4种纳米颗粒对人胃癌BGC-823细胞的生物学效应
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摘要
目的:制备特定粒径范围的纳米活性炭(activated carbon nanoparticles,ACNP),探讨ACNP对人胃癌BGC-823细胞的作用及相关机制;同时,选择与ACNP在同一粒径范围的其他三种纳米颗粒(纳米SiO_2、纳米TiO_2、纳米ZnO),探讨不同化学组成的纳米颗粒对人胃癌BGC-823细胞的生物学效应及作用机制。
方法:采用球磨法结合悬浮分离制备ACNP,用原子力显微镜和透射电镜对制备的ACNP进行表征,用透射电镜对其他三种纳米颗粒(纳米SiO_2、纳米TiO_2、纳米ZnO)进行表征;采用MTT法及乳酸脱氢酶(LDH)漏出量测定初步评价ACNP、纳米SiO_2、纳米ZiO_2以及纳米ZnO对BGC-823细胞的毒性;采用甲基绿-派洛宁染色法和透射电镜观察4种纳米颗粒对细胞形态及超微结构的影响;采用流式细胞术(FCM)检测细胞凋亡率和细胞周期;用流式细胞仪以罗丹明123(Rh123)作为荧光指示剂检测细胞线粒体膜电位(MMP)的改变;不同浓度ACNP、纳米SiO_2、纳米TiO_2作用24 h后,用活性氧(ROS)捕获剂双氢罗丹明123(DHR123)孵育细胞,DHR123在细胞内被ROS氧化为可发出荧光的Rh123,通过流式细胞仪检测细胞内Rh123的平均荧光强度(MFI)而测得细胞内ROS水平。
结果:ACNP、纳米SiO_2、纳米ZnO为球状颗粒,平均粒径分别为(34±6)nm,(30±5)nm,(50±10)nm;纳米TiO_2为近球状颗粒,平均粒径为(20±5)nm。4种纳米颗粒均能明显抑制BGC-823细胞的增殖,呈剂量和时间依赖性。ACNP作用48,72h后的半数抑制浓度(IC_(50))分别为1.18,0.87mg/ml;纳米SiO_2作用24,48,72h后的IC_(50)分别为1.26,0.95,0.68mg/ml;纳米TiO_2作用72 h后的IC_(50)为0.88mg/ml;纳米ZnO作用24,48,72h后的IC_(50)分别为39.52,30.71,20.61μg/mL。4种纳米颗粒对BGC-823细胞毒性比较:纳米ZnO＞纳米SiO_2＞ACNP＞纳米TiO_2。ACNP作用24h后,低浓度(0.1,0.2 mg/ml)组LDH漏出量与对照组相比没有显著差异,0.4mg/ml和0.8mg/ml浓度组LDH漏出量高于对照组(P＜0.05);作用48h后,各ACNP组LDH漏出量均显著高于对照组。纳米SiO_2各浓度作用组LDH漏出量均显著高于对照组,在0.1～0.8mg/ml浓度范围内呈剂量依赖性。0.1mg/ml的纳米TiO_2作用24h,LDH漏出量与对照组相比没有显著差异,此后随着作用浓度的增加和作用时间的延长,各组LDH漏出量高于对照组(P＜0.05)。纳米ZnO各浓度作用组LDH漏出量均显著高于对照组,在12.5～100.0μg/ml浓度范围内呈剂量依赖性。0.1 mg/ml的ACNP作用24 h后,细胞出现凋亡特征的形态学改变;凋亡细胞体积变小,细胞质浓缩,细胞核固缩、裂解。经0.2 mg/ml纳米SiO_2、0.2 mg/ml纳米TiO_2、12.5μg/ml纳米ZnO作用24 h后的细胞均出现坏死特征的形态学改变;坏死细胞膜破裂,核浓缩或溶解。在4种纳米颗粒作用组均可见纳米颗粒进入细胞及线粒体损伤。0.1,0.2 mg/ml的ACNP作用24 h后,细胞凋亡率分别为(5.01±1.16)%、(8.21±1.63)%,与对照组(2.48±0.58)%比较均有显著性差异;坏死率与对照组相比没有显著差异。