Resistance to UV‐induced apoptosis by β‐HPV5 E6 involves targeting of activated BAK for proteolysis by recruitment of the HERC1 ubiquitin ligase

UV exposure is the main etiological agent in the development of non‐melanoma skin cancer (NMSC), but mounting evidence suggests a co‐factorial role for β‐genus HPV types early in tumor initiation or progression. UV damage initiates an apoptotic response, driven at the mitochondrial level by BCL‐2 family proteins, that eliminates damaged cells that may accumulate deleterious mutations and acquire tumorigenic properties. BAK is a pro‐apoptotic BCL‐2 protein that functions ultimately to form pores that permeabilize the mitochondrial outer membrane, thereby committing a cell to death, a process involving changes in BAK phosphorylation and conformation. The E6 protein of β‐type HPV5 signals BAK for proteasomal degradation, a function that confers protection from UV‐induced apoptosis. We find that HPV5 E6 does not constitutively target BAK for proteolysis, but targets the latter stages of BAK activation, following changes in phosphorylation and conformation. A mutational analysis identified the lysine residue on BAK required for proteolysis, and a functional siRNA screen identified the HECT domain E3 ubiquitin ligase HERC1 as being required for E6‐mediated BAK degradation. We show that HERC1 interacts with BAK in E6‐expressing cells that have been damaged by UV, and provide evidence that the interaction of HERC1 with BAK requires access to a hydrophobic surface on BAK that binds BH3 domains of BCL‐2 proteins. We also show that HERC1 contains a putative BH3 domain that can bind to BAK. These findings reveal a specific and unique mechanism used by the HPV5 E6 protein to target BAK.     

Preservative-free tafluprost/timolol fixed combination: comparative 24-h efficacy administered morning or evening in open-angle glaucoma patients

ABSTRACT

Background

Ideal dosing for the preservative-free (PF) tafluprost/timolol fixed combination (TTFC) remains to be elucidated.     

17β-estradiol mediates protection against microvascular endothelial cell hyperpermeability

Background

Previous work from our laboratory demonstrated the involvement of “intrinsic” mitochondrial apoptotic signaling in vascular hyperpermeability. The objective of this study was to determine if 17β-estradiol, a known inhibitor of apoptosis, would attenuate microvascular endothelial cell hyperpermeability.

Methods

Rat lung microvascular endothelial cell monolayers were treated with 17β-estradiol or estrogen-receptor antagonist ICI 182780 after transfection with BAK peptide (5 μg/mL). Fluorescein isothiocyanate (FITC)-albumin was used to determine the change in permeability. Mitochondrial reactive oxygen species (ROS) formation and transmembrane potential were determined using 123 dihydrorhodamine and JC-1, respectively. Cytosolic cytochrome c levels and caspase-3 activity were determined using enzyme-linked immunosorbent assay and fluorometric assay respectively.

Results

17β-estradiol (10 nm) attenuated BAK-induced hyperpermeability (P < .05), ROS formation, cytochrome c release, and caspase-3 activation. The estrogen receptor antagonist ICI 182780 blocked the protective effect of 17β-estradiol on hyperpermeability (P < .05).

Conclusions

17β-estradiol attenuates BAK-induced hyperpermeability in rat lung microvascular endothelial cells by way of an estrogen-receptor mediated pathway.     

椎弓根钉系统联合单枚BAK后路腰椎体间融合术

目的 介绍椎弓根钉系统联合单枚BAK在腰椎椎体间融合术中的应用体会。方法 对26例患者(椎间盘突出伴退变性滑脱18例，Ⅰ～Ⅱ度脊柱滑脱伴椎弓崩解3例，椎间盘摘除术后失败5例)行相关节段椎弓根钉并联合单枚BAK内固定手术。结果 经平均28．6个月(10～46个月)随访，所有患者临床症状缓解，无断钉及融合器移位现象，术后融合率达100％。结论 椎弓根钉联合单枚BAK后路腰椎椎体间融合术具有操作简单，对椎管干扰小，术后脊柱稳定性好等优点。     

Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells,thereby enhancing the response to anti-ERBB1/ERBB2 therapy

The present studies focused on defining the mechanisms by which anoikis-resistant (AR) mammary carcinoma cells can be reverted to a therapy-sensitive phenotype. AR mammary carcinoma cells had reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA. In AR cells expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. AR cells were resistant to cell killing by multiple anti-tumor cell therapies, including ERBB1/2 inhibitor + MCL-1 inhibitor treatment, and had a reduced autophagic flux response to these therapies, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL caused necro-apoptosis in AR cells to a greater extent than in parental cells. Pre-treatment of anoikis-resistant cells with histone deacetylase inhibitors (HDACIs) for 24 h increased the levels of toxic BH3 domain proteins, reduced MCL-1 levels, and restored/re-sensitized the cell death response of AR tumor cells to multiple toxic therapies. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored the chemo-sensitivity of the tumors and prolonged animal survival. These data argue that one mechanism to enhance the anti-tumor effect of chemotherapy could be HDACI pre-treatment.     

