Genistein对环境内分泌干扰物促人神经母细胞瘤细胞增殖的抑制作用
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摘要
研究背景:环境内分泌干扰物(Environmental Endocrine Disruptors,EED)是环境污染物中的重要成分,在机体内具有激素样作用;其主要来源于杀虫剂、塑料、洗涤剂、燃烧产物及工农业产物。近年来,EED的污染已成为全球关注的重要议题,EED对性激素敏感性恶性肿瘤的影响成为研究热点。神经母细胞瘤是最常见的小儿恶性实体肿瘤之一,其病因及机制不清。已发现环境内分泌干扰物是神经母细胞瘤重要的促瘤因素,选择一种能有效抑制环境内分泌干扰物促瘤作用的物质对于肿瘤的防治有着重要的意义。
PI3K/Akt信号转导通路广泛存在细胞中,是参与细胞生长、增殖、分化调节的信号转导通路,可被G蛋白偶联受体和(或)蛋白酪氨酸激酶受体激活,也可被Ras蛋白激活。多种生长因子主要通过PI3K/Akt信号转导通路而发挥作用。业已证实PI3K/Akt通路在人类某些恶性肿瘤的发生、发展中起着重要的作用。
5,7,4'-三羟异黄酮(Genistein,GEN)是一种蛋白酪氨酸激酶(PTK)抑制剂,为常见的黄酮类化合物,是大豆的重要成份;是当前研究大豆类异黄酮生物学效应的主要代表物。GEN作为一种植物雌激素,与17β-雌二醇结构相似,能够与17β-雌二醇竞争和雌激素-β受体相结合,发挥弱雌激素或抗雌激素作用,在肿瘤的发生、发展阶段存在多重抑制效应。GEN对环境内分泌干扰物引起的肿瘤细胞增殖效应是否具有抑制作用,目前国内外尚未见报道。
研究目的:本研究观察GEN对环境内分泌干扰物双酚A(BPA)和邻苯二甲酸二(2-乙基)己酯(DEHP)诱导的神经母细胞瘤细胞增殖作用的影响,并初步探讨其作用机制及PI3K/Akt信号转导通路在其中的变化,为暴露于环境污染中的儿童预防神经母细胞瘤开辟新的途径。
材料与方法:
一、GEN抑制BPA、DEHP诱导的人神经母细胞瘤SK-N-SH细胞增殖效应实验体系的建立:SK-N-SH细胞经常规培养扩增后分为24组:组1,RPMI1640无酚红培液加无水乙醇(对照组);组2,加GEN1(浓度为2μmol/L):组3,加GEN2(浓度为5μmol/L);组4,加GNE3(浓度为12.5μmol/L);组5,加GEN4(浓度为25μmol/L);组6,加GEN5(浓度为50μmol/L);组7,加E2;组8,加BPA;组9,加DEHP;组10,同时加入E2+GEN1;组11,同时加入E2+GEN2;组12,同时加入E2+GEN3;组13,同时加入E2+GEN4;组14,同时加入E2+GEN5;组15,同时加入BPA+GEN1;组16,同时加入BPA+GEN2;组17,同时加入BPA+GEN3;组18,同时加入BPA+GEN4;组19,同时加入BPA+GEN5;组20,同时加入DEHP+GEN1;组21,同时加入DEHP+GEN2组;22,同时加入DEHP+GEN3;组23,同时加入DEHP+GEN4;组24,同时加入DEHP+GEN5。培养72 h检测细胞光吸收度值(AV)观察细胞增殖情况。
二、GEN对BPA、DEHP诱导的SK-N-SH细胞增殖效应影响的体外增殖实验:SK-N-SH细胞经常规培养扩增后分为8组:组1,RPMI1640无酚红培液加无水乙醇(对照组);组2,加GEN(GEN组);组3,加E2(E2组);组4,同时加E2和GEN(E2+GEN组);组5,加BPA(BPA组);组6,同时加BPA和GEN(BPA+GEN组);组7,加DEHP(DEHP组);组8,同时加DEHP和GEN(DEHP+GEN组)。分别在24、48和72 h检测细胞光吸收度值(AV)。同时72 h用流式细胞仪检测细胞周期及细胞凋亡指数(AI)。并在72 h时采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)观察细胞凋亡情况,Western blot分析法检测Caspase-3蛋白表达。
三、PI3K/Akt信号转导通路在GEN抑制环境内分泌干扰物促神经母细胞瘤增殖效应中变化的实验研究:SK-N-SH细胞经常规培养扩增后分为8组:组1,RPMI1640无酚红培液加无水乙醇(对照组);组2,加GEN(GEN组);组3,加E2(E2组);组4,同时加E2和GEN(E2+GEN组);组5,加BPA(BPA组);组6,同时加BPA和GEN(BPA+GEN组);组7,加DEHP(DEHP组);组8,同时加DEHP和GEN(DEHP+GEN组)。72 h检测细胞光吸收度值(AV)。同时采用Western blot分析法检测Akt蛋白及其磷酸化蛋白(p-Akt)的表达。
结果:
