dose limiting. Multi‐organ failure (MOF) was dose limiting on schedule B. The overall complete (CR) and partial (PR) response rate in patients treated at or below the MTD was 17% (4/23, 2 CRs, 2 PRs) for schedule A and 10% (2/19, 2 PRs) for schedule B. Pevonedistat plasma concentrations peaked after infusion followed by elimination in a biphasic pattern. Pharmacodynamic studies of biological correlates of NAE inhibition demonstrated target‐specific activity of pevonedistat. In conclusion, administration of the first‐in‐class agent, pevonedistat, was feasible in patients with MDS and AML and modest clinical activity was observed.     

Preventive antitumor activity against hepatocellular carcinoma (HCC) induced by immunization with fusions of dendritic cells and HCC cells in mice

BACKGROUND: The prevention of recurrence of hepatocellular carcinoma (HCC) after treatment is very important for improvement of the prognosis of HCC patients. Dendritic cells (DCs) are potent antigen-presenting cells that can prime naive T cells to induce a primary immune response. We attempted to induce preventive antitumor immunity against HCC by immunizing BALB/c mice with fusions of DCs and HCC cells. METHODS: Murine bone marrow-derived DCs and a murine HCC cell line. BNL cells, were fused by treatment with 50% polyethyleneglvcol (PEG). Fusion efficacy was assessed by the analysis of fusions of BNL cells stained with red fluorescent dye and DCs stained with green fluorescent dye. Mice injected intravenously with DC/BNL fusions were challenged by BNL cell inoculation. RESULTS: About 30% of the PEG-treated non-adherent cells with both fluorescences were considered to be fusion cells. The cell fraction of DC/BNL fusions showed phenotypes of DCs, MHC class II, CD80, CD86, and intercellular adhesion molecule (ICAM)-1, which were not expressed on BNL cells. Mice immunized with the fusions were protected against the inoculation of BNL tumor cells, whereas injection with a mixture of DCs and BNL cells not treated with PEG did not provide significant resistance against BNL cell inoculation. Splenocytes from DC/BNL fusion-immunized mice showed lytic activity against BNL cells. CONCLUSIONS: These results demonstrate that immunization with fusions of DCs and HCC cells is capable of inducing preventive antitumor immunity against HCC.     

Structural mechanism of ubiquitin and NEDD8 deamidation catalyzed by bacterial effectors that induce macrophage-specific apoptosis

Targeting eukaryotic proteins for deamidation modification is increasingly appreciated as a general bacterial virulence mechanism. Here, we present an atomic view of how a bacterial deamidase effector, cycle-inhibiting factor homolog in Burkholderia pseudomallei (CHBP), recognizes its host targets, ubiquitin (Ub) and Ub-like neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), and catalyzes site-specific deamidation. Crystal structures of CHBP–Ub/NEDD8 complexes show that Ub and NEDD8 are similarly cradled by a large cleft in CHBP with four contacting surfaces. The pattern of Ub/NEDD8 recognition by CHBP resembles that by the E1 activation enzyme, which critically involves the Lys-11 surface in Ub/NEDD8. Close examination of the papain-like catalytic center reveals structural determinants of CHBP being an obligate glutamine deamidase. Molecular-dynamics simulation identifies Gln-31/Glu-31 of Ub/NEDD8 as one key determinant of CHBP substrate preference for NEDD8. Inspired by the idea of using the unique bacterial activity as a tool, we further discover that CHBP-catalyzed NEDD8 deamidation triggers macrophage-specific apoptosis, which predicts a previously unknown macrophage-specific proapoptotic signal that is negatively regulated by neddylation-mediated protein ubiquitination/degradation.     

肝细胞癌诊治中的若干问题

随着肝细胞癌（HCC）肿瘤标志物和CT／MRI诊断的应用、手术切除及局部消融等治疗方法的进步,使HCC的5年生存率达到了63．4％。但是由于我国HCC早期诊断水平的不均衡,可进行手术切除的病例仅仅有20％～30％。对于高危人群定期开展血清肿瘤标志物和肝脏超声检查;提高三期动态增强CT和Gd—DTPA增强MRI等影像学诊断水平,同时积极开展多学科会诊,制定个性化治疗方案和减少术后肝功能衰竭发生等若干问题,是提高我国HCC早期诊断水平,提高治疗效果,延长生存期的有效手段。     

The investigational agent MLN2238 induces apoptosis and is cytotoxic to CLL cells in vitro,as a single agent and in combination with other drugs

Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in the U.S. The course of the disease has been shown to be negatively impacted by increased levels of BCL2. Strategies to downregulate BCL2 and shift the balance towards cellular demise are actively being explored. Therefore, we examined whether the investigational agent MLN2238 could inhibit the proteasomal machinery and induce CLL cell death while also downregulating BCL2. MLN2238‐induced cell death was studied in peripheral blood mononuclear cells from 28 CLL patients. MLN2238 produced a dose‐dependent reduction in BCL2 and CLL cell viability with maximum cell death observed at a 50 nmol/l concentration by 48 h. Annexin‐V staining, PARP1 and caspase‐3 cleavage along with an increase in mitochondrial membrane permeability were noted after cells were treated with MLN2238; however, apoptosis was only partially blocked by the pan‐caspase inhibitor z‐VAD.fmk. Furthermore, we observed enhanced anti‐CLL effects in tumour cells treated with either a combination of MLN2238 and the BH3 mimetic AT‐101 or MLN2238 and fludarabine. Together, our data suggest the potential for proteasome inhibitor based therapy in CLL and the rationale design of drug combination strategies based on CLL biology.     

Liver transplantation for HCC: its role

Liver transplantation (LT) is the only treatment that offers a chance of cure for hepatocellular carcinoma (HCC) and the underlying liver cirrhosis simultaneously, but the availability of liver grafts and the aggressiveness of tumor recurrence are critical limiting factors of LT for patients with HCC. In most Asian countries, the serious shortage of deceased donors and the strong demand for LT has lead to the development of living-donor LT (LDLT) as a practical alternative replacing deceased-donor LT (DDLT). Grafts in Western countries are issued from DDLT and graft allocations are under the responsibilities of state agencies which apply strict rules based on the MELD (model for end-stage liver disease) score. Considering that HCC recurrence is the most common cause of post-transplant patient death, recipient candidates should be prudently selected through objectively established criteria. Points in addition to the MELD score can be allotted to patients with HCC providing that the HCC remains within the Milan criteria. The increasing number of LT candidates with HCC results in increasing waiting periods, which necessitate the consideration of pretransplant treatment of HCC, including partial liver resection. Both specific Western units and some Asian major LDLT centers have challenged the Milan criteria. The eligibility criteria of both DDLT and LDLT for HCC are likely to be expanded more than before, but this still requires further qualified risk–benefit analyses. The development of new effective treatment modalities before LT and for HCC recurrence might expand the selection criteria further without incurring an increased recurrence rate.