Epigenetic modulators from “The Big Blue”: A treasure to fight against cancer

Cancer remains a major public health problem in our society. The development of potent novel anti-cancer drugs selective for tumor cells is therefore still required. Deregulation of the epigenetic machinery including DNA methylation, histone modifications and non-coding RNAs is a hallmark of cancer, which provides potential new therapeutic targets. Natural products or their derivatives represent a major class of anti-cancer drugs in the arsenal available to the clinician. However, regarding epigenetically active anti-cancer agents for clinics, the oceans represent a largely untapped resource. This review focuses on marine natural compounds with epigenetic activities and their synthetic derivatives displaying anti-cancer properties including largazole, psammaplins, trichostatins and azumamides.     

姜黄素抑制k562细胞生长并诱导凋亡机制的研究

目的探讨姜黄素抑制人类红白血病细胞（k562细胞）生长并诱导凋亡的机制及其对细胞凋亡相关基因BCL-XL和BAK表达的影响。方法把不同浓度姜黄素加入k562细胞,流式细胞仪检测其凋亡及其细胞周期的变化,RT-PCR方法检测BCL-XL和BAK mRNA的表达,免疫组化方法检测BCL—XL和BAK蛋白的表达。结果姜黄素可以对k562细胞有较明显的增殖抑制作用,且呈剂量依赖,流式细胞术结果显示姜黄素浓度在100μmol／L时作用24、48h后,凋亡率高于对照组（P〈0．05）;随着姜黄素浓度增加,作用于k562细胞后可以使其倍增时间明显延长,G1期细胞增多,S期细胞减少;RT-PCR结果显示姜黄素能下调k562细胞BCL-XL表达,上调BAK mRNA的表达（P〈0．05）,免疫组化法发现姜黄素可以降低BCL-XL表达,升高BAK的表达（P〈0．05）。结论姜黄素对k562细胞的生长有较明显的抑制作用,且呈一定时间与剂量关系,并促进k562细胞凋亡,可能和下调BCL—XL的表达和上调BAK的表达相关。     

Complications associated with posterior lumbar interbody fusion using Bagby and Kuslich method for treatment of spondylolisthesis

Objective To analyze complications associated with posterior lumbar interbody fusion(PLIF)in which two Bagby and Kuslich(BAK)interbody fusion cages were implanted.Methods A total of 118 patients with spondylolytic spondylolisthesis underwent single-level PLIF using two BAK cages filled with morselized autogenous bone.The major clinical and ragiographic complications were analyzed after a follow-up with an average time of 2 years and 9 months.Results Complications were divided into intraoperative and postoperative complications.Intraoperative complications mainly included dural tear(4 patients,3.4?,nerve root injury(3 patients,2.5?and suboptimal cage position(9 patients,7.5?.No death was caused by the operation.Postoperative complications chiefly consisted of cage retropulsion(3 patients,2.5?,cage subsidence(4 patients,3.4?,and postlaminectomy arachnoiditis(2 patients,1.7?,Pseudarthrosis was noted radiographically with evidence of motion between adjacent vertebra on lateral flexion-extension films and luciencies around the cages(2 patient,1.7?.continuous posterior cage migration(2 patients,1.7?or continuous cage subsidence(2 patients,1.7?.Two patients died,one from a traffic accident and the other from metastatic cancer 1 year postoperatively.Conclusions The results of this study indicate that PLIF with BAK cages is and effective but also technically difficult procedure.The relatively high incidence of complications reminds us of the importance of surgical indications and proper manipulations.     

经腹腔镜BAK腰5—骶1椎体间融合术的应用解剖

目的为经腹腔镜BAK椎体间融合术提供解剖学基础.方法在30具成人尸体标本上解剖观察了L5～S1椎间盘的形态及其前侧的毗邻关系.结果①脊柱L5～S1段前方有两侧髂血管围成的“∧'形区,内有骶中动、静脉,上腹下神经丛走行.②椎间盘平面正对腹前壁耻骨联合上方(6.7±2.8)cm处.结论经腹腔镜BAK椎体间融合术是安全、可行的术式.