纳米SiO_2、纳米TiO_2及纳米ZnO作用24 h,各浓度组细胞坏死率高于对照组(P＜0.05),并呈明显剂量依赖性;凋亡率与对照组相比没有显著差异。经0.1,0.2 mg/ml ACNP作用24,48,72 h后,均可见细胞被明显阻滞在S期,G_0/G_1期细胞明显减少;DNA含量直方图上可见亚二倍体细胞峰,即凋亡细胞峰。纳米ZnO作用组也在24 h后即出现S期细胞增多,G_0/G_1期细胞减少;而纳米SiO_2和纳米TiO_2在作用48 h后才出现明显的S期细胞增多现象;在纳米SiO_2、纳米TiO_2及纳米ZnO作用组的DNA含量直方图上可见碎片峰,未见亚二倍体细胞峰(凋亡细胞峰)。ACNP(0.1,0.2,0.4 mg/ml)作用24h后,BGC-823细胞的MMP呈剂量依赖性降低;纳米SiO_2(0.1,0.2,0.4 mg/ml)及纳米TiO_2(0.1,0.2,0.4 mg/ml)作用于BGC-823细胞24 h后,MMP与对照组比较也有显著降低,并呈剂量依赖性;3种纳米颗粒引起细胞MMP降低的幅度比较:ACNP＞纳米SiO_2＞纳米TiO_2。0.1,0.2 mg/ml ACNP作用24 h后,代表细胞内ROS水平的Rh123平均荧光强度(MFI)分别为101.11±3.63、82.21±2.70,较对照组(62.18±2.05)明显升高(P＜0.05);纳米SiO_2(0.1,0.2,0.4 mg/ml)作用24 h后,细胞内ROS水平显著降低,并呈剂量依赖性;0.1,0.2 mg/ml纳米TiO_2作用24 h后,细胞内ROS水平高于对照组(P＜0.05)。
结论:ACNP在体外能抑制人胃癌BGC-823细胞的增殖,可使细胞阻滞于S期,诱导细胞凋亡,显示出较强的抗肿瘤活性。ACNP诱导肿瘤细胞凋亡的可能机制是诱导细胞发生氧化应激,生成ROS,并进一步通过活化线粒体信号转导途径诱导细胞凋亡;ACNP可进入细胞核,可能直接作用于核内遗传物质,阻断细胞有丝分裂,诱导细胞凋亡。纳米SiO_2、纳米TiO_2、纳米ZnO能对BGC-823细胞造成以细胞坏死为主的毒性损伤。纳米SiO_2可以产生Si和SiO自由基,诱导细胞氧化损伤,使其膜性结构通透性增加,细胞内容物(包括细胞内酶)漏出至膜外,造成细胞坏死。纳米TiO_2作为一种常见的光催化纳米材料,能够通过产生ROS或使BGC-823细胞内氧化应激增加,使细胞内ROS水平升高,ROS引起膜脂质过氧化,导致细胞膜及线粒体膜通透性增大。富集可溶性金属成分的纳米ZnO对膜结构的损伤作用明显强于其他三种纳米颗粒,能破坏细胞膜,导致细胞肿胀、坏死,其细胞毒性明显大于其他三种纳米颗粒。化学组成不同的纳米颗粒对细胞的生物学效应既有共性又有个性。纳米颗粒的细胞生物学效应可能受粒径、形状、表面特征、化学组成等多种因素的影响。
Objective:To prepare the activated carbon nanoparticles(ACNP) of specific particle size and investigate the effects of ACNP on the human gastric carcinoma cell line BGC-823 in vitro.The silicon dioxide nanoparticles(nano-SiO_2),titanium dioxide nanoparticles(nano-TiO_2) and zinc oxide nanoparticles(nano-ZnO) were also used to investigate the effects of nanoparticles with different chemical composition on the human gastric carcinoma cell line BGC-823 in vitro.
