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.     

补肾活血方对血管性痴呆大鼠海马神经细胞自噬的影响＊

目的 探究补肾活血方对血管性痴呆（Vascular Dementia， VD）大鼠模型自噬的影响。方法 52周龄SD雄性大鼠50只，随机分为假手术组（Sham组）、模型组（VD组）、模型+补肾活血方组（BSHX组）、模型+雷帕霉素组（Rap组）和模型+3-甲基腺嘌呤组（3-MA组），每组10只大鼠。除Sham组外其余各组采用两血管阻断法（2-VO）建立VD模型。BSHX组中药灌胃治疗28天；自噬干预组于造模前30 min侧脑室给药。运用Morris水迷宫测试各组大鼠的学习记忆能力；通过尼氏染色和透射电子显微镜（Transmission electron microscope，TEM）观察大鼠海马区病理形态及自噬变化；采用蛋白免疫印迹（Western blot）法和反转录实时荧光定量PCR（RT-qPCR）检测大鼠海马Beclin-1、P62以及微管相关蛋白1轻链3（LC3）蛋白及mRNA表达情况。结果 与VD组比较，BSHX组大鼠学习记忆能力显著提升（P<0.05），镜下观察BSHX组和3-MA组的细胞形态及数量均有改善，自噬小体鲜见，Beclin-1以及LC3蛋白和mRNA表达水平显著降低（P<0.05），P62蛋白和mRNA表达水平明显升高（P<0.05）结论 补肾活血方可以降低VD大鼠海马区Beclin-1和LC3蛋白及mRNA的表达，提高P62的表达，通过抑制自噬的发生，减轻对神经细胞的损伤，改善学习记忆能力。     

Rapamycin prevents thoracic aortic aneurysm and dissection in mice

Objective

The purpose of this study was to investigate whether rapamycin inhibits the development of thoracic aortic aneurysm and dissection (TAAD) in mice.

mTOR通路抑制剂雷帕霉素对真菌性角膜炎小鼠角膜瘢痕化的影响

目的　探讨mTOR通路抑制剂雷帕霉素对真菌性角膜炎小鼠角膜瘢痕化的影响。方法　取96只SPF级C57BL/6J雄性小鼠,随机分为雷帕霉素组和对照组，每组各48只。两组小鼠同时建立真菌性角膜炎模型。雷帕霉素组模型制作前1 d按6.0 mg·kg^-1雷帕霉素对小鼠进行腹腔注射预处理，之后按0.2 g·L^-1浓度在结膜下注射5 μL，持续3 d；对照组注射PBS溶液。造模后对各组小鼠进行角膜临床评分，Western blot和实时荧光定量PCR分别检测造模后各组小鼠不同时间角膜LC-3Ⅱ、α-SMA和 TGF-β1表达情况。结果　造模后24 h、48 h、72 h、96 h、120 h、144 h、336 h，对照组小鼠角膜临床评分均明显高于雷帕霉素组，差异均有统计学意义(均为P＜0.05) 。造模后144 h、216 h雷帕霉素组小鼠角膜LC-3Ⅱ表达上调，LC-3Ⅱ蛋白和mRNA相对表达量与对照组相比，差异均有统计学意义（均为P<0.05）；而在造模后72 h及336 h两组LC-3Ⅱ蛋白和mRNA相对表达量差异均无统计学意义（均为P>0.05）。与对照组相比，造模后144 h、216 h雷帕霉素组小鼠角膜α-SMA蛋白及 mRNA相对表达量下降，差异均有统计学意义（均为P<0.05）；造模后144 h、336 h雷帕霉素组TGF-β1 mRNA相对表达量亦下降，与对照组相比，差异均有统计学意义（均为P＜0.05）。结论　雷帕霉素通过促进自噬作用下调角膜瘢痕化相关因子的表达，减轻了真菌性角膜炎模型小鼠角膜瘢痕化程度。     