一、GEN抑制BPA、DEHP诱导的人神经母细胞瘤SK-N-SH细胞增殖效应实验体系的建立:SK-N-SH细胞经不同浓度的GEN作用72 h时,GEN不同浓度组对细胞生长的影响不同。与对照组(AV为0.79±0.14)比较,2μmol/L和5μmol/L浓度组AV分别为0.97±0.05和0.94±0.08,具有促增殖作用(p＜0.01);12.5μmol/L浓度组的AV为0.77±0.07,与对照组比较,有抑制生长趋势,但无统计学差异;而25μmol/L、50μmol/L浓度组的AV分别为0.63±0.02和0.43±0.02,具有明显抑制作用(p＜0.05)。当加入E2及BPA、DEHP后,GEN对各组细胞生长的影响有所不同。对E2诱导的细胞增殖,同时加入GEN和E2时各组的细胞AV分别为0.93±0.03、0.89±0.03、0.82±0.06和0.72±0.05,较单独加入E2组有降低,具有明显的统计学差异(p＜0.01)。对BPA诱导的细胞增殖,同时加入GEN和BPA时各组细胞AV分别为0.97±0.07,0.92±0.02,0.85±0.07,0.84±0.07,0.83±0.03,较单独BPA组均有下降,具有明显统计学差异(p＜0.001)。而对DEHP诱导的细胞增殖,加入GEN的组22、23、24的AV为0.88±0.04,0.87±0.01和0.72±0.11,较单独DEHP组有降低,具有统计学差异(p＜0.01),而组20、21与未加入GEN组比较无统计学差异。
二、GEN对BPA、DEHP诱导的SK-N-SH细胞增殖效应影响的体外实验:SK-N-SH细胞经GEN及E2、BPA、DEHP作用后,各时点的AV有所不同,24h时,加入GEN组与未加入GEN组比较,细胞AV略有降低,但其差别无统计学意义。作用48h时,各EEDs组联合GEN作用后的细胞AV较单独EEDs组有降低。其中组4(E2+GEN组)的AV是组3(E2组)的75%,降低了25%,两者比较具有显著的统计学差异(p＜0.001)。对BPA而言,同时加入GEN和BPA的细胞AV降低为0.58±0.09,是单独BPA作用组(0.75±0.02)的77%,两者比较具有显著的统计学差异(p＜0.001);同样,组8(DEHP+GEN组)的AV较组7(DEHP组)也有降低,两者之间的差异具有显著统计学意义(p＜0.001)。作用72h时,结果与48h时类似;联合GEN作用的各EEDs组细胞AV较单独EEDs组有降低,两者比较具有明显统计学差异(p＜0.001)。72h时对各组细胞周期进行分析,EEDS联合GEN各组的G2/M期细胞所占比例明显增加,与单独EEDs组比较具有统计学意义(p＜0.01),而sub-G0期(凋亡亚二倍体峰)、凋亡指数及Caspase-3蛋白表达各组之间无统计学差异(p＞0.05)。
三、PI3K/Akt信号转导通路在GEN抑制环境内分泌干扰物促神经母细胞瘤增殖效应中变化的实验研究:72h时Western blot检测发现EEDs联合GEN的各组较单独EEDs组的Akt及p-Akt的蛋白表达降低,两者之间的差异具有统计学意义(p＜0.05)。
结论:
1.GEN对神经母细胞瘤细胞生长呈双向作用,具有浓度依赖性。
2.GEN可通过细胞周期G2/M期阻滞抑制环境内分泌干扰物对人神经母细胞瘤细胞促增殖作用。
3.GEN可能干扰PI3K/Akt信号转导通路的细胞信号转导,从而抑制环境内分泌干扰物促人神经母细胞瘤细胞的增殖。
4.GEN对环境内分泌干扰物促神经母细胞瘤细胞生长的抑制作用,凋亡可能不是主要途径。
Backgrounds and Purpose:Environmental endocrine disruptors(EEDs) are the considerable elements of the environmental pollution.Such contaminants are derived from pesticides,plastics,combustion by-products,plants and agricultural products. They enter the body and disrupt normal endocrine functions,having adverse effects on wildlife and human health.In recent years,widespread attention has been paid to EEDs.There has been increasing interest in the effects of EEDs on estrogen-sensitive malignancies.Neuroblastoma(NB) is a common and embryonal pediatric malignant solid tumor.The cause of NB remains unknown.The promoting effect of EEDs on NB has been proved in vivo and vitro,and it is necessary and urgent to explore inhibitions to prevent this growth-promoting effects.