Methods:ACNP were prepared by ball-mill grinding and floating-filtrating method. The particle size and morphology of ACNP were studied by atomic force microscopy (AFM) and transmission electron microscopy(TEM).The particle sizes and morphology of nano-SiO_2,nano-TiO_2 and nano-ZnO were studied by TEM.BGC-823 cells were treated with ACNP,nano-SiO_2,nano-TiO_2 and nano-ZnO at various concentrations for different durations.The inhibitory effects of the nanoparticles on the proliferation of BGC-823 cells were observed by MTT assay.The lactate dehydrogenase(LDH) leakages in the culture medium were determined with LDH activity detection kit.LDH leakage reflects the integrality of cell membrane.The alterations in morphology were observed by Methyl Green-Pyronin staining and TEM. The apoptotic rate and the cell cycle of BGC-823 cells were examined by flow cytometry(FCM).After being treated with ACNP,nano-SiO_2,nano-TiO_2 for 24 hours, the mitochondrial membrane potential(MMP) was labeled by rhodamine123(Rh123) and examined by FCM.Dihydrorhodamine123(DHR123) was used as a reactive oxygen species(ROS) capture.The mean fluorescent intensity(MFI) of Rh123 which was the product of intracellular oxidation was examined by FCM,and the level of ROS was thus measured.
Results:ACNP,nano-SiO_2,and nano-ZnO particles were spherical.The average diameters of ACNP,nano-SiO_2,and nano-ZnO were(34±6)nm,(30±5)nm and(50±10) nm,respectively.Nano-TiO_2 particles were nearly spherical.The average diameter of nano-TiO_2 was(20±5) nm.ACNP,nano-SiO_2,nano-TiO_2 and nano-ZnO significantly inhibited the proliferation of BGC-823 cells in dose-and time-dependent manners.The 50%inhibitory concentrations(IC_(50)) of ACNP after 48 and 72 hours were 1.18 and 0.87 mg/ml,respectively,while those of nano-SiO_2 after 24,48 and 72 hours were 1.26,0.95 and 0.68 mg/ml,those of nano-ZnO after 24,48 and 72 hours were 39.52,30.71 and 20.61μg/mL,respectively.The IC_(50) of nano-TiO_2 after 72 hours was 0.88mg/ml.The comparison of the cytotoxicities of four kinds of nanoparticles was nano-ZnO＞nano-SiO_2＞ACNP＞nano-TiO_2.At 24 hours after treated with 0.1mg/ml and 0.2mg/ml ACNP,the LDH leakages were not higher than that of the control group. However,the LDH leakages at concentrations of 0.4 mg/ml and 0.8 mg/ml were higher than that of the control group(P＜0.05).At 48 hours after treated with ACNP,the LDH leakages of four ACNP groups were significantly higher than that of the control group. The LDH leakages of nano-SiO_2(0.1mg/ml to 0.8mg/ml) groups significantly increased in a dose-dependent manner.At 24 hours after treated with 0.1 mg/ml nano-TiO_2,the LDH leakage was not higher than that of the control group.As the dose of nano-TiO_2 increased and the time prolonged,the LDH leakages of nano-TiO_2 groups increased.The LDH leakages of nano-ZnO(12.5μg/ml to 100.0μg/ml) groups significantly increased in a dose-dependent manner.The cells treated with 0.1mg/ml ACNP for 24 hours exhibited morphological characteristics of apoptosis including condensation of the cytoplasm,condensation and