Sorafenib,rapamycin, and venetoclax attenuate doxorubicin-induced senescence and promote apoptosis in HCT116 cells

Emerging evidence has shown that the therapy-induced senescent growth arrest in cancer cells is of durable nature whereby a subset of cells can reinstate proliferative capacity. Promising new drugs named senolytics selectively target senescent cells and commit them into apoptosis. Accordingly, senolytics have been proposed as adjuvant cancer treatment to cull senescent tumor cells, and thus, screening for agents that exhibit senolytic properties is highly warranted. Our study aimed to investigate three agents, sorafenib, rapamycin, and venetoclax for their senolytic potential in doxorubicin-induced senescence in HCT116 cells. HCT116 cells were treated with one of the three agents, sorafenib (5 µM), rapamycin (100 nM), or venetoclax (10 µM), in the absence or presence of doxorubicin (1 µM). Senescence was evaluated using microscopy-based and flow cytometry-based Senescence-associated-β-galactosidase staining (SA-β-gal), while apoptosis was assessed using annexin V-FITC/PI, and Muse caspase-3/-7 activity assays. We screened for potential genes through which the three drugs exerted senolytic-like action using the Human Cancer Pathway Finder PCR array. The three agents reduced doxorubicin-induced senescent cell subpopulations and significantly enhanced the apoptotic effect of doxorubicin compared with those treated only with doxorubicin. The senescence genes IGFBP5 and BMI1 and the apoptosis genes CASP7 and CASP9 emerged as candidate genes through which the three drugs exhibited senolytic-like properties. These results suggest that the attenuation of doxorubicin-induced senescence might have shifted HCT116 cells to apoptosis by exposure to the tested pharmacological agents. Our work argues for the use of senolytics to reduce senescence-mediated resistance in tumor cells and to enhance chemotherapy efficacy.     

Hsa-miR-4282对肝癌细胞系SMMC-7721生长的影响

目的  探讨Hsa-miR-4282在肝癌细胞系SMMC-7721中的表达及其对细胞生长的影响。方法 采用实时荧光定量PCR法检测Hsa-miR-4282在人正常肝上皮细胞系HL-7702和人肝癌细胞系MHCC97-H、SMMC-7721，以及 20例肝癌组织及其相应癌旁组织中的表达。采用瞬时转染法将Hsa-miR-4282 mimics（上调组）和Hsa-miR-4282 inhibitor（下调组）分别转染肝癌SMMC-7721细胞，上调组和下调组分别设置相应阴性对照组。转染后采用MTT法检测细胞增殖能力，平板克隆形成实验检测细胞克隆形成能力，流式细胞仪检测细胞凋亡能力。结果 Hsa-miR-4282在肝癌组织、肝癌细胞MHCC97-H及肝癌细胞SMMC-7721中的表达均低于癌旁组织及正常肝细胞HL-7702（P＜0.05）。MTT实验结果显示，Hsa-miR-4282上调后肝癌SMMC-7721细胞的OD值低于其阴性对照组，而下调后OD值高于其阴性对照组 （P＜0.05）。平板克隆形成实验显示，下调组的细胞克隆数高于其阴性对照组［（240±7） 个 vs （191±10） 个，P=0.005）］，而上调组细胞克隆数低于基阴性对照组［（146±10） 个 vs （193±12） 个，P=0.013）］。流式细胞仪检测结果显示，Hsa-miR-4282上调组细胞凋亡率较其阴性对照组升高［（23.89±1.89）% vs（16.6±1.14）%，P=0.009）］，下调组细胞凋亡率较期阴性对照组降低［（14.98±0.46）% vs （17.79±0.73）%，P=0.010］。结论 Hsa-miR-4282上调可抑制肝癌SMMC-7721细胞增殖，促进细胞凋亡，可能与肝癌的发病机制有关。     

