盐霉素抑制肝癌细胞增殖、侵袭和转移的机制研究
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摘要
目的：探讨盐霉素对人肝癌细胞株增殖、侵袭和转移能力影响的作用机制。构建靶向c-Src基因的ShRNA慢病毒表达载体以稳定抑制c-Src基因的表达,建立稳定干扰c-Src基因的肝癌细胞株；研究c-Src基因稳定抑制对肝癌细胞株HepG2生物学特性及对盐霉素敏感性的影响及其机制。
方法：1.MTT法检测盐霉素对肝癌细胞和正常肝细胞L02增殖的影响；倒置显微镜观察细胞形态改变；利用FITC标记的鬼笔环肽进行微丝荧光染色,激光共聚焦技术获得盐霉素作用下F-actin细胞骨架的形态变化；Transwell小室法测定盐霉素对肝癌细胞体外迁移、侵袭能力的变化。流式细胞术检测细胞周期和凋亡情况Western Blotting法检测盐霉素对肝癌细胞中Src/PI3K/Akt通路中相关蛋白表达的影响。
2.采用慢病毒构建的靶向c-Src基因的ShRNA,感染HepG2肝癌细胞,利用Western Blotting方法鉴定胞内Src蛋白的敲减效率筛选建立稳定沉默株。实验分为六组：CON组(空白组,不作转染),NC组(阴性对照空白质粒转染组,HepG2-shVector转染组),KD组(HepG2-shSrc转染组),CON+Sal组(CON+4μM Salinomycin作用组),NC+Sal组(HepG2-shSrc+4μM Salinomycin),KD+Sal组(HepG2-shSrc+4μ M Salinomycin).MTT检测各组细胞的增殖情况；流式细胞术检测细胞周期、凋亡；Western Blotting检测Src. MDR1、Akt、cyclinD1. pro-Caspase-9蛋白的表达情况
结果：1、MTT实验证实盐霉素抑制肝癌细胞的增殖,并呈现出时间、剂量依赖效应；而盐霉素对正常肝细胞L02的增殖抑制作用不明显。倒置显微镜显示肝癌细胞密度呈剂量依赖性降低,同时观察到凋亡细胞所具有的形态学改变。经过盐霉素(1μM和4μM)处理的肝癌细胞,迁移和侵袭实验中穿膜细胞数均明显低于对照组(P<0.05),且以F-actin为基础的微丝骨架结构发生紊乱。FCM(PI单染和AnnexinV-PI双染法)检测发现盐霉素作用于肝癌细胞24h后凋亡细胞数量呈剂量依赖性增加,G0／G1期细胞明显增多。Western Blotting显示盐霉素(4u M和8u M)作用肝癌细胞48h后,c-Src, Akt,Bcl-2, Bcatenin,c-myc,cyclinD1,survivin,MMP-2, MMP-9的表达水平降低；E-cadherin,Bad, Bax的表达水平增加,GSK-3β,caspase-9,caspase-3的活性增强,PARP的裂解增加(P<0.05).2. We stern Blott ing结果显示Lv-shRNA-Src对c-Src基因的表达有显著抑制作用,抑制效率达90%以上,与CON组和NC组相比差异具有统计学意义(P<0.05),而CON和NC组相比差异无统计学意义(P>0.05)。MTT实验证实盐霉素抑制CON, NC, KD三组细胞的增殖,并呈现出时间、剂量依赖效应。KD组细胞增殖活性低于CON,NC组(P<0.05),而CON,NC组比较差异无统计学意义(P>0.05)。与CON+Sal组、NC+Sal组、KD组相比,KD+Sal组细胞增殖活性明显降低,且随着时间延长,细胞的抑制作用增强(P<0.05),CON+Sal组细胞增殖抑制作用强于K D组(P<0.05),而CON+Sal组与NC+Sal组比较差异无统计学意义(P>0.05)。干预各组细胞48h后,KD组G0／G1期细胞比例增加高于CON、NC组(P<0.05),而CON、NC组相比差异无统计学意义(P>0.05)。与CON+Sal组细胞G0／G1期比例相比,KD+Sal组比例增高(P<0.05),而NC+Sal组细胞的G0／G1期比例变化不明显(P>0.05)。干预各组细胞48h后,与NC+Sal组相比,KD+Sal组细胞凋亡率增高(P<0.05)。与KD组相比,NC+Sal组及KD+Sal组细胞凋亡率增高(P<0.05)。与NC组相比,KD组、NC+Sal组细胞凋亡率增高(P<0.05)。干预各组细胞48h后,与NC组相比,KD组细胞MDR1、Akt的磷酸化水平(p-Akt(Ser473) p-Akt(Thr308))和cyclinD1蛋白表达量显著降低,而pro-Caspase-9蛋白表达水平虽然有所降低,但并不显著。与NC. NC+Sal、KD组相比,KD+Sal组细胞中MDR1、p-Akt(Ser473). p-Akt(Thr308)、Pro-Caspase-9和cvclinD1蛋白表达明显降低(P<0.05),而Ak t蛋白表达无明显改变(P>0.05)。
结论：1、盐霉素能有效抑制人肝癌细胞增殖、迁移和侵袭,其作用机制可能与抑制Src/PI3K/Akt信号通路的活性有关。
2.RNAi能有效抑制c-Src基因表达,抑制肝癌细胞增殖活性,诱导其凋亡,并增加其对盐霉素的敏感性,其机制可能与抑制MDR1和Src/PI3K/Akt信号通路活性及活化Caspase家族凋亡通路有关。
目的：研究姜黄素在体外对膀胱癌EJ细胞的生长抑制作用和凋亡诱导效应,同时探讨姜黄素抗膀胱癌的分子机制。观察姜黄素联合PI3k抑制剂LY294002对人膀胱癌EJ细胞生长及凋亡的影响。