fragmentation of the nuclear chromatin.The cells treated with 0.2 mg/ml nano-SiO_2,0.2mg/ml nano-TiO_2 or 12.5μg/ml nano-ZnO for 24 hours exhibited morphological characteristics of necrosis including disintegration of the plasma membranes,nuclear condensation and nuclear lysis.Mitochondrial damage was observed in the cells treated with ACNP,nano-SiO_2, nano-TiO_2 or nano-ZnO.These nanoparticles could enter the cells.At 24 hours after treated with 0.1 mg/ml and 0.2 mg/ml ACNP,the apoptotic rates were(5.01±1.16)% and(8.21±1.63)%,respectively,both of which were significantly higher than that of the control group(2.48±0.58)%.The necrotic rates were not higher than that of the control group.At 24 hours after treated with nano-SiO_2,nano-TiO_2 or nano-ZnO,the necrotic rates significantly increased in a dose-dependent manner while the apoptotic rates were not higher than that of the control group.In ACNP group,the percentage of cells at S phase was higher than that in control group,while the percentage of G_0/G_1-phase cells decreased significantly after 24 hours exposure.The typical "Sub-G_1 peak"(apoptotic peak) was also detected in ACNP group.In nano-ZnO group,the percentage of S-phase cells increased while the percentage of G_0/G_1-phase cells decreased significantly after 24 hours exposure.In nano-SiO_2 and nano-TiO_2 groups,the percentage of S-phase cells increased after 48 hours exposure.After treated with 0.1, 0.2,0.4 mg/ml ACNP for 24 hours,the MMP of BGC-823 cells decreased in a dose-dependent manner.After treated with 0.1,0.2,0.4 mg/ml nano-SiO_2 or nano-TiO_2 for 24 hours,the MMP of BGC-823 cells decreased in a dose-dependent manner,too. After treated with 0.1 mg/ml and 0.2 mg/ml ACNP for 24 hours and incubated with 5μmol/L DHR123 for 1 hour,the MFI of oxidized Rh123(or the cellular ROS levels) were 101.11±3.63 and 82.21±2.70,respectively,both of which were significantly higher than that of the control group(62.18±2.05)(P＜0.05).After treated with 0.1, 0.2,0.4 mg/ml nano-SiO_2 for 24 hours,the cellular ROS levels deceased in a dose-dependent manner.After treated with 0.1 mg/ml and 0.2 mg/ml nano-TiO_2 for 24 hours,the cellular ROS levels were higher than that of the control group(P＜0.05).
Conclusion:ACNP could inhibit proliferation,cause S-phase arrest and induce apoptosis of BGC-823 cells which demonstrated strong cytotoxicity in vitro.ACNP may induce oxidative stress in BGC-823 cells by overproduction of ROS,and then induce apoptosis of BGC-823 cells via activating mitochondrial signal transduction pathway. Furthermore,Fine activated carbon particles entered the nuclei of tumor cells,which would damage DNA,inhibit DNA replication,and induce cell apoptosis.Nano-SiO_2, nano-TiO_2 and nano-ZnO could directly induce necrosis of BGC-823 cells via disintegrating plasma membranes.In conclusion,nanoparticles with different chemical composition could induce cytotoxicity in BGC-823 cells.The biological effects of nanoparticles may be attributed to particle size and shape,surface characteristics, chemical composition,and some other more factors.