健脾益肾方对非小细胞肺癌细胞体外增殖凋亡作用的实验研究

目的:探讨健脾益肾方对非小细胞肺癌(NSCLC)细胞体外增殖凋亡的作用。方法:人NSCLC细胞系A549分为四组:空白对照组(仅加入细胞培养液)、阴性对照组(加入细胞,不进行中药处理)、实验组(加细胞加中药处理)。荧光定量PCR和Western blot分别检测Survivin、Bcl-2和Caspase-3的mRNA和蛋白表达。MTT检测细胞增殖;流式细胞术检测细胞凋亡。结果:与空白对照组相比,阴性对照组细胞在24、48、72 h的吸光度值明显升高,细胞凋亡率下降,Survivin和Bcl-2 mRNA和蛋白相对表达量上调,Caspase-3 mRNA和蛋白相对表达量下调,组间比较差异有统计学意义(P<0.05);而健脾益肾方处理的实验组24、48、72 h的吸光度值均显著降低,细胞凋亡率显著上升,Survivin和Bcl-2 mRNA和蛋白相对表达量下调,Caspase-3 mRNA和蛋白相对表达量上调,与空白对照组相比差异有统计学意义(P<0.05)。结论:健脾益肾方可通过下调Survivin和Bcl-2、上调Caspase-3表达诱导NSCLC细胞凋亡,并抑制肿瘤细胞的增殖,进而抑制NSCLC的发展。     

死亡结构域相关蛋白及其在宫颈病变中的研究进展

宫颈癌对妇女健康构成严重威胁,人乳头瘤病毒感染与宫颈病变及宫颈癌的发生密切相关。关于宫颈癌发生发展的机制仍在研究中。近年研究发现一种多功能核蛋白,即死亡结构域相关蛋白(death domain associated protein,Daxx),其与细胞内蛋白或病毒蛋白相互作用,参与调节细胞凋亡、转录调控、抗病毒等细胞活动,在不同途径中发挥不同的生理或病理作用。通过对Daxx功能及其作用机制的研究有助于进一步阐明宫颈癌发生发展的机制,有助于发现新的预防和治疗方法。综述Daxx的一般特性和研究现况及其在宫颈病变的研究进展。     

Cellular mechanisms of hereditary photoreceptor degeneration – Focus on cGMP

The cellular mechanisms underlying hereditary photoreceptor degeneration are still poorly understood, a problem that is exacerbated by the enormous genetic heterogeneity of this disease group. However, the last decade has yielded a wealth of new knowledge on degenerative pathways and their diversity. Notably, a central role of cGMP-signalling has surfaced for photoreceptor cell death triggered by a subset of disease-causing mutations.In this review, we examine key aspects relevant for photoreceptor degeneration of hereditary origin. The topics covered include energy metabolism, epigenetics, protein quality control, as well as cGMP- and Ca²⁺-signalling, and how the related molecular and metabolic processes may trigger photoreceptor demise. We compare and integrate evidence on different cell death mechanisms that have been associated with photoreceptor degeneration, including apoptosis, necrosis, necroptosis, and PARthanatos. A special focus is then put on the mechanisms of cGMP-dependent cell death and how exceedingly high photoreceptor cGMP levels may cause activation of Ca²⁺-dependent calpain-type proteases, histone deacetylases and poly-ADP-ribose polymerase. An evaluation of the available literature reveals that a large group of patients suffering from hereditary photoreceptor degeneration carry mutations that are likely to trigger cGMP-dependent cell death, making this pathway a prime target for future therapy development.Finally, an outlook is given into technological and methodological developments that will with time likely contribute to a comprehensive overview over the entire metabolic complexity of photoreceptor cell death. Building on such developments, new imaging technology and novel biomarkers may be used to develop clinical test strategies, that fully consider the genetic heterogeneity of hereditary retinal degenerations, in order to facilitate clinical testing of novel treatment approaches.     