Phosphatidylinositol-3-Kinase/Akt(PI3K/Akt) signaling pathway is one of the common pathways for cell proliferation and differentiation,and can be activated by the G-protein-coupled receptor(GPCR),receptor of the protein tyrosine kinase(PTK) or the Ras protein.It has been proved that PI3K/Akt pathway is one of the most general ways for many growth factors.It is confirmed that PI3K/Akt signaling pathway plays an important role in the initiation and promotion of some human malignant tumors.
Genistein(4',5,7-trihydroxyisoflavone,GEN) as an inhibitor of PTK,is a general flavone derived from soybean,and a popular isoflavone for its anticancer biological activity.As a phytoestrogen,it can bind with estrogen receptor(ER),especially ER-βto educe biological activities as an agonistic and antagonistic properties of estrogen with respect to cell growth and differentiation.However,few researches have been made to observe the effect of GEN on the biologic activity of EEDs.In order to find the effects of GEN on the growth promotion of EEDs,we first devised several EEDs as intervention substances to observe their effects on proliferation of SK-N-SH human neuroblastoma cells.Then we use the mixture of GEN and selected EEDs to observe the depression effects of GEN.Furthermore,the cell cycle,cell apoptosis,the protein caspase-3,Akt and its phosphorylation were investigated to observe the underlying mechanisms.
Materials and Methods:
1.Experiment system establishment of the effect of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:NB cells were cultured in estrogen-free RPMI 1640 without phenol red and divided into twenty-four groups based on different concentration of GEN treatments:group 1,no treatment(control);group 2,treated with GEN1(CO:2μmol/L);group 3,treated with GEN2(CO:5μmol/L);group 4, treated with GEN3(CO:12.5μmol/L);group 5,treated with GEN4(CO:25μmol/L); group 6,treated with GEN5(CO:50μmol/L );group 7,treated with 17β-estradiol(E2); group 8,treated with bisphenol A(BPA);group 9,treated with Di-2-Ethylhexl Phthalate(DEHP);group 10,treated with both E2 and GEN1;group 11,treated with both E2 and GEN2;group 12,treated with both E2 and GEN3;group 13,treated with both E2 and GEN4;group 14,treated with both E2 and GENS;group 15,treated with both BPA and GEN1;group 16,treated with both BPAand GEN2;group 17,treated with both BPA and GEN3;group 18,treated with both BPA and GEN4;group 19, treated with both BPA and GENS;group20,treated with both DEHP and GEN1; group21,treated with both DEHP and GEN2;group22,treated with both DEHP and GEN3;group23,treated with both DEHP and GEN4;group24,treated with both DEHP and GEN5.Absorbance value(AV) was determined at time 72 hours.
2.Inhibitory action of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:NB cells were cultured in estrogen-free RPMI1640 without phenol red and divided into eight groups based on different treatments:group 1,no treatment (control);group 2,treated with GEN(CO:12.5μmol/L);group 3,treated with E2; group 4,treated with both E2 and GEN;group 5,treated with BPA;group 6,treated with both BPA and GEN;group 7,treated with DEHP;group 8,treated with both DEHP and GEN.AV was determined at time 24,48 and 72 hours.Flow cytometer was employed to monitor cell cycles,terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling(TUNEL) were employed to monitor cell apoptosis.Western blot analysis was employed to monitor the expression of caspase-3.
3.Interruption of GEN to PI3K/Akt signaling pathway when combined with EEDs: NB cells were cultured in estrogen-free RPMI1640 without phenol red and divided into eight groups based on different treatments:group 1,no treatment(control);group 2,treated with GEN(CO:12.5μmol/L);group 3,treated with E2;group 4,treated with both E2 and GEN;group 5,treated with BPA;group 6,treated with both BPA and GEN;group 7,treated with DEHP;group 8,treated with both DEHP and GEN. Western blot analysis was employed to monitor the expression of Akt and its phosphorylation,p-Akt.
Results:
1.Experiment system establishment of the effect of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:
At 72 hours,AV was 0.97±0.05 for group 2,0.94±0.08 for group 3;both of them were increased when compared with that of control group(p＜0.01).There was no difference between group 4(AV:0.77±0.07) and the control group.Compared with that of the control group,the AV of group5(AV:0.63±0.02) and group6(AV: 0.43±0.02) were definitely decreased with obvious difference.When treated with E2 and GEN,the AV of group12,13,14 and 15 were 93±0.03,0.89±0.03,0.82±0.06 and 0.72±0.05;compared with that of the group treated with E2 only,the AV was decreased and had statistical difference(p＜0.01).When treated with BPA and GEN, the AV each group was also decreased,compared with that of the group treated with BPA only,statistical difference existed(p＜0.001).When treated with both DEHP and GEN,the AV of group22,23 and 24 were decreased,compared with that of the group treated with DEHP only,the results had statistical significance(p＜0.01),however there were no statistical significances between the group20,21 and group9.
