【目的】探讨长梗秦艽酮、微小RNA(miR)-495-3p质粒对肾癌细胞增殖、侵袭、凋亡及顺铂敏感性的影响及协同作用。【方法】(1)体外研究:将人肾癌caki-1细胞分为空白组(未处理),长梗秦艽酮低、中、高剂量组,miR阴性对照(miRNC)组和miR-495...【目的】探讨长梗秦艽酮、微小RNA(miR)-495-3p质粒对肾癌细胞增殖、侵袭、凋亡及顺铂敏感性的影响及协同作用。【方法】(1)体外研究:将人肾癌caki-1细胞分为空白组(未处理),长梗秦艽酮低、中、高剂量组,miR阴性对照(miRNC)组和miR-495-3p组(转染组)等6组。细胞计数试剂盒8(CCK-8)法检测不同浓度顺铂处理的caki-1细胞活力、半数抑制浓度(IC50),膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙啶(PI)法测定caki-1细胞凋亡情况,Transwell实验测定caki-1细胞侵袭能力,实时定量聚合酶链反应(qRT-PCR)法检测细胞中miR-495-3p和X染色体连锁凋亡抑制蛋白(XIAP)mRNA表达。(2)体内研究:构建高度免疫缺陷NSG小鼠肾癌移植瘤模型。将40只NSG小鼠分为阴性组(腋部皮下注射转染miR-NC质粒的肾癌caki-1细胞)、转染组(腋部皮下注射转染miR-495-3p质粒的肾癌caki-1细胞)、药物组(腋部皮下注射肾癌caki-1细胞,灌胃长梗秦艽酮)和联合组(腋部皮下注射转染miR-495-3p质粒的肾癌caki-1细胞,灌胃长梗秦艽酮),检测并比较各组小鼠的肿瘤质量和体积。【结果】(1)与空白组比较,长梗秦艽酮低、中、高剂量组caki-1细胞活力及对顺铂的IC50值均显著降低,凋亡率显著增加,细胞侵袭能力显著降低,miR-495-3p表达水平升高,XIAP m RNA表达水平显著降低(均P<0.05);与miR-NC组比较,miR-495-3p组caki-1细胞活力、对顺铂的IC50值均显著降低,凋亡率显著增加,细胞侵袭能力显著降低,miR-495-3p表达水平升高,XIAP mRNA表达水平显著降低(均P<0.05);长梗秦艽酮高剂量组与miR-495-3p组上述各指标水平比较,差异均无统计学意义(P>0.05)。(2)与阴性组比较,转染组、药物组和联合组小鼠肿瘤质量和肿瘤体积均显著减小(P<0.05);联合组小鼠肿瘤质量和肿瘤体积均小于转染组和药物组(P<0.05);转染组小鼠肿瘤质量和肿瘤体积与药物组比较,差异无统计学意义(P>0.05)。【结论】长梗秦艽酮、miR-495-3p质粒可靶向抑制XIAP降低肾癌细胞生物活性,促进细胞凋亡,抑制细胞侵袭,增加细胞的顺铂敏感性,且二者有一定的协同作用。展开更多
Background: The endocannabinoid (EC) system is well characterized in the central nervous system but scarcely studied in peripheral organs. In this paper, we newly identify the effect of the EC anandamide (AEA) upon re...Background: The endocannabinoid (EC) system is well characterized in the central nervous system but scarcely studied in peripheral organs. In this paper, we newly identify the effect of the EC anandamide (AEA) upon renal proximal tubule cells. Methods: Measurement of lactate dehydrogenase (LDH) release after treatment of primary renal proximal tubule cells (RPTEC) and renal carcinoma cell line (Caki-1) with AEA, arachidonic acid (AA), ethanolamide (EtAm), EC receptor CB1 antagonist (AM251), CB2 receptor antagonist (SR144528), TRPV1 receptor antagonist (capsazepine), degradation enzyme fatty acid amide hydrolase (FAAH) antagonist (URB597), antioxidants GSH-EE;Trolox, GSH depletor BSO, membrane cholesterol depletor (MCD), apoptosis inhibitor zVAD, necroptosis inhibitor Nec-1 or ferroptosis inhibitor Fer-1. Western blot and qRT-PCR analysis plus determination of reactive oxygen species (ROS) via H2-DCFDA were performed. Histology for EC enzymes, N-acetylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) and FAAH, as well as the determination of physiological levels of ECs in human and rat renal tissue via liquid chromatography were conducted. Results: AEA both dose- and time-dependently induces cell death in RPTEC and Caki-1 within hours, characterized by cell blebbing, not influenced by blocking the described EC receptors by AM251, SR144528, capsazepine or FAAH by URB597 or MCD. Cell death is mediated via ROS. There is no difference found in the histology of the enzymes FAAH and NAPE-PLD in human renal tissue with interstitial nephritis. Blocking of apoptotic, necroptotic or ferroptotic cell death does not lead to a reduction in LDH release in vitro. Conclusion: The endocannabinoid anandamide induces cell death in renal proximal tubule cell in a time- and dose-dependent manner. This pathway is mediated via ROS and is independent of cannabinoid receptors, membrane cholesterol or FAAH activity.展开更多