丹参二萜醌类活性成分通过PKCδ/PKD1μ信号通路抗胰腺癌的作用研究

摘要 目的 验证丹参二萜醌类活性成分对胰腺癌和多发性骨髓瘤的抑制效应，阐明其诱导胰腺癌和多发性骨髓瘤凋亡的作用机制。方法 胰腺癌细胞AsPC-1、BxPC-3用含10% gibco胎牛血清的RPMI1640培养液培养，按隐丹参酮组（30μM）、丹参新酮组（15μM）、去氢丹参新酮组（15μM）分别加药处理。cell-counting-kit-8（CCK8）法检测细胞存活率；AnnexinⅤ-FITC/PI流式细胞术检测细胞凋亡；Western Blot检测相关PKC同工酶磷酸化水平；对AsPC-1和BxPC-3细胞进行siRNA转染，Western Blot检测相关PKC同工酶磷酸化水平。结果 隐丹参酮组AsPC-1、BxPC-3细胞存活率分别为40.1%±5.0%、36.2%±5.4%；丹参新酮组AsPC-1、BxPC-3细胞存活率分别为52.1%±5.1%、47.2%±5.7%；去氢丹参新组AsPC-1、BxPC-3细胞存活率分别为46.1%±5.0%、42.2%±5.4%(P<0.01)。流式细胞术结果显示：AsPC-1组内空白对照组、隐丹参酮组、丹参新酮组、去氢丹参新酮组细胞的凋亡率分别为4.71%、30.10%、52.26%、42.30%；BxPC-3组内空白对照组、隐丹参酮组、丹参新酮组、去氢丹参新酮组细胞的凋亡率分别为5.10%、30.66%、33.76%、51.76%（P<0.01）。Western Blot检测显示隐丹参酮组、丹参新酮组、去氢丹参新酮组较空白对照组，胰腺癌AsPC-1、BxPC-3细胞p-PKD/PKCμ ser916、p-PKCδ thr505、p-PKD/PKCμ ser744/748的水平降低。Western Blot检测显示，siRNA沉默胰腺癌AsPC-1、BxPC-3细胞PKCδ，胰腺癌AsPC-1、BxPC-3细胞PKD/PKCμ ser744/748的磷酸化水平下调。结论 隐丹参酮、丹参新酮、去氢丹参新酮通过抑制PKCδthr505的磷酸化水平，继而PKD1μser744/748磷酸化水平下调，从而显著促进胰腺癌AsPC-1和BxPC-3细胞凋亡发生。     

Deoxynivalenol induces apoptosis in PC12 cells via the mitochondrial pathway

Deoxynivalenol (DON) has broad toxicity in animals and humans. In this study the impact of DON treatment on apoptotic pathways in PC12 cells was determined. The effects of DON were evaluated on (i) typical indicators of apoptosis, including cellular morphology, cell activity, lactate dehydrogenase (LDH) release, and apoptosis ratio in PC12 cells, and on (ii) the expression of key genes and proteins related to apoptosis, including Bcl-2, Bax, Bid, cytochrome C (Cyt C), apoptosis inducing factor (AIF), cleaved-Caspase9, and cleaved-Caspase3. DON treatment inhibited proliferation of PC12 cells, induced significant morphological changes and apoptosis, promoted the release of Cyt C and AIF from the mitochondria, and increased the activities of cleaved-Caspase9 and cleaved-Caspase3. Bcl-2 expression decreased with increasing DON concentrations, in contrast to Bax and Bid, which were increased with increasing DON concentration. These data demonstrate that DON induces apoptosis in PC12 cells through the mitochondrial apoptosis pathway.     