2.Inhibitory action of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:
At 24 hours,there's no difference of the AV between the group treated with GEN and without GEN.However,at 48 hours,the differences were found.The AV of group 4 was decreased by 25%,compared to the group 3 treated with E2 only(p＜0.001). The same phenomenon could be found in the left group.Comparing with group 5,the AV of group 6 was decreased to 77%;the differences between the two groups had statistical significance(p＜0.001).When treated with both DEHP and GEN,the results also had statistical significance(p＜0.001).The same phenomenon could be found at 72 hours,and the differences between the groups treated with GEN and with EEDs only also had statistical significance.The percentage of cells in G2/M at 72 hours treated with GEN was also different from the ones treated with no GEN,and the results had statistical significance(p＜0.05).However,the percentage of cells in sub-G0 of each group had no difference.The result of TUNEL was similar to flow cytometer.We also monitored the expression of Caspase-3 by Western blot analysis, but there was no difference of expression between the groups(p＞0.05).
3.Interruption of GEN to PI3K/Akt signaling pathway combined with EEDs:
At 72 hours,the expression of Akt and p-Akt was abundant in E2,BPA,and DEHP groups,when treated with the mixture of GEN and EEDs,the expression of the two proteins was decreased.The differences between the group with the mixture and the EEDs only had statistical significance(p＜0.05).
Conclusions:
1.GEN can inhibit the growth of SK-N-SH cells in a dose-dependent manner.
2.GEN can inhibit the growth-promoting effect of the EEDs on SK-N-SH cells.
3.The biological effects of GEN may be activated by blocking the G2/M of cell cycle and by disrupting the PI3K/Akt signaling pathway.And the activation exists even though in the environment with high level of estrogen made by EEDs.
4.No obvious apoptosis can be found in this experiment.
PI3K/Akt信号转导通路广泛存在细胞中,是参与细胞生长、增殖、分化调节的信号转导通路,可被G蛋白偶联受体和(或)蛋白酪氨酸激酶受体激活,也可被Ras蛋白激活。多种生长因子主要通过PI3K/Akt信号转导通路而发挥作用。业已证实PI3K/Akt通路在人类某些恶性肿瘤的发生、发展中起着重要的作用。
5,7,4'-三羟异黄酮(Genistein,GEN)是一种蛋白酪氨酸激酶(PTK)抑制剂,为常见的黄酮类化合物,是大豆的重要成份;是当前研究大豆类异黄酮生物学效应的主要代表物。GEN作为一种植物雌激素,与17β-雌二醇结构相似,能够与17β-雌二醇竞争和雌激素-β受体相结合,发挥弱雌激素或抗雌激素作用,在肿瘤的发生、发展阶段存在多重抑制效应。GEN对环境内分泌干扰物引起的肿瘤细胞增殖效应是否具有抑制作用,目前国内外尚未见报道。
研究目的:本研究观察GEN对环境内分泌干扰物双酚A(BPA)和邻苯二甲酸二(2-乙基)己酯(DEHP)诱导的神经母细胞瘤细胞增殖作用的影响,并初步探讨其作用机制及PI3K/Akt信号转导通路在其中的变化,为暴露于环境污染中的儿童预防神经母细胞瘤开辟新的途径。
材料与方法:
一、GEN抑制BPA、DEHP诱导的人神经母细胞瘤SK-N-SH细胞增殖效应实验体系的建立:SK-N-SH细胞经常规培养扩增后分为24组:组1,RPMI1640无酚红培液加无水乙醇(对照组);组2,加GEN1(浓度为2μmol/L):组3,加GEN2(浓度为5μmol/L);组4,加GNE3(浓度为12.5μmol/L);组5,加GEN4(浓度为25μmol/L);组6,加GEN5(浓度为50μmol/L);组7,加E2;组8,加BPA;组9,加DEHP;组10,同时加入E2+GEN1;组11,同时加入E2+GEN2;组12,同时加入E2+GEN3;组13,同时加入E2+GEN4;组14,同时加入E2+GEN5;组15,同时加入BPA+GEN1;组16,同时加入BPA+GEN2;组17,同时加入BPA+GEN3;组18,同时加入BPA+GEN4;组19,同时加入BPA+GEN5;组20,同时加入DEHP+GEN1;组21,同时加入DEHP+GEN2组;22,同时加入DEHP+GEN3;组23,同时加入DEHP+GEN4;组24,同时加入DEHP+GEN5。培养72 h检测细胞光吸收度值(AV)观察细胞增殖情况。