方法:采用球磨法结合悬浮分离制备ACNP,用原子力显微镜和透射电镜对制备的ACNP进行表征,用透射电镜对其他三种纳米颗粒(纳米SiO_2、纳米TiO_2、纳米ZnO)进行表征;采用MTT法及乳酸脱氢酶(LDH)漏出量测定初步评价ACNP、纳米SiO_2、纳米ZiO_2以及纳米ZnO对BGC-823细胞的毒性;采用甲基绿-派洛宁染色法和透射电镜观察4种纳米颗粒对细胞形态及超微结构的影响;采用流式细胞术(FCM)检测细胞凋亡率和细胞周期;用流式细胞仪以罗丹明123(Rh123)作为荧光指示剂检测细胞线粒体膜电位(MMP)的改变;不同浓度ACNP、纳米SiO_2、纳米TiO_2作用24 h后,用活性氧(ROS)捕获剂双氢罗丹明123(DHR123)孵育细胞,DHR123在细胞内被ROS氧化为可发出荧光的Rh123,通过流式细胞仪检测细胞内Rh123的平均荧光强度(MFI)而测得细胞内ROS水平。
结果:ACNP、纳米SiO_2、纳米ZnO为球状颗粒,平均粒径分别为(34±6)nm,(30±5)nm,(50±10)nm;纳米TiO_2为近球状颗粒,平均粒径为(20±5)nm。4种纳米颗粒均能明显抑制BGC-823细胞的增殖,呈剂量和时间依赖性。ACNP作用48,72h后的半数抑制浓度(IC_(50))分别为1.18,0.87mg/ml;纳米SiO_2作用24,48,72h后的IC_(50)分别为1.26,0.95,0.68mg/ml;纳米TiO_2作用72 h后的IC_(50)为0.88mg/ml;纳米ZnO作用24,48,72h后的IC_(50)分别为39.52,30.71,20.61μg/mL。4种纳米颗粒对BGC-823细胞毒性比较:纳米ZnO＞纳米SiO_2＞ACNP＞纳米TiO_2。ACNP作用24h后,低浓度(0.1,0.2 mg/ml)组LDH漏出量与对照组相比没有显著差异,0.4mg/ml和0.8mg/ml浓度组LDH漏出量高于对照组(P＜0.05);作用48h后,各ACNP组LDH漏出量均显著高于对照组。纳米SiO_2各浓度作用组LDH漏出量均显著高于对照组,在0.1～0.8mg/ml浓度范围内呈剂量依赖性。0.1mg/ml的纳米TiO_2作用24h,LDH漏出量与对照组相比没有显著差异,此后随着作用浓度的增加和作用时间的延长,各组LDH漏出量高于对照组(P＜0.05)。纳米ZnO各浓度作用组LDH漏出量均显著高于对照组,在12.5～100.0μg/ml浓度范围内呈剂量依赖性。0.1 mg/ml的ACNP作用24 h后,细胞出现凋亡特征的形态学改变;凋亡细胞体积变小,细胞质浓缩,细胞核固缩、裂解。经0.2 mg/ml纳米SiO_2、0.2 mg/ml纳米TiO_2、12.5μg/ml纳米ZnO作用24 h后的细胞均出现坏死特征的形态学改变;坏死细胞膜破裂,核浓缩或溶解。在4种纳米颗粒作用组均可见纳米颗粒进入细胞及线粒体损伤。0.1,0.2 mg/ml的ACNP作用24 h后,细胞凋亡率分别为(5.01±1.16)%、(8.21±1.63)%,与对照组(2.48±0.58)%比较均有显著性差异;坏死率与对照组相比没有显著差异。纳米SiO_2、纳米TiO_2及纳米ZnO作用24 h,各浓度组细胞坏死率高于对照组(P＜0.05),并呈明显剂量依赖性;凋亡率与对照组相比没有显著差异。经0.1,0.2 mg/ml ACNP作用24,48,72 h后,均可见细胞被明显阻滞在S期,G_0/G_1期细胞明显减少;DNA含量直方图上可见亚二倍体细胞峰,即凋亡细胞峰。纳米ZnO作用组也在24 h后即出现S期细胞增多,G_0/G_1期细胞减少;而纳米SiO_2和纳米TiO_2在作用48 h后才出现明显的S期细胞增多现象;在纳米SiO_2、纳米TiO_2及纳米ZnO作用组的DNA含量直方图上可见碎片峰,未见亚二倍体细胞峰(凋亡细胞峰)。ACNP(0.1,0.2,0.4 mg/ml)作用24h后,BGC-823细胞的MMP呈剂量依赖性降低;纳米SiO_2(0.1,0.2,0.4 mg/ml)及纳米TiO_2(0.1,0.2,0.4 mg/ml)作用于BGC-823细胞24 h后,MMP与对照组比较也有显著降低,并呈剂量依赖性;3种纳米颗粒引起细胞MMP降低的幅度比较:ACNP＞纳米SiO_2＞纳米TiO_2。0.1,0.2 mg/ml ACNP作用24 h后,代表细胞内ROS水平的Rh123平均荧光强度(MFI)分别为101.11±3.63、82.21±2.70,较对照组(62.18±2.05)明显升高(P＜0.05);纳米SiO_2(0.1,0.2,0.4 mg/ml)作用24 h后,细胞内ROS水平显著降低,并呈剂量依赖性;0.1,0.2 mg/ml纳米TiO_2作用24 h后,细胞内ROS水平高于对照组(P＜0.05)。
结论:ACNP在体外能抑制人胃癌BGC-823细胞的增殖,可使细胞阻滞于S期,诱导细胞凋亡,显示出较强的抗肿瘤活性。ACNP诱导肿瘤细胞凋亡的可能机制是诱导细胞发生氧化应激,生成ROS,并进一步通过活化线粒体信号转导途径诱导细胞凋亡;ACNP可进入细胞核,可能直接作用于核内遗传物质,阻断细胞有丝分裂,诱导细胞凋亡。纳米SiO_2、纳米TiO_2、纳米ZnO能对BGC-823细胞造成以细胞坏死为主的毒性损伤。纳米SiO_2可以产生Si和SiO自由基,诱导细胞氧化损伤,使其膜性结构通透性增加,细胞内容物(包括细胞内酶)漏出至膜外,造成细胞坏死。纳米TiO_2作为一种常见的光催化纳米材料,能够通过产生ROS或使BGC-823细胞内氧化应激增加,使细胞内ROS水平升高,ROS引起膜脂质过氧化,导致细胞膜及线粒体膜通透性增大。富集可溶性金属成分的纳米ZnO对膜结构的损伤作用明显强于其他三种纳米颗粒,能破坏细胞膜,导致细胞肿胀、坏死,其细胞毒性明显大于其他三种纳米颗粒。化学组成不同的纳米颗粒对细胞的生物学效应既有共性又有个性。纳米颗粒的细胞生物学效应可能受粒径、形状、表面特征、化学组成等多种因素的影响。
Objective:To prepare the activated carbon nanoparticles(ACNP) of specific particle size and investigate the effects of ACNP on the human gastric carcinoma cell line BGC-823 in vitro.The silicon dioxide nanoparticles(nano-SiO_2),titanium dioxide nanoparticles(nano-TiO_2) and zinc oxide nanoparticles(nano-ZnO) were also used to investigate the effects of nanoparticles with different chemical composition on the human gastric carcinoma cell line BGC-823 in vitro.
