【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
Objective: Aldehyde dehydrogenase (ALDH) enzymatic activity identifies ovarian cancer stem-like cells. We investigated the antineoplastic activity of the ALDH inhibitor Disulfiram on bulk ovarian cancer cells and CD13...Objective: Aldehyde dehydrogenase (ALDH) enzymatic activity identifies ovarian cancer stem-like cells. We investigated the antineoplastic activity of the ALDH inhibitor Disulfiram on bulk ovarian cancer cells and CD133+/ALDH+ cancer stem-like cells. Study Design: Ovarian cancer cell lines, human ovarian surface epithelial cells, and mesenchymal stem cells were treated with increasing concentrations of Disulfiram and/or Cisplatin in vitro. Treated cells were assessed for viability or FACS-analyzed for either percentage of ovarian cancer stem-like cells or induction of apoptosis. Disulfiram’s impact on cancer stem-like cells was tested in vitro using tumor sphere formation assays and in vivo using tumor initiation assays with in vitro-treated A2780 cells in NSG mice. Finally, Disulfiram’s in vivo activity was assessed versus CD133+/ALDH+ cell-initiated tumor xenografts. Results: Disulfiram demonstrated antineoplastic activity against multiple ovarian cancer cell lines. While Disulfiram had limited in vitro toxicity against human ovarian surface epithelial cells or mesenchymal stem cells (IC50 of ~15 μM and >30 μM, respectively), its antineoplastic activity against cell lines was comparable to Cisplatin (IC50 ~1.5 μM). Disulfiram-mediated cell death was due, at least in part, to induction of apoptosis. Disulfiram activity was additive with chemotherapy. Disulfiram demonstrated selective depletion of CD44+ cells but not the CD133+ cancer stem-like cells. Disulfiram had no therapeutic impact on tumor initiation studies or in vivo therapy of whole cell line or stem cell-initiated tumor xenografts. Conclusions: In biologically relevant concentrations, Disulfiram has clear antineoplastic activity against ovarian cancer cells in vitro. Disulfiram selectively depleted CD44+ but not CD133+ ovarian cancer stem-like cells in vitro. However, Disulfiram had no significant activity in vivo. Thus, improved and more selective ALDH inhibitors may be required to target ovarian cancer stem cells.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。
文摘Objective: Aldehyde dehydrogenase (ALDH) enzymatic activity identifies ovarian cancer stem-like cells. We investigated the antineoplastic activity of the ALDH inhibitor Disulfiram on bulk ovarian cancer cells and CD133+/ALDH+ cancer stem-like cells. Study Design: Ovarian cancer cell lines, human ovarian surface epithelial cells, and mesenchymal stem cells were treated with increasing concentrations of Disulfiram and/or Cisplatin in vitro. Treated cells were assessed for viability or FACS-analyzed for either percentage of ovarian cancer stem-like cells or induction of apoptosis. Disulfiram’s impact on cancer stem-like cells was tested in vitro using tumor sphere formation assays and in vivo using tumor initiation assays with in vitro-treated A2780 cells in NSG mice. Finally, Disulfiram’s in vivo activity was assessed versus CD133+/ALDH+ cell-initiated tumor xenografts. Results: Disulfiram demonstrated antineoplastic activity against multiple ovarian cancer cell lines. While Disulfiram had limited in vitro toxicity against human ovarian surface epithelial cells or mesenchymal stem cells (IC50 of ~15 μM and >30 μM, respectively), its antineoplastic activity against cell lines was comparable to Cisplatin (IC50 ~1.5 μM). Disulfiram-mediated cell death was due, at least in part, to induction of apoptosis. Disulfiram activity was additive with chemotherapy. Disulfiram demonstrated selective depletion of CD44+ cells but not the CD133+ cancer stem-like cells. Disulfiram had no therapeutic impact on tumor initiation studies or in vivo therapy of whole cell line or stem cell-initiated tumor xenografts. Conclusions: In biologically relevant concentrations, Disulfiram has clear antineoplastic activity against ovarian cancer cells in vitro. Disulfiram selectively depleted CD44+ but not CD133+ ovarian cancer stem-like cells in vitro. However, Disulfiram had no significant activity in vivo. Thus, improved and more selective ALDH inhibitors may be required to target ovarian cancer stem cells.