二、GEN对BPA、DEHP诱导的SK-N-SH细胞增殖效应影响的体外增殖实验:SK-N-SH细胞经常规培养扩增后分为8组:组1,RPMI1640无酚红培液加无水乙醇(对照组);组2,加GEN(GEN组);组3,加E2(E2组);组4,同时加E2和GEN(E2+GEN组);组5,加BPA(BPA组);组6,同时加BPA和GEN(BPA+GEN组);组7,加DEHP(DEHP组);组8,同时加DEHP和GEN(DEHP+GEN组)。分别在24、48和72 h检测细胞光吸收度值(AV)。同时72 h用流式细胞仪检测细胞周期及细胞凋亡指数(AI)。并在72 h时采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)观察细胞凋亡情况,Western blot分析法检测Caspase-3蛋白表达。
三、PI3K/Akt信号转导通路在GEN抑制环境内分泌干扰物促神经母细胞瘤增殖效应中变化的实验研究:SK-N-SH细胞经常规培养扩增后分为8组:组1,RPMI1640无酚红培液加无水乙醇(对照组);组2,加GEN(GEN组);组3,加E2(E2组);组4,同时加E2和GEN(E2+GEN组);组5,加BPA(BPA组);组6,同时加BPA和GEN(BPA+GEN组);组7,加DEHP(DEHP组);组8,同时加DEHP和GEN(DEHP+GEN组)。72 h检测细胞光吸收度值(AV)。同时采用Western blot分析法检测Akt蛋白及其磷酸化蛋白(p-Akt)的表达。
结果:
一、GEN抑制BPA、DEHP诱导的人神经母细胞瘤SK-N-SH细胞增殖效应实验体系的建立:SK-N-SH细胞经不同浓度的GEN作用72 h时,GEN不同浓度组对细胞生长的影响不同。与对照组(AV为0.79±0.14)比较,2μmol/L和5μmol/L浓度组AV分别为0.97±0.05和0.94±0.08,具有促增殖作用(p＜0.01);12.5μmol/L浓度组的AV为0.77±0.07,与对照组比较,有抑制生长趋势,但无统计学差异;而25μmol/L、50μmol/L浓度组的AV分别为0.63±0.02和0.43±0.02,具有明显抑制作用(p＜0.05)。当加入E2及BPA、DEHP后,GEN对各组细胞生长的影响有所不同。对E2诱导的细胞增殖,同时加入GEN和E2时各组的细胞AV分别为0.93±0.03、0.89±0.03、0.82±0.06和0.72±0.05,较单独加入E2组有降低,具有明显的统计学差异(p＜0.01)。对BPA诱导的细胞增殖,同时加入GEN和BPA时各组细胞AV分别为0.97±0.07,0.92±0.02,0.85±0.07,0.84±0.07,0.83±0.03,较单独BPA组均有下降,具有明显统计学差异(p＜0.001)。而对DEHP诱导的细胞增殖,加入GEN的组22、23、24的AV为0.88±0.04,0.87±0.01和0.72±0.11,较单独DEHP组有降低,具有统计学差异(p＜0.01),而组20、21与未加入GEN组比较无统计学差异。
二、GEN对BPA、DEHP诱导的SK-N-SH细胞增殖效应影响的体外实验:SK-N-SH细胞经GEN及E2、BPA、DEHP作用后,各时点的AV有所不同,24h时,加入GEN组与未加入GEN组比较,细胞AV略有降低,但其差别无统计学意义。作用48h时,各EEDs组联合GEN作用后的细胞AV较单独EEDs组有降低。其中组4(E2+GEN组)的AV是组3(E2组)的75%,降低了25%,两者比较具有显著的统计学差异(p＜0.001)。对BPA而言,同时加入GEN和BPA的细胞AV降低为0.58±0.09,是单独BPA作用组(0.75±0.02)的77%,两者比较具有显著的统计学差异(p＜0.001);同样,组8(DEHP+GEN组)的AV较组7(DEHP组)也有降低,两者之间的差异具有显著统计学意义(p＜0.001)。作用72h时,结果与48h时类似;联合GEN作用的各EEDs组细胞AV较单独EEDs组有降低,两者比较具有明显统计学差异(p＜0.001)。72h时对各组细胞周期进行分析,EEDS联合GEN各组的G2/M期细胞所占比例明显增加,与单独EEDs组比较具有统计学意义(p＜0.01),而sub-G0期(凋亡亚二倍体峰)、凋亡指数及Caspase-3蛋白表达各组之间无统计学差异(p＞0.05)。
三、PI3K/Akt信号转导通路在GEN抑制环境内分泌干扰物促神经母细胞瘤增殖效应中变化的实验研究:72h时Western blot检测发现EEDs联合GEN的各组较单独EEDs组的Akt及p-Akt的蛋白表达降低,两者之间的差异具有统计学意义(p＜0.05)。
结论:
1.GEN对神经母细胞瘤细胞生长呈双向作用,具有浓度依赖性。
2.GEN可通过细胞周期G2/M期阻滞抑制环境内分泌干扰物对人神经母细胞瘤细胞促增殖作用。
3.GEN可能干扰PI3K/Akt信号转导通路的细胞信号转导,从而抑制环境内分泌干扰物促人神经母细胞瘤细胞的增殖。
4.GEN对环境内分泌干扰物促神经母细胞瘤细胞生长的抑制作用,凋亡可能不是主要途径。
Backgrounds and Purpose:Environmental endocrine disruptors(EEDs) are the considerable elements of the environmental pollution.Such contaminants are derived from pesticides,plastics,combustion by-products,plants and agricultural products. They enter the body and disrupt normal endocrine functions,having adverse effects on wildlife and human health.In recent years,widespread attention has been paid to EEDs.There has been increasing interest in the effects of EEDs on estrogen-sensitive malignancies.Neuroblastoma(NB) is a common and embryonal pediatric malignant solid tumor.The cause of NB remains unknown.The promoting effect of EEDs on NB has been proved in vivo and vitro,and it is necessary and urgent to explore inhibitions to prevent this growth-promoting effects.