方法1、采用MTT法测定不同浓度姜黄素(0-45μuM)经过24h和48h作用后,EJ细胞增殖的变化,同时观察细胞形态学改变。收集不同浓度姜黄素(0,12.5、25and50μM)作用24h后的EJ细胞,经流式细胞仪检测膀胱癌EJ细胞的DNA含量及凋亡细胞数。Westernblotting法测定不同浓度姜黄素(0,12.5、25and50μM)作用EJ细胞后,c-myc, Bcl-2家族,caspase家族及PI3K/Akt信号通路的凋亡相关蛋白的表达变化。2、用20μ M LY294002提前作用于EJ细胞1h,然后加入不同浓度的姜黄素(0,12.5、25and50μM)共同培养至24h。MTT法检测单独使用姜黄素(0,12.5、25an d50μ M). LY294002(20μM)及二者联合,对体外培养的膀胱癌EJ细胞的生长抑制作用；倒置显微镜观察细胞形态改变；流式细胞仪(AnnexinV-PI双染法)检测各组细胞凋亡率；Westernblotting法测定不同浓度姜黄素(25and50μM)单独或联合20μMLY294002作用EJ细胞后,凋亡相关蛋白caspase-9,-7,-3及PARP的表达变化。
结果1、MTT、实验证实姜黄素以浓度及时间依赖的方式抑制EJ细胞的增殖。倒置显微镜显示EJ细胞密度呈剂量依赖性降低,同时观察到凋亡细胞所具有的形态学改变。FCM(PI单染和AnnexinV-PI双染法)检测发现姜黄素作用于EJ细胞24h后sub-G1峰及凋亡细胞数量呈剂量依赖性增加。Westernblotting显示不同浓度姜黄素(0,25and50μ M)作用EJ细胞后,c-myc, Bad, Bax的表达水平增加,PTEN, GSK-3β, c-Raf, caspase-9, caspase-7, caspase-3的活性增强,PARP的裂解增加和Bc1-2,Akt的表达水平降低。2、MTT实验证实姜黄素联合LY294002组明显抑制EJ细胞的增殖,优于LY294002、姜黄素单独作用组,具有浓度及时间依赖的特点。倒置显微镜显示不同浓度姜黄素联合LY294002组EJ细胞密度的降低和典型凋亡细胞形态的出现明显于LY294002、相应浓度的姜黄素单独作用组。FCM(AnnexinV-PI双染法)检测发现处理24h后,不同浓度姜黄素联合LY294002组凋亡的EJ细胞数量明显多于LY294002、相应浓度的姜黄素单独作用组,且呈剂量依赖性增加。Western Blotting显示不同浓度姜黄素联合LY294002组凋亡相关蛋白caspase-9,caspase-7, caspase-3的活性增强,PARP的裂解增加出现的时间早于相应浓度的姜黄素单独作用组。
结论：1、姜黄素对膀胱癌EJ细胞具有增殖抑制作用和凋亡诱导效应。姜黄素能显著增加c-myc的表达,进而引起Bax的上调,Bcl-2的下调和caspase-9,-7,-3的增加。姜黄素抑制EJ细胞PI3K/Akt信号通路的激活,继之激活下游GSK-3β,caspase-9和Bad等多种促凋亡分子的表达,最终诱导EJ细胞凋亡发生。C-myc和PI3K/Akt信号通路在姜黄素诱导的膀胱癌EJ细胞的凋亡中起了重要的作用。2、LY294002与姜黄素单独应用均可抑制EJ细胞增殖LY294002能有效提高姜黄素体外对膀胱癌EJ细胞的抑制作用与单独用药组相比,联合用药组具有明显的时间依赖性协同增强EJ细胞凋亡的作用。二者联合用药增强诱导细胞凋亡主要是激活caspase家族,通过线粒体途径促进膀胱癌EJ细胞的凋亡裂解PARP来调控的。LY294002可提高姜黄素治疗膀胱癌的效果,为临床治疗膀胱癌提供新的思路和方法
Objective:To investigate effects of salinomycin on the proliferative, invisive and metastatic abilities of hepatoma cell line in vitro and analyze the possible mechanisms. To construct lentivirus-mediated shRNA expression vector targeting c-Src and to establish stable hepatoma cell line HepG2. To explore the effect RNAi-mediated inhibition of c-Src gene on apoptosis and chemosensitivity to Salinomycin in hepatoma HepG2cells and its underlying mechanism.
Methods:1、MTT was used for determining the proliferative abilities of hepatoma cells and human normal liver L02cells. Morphological changes were observed under the inverted phase-contrast microscope and photographed. Cell apoptosis was detected by Hoechst33258staining and flowcytometry analysis. The morphological alteration was observed by Hoechst33258staining. The cytoskeleton was observed using confocal laser scanning microscopy after F-actin staining by FITC-labeled phalloidin. The invasive and migratory abilities were detected by Transwell assay. Cell-cycle analysis was detected by flowcytometry. The relative protein expressions of Src/PI3K/Akt signal