Methods:ACNP were prepared by ball-mill grinding and floating-filtrating method. The particle size and morphology of ACNP were studied by atomic force microscopy (AFM) and transmission electron microscopy(TEM).The particle sizes and morphology of nano-SiO_2,nano-TiO_2 and nano-ZnO were studied by TEM.BGC-823 cells were treated with ACNP,nano-SiO_2,nano-TiO_2 and nano-ZnO at various concentrations for different durations.The inhibitory effects of the nanoparticles on the proliferation of BGC-823 cells were observed by MTT assay.The lactate dehydrogenase(LDH) leakages in the culture medium were determined with LDH activity detection kit.LDH leakage reflects the integrality of cell membrane.The alterations in morphology were observed by Methyl Green-Pyronin staining and TEM. The apoptotic rate and the cell cycle of BGC-823 cells were examined by flow cytometry(FCM).After being treated with ACNP,nano-SiO_2,nano-TiO_2 for 24 hours, the mitochondrial membrane potential(MMP) was labeled by rhodamine123(Rh123) and examined by FCM.Dihydrorhodamine123(DHR123) was used as a reactive oxygen species(ROS) capture.The mean fluorescent intensity(MFI) of Rh123 which was the product of intracellular oxidation was examined by FCM,and the level of ROS was thus measured.
Results:ACNP,nano-SiO_2,and nano-ZnO particles were spherical.The average diameters of ACNP,nano-SiO_2,and nano-ZnO were(34±6)nm,(30±5)nm and(50±10) nm,respectively.Nano-TiO_2 particles were nearly spherical.The average diameter of nano-TiO_2 was(20±5) nm.ACNP,nano-SiO_2,nano-TiO_2 and nano-ZnO significantly inhibited the proliferation of BGC-823 cells in dose-and time-dependent manners.The 50%inhibitory concentrations(IC_(50)) of ACNP after 48 and 72 hours were 1.18 and 0.87 mg/ml,respectively,while those of nano-SiO_2 after 24,48 and 72 hours were 1.26,0.95 and 0.68 mg/ml,those of nano-ZnO after 24,48 and 72 hours were 39.52,30.71 and 20.61μg/mL,respectively.The IC_(50) of nano-TiO_2 after 72 hours was 0.88mg/ml.The comparison of the cytotoxicities of four kinds of nanoparticles was nano-ZnO＞nano-SiO_2＞ACNP＞nano-TiO_2.At 24 hours after treated with 0.1mg/ml and 0.2mg/ml ACNP,the LDH leakages were not higher than that of the control group. However,the LDH leakages at concentrations of 0.4 mg/ml and 0.8 mg/ml were higher than that of the control group(P＜0.05).At 48 hours after treated with ACNP,the LDH leakages of four ACNP groups were significantly higher than that of the control group. The LDH leakages of nano-SiO_2(0.1mg/ml to 0.8mg/ml) groups significantly increased in a dose-dependent manner.At 24 hours after treated with 0.1 mg/ml nano-TiO_2,the LDH leakage was not higher than that of the control group.As the dose of nano-TiO_2 increased and the time prolonged,the LDH leakages of nano-TiO_2 groups increased.The LDH leakages of nano-ZnO(12.5μg/ml to 100.0μg/ml) groups significantly increased in a dose-dependent manner.The cells treated with 0.1mg/ml ACNP for 24 hours exhibited morphological characteristics of apoptosis including condensation of the cytoplasm,condensation and fragmentation of the nuclear chromatin.The cells treated with 0.2 