Phosphatidylinositol-3-Kinase/Akt(PI3K/Akt) signaling pathway is one of the common pathways for cell proliferation and differentiation,and can be activated by the G-protein-coupled receptor(GPCR),receptor of the protein tyrosine kinase(PTK) or the Ras protein.It has been proved that PI3K/Akt pathway is one of the most general ways for many growth factors.It is confirmed that PI3K/Akt signaling pathway plays an important role in the initiation and promotion of some human malignant tumors.
Genistein(4',5,7-trihydroxyisoflavone,GEN) as an inhibitor of PTK,is a general flavone derived from soybean,and a popular isoflavone for its anticancer biological activity.As a phytoestrogen,it can bind with estrogen receptor(ER),especially ER-βto educe biological activities as an agonistic and antagonistic properties of estrogen with respect to cell growth and differentiation.However,few researches have been made to observe the effect of GEN on the biologic activity of EEDs.In order to find the effects of GEN on the growth promotion of EEDs,we first devised several EEDs as intervention substances to observe their effects on proliferation of SK-N-SH human neuroblastoma cells.Then we use the mixture of GEN and selected EEDs to observe the depression effects of GEN.Furthermore,the cell cycle,cell apoptosis,the protein caspase-3,Akt and its phosphorylation were investigated to observe the underlying mechanisms.
Materials and Methods:
1.Experiment system establishment of the effect of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:NB cells were cultured in estrogen-free RPMI 1640 without phenol red and divided into twenty-four groups based on different concentration of GEN treatments:group 1,no treatment(control);group 2,treated with GEN1(CO:2μmol/L);group 3,treated with GEN2(CO:5μmol/L);group 4, treated with GEN3(CO:12.5μmol/L);group 5,treated with GEN4(CO:25μmol/L); group 6,treated with GEN5(CO:50μmol/L );group 7,treated with 17β-estradiol(E2); group 8,treated with bisphenol A(BPA);group 9,treated with Di-2-Ethylhexl Phthalate(DEHP);group 10,treated with both E2 and GEN1;group 11,treated with both E2 and GEN2;group 12,treated with both E2 and GEN3;group 13,treated with both E2 and GEN4;group 14,treated with both E2 and GENS;group 15,treated with both BPA and GEN1;group 16,treated with both BPAand GEN2;group 17,treated with both BPA and GEN3;group 18,treated with both BPA and GEN4;group 19, treated with both BPA and GENS;group20,treated with both DEHP and GEN1; group21,treated with both DEHP and GEN2;group22,treated with both DEHP and GEN3;group23,treated with both DEHP and GEN4;group24,treated with both DEHP and GEN5.Absorbance value(AV) was determined at time 72 hours.
2.Inhibitory action of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:NB cells were cultured in estrogen-free RPMI1640 without phenol red and divided into eight groups based on different treatments:group 1,no treatment (control);group 2,treated with GEN(CO:12.5μmol/L);group 3,treated with E2; group 4,treated with both E2 and GEN;group 5,treated with BPA;group 6,treated with both BPA and GEN;group 7,treated with DEHP;group 8,treated with both DEHP and GEN.AV was determined at time 24,48 and 72 hours.Flow cytometer was employed to monitor cell cycles,terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling(TUNEL) were employed to monitor cell apoptosis.Western blot analysis was employed to monitor the expression of caspase-3.
3.Interruption of GEN to PI3K/Akt signaling pathway when combined with EEDs: NB cells were cultured in estrogen-free RPMI1640 without phenol red and divided into eight groups based on different treatments:group 1,no treatment(control);group 2,treated with GEN(CO:12.5μmol/L);group 3,treated with E2;group 4,treated with both E2 and GEN;group 5,treated with BPA;group 6,treated with both BPA and GEN;group 7,treated with DEHP;group 8,treated with both DEHP and GEN. Western blot analysis was employed to monitor the expression of Akt and its phosphorylation,p-Akt.