pathway were evaluated by Western Blotting.2、Lentiviral shRNA targeting c-Src was applied to construct stable knock-down hepatoma cell line HepG2, Western Blotting techniques were used to detect the alteration of protein level. HepG2cells were conventionally cultured and divided into6groups:control group (CON), negative control group (NC), RNAi c-Src group (KD), control combined with Salinomycin group (CON+Sal), negative control combined with Salinomycin group (NC+Sal), RNAi Src combined with Salinomycin group (KD+Sal). The proliferation of above mentioned HepG2cells was examined by MTT assay. Flow cytometry (FCM) was employed to detect the cell apoptosis and cell cycle. The protein expressions of MDRland Src/PI3K/Akt signal pathway were evaluated by Western Blotting.
Results:1、The treatment of L02cells with salinomycin (0～8μmol/L) for24h and48h had no significant effects on cell viability. However, treatment of similar doses of salinomycin suppressed the growth of hepatoma cells in a time and concentration dependent manner. Salinomycin could lead to characteristic morphologieal changes of apoptosis in human hepatoma cells after24h and48h in a concentration dependent manner. Hepatoma cells was treated by salinomycin (1μmol/L,4μmol/L) for24h to detect the invasive and migratory abilities. Compared to that in the control group, the number of invasive and migratory cells was remarkably decreased (P<0.01), the structure of F-actin microfilament cytoskeleton was significantly changed. Salinomycin could also induce a cell cycle arrest of hepatoma cells at the phase of G0/G1. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after salinomycin treatment. In addition, Immunoblot showed that salinomycin inhibited the activation of Src/PI3K/Akt pathway in a dose-dependent manner in hepatoma cells. Activation of GSK-3β, caspase-9, caspase-3, cleavage of PARP, upregulation of Bad, Bax, E-cadherin, downregulation of c-Src, Akt, Bcl-2, c-myc, cyclin Dl, survivin, MMP-2and MMP-9were also found in salinomycin-treated hepatoma cells.2、KD group had a significantly lower protein level of c-Src Src and MTT value than CON group and NC group (P<0.05), and had a higher apoptotic rate and larger percentage of G0/G1arrested cells and lower MDR1, Akt, cyclinDl expression than NC group (P<0.05). In KD+Sal group, MTT value was more lower and stronger caspase-9expression and lower MDR1, Akt, cyclinDl expression and larger percentage of G0/G1arrested cells than in KD group and NC+Sal group (P<0.05).