雷帕霉素对去甲氧柔红霉素诱导急性髓系白血病THP-1细胞凋亡的影响

目的:探讨雷帕霉素（Rapa）对去甲氧柔红霉素（IDA）诱导急性髓系白血病THP-1细胞凋亡的影响及其分子机制。方法:分别用10、20、40、80 nmol/L Rapa处理THP-1细胞1 h，另设未经Rapa处理的细胞。采用蛋白质印迹法检测THP-1细胞自噬标志物LC3蛋白的转换情况（LC3Ⅱ/LC3Ⅰ），采用流式细胞术检测细胞凋亡，确定Rapa处理浓度。用不同浓度IDA作用THP-1细胞24 h，采用CCK-8法检测IDA对THP-1细胞的增殖抑制率，计算半数抑制浓度（ IC50）。以低于 IC50的IDA作用Rapa处理或未处理的THP-1细胞24 h，CCK-8法检测细胞增殖抑制率，流式细胞术检测细胞凋亡情况，实时荧光定量聚合酶链反应检测自噬相关基因Beclin-1、LC3和p62的表达变化，蛋白质印迹法检测自噬标志物LC3蛋白的转换情况。 结果:20 nmol/L Rapa处理的THP-1细胞LC3Ⅱ/LC3Ⅰ高于未处理的细胞（ P=0.002 4）；80 nmol/L Rapa处理的细胞凋亡率高于未处理的细胞（ P=0.007 3）。根据蛋白质印迹法和流式细胞术检测结果，选取20 nmol/L Rapa作为预处理浓度。IDA对THP-1细胞作用24 h的 IC50为59.874 nmol/L。50 nmol/L IDA作用24 h后，Rapa预处理的THP-1细胞增殖抑制率[（69.67±5.03）%比（41.67±3.51）%]和细胞凋亡率[（74.35±4.83）%比（41.25±5.24）%]均高于未预处理的细胞（均 P<0.05）；Rapa预处理的THP-1细胞Beclin-1、LC3 mRNA表达水平及LC3Ⅱ/LC3Ⅰ均高于未预处理的细胞，p62 mRNA表达水平低于未预处理的细胞（均 P<0.05）。 结论:Rapa能增强较低剂量IDA诱导的THP-1细胞凋亡，此效应可能是通过其引起THP-1细胞过度自噬实现的。     

他克莫司和雷帕霉素对肝癌肝移植患者Foxp3+ Treg的影响及防治排斥反应的研究

目的 探讨免疫抑制剂他克莫司和雷帕霉素对肝癌肝移植受者Foxp3+ Treg产生的影响及其防治排斥反应的疗效.方法 自移植后第2个月到第12个月,每月采血,采用实时荧光定量PCR法检测他克莫司组和雷帕霉素组肝癌肝移植受者新鲜外周血单个核细胞中Foxp3 mRNA的表达水平,通过同期术后观察和实验室检查,比较两组受者间Foxp3 mRNA表达水平和急性排斥反应发生率的差异.结果 他克莫司组受者的外周血单个核细胞中Foxp3 mRNA表达水平(0.1032±0.0943)明显低于雷帕霉素组受者(1.2136±0.6738),差异有统计学意义(t=5.1610,P＜0.01);雷帕霉素组患者比同期他克莫司组受者术后急性排斥反应发生率明显减低,差异有统计学意义(x2=2.2222,P＜0.05).结论 他克莫司抑制了肝癌肝移植术后免疫耐受的诱导,而雷帕霉素可能参与了免疫耐受的诱导和维持;雷帕霉素对肝癌肝移植受者防治排斥反应的效果更好.     

Rapamycin inhibiting Jurkat T cells viability through changing mRNA expression of serine/threonine protein phosphatase 2A

Aims

In this study, we analyzed the mRNA expression of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) in the human leukemic T-cell line Jurkat cells treated with rapamycin, to determine whether rapamycin inhibiting cell viability is accompanied with the change of mRNA expression of PP2A.

Methods and results

Jurkat cells were incubated with various concentrations of rapamycin and cultured for different hours. Cell viability was assessed by MTT assay. The mRNA expressions of PP2A subunits were measured by quantitative real-time polymerase chain reaction (PCR). We found that rapamycin had an inhibitory effect on cell viability. IC50 was 343.3 nM at 48 h.We also found rapamycin had a dose and time-dependent effect on the gene expression of PP2A. When setting the concentration of rapamycin 500 nM, the mRNA expressions of PP2A subunits (Aa, Aβ, PR55a, PR55δ, PR61γ, PR70, Ca and Cβ) were declined significantly at 48 h. When treated with various concentrations of rapamycin for 48 h, the mRNA expressions of PP2A subunits were down-regulated in the range from 10 nM to500 nM.

Conclusions

Rapamycin inhibiting Jurkat T cells viability may be related to the reduction of PP2A mRNA expressions.     

从雷帕霉素与肝癌的关系浅谈转化医学的研究思路

Translational medicine is a new concept in China.It sets up a bridge between basic research and clinical work,and aims to transfer the basic study results to clinical practice as soon as possible.It is...