Results:
1.Experiment system establishment of the effect of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:
At 72 hours,AV was 0.97±0.05 for group 2,0.94±0.08 for group 3;both of them were increased when compared with that of control group(p＜0.01).There was no difference between group 4(AV:0.77±0.07) and the control group.Compared with that of the control group,the AV of group5(AV:0.63±0.02) and group6(AV: 0.43±0.02) were definitely decreased with obvious difference.When treated with E2 and GEN,the AV of group12,13,14 and 15 were 93±0.03,0.89±0.03,0.82±0.06 and 0.72±0.05;compared with that of the group treated with E2 only,the AV was decreased and had statistical difference(p＜0.01).When treated with BPA and GEN, the AV each group was also decreased,compared with that of the group treated with BPA only,statistical difference existed(p＜0.001).When treated with both DEHP and GEN,the AV of group22,23 and 24 were decreased,compared with that of the group treated with DEHP only,the results had statistical significance(p＜0.01),however there were no statistical significances between the group20,21 and group9.
2.Inhibitory action of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:
At 24 hours,there's no difference of the AV between the group treated with GEN and without GEN.However,at 48 hours,the differences were found.The AV of group 4 was decreased by 25%,compared to the group 3 treated with E2 only(p＜0.001). The same phenomenon could be found in the left group.Comparing with group 5,the AV of group 6 was decreased to 77%;the differences between the two groups had statistical significance(p＜0.001).When treated with both DEHP and GEN,the results also had statistical significance(p＜0.001).The same phenomenon could be found at 72 hours,and the differences between the groups treated with GEN and with EEDs only also had statistical significance.The percentage of cells in G2/M at 72 hours treated with GEN was also different from the ones treated with no GEN,and the results had statistical significance(p＜0.05).However,the percentage of cells in sub-G0 of each group had no difference.The result of TUNEL was similar to flow cytometer.We also monitored the expression of Caspase-3 by Western blot analysis, but there was no difference of expression between the groups(p＞0.05).
3.Interruption of GEN to PI3K/Akt signaling pathway combined with EEDs:
At 72 hours,the expression of Akt and p-Akt was abundant in E2,BPA,and DEHP groups,when treated with the mixture of GEN and EEDs,the expression of the two proteins was decreased.The differences between the group with the mixture and the EEDs only had statistical significance(p＜0.05).
Conclusions:
1.GEN can inhibit the growth of SK-N-SH cells in a dose-dependent manner.
2.GEN can inhibit the growth-promoting effect of the EEDs on SK-N-SH cells.
3.The biological effects of GEN may be activated by blocking the G2/M of cell cycle and by disrupting the PI3K/Akt signaling pathway.And the activation exists even though in the environment with high level of estrogen made by EEDs.
4.No obvious apoptosis can be found in this experiment.
引文
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25. Ismail A I, Kang K S, lee H A, et al. Genistein-induced neuronal apoptosis and G2/M cell cycle arrestis associated with MDC1 up-regulation and P1K1 down-regulation [J]. Eur J Pharmacology 2007, 7:1-9.
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9. Fortes E M, Sugawara, Eduardo K V, et al. High intake of phytoestrogens and precocious thelarche: case report with a possible correlation [J]. Arq Bras Endocrinol Metabol, 2007, 51(3): 500-503.
10. Garcia-Garro A J,Gernandez F, Maria G R, et al. Management with soya of 1-4 years-old suffering malnutrition [J]. Aten Primaria, 2007,39(2): 69-73.
11. Paolo G G, Brunetti E C, Alessandra G, et al. Soy protein formulas in children: no hormonal effects in long-term feeding [J]. J Pediatr Endocrinol Metab, 2004, 17(2): 191-196.
12. Coral A L, Zhang J X, Michelle S C. Genistein studies in rats: potential for breast cancer prevention and reproductive and developmental toxicity [J]. Am J Clin Nutr1998;68(s):1400-1405.
13. Smith 1 J, Blumenthal, M W, et al. Dietary intake of soy genistein is associated with lung function in patients with asthma [J]. J Asthma, 2005,41(8): 833-843.
14. Miyake Yoshihiro, Miyamoto, Shoichi Hirota,et al. Soy, isoflavones, and prevalence of allergic rhinitis in Japanese women: the Osaka Maternal and Child Health Study [J]. J Allergy Clin Immunol. 2005, 115(6): 1176-1183.
15. Osborn D A, Sinn J. Soy formula for prevention of allergy and food intolerance in infants [J]. Cochrane Database of Systematic Reviews 2006,4.
16. Tuohy PG. Soy infant formula and phytoestrogens [J]. J. Paediatr. Child Health, 2003,39:401-405.
17. Hirzel S. Genistein: model substance for describing endocrine effects of phytoestrogrns. BUA Report 222. German Chemical Society Advisory Committee on Existing Chemicals, Stuttgart, 2000.