Conclusion:1、The results suggested that the proliferative, invasive and migratory abilities of hepatoma cells can be inhibited by salinomycin, which may be related to blocking the Src/PI3K/AKT kinase cascade signaling pathway.2、RNAi could specifically supress c-Src gene expression, effectively inhibit the proliferation and increase G0/G1cell population and accelerate the apoptosis of HepG2cells, and enhance its chemosensitivity to Salinomycin. The mechanism may be related to the suppression of the activation of MDRland Src/PI3K/Akt pathway and the activation of Caspase signaling pathways.
Objective:Cancer chemopreventive agent curcumin have been shown to possess inhibition cell growtlh and induction apoptosis properties in several types of cancer. However, the detailed molecular mechanisms of the compound remain far from clear in EJ bladder cancer cells. In order to explore the possible mechanism and offer experimental evidence of anti-cancer effcet of curcumin combined with LY294002, we investigated the anti-proliferation and apoptosis induction effcet of curcumin combined with LY294002on human bladder cancer cells.
Methods:1. The effect of curcumin on EJ cells growth inhibition and apoptosis was detected by MTT assays and flow cytometry. The phosphorylation levels of PTEN, PDK1, Akt, GSK-3β, c-Raf, Bad and the expression levels of c-myc, Bax, Bcl-2, caspase-9, caspase-7, caspase-3, PARP by curcumin administration were examined by Western Blotting.2. After EJ cells were pretreated with the PI3k inhibitor LY294002(20μM) in complete medium for1h, and then various doses of Curcumin (0,12.5、25and50μM) was added to co-culture for the indicated time periods. The growth inhibition of curcumin and LY294002alone or in combination on bladder cancer EJ cells by MTT assay. Morphological changes were observed under the inverted phase-contrast microscope and photographed. Apoptotic rates of human EJ bladder carcinoma cells were determined by flow cytometry (FCM). Curcumin and LY294002alone or in combination regulated caspase-9、caspase-7、caspase-3and PARP protein expression in EJ cells by Western Blotting analysis.
Results:1. Curcumin suppressed the growth of EJ cells in a time-and concentration-dependent manners. Immunoblot showed that curcumin increased expression levels of c-myc and inhibited the activation of PI3K/Akt pathway in a time-dependent manner in EJ cells. Activation of PTEN, GSK-3(3, c-Raf, caspase-9, caspase-7, caspase-3, cleavage of PARP, upregulation of Bad, Bax, downregulation of Akt, Bcl-2were also found in curcumin-treated EJ cells.2. EJ cells viability showed remarkable decrease in a concentration-and time-dependented manner in the curcumin (12.5,25and50μM)+LY294002(20μM) group as compared with curcumin (12.5,25and50μM)-treated control group or with LY294002(20μM) alone. Curcumin and LY294002could lead to characteristic morphologieal changes of apoptosis in human bladder carcinoma cells after24h including cellular rounding, shrinkage, chromatin condensation, and membrane blebbing, the appearance of membrane-associated apoptotic bodies and separation from neighboring cells. Combined treatment with LY294002and curcumin increased the percentage of apoptotic cell death in EJ cells as compared with either compound alone. The presenting time of the cleavage of caspase-9,-7,-3and PARP following the LY294002(20μM)/curcumin (25or50μM) combination administration is earlier than that of curcumin (25or50μM) alone treatment in EJ cells by Western Blotting analysis.
Conclusion:1.These findings esteblish a mechanistic linkup or interaction between c-myc, Bax, Bad, Bcl-2, caspase cascades, PI3K/Akt pathway and curcumin-induced apoptosis of EJ cells, suggest that c-myc and PI3K/Akt signaling pathway plays an important roles in curcumin-induced apoptosis of EJ bladder cancer cells.2. Curcumin or LY294002could significantly inhibit the growth of human EJ bladder carcinoma cells, LY294002could effectively increase the growth inhibition of curcumin in EJ cells in vitro. The curcumin and LY294002can all induce human EJ carcinoma cells apoptosis. When the two drugs combined, the inductive effect for cell apoptosis became more obvious, which suggest a possible underlying molecular mechanism Where by curcumin and LY294002could induce the apoptosis signaling pathway in human EJ bladder carcinoma cells by caspase family protein activation and cleavage of PARP protein through mitochondrion-dependent pathway. LY294002could enhance the anti-tumor effect of curcumin, which provide the new thread and methods for the clinical thrapy of bladder cancer.