Aloe-Emodin Protects RIN-5F (Pancreatic β-cell) Cell from Glucotoxicity via Regulation of Pro-Inflammatory Cytokine and Downregulation of Bax and Caspase 3

To determine the protective effect of aloe-emodin (AE) from high glucose induced toxicity in RIN-5F (pancreatic β-cell) cell and restoration of its function was analyzed. RIN-5F cells have been cultured in high glucose (25 mM glucose) condition, with and without AE treatment. RIN-5F cells cultured in high glucose decreased cell viability and increased ROS levels after 48 hr compared with standard medium (5.5 mM glucose). Glucotoxicity was confirmed by significantly increased ROS production, increased pro-inflammatory (IFN-γ, IL-1β,) & decreased anti-inflammatory (IL-6&IL-10) cytokine levels, increased DNA fragmentation. In addition, we found increased Bax, caspase 3, Fadd, and Fas and significantly reduced Bcl-2 expression after 48 hr. RIN-5F treated with both high glucose and AE (20 μM) decreased ROS generation and prevent RIN-5F cell from glucotoxicity. In addition, AE treated cells cultured in high glucose were transferred to standard medium, normal responsiveness to glucose was restored within 8hr and normal basal insulin release within 24 hr was achieved when compared to high glucose.     

灯盏乙素介入IP3R-Ca2+途径抑制β淀粉样蛋白诱导的神经细胞凋亡

目的：观察灯盏乙素（Scu）对β淀粉样蛋白（Aβ）诱导的细胞模型中1，4，5-三磷酸肌醇受体（IP₃R）-Ca²⁺途径的影响，探讨其在阿尔茨海默病（AD）病程中可能发挥的积极作用。方法：选用神经母细胞瘤细胞分为对照组、Scu处理组、Aβ处理组、Aβ+Scu （高、中、低）处理组及Aβ+IP₃R拮抗剂组，用CCK-8法筛选药物浓度并检测各组细胞生存率；用酶联免疫吸附法检测各组细胞中1，4，5-三磷酸肌醇（IP₃）的含量；用蛋白印迹和实时荧光定量聚合酶链反应方法检测各组细胞IP₃R和凋亡相关因子Caspase-3、Bcl-2、Bax的蛋白及mRNA的表达水平；用激光共聚焦显微镜观察各组细胞内Ca²⁺浓度的变化；用AnnexinV/PI双染法测定各组细胞的凋亡率。结果：与对照组和Scu处理组相比，Aβ处理组细胞存活率下降，IP₃含量升高，IP₃R、Bax和Caspase-3的蛋白及mRNA表达上调，Bcl-2蛋白及mRNA的表达下调，细胞胞浆内Ca²⁺浓度及细胞凋亡率升高；Aβ+Scu处理组细胞中各检测指标的变化与Aβ处理组的结果正好相反，IP₃R通道下游指标的变化与Aβ+IP₃R拮抗剂组基本一致。结论：Scu能够下调通路蛋白IP₃、IP₃R的表达，抑制Aβ介导的Ca²⁺内流所致的细胞凋亡，可能通过对IP₃R-Ca²⁺途径的调控来影响AD病程。     

莱菔硫烷促进神经胶质瘤细胞凋亡的机制探讨

目的探讨莱菔硫烷(SFN)促进神经胶质瘤细胞凋亡的作用机制。方法采用噻唑蓝(MTT)法与流式细胞法检测不同浓度的SFN对神经胶质瘤U251细胞系生长的抑制作用,测定半数抑制浓度,空白对照设为对照组;采用Western blot研究SFN对U251细胞内Cyt-c的表达情况。结果不同浓度SFN组A值均低于对照组,差异有统计学意义(P<0.05)。不同浓度SFN均对U251细胞生长有一定抑制作用,12.5μmol/l、25μmol/l、50μmol/l、100μmol/l浓度的SFN对U251细胞生长抑制率逐渐增强,各组间比较差异均有统计学意义(P<0.05)。但100μmol/l与200μmol/l浓度的SFN对U251细胞生长抑制率比较无统计学意义(P>0.05)。不同浓度SFN诱导细胞凋亡率及Cyt-c蛋白表达均显著高于对照组,且随着SFN浓度递增,U251细胞凋亡率、Cyt-c蛋白表达逐渐增加,各组间比较差异均有统计学意义(P<0.05)。结论SFN可能参与神经胶质瘤细胞的凋亡过程,表现为诱导凋亡U251细胞,可能与Cyt-c表达增高存在关系。