18. Jana M, Jarmila C, Vaclav Z, et al. Actual levels of soy phytoestrogens in children correlate with thyroid laboratory parameters [J]. Clin Chem lab Med 2006, 44(2):171-174.
19. 李丽,刘兆平,严卫星. 大豆异黄酮毒性作用研究进展[J]. 国外医学卫生学分册 2005, 32(6):338-342.
20. Heather B P, Anne E F, Eva K P. Neonatal genistein or bisphenol-A exposure alters sexual differentiation of the AVPV [J]. Neurotoxicology and Teratology, 2006,28:111-118.
21. Yiannis K, Gordon C B, Tray 1 M. Effectsof thelsoflavones Genistein and Equol on the Gonadal Development of Japanese medaka (Oryzias latipes) [J]. Environ Health Perspect, 2003, 111:1158-1163.
22. McClain R M. Wolz E, Davidovich A, et al. Reproductive safety studies with genistein in rats [J]. Food and Chemical Toxicology, 2007, 45: 1319-1332.
23. Schweigerer 1, Fotsis T. Increased expression of the MYCN oncogene in human neuroblastoma cells and possible, new therapeutic approaches [J]. Klin Padiatr, 1991, 203(4): 319-22.
24. Sarkar F H, li Y. Mechanisms of cancer chemoprevention by soy isoflavone genistein. Cancer Metastasis Rev, 2002, 21: 265-280.
25. Ismail A I, Kang K S, lee H A, et al. Genistein-induced neuronal apoptosis and G2/M cell cycle arrestis associated with MDC1 up-regulation and P1K1 down-regulation [J]. Eur J Pharmacology 2007, 7:1-9.
26. Alison Brown, Pauline Jolly, Huachen Weil. Genistein modulates neuroblastoma cell proliferation and differentiation through induction of apoptosis and regulation of tyrosine kinase activity and N-myc expression [J]. Carcinogenesis 1998, 19(6): 991-997.
27. Rocio G V, lothar S, Miguel A M. Matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 expression in paediatric tumour cells. Effects of tumour cell proliferation modulators on gelatinolytic activity [J]. J Cancer Res Clin Oncol, 1995,121:275-278.
28. Shalizeh N, Sami K, Joseph A, et al. Receptor tyrosine kinase inhibition suppresses growth of pediatric renal tumor cells in vitro [J]. J Pedia Surg, 2000, 35(6):884-890.
29. Yamane M, Abe A. omega-hydroxylation activity toward leukotriene B(4) and polyunsaturated fatty acids in the human hepatoblastoma cell line, HepG2, and human lung adenocarcinoma cell line, A549 [J]. J Biochem, 2001, 128(5): 827-35.
30. Kim J A, lee, Y S. Role of reactive oxygen species generated by NADPH oxidase in the mechanism of activation of K(+)-Cl(-)-cotransport by N-ethylmaleimide in HepG2 human hepatoma cells [J]. Free Radic Res, 2002, 35(1): 43-53.
31. Dwayne M B, Fayth K. Yoshimura. Genistein reduces NF-kB in T lymphoma cells via a caspase-mediated cleavage of IkBa [J]. Biochemical Pharmacology, 2003,66:1009-1018.
32. Mansour A, Harrison, Jonathan R, et al. Correlation of ZAP-70 expression in B cell leukemias to the ex vivo response to a combination of fludarabine/genistein [J]. Cancer Immunol Immunother, 2007, 56(4):501-514.
33. Jeong M H, Jo, Wol S Y, et al. The modulation of radiation-induced cell death by genistein in K562 cells: activation of thymidine kinase 1 [J]. Cell Res, 2005, 14(4): 295-302.
34. Miguel 1 1, Elaine W, Caroline A A. Cells lacking DNA Topoisomerase II are Resistant to Genistein [J]. J Nat Prod, 2007, 70:763-767.
35. Angela P P, Chen N G, Venkataraman C S, et al. Therapeutic targeting of nuclear receptor corepressor misfolding in acute promyelocytic leukemia cells with genistein [J]. Mol Cancer Ther. 2007, 6(8): 2240-2248.
36. Shen J, Tai YC, Zhou J B, et al. Synergistic antileukemia effect of genistein and chemotherapy in mouse xenograft model and potential mechanism through MAPK signaling [J]. Experimental Hematology, 2007, 35: 75-83.
37. Fatih M U, Yoav M, Chen C, et al. Treatment of Therapy-Refractory B-lineage Acute lymphoblastic leukemia with an Apoptosis-inducing CD19-directed Tyrosine Kinase Inhibitor [J]. Clinical Cancer Research, 1999, 5(12) 3906-3913.
38. Ramzi M. Mohammad A, Amro A, et al. Genistein sensitizes diffuse large cell lymphoma to CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy [J]. Mol Cancer Ther, 2003, 2(12):1361-1368.