方法：1.MTT法检测盐霉素对肝癌细胞和正常肝细胞L02增殖的影响；倒置显微镜观察细胞形态改变；利用FITC标记的鬼笔环肽进行微丝荧光染色,激光共聚焦技术获得盐霉素作用下F-actin细胞骨架的形态变化；Transwell小室法测定盐霉素对肝癌细胞体外迁移、侵袭能力的变化。流式细胞术检测细胞周期和凋亡情况Western Blotting法检测盐霉素对肝癌细胞中Src/PI3K/Akt通路中相关蛋白表达的影响。
2.采用慢病毒构建的靶向c-Src基因的ShRNA,感染HepG2肝癌细胞,利用Western Blotting方法鉴定胞内Src蛋白的敲减效率筛选建立稳定沉默株。实验分为六组：CON组(空白组,不作转染),NC组(阴性对照空白质粒转染组,HepG2-shVector转染组),KD组(HepG2-shSrc转染组),CON+Sal组(CON+4μM Salinomycin作用组),NC+Sal组(HepG2-shSrc+4μM Salinomycin),KD+Sal组(HepG2-shSrc+4μ M Salinomycin).MTT检测各组细胞的增殖情况；流式细胞术检测细胞周期、凋亡；Western Blotting检测Src. MDR1、Akt、cyclinD1. pro-Caspase-9蛋白的表达情况
结果：1、MTT实验证实盐霉素抑制肝癌细胞的增殖,并呈现出时间、剂量依赖效应；而盐霉素对正常肝细胞L02的增殖抑制作用不明显。倒置显微镜显示肝癌细胞密度呈剂量依赖性降低,同时观察到凋亡细胞所具有的形态学改变。经过盐霉素(1μM和4μM)处理的肝癌细胞,迁移和侵袭实验中穿膜细胞数均明显低于对照组(P<0.05),且以F-actin为基础的微丝骨架结构发生紊乱。FCM(PI单染和AnnexinV-PI双染法)检测发现盐霉素作用于肝癌细胞24h后凋亡细胞数量呈剂量依赖性增加,G0／G1期细胞明显增多。Western Blotting显示盐霉素(4u M和8u M)作用肝癌细胞48h后,c-Src, Akt,Bcl-2, Bcatenin,c-myc,cyclinD1,survivin,MMP-2, MMP-9的表达水平降低；E-cadherin,Bad, Bax的表达水平增加,GSK-3β,caspase-9,caspase-3的活性增强,PARP的裂解增加(P<0.05).2. We stern Blott ing结果显示Lv-shRNA-Src对c-Src基因的表达有显著抑制作用,抑制效率达90%以上,与CON组和NC组相比差异具有统计学意义(P<0.05),而CON和NC组相比差异无统计学意义(P>0.05)。MTT实验证实盐霉素抑制CON, NC, KD三组细胞的增殖,并呈现出时间、剂量依赖效应。KD组细胞增殖活性低于CON,NC组(P<0.05),而CON,NC组比较差异无统计学意义(P>0.05)。与CON+Sal组、NC+Sal组、KD组相比,KD+Sal组细胞增殖活性明显降低,且随着时间延长,细胞的抑制作用增强(P<0.05),CON+Sal组细胞增殖抑制作用强于K D组(P<0.05),而CON+Sal组与NC+Sal组比较差异无统计学意义(P>0.05)。干预各组细胞48h后,KD组G0／G1期细胞比例增加高于CON、NC组(P<0.05),而CON、NC组相比差异无统计学意义(P>0.05)。与CON+Sal组细胞G0／G1期比例相比,KD+Sal组比例增高(P<0.05),而NC+Sal组细胞的G0／G1期比例变化不明显(P>0.05)。干预各组细胞48h后,与NC+Sal组相比,KD+Sal组细胞凋亡率增高(P<0.05)。与KD组相比,NC+Sal组及KD+Sal组细胞凋亡率增高(P<0.05)。与NC组相比,KD组、NC+Sal组细胞凋亡率增高(P<0.05)。干预各组细胞48h后,与NC组相比,KD组细胞MDR1、Akt的磷酸化水平(p-Akt(Ser473) p-Akt(Thr308))和cyclinD1蛋白表达量显著降低,而pro-Caspase-9蛋白表达水平虽然有所降低,但并不显著。与NC. NC+Sal、KD组相比,KD+Sal组细胞中MDR1、p-Akt(Ser473). p-Akt(Thr308)、Pro-Caspase-9和cvclinD1蛋白表达明显降低(P<0.05),而Ak t蛋白表达无明显改变(P>0.05)。
结论：1、盐霉素能有效抑制人肝癌细胞增殖、迁移和侵袭,其作用机制可能与抑制Src/PI3K/Akt信号通路的活性有关。
2.RNAi能有效抑制c-Src基因表达,抑制肝癌细胞增殖活性,诱导其凋亡,并增加其对盐霉素的敏感性,其机制可能与抑制MDR1和Src/PI3K/Akt信号通路活性及活化Caspase家族凋亡通路有关。