mg/ml nano-SiO_2,0.2mg/ml nano-TiO_2 or 12.5μg/ml nano-ZnO for 24 hours exhibited morphological characteristics of necrosis including disintegration of the plasma membranes,nuclear condensation and nuclear lysis.Mitochondrial damage was observed in the cells treated with ACNP,nano-SiO_2, nano-TiO_2 or nano-ZnO.These nanoparticles could enter the cells.At 24 hours after treated with 0.1 mg/ml and 0.2 mg/ml ACNP,the apoptotic rates were(5.01±1.16)% and(8.21±1.63)%,respectively,both of which were significantly higher than that of the control group(2.48±0.58)%.The necrotic rates were not higher than that of the control group.At 24 hours after treated with nano-SiO_2,nano-TiO_2 or nano-ZnO,the necrotic rates significantly increased in a dose-dependent manner while the apoptotic rates were not higher than that of the control group.In ACNP group,the percentage of cells at S phase was higher than that in control group,while the percentage of G_0/G_1-phase cells decreased significantly after 24 hours exposure.The typical "Sub-G_1 peak"(apoptotic peak) was also detected in ACNP group.In nano-ZnO group,the percentage of S-phase cells increased while the percentage of G_0/G_1-phase cells decreased significantly after 24 hours exposure.In nano-SiO_2 and nano-TiO_2 groups,the percentage of S-phase cells increased after 48 hours exposure.After treated with 0.1, 0.2,0.4 mg/ml ACNP for 24 hours,the MMP of BGC-823 cells decreased in a dose-dependent manner.After treated with 0.1,0.2,0.4 mg/ml nano-SiO_2 or nano-TiO_2 for 24 hours,the MMP of BGC-823 cells decreased in a dose-dependent manner,too. After treated with 0.1 mg/ml and 0.2 mg/ml ACNP for 24 hours and incubated with 5μmol/L DHR123 for 1 hour,the MFI of oxidized Rh123(or the cellular ROS levels) were 101.11±3.63 and 82.21±2.70,respectively,both of which were significantly higher than that of the control group(62.18±2.05)(P＜0.05).After treated with 0.1, 0.2,0.4 mg/ml nano-SiO_2 for 24 hours,the cellular ROS levels deceased in a dose-dependent manner.After treated with 0.1 mg/ml and 0.2 mg/ml nano-TiO_2 for 24 hours,the cellular ROS levels were higher than that of the control group(P＜0.05).
Conclusion:ACNP could inhibit proliferation,cause S-phase arrest and induce apoptosis of BGC-823 cells which demonstrated strong cytotoxicity in vitro.ACNP may induce oxidative stress in BGC-823 cells by overproduction of ROS,and then induce apoptosis of BGC-823 cells via activating mitochondrial signal transduction pathway. Furthermore,Fine activated carbon particles entered the nuclei of tumor cells,which would damage DNA,inhibit DNA replication,and induce cell apoptosis.Nano-SiO_2, nano-TiO_2 and nano-ZnO could directly induce necrosis of BGC-823 cells via disintegrating plasma membranes.In conclusion,nanoparticles with different chemical composition could induce cytotoxicity in BGC-823 cells.The biological effects of nanoparticles may be attributed to particle size and shape,surface characteristics, chemical composition,and some other more factors.
引文
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