文摘【目的】探讨长梗秦艽酮、微小RNA(miR)-495-3p质粒对肾癌细胞增殖、侵袭、凋亡及顺铂敏感性的影响及协同作用。【方法】(1)体外研究:将人肾癌caki-1细胞分为空白组(未处理),长梗秦艽酮低、中、高剂量组,miR阴性对照(miRNC)组和miR-495-3p组(转染组)等6组。细胞计数试剂盒8(CCK-8)法检测不同浓度顺铂处理的caki-1细胞活力、半数抑制浓度(IC50),膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙啶(PI)法测定caki-1细胞凋亡情况,Transwell实验测定caki-1细胞侵袭能力,实时定量聚合酶链反应(qRT-PCR)法检测细胞中miR-495-3p和X染色体连锁凋亡抑制蛋白(XIAP)mRNA表达。(2)体内研究:构建高度免疫缺陷NSG小鼠肾癌移植瘤模型。将40只NSG小鼠分为阴性组(腋部皮下注射转染miR-NC质粒的肾癌caki-1细胞)、转染组(腋部皮下注射转染miR-495-3p质粒的肾癌caki-1细胞)、药物组(腋部皮下注射肾癌caki-1细胞,灌胃长梗秦艽酮)和联合组(腋部皮下注射转染miR-495-3p质粒的肾癌caki-1细胞,灌胃长梗秦艽酮),检测并比较各组小鼠的肿瘤质量和体积。【结果】(1)与空白组比较,长梗秦艽酮低、中、高剂量组caki-1细胞活力及对顺铂的IC50值均显著降低,凋亡率显著增加,细胞侵袭能力显著降低,miR-495-3p表达水平升高,XIAP m RNA表达水平显著降低(均P<0.05);与miR-NC组比较,miR-495-3p组caki-1细胞活力、对顺铂的IC50值均显著降低,凋亡率显著增加,细胞侵袭能力显著降低,miR-495-3p表达水平升高,XIAP mRNA表达水平显著降低(均P<0.05);长梗秦艽酮高剂量组与miR-495-3p组上述各指标水平比较,差异均无统计学意义(P>0.05)。(2)与阴性组比较,转染组、药物组和联合组小鼠肿瘤质量和肿瘤体积均显著减小(P<0.05);联合组小鼠肿瘤质量和肿瘤体积均小于转染组和药物组(P<0.05);转染组小鼠肿瘤质量和肿瘤体积与药物组比较,差异无统计学意义(P>0.05)。【结论】长梗秦艽酮、miR-495-3p质粒可靶向抑制XIAP降低肾癌细胞生物活性,促进细胞凋亡,抑制细胞侵袭,增加细胞的顺铂敏感性,且二者有一定的协同作用。
基金supported by Koeln Fortune Program/Faculty of Medicine,University of Cologne and excellence cluster initiative supported by University of Cologne and DFG.
文摘Background: The endocannabinoid (EC) system is well characterized in the central nervous system but scarcely studied in peripheral organs. In this paper, we newly identify the effect of the EC anandamide (AEA) upon renal proximal tubule cells. Methods: Measurement of lactate dehydrogenase (LDH) release after treatment of primary renal proximal tubule cells (RPTEC) and renal carcinoma cell line (Caki-1) with AEA, arachidonic acid (AA), ethanolamide (EtAm), EC receptor CB1 antagonist (AM251), CB2 receptor antagonist (SR144528), TRPV1 receptor antagonist (capsazepine), degradation enzyme fatty acid amide hydrolase (FAAH) antagonist (URB597), antioxidants GSH-EE;Trolox, GSH depletor BSO, membrane cholesterol depletor (MCD), apoptosis inhibitor zVAD, necroptosis inhibitor Nec-1 or ferroptosis inhibitor Fer-1. Western blot and qRT-PCR analysis plus determination of reactive oxygen species (ROS) via H2-DCFDA were performed. Histology for EC enzymes, N-acetylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) and FAAH, as well as the determination of physiological levels of ECs in human and rat renal tissue via liquid chromatography were conducted. Results: AEA both dose- and time-dependently induces cell death in RPTEC and Caki-1 within hours, characterized by cell blebbing, not influenced by blocking the described EC receptors by AM251, SR144528, capsazepine or FAAH by URB597 or MCD. Cell death is mediated via ROS. There is no difference found in the histology of the enzymes FAAH and NAPE-PLD in human renal tissue with interstitial nephritis. Blocking of apoptotic, necroptotic or ferroptotic cell death does not lead to a reduction in LDH release in vitro. Conclusion: The endocannabinoid anandamide induces cell death in renal proximal tubule cell in a time- and dose-dependent manner. This pathway is mediated via ROS and is independent of cannabinoid receptors, membrane cholesterol or FAAH activity.