目的：研究姜黄素在体外对膀胱癌EJ细胞的生长抑制作用和凋亡诱导效应,同时探讨姜黄素抗膀胱癌的分子机制。观察姜黄素联合PI3k抑制剂LY294002对人膀胱癌EJ细胞生长及凋亡的影响。
方法1、采用MTT法测定不同浓度姜黄素(0-45μuM)经过24h和48h作用后,EJ细胞增殖的变化,同时观察细胞形态学改变。收集不同浓度姜黄素(0,12.5、25and50μM)作用24h后的EJ细胞,经流式细胞仪检测膀胱癌EJ细胞的DNA含量及凋亡细胞数。Westernblotting法测定不同浓度姜黄素(0,12.5、25and50μM)作用EJ细胞后,c-myc, Bcl-2家族,caspase家族及PI3K/Akt信号通路的凋亡相关蛋白的表达变化。2、用20μ M LY294002提前作用于EJ细胞1h,然后加入不同浓度的姜黄素(0,12.5、25and50μM)共同培养至24h。MTT法检测单独使用姜黄素(0,12.5、25an d50μ M). LY294002(20μM)及二者联合,对体外培养的膀胱癌EJ细胞的生长抑制作用；倒置显微镜观察细胞形态改变；流式细胞仪(AnnexinV-PI双染法)检测各组细胞凋亡率；Westernblotting法测定不同浓度姜黄素(25and50μM)单独或联合20μMLY294002作用EJ细胞后,凋亡相关蛋白caspase-9,-7,-3及PARP的表达变化。
结果1、MTT、实验证实姜黄素以浓度及时间依赖的方式抑制EJ细胞的增殖。倒置显微镜显示EJ细胞密度呈剂量依赖性降低,同时观察到凋亡细胞所具有的形态学改变。FCM(PI单染和AnnexinV-PI双染法)检测发现姜黄素作用于EJ细胞24h后sub-G1峰及凋亡细胞数量呈剂量依赖性增加。Westernblotting显示不同浓度姜黄素(0,25and50μ M)作用EJ细胞后,c-myc, Bad, Bax的表达水平增加,PTEN, GSK-3β, c-Raf, caspase-9, caspase-7, caspase-3的活性增强,PARP的裂解增加和Bc1-2,Akt的表达水平降低。2、MTT实验证实姜黄素联合LY294002组明显抑制EJ细胞的增殖,优于LY294002、姜黄素单独作用组,具有浓度及时间依赖的特点。倒置显微镜显示不同浓度姜黄素联合LY294002组EJ细胞密度的降低和典型凋亡细胞形态的出现明显于LY294002、相应浓度的姜黄素单独作用组。FCM(AnnexinV-PI双染法)检测发现处理24h后,不同浓度姜黄素联合LY294002组凋亡的EJ细胞数量明显多于LY294002、相应浓度的姜黄素单独作用组,且呈剂量依赖性增加。Western Blotting显示不同浓度姜黄素联合LY294002组凋亡相关蛋白caspase-9,caspase-7, caspase-3的活性增强,PARP的裂解增加出现的时间早于相应浓度的姜黄素单独作用组。
结论：1、姜黄素对膀胱癌EJ细胞具有增殖抑制作用和凋亡诱导效应。姜黄素能显著增加c-myc的表达,进而引起Bax的上调,Bcl-2的下调和caspase-9,-7,-3的增加。姜黄素抑制EJ细胞PI3K/Akt信号通路的激活,继之激活下游GSK-3β,caspase-9和Bad等多种促凋亡分子的表达,最终诱导EJ细胞凋亡发生。C-myc和PI3K/Akt信号通路在姜黄素诱导的膀胱癌EJ细胞的凋亡中起了重要的作用。2、LY294002与姜黄素单独应用均可抑制EJ细胞增殖LY294002能有效提高姜黄素体外对膀胱癌EJ细胞的抑制作用与单独用药组相比,联合用药组具有明显的时间依赖性协同增强EJ细胞凋亡的作用。二者联合用药增强诱导细胞凋亡主要是激活caspase家族,通过线粒体途径促进膀胱癌EJ细胞的凋亡裂解PARP来调控的。LY294002可提高姜黄素治疗膀胱癌的效果,为临床治疗膀胱癌提供新的思路和方法
Objective:To investigate effects of salinomycin on the proliferative, invisive and metastatic abilities of hepatoma cell line in vitro and analyze the possible mechanisms. To construct lentivirus-mediated shRNA expression vector targeting c-Src and to establish stable hepatoma cell line HepG2. To explore the effect RNAi-mediated inhibition of c-Src gene on apoptosis and chemosensitivity to Salinomycin in hepatoma HepG2cells and its underlying mechanism.
Methods:1、MTT was used for determining the proliferative abilities of hepatoma cells and human normal liver L02cells. Morphological changes were observed under the inverted phase-contrast microscope and photographed. Cell apoptosis was detected by Hoechst33258staining and flowcytometry analysis. The morphological alteration was observed by Hoechst33258staining. The cytoskeleton was observed using confocal laser scanning microscopy after F-actin staining by FITC-labeled phalloidin. The invasive and migratory abilities were detected by Transwell assay. Cell-cycle analysis was detected by flowcytometry. The relative protein expressions of Src/PI3K/Akt signal pathway were evaluated by Western Blotting.2、Lentiviral shRNA targeting c-Src was applied to construct stable knock-down hepatoma cell line HepG2, Western Blotting techniques were used to detect the alteration of protein level. HepG2cells were conventionally cultured and divided into6groups:control group (CON), negative control group (NC), RNAi c-Src group (KD), control combined with Salinomycin group (CON+Sal), negative control combined with Salinomycin group (NC+Sal), RNAi Src combined with Salinomycin group (KD+Sal). The proliferation of above mentioned HepG2cells was examined by MTT assay. Flow cytometry (FCM) was employed to detect the cell apoptosis and cell cycle. The protein expressions of MDRland Src/PI3K/Akt signal pathway were evaluated by Western Blotting.
Results:1、The treatment of L02cells with salinomycin (0～8μmol/L) for24h and48h had no significant effects on cell viability. However, treatment of similar doses of salinomycin suppressed the growth of hepatoma cells in a time and concentration dependent manner. Salinomycin could lead to characteristic morphologieal changes of apoptosis in human hepatoma cells after24h and48h in a concentration dependent manner. Hepatoma cells was treated by salinomycin (1μmol/L,4μmol/L) for24h to detect the invasive and migratory abilities. Compared to that in the control group, the number of invasive and migratory cells was remarkably decreased (P<0.01), the structure of F-actin microfilament cytoskeleton was significantly changed. Salinomycin could also induce a cell cycle arrest of hepatoma cells at the phase of G0/G1. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after salinomycin treatment. In addition, Immunoblot showed that salinomycin inhibited the activation of Src/PI3K/Akt pathway in a dose-dependent manner in hepatoma cells. Activation of GSK-3β, caspase-9, caspase-3, cleavage of PARP, upregulation of Bad, Bax, E-cadherin, downregulation of c-Src, Akt, Bcl-2, c-myc, cyclin Dl, survivin, MMP-2and MMP-9were also found in salinomycin-treated hepatoma cells.2、KD group had a significantly lower protein level of c-Src Src and MTT value than CON group and NC group (P<0.05), and had a higher apoptotic rate and larger percentage of G0/G1arrested cells and lower MDR1, Akt, cyclinDl expression than NC group (P<0.05). In KD+Sal group, MTT value was more lower and stronger caspase-9expression and lower MDR1, Akt, cyclinDl expression and larger percentage of G0/G1arrested cells than in KD group and NC+Sal group (P<0.05).
Conclusion:1、The results suggested that the proliferative, invasive and migratory abilities of hepatoma cells can be inhibited by salinomycin, which may be related to blocking the Src/PI3K/AKT kinase cascade signaling pathway.2、RNAi could specifically supress c-Src gene expression, effectively inhibit the proliferation and increase G0/G1cell population and accelerate the apoptosis of HepG2cells, and enhance its chemosensitivity to Salinomycin. The mechanism may be related to the suppression of the activation of MDRland Src/PI3K/Akt pathway and the activation of Caspase signaling pathways.
Objective:Cancer chemopreventive agent curcumin have been shown to possess inhibition cell growtlh and induction apoptosis properties in several types of cancer. However, the detailed molecular mechanisms of the compound remain far from clear in EJ bladder cancer cells. In order to explore the possible mechanism and offer experimental evidence of anti-cancer effcet of curcumin combined with LY294002, we investigated the anti-proliferation and apoptosis induction effcet of curcumin combined with LY294002on human bladder cancer cells.
Methods:1. The effect of curcumin on EJ cells growth inhibition and apoptosis was detected by MTT assays and flow cytometry. The phosphorylation levels of PTEN, PDK1, Akt, GSK-3β, c-Raf, Bad and the expression levels of c-myc, Bax, Bcl-2, caspase-9, caspase-7, caspase-3, PARP by curcumin administration were examined by Western Blotting.2. After EJ cells were pretreated with the PI3k inhibitor LY294002(20μM) in complete medium for1h, and then various doses of Curcumin (0,12.5、25and50μM) was added to co-culture for the indicated time periods. The growth inhibition of curcumin and LY294002alone or in combination on bladder cancer EJ cells by MTT assay. Morphological changes were observed under the inverted phase-contrast microscope and photographed. Apoptotic rates of human EJ bladder carcinoma cells were determined by flow cytometry (FCM). Curcumin and LY294002alone or in combination regulated caspase-9、caspase-7、caspase-3and PARP protein expression in EJ cells by Western Blotting analysis.
Results:1. Curcumin suppressed the growth of EJ cells in a time-and concentration-dependent manners. Immunoblot showed that curcumin increased expression levels of c-myc and inhibited the activation of PI3K/Akt pathway in a time-dependent manner in EJ cells. Activation of PTEN, GSK-3(3, c-Raf, caspase-9, caspase-7, caspase-3, cleavage of PARP, upregulation of Bad, Bax, downregulation of Akt, Bcl-2were also found in curcumin-treated EJ cells.2. EJ cells viability showed remarkable decrease in a concentration-and time-dependented manner in the curcumin (12.5,25and50μM)+LY294002(20μM) group as compared with curcumin (12.5,25and50μM)-treated control group or with LY294002(20μM) alone. Curcumin and LY294002could lead to characteristic morphologieal changes of apoptosis in human bladder carcinoma cells after24h including cellular rounding, shrinkage, chromatin condensation, and membrane blebbing, the appearance of membrane-associated apoptotic bodies and separation from neighboring cells. Combined treatment with LY294002and curcumin increased the percentage of apoptotic cell death in EJ cells as compared with either compound alone. The presenting time of the cleavage of caspase-9,-7,-3and PARP following the LY294002(20μM)/curcumin (25or50μM) combination administration is earlier than that of curcumin (25or50μM) alone treatment in EJ cells by Western Blotting analysis.
Conclusion:1.These findings esteblish a mechanistic linkup or interaction between c-myc, Bax, Bad, Bcl-2, caspase cascades, PI3K/Akt pathway and curcumin-induced apoptosis of EJ cells, suggest that c-myc and PI3K/Akt signaling pathway plays an important roles in curcumin-induced apoptosis of EJ bladder cancer cells.2. Curcumin or LY294002could significantly inhibit the growth of human EJ bladder carcinoma cells, LY294002could effectively increase the growth inhibition of curcumin in EJ cells in vitro. The curcumin and LY294002can all induce human EJ carcinoma cells apoptosis. When the two drugs combined, the inductive effect for cell apoptosis became more obvious, which suggest a possible underlying molecular mechanism Where by curcumin and LY294002could induce the apoptosis signaling pathway in human EJ bladder carcinoma cells by caspase family protein activation and cleavage of PARP protein through mitochondrion-dependent pathway. LY294002could enhance the anti-tumor effect of curcumin, which provide the new thread and methods for the clinical thrapy of bladder cancer.
引文
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