两种植物黄酮抗白血病细胞HL-60效应及机制研究
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摘要
白血病是造血系统的恶性肿瘤,发病率在各种人类肿瘤中占第六位,居年轻人恶性疾病的首位,是一种严重威胁人类生命健康的恶性疾病,努力寻找天然、高效的防治药物以预防和治疗白血病,是一项具有重要的现实和理论意义的研究课题。植物黄酮是膳食中一类较为重要的植物化学物,具有抗氧化、抗炎症、抗肿瘤、抗动脉粥样硬化等广泛、优良的生物学效应,特别是对于肿瘤的发生、增殖、迁移、侵袭、血管生成以及耐药性等各阶段、各方面都具有显著的抑制作用,植物黄酮抗肿瘤研究一直是国内外研究的一个热点。本课题组前期研究选用23种分子结构明确的常见植物黄酮,对其抗肿瘤效应进行了评价和筛选,结果显示,3,6-二羟黄酮和2’-羟基二氢黄酮具有较强的抗肿瘤效应,其抗肿瘤作用及其机制值得深入探讨,本课题是在此研究基础上的进一步深入。
肿瘤细胞最显著的特点是不受控制的快速增殖特性,能够有效抑制肿瘤细胞的增殖是抗肿瘤作用的最基本表现。细胞存活率,即活细胞的比例,反映出该群细胞的整体增殖活力状况,细胞存活率越高反映出该群细胞的增殖活性越好,反之则表明该群细胞中的死亡细胞、凋亡细胞比例高,细胞整体增殖活性低。细胞凋亡是以细胞核浓缩、染色体DNA被以核小体为单位切成梯状片段、细胞缩小,最终形成细胞凋亡小体等形态变化为特征。研究表明,细胞凋亡发生的关键环节在于线粒体膜功能发生改变,内膜跨膜电位消失和线粒体内蛋白酶活化物(如细胞色素C)的释放,激发各种凋亡相关的代谢变化,从而启动凋亡程序。线粒体跨膜电位消失与细胞色素C从线粒体的释放被认为是细胞凋亡启动的关键步骤:释放到细胞浆的细胞色素C在dATP存在的条件下激活caspase家族,从而启动和执行凋亡信号转导。
细胞中抗氧化酶系统的功能对于调控活性氧(ROS)以及调节包括增殖、凋亡、炎症反应的细胞生命活动具有极其重要的作用。肿瘤细胞内的抗氧化酶活性都显著低于正常细胞,肿瘤细胞清除ROS和生成GSH的能力较低,而肿瘤细胞内的ROS水平往往高于正常细胞,降低细胞内抗氧化酶活性能够选择性杀伤肿瘤细胞,这可能是抗肿瘤药物发挥效应的重要机制。促分裂素原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号通路是真核生物信号传递网络中的重要途径之一,在基因表达调控和细胞质功能活动中发挥关键作用。近年来一些研究表明,ROS和氧化应激可介导MAPKs信号途径的改变,诱导细胞凋亡,其中JNK/SAPK和p38MAPK两条途径在氧化应激的研究中引起广泛关注。前期研究表明3,6-二羟黄酮可显著影响HL-60细胞胞内ROS水平,3,6-二羟黄酮是否因此影响MAPK信号通路,值得探讨。
基于以上分析,本课题选用前期药效实验筛选出的具有较强抗肿瘤效应的3,6-二羟黄酮和2’-羟基二氢黄酮,观察两种植物黄酮化合物对白血病HL-60细胞增殖和凋亡的影响,利用流式细胞分析、激光共聚焦扫描显微镜、Western blot等分子生物学检测分析技术,检测3,6-二羟黄酮对HL-60细胞线粒体膜电位水平降低、细胞色素C释放以及caspase家族激活这一凋亡启动和执行信号转导的影响,同时探讨3,6-二羟黄酮诱导HL-60细胞凋亡过程中,对细胞抗氧化酶活性和MAPKs信号通路的影响。
本研究主要实验结果和结论如下:
1.细胞生长曲线结果显示,不同剂量的3,6-二羟黄酮和2’-羟基二氢黄酮作用于HL-60细胞,细胞增殖能力产生不同程度降低,显示出浓度-依赖性和时间-依赖性效应关系;10μM浓度的3,6-二羟黄酮和20μM的2’-羟基二氢黄酮能够有效抑制HL-60细胞的快速增殖,20μM以上浓度的3,6-二羟黄酮则可有效阻止HL-60细胞的增殖,显示出杀灭HL-60细胞的抗肿瘤活性。与此一致,细胞活力分析结果表明,3,6-二羟黄酮和2’-羟基二氢黄酮能够剂量、时间-依赖性的降低HL-60细胞的细胞活力。以上结果表明,3,6-二羟黄酮和2’-羟基二氢黄酮能够显著降低HL-60细胞的细胞活力,抑制其增殖。
2.细胞形态学观察可见,正常HL-60细胞生长状态良好,形态完整,细胞呈珍珠(圆球)形,悬浮生长,细胞排列紧密,大小均匀,组织完整,未见破裂细胞和细胞碎片;3,6-二羟黄酮和2’-羟基二氢黄酮处理24h后,HL-60细胞形态随处理剂量的增加逐渐呈现出显著异常,表现为细胞排列稀疏、紊乱,大小不均匀,形态异常,大量细胞皱缩变圆、胞质凝缩或肿大破裂,肿胀细胞、破裂细胞和细胞碎片逐渐增多,这些形态变化都是凋亡细胞或死亡细胞的表现特征。这表明,3,6-二羟黄酮和2’-羟基二氢黄酮作用HL-60细胞后,细胞中死亡细胞、凋亡细胞和细胞碎片大量增加。
3.流式细胞凋亡分析结果表明,20μM的3,6-二羟黄酮和2’-羟基二氢黄酮处理,能够显著诱导HL-60细胞发生凋亡,凋亡细胞比例显著增高,显示出良好的抗肿瘤效应。进一步研究表明,20μM的3,6-二羟黄酮作用于HL-60细胞,细胞线粒体膜电位水平随作用时间增加而逐渐降低,并在作用8 h后显著低于正常对照组水平;Western blot方法检测胞浆蛋白中细胞色素C的含量,结果表明,20μM的3,6-二羟黄酮作用后2 h即见细胞胞浆中的细胞色素C含量显著升高,并随作用时间的增加逐渐增高;这表明3,6-二羟黄酮可诱导HL-60细胞线粒体膜电位显著降低,并导致线粒体中的细胞色素C大量释放至胞浆,启动细胞凋亡。
4.细胞抗氧化酶活性检测结果显示,3,6-二羟黄酮可显著降低HL-60细胞胞内抗氧化酶SOD、CAT、GSH-Px的活性,并显著降低SOD的表达,细胞中脂质过氧化物MDA水平显著升高。表明3,6-二羟黄酮可通过降低胞内抗氧化酶活性增加HL-60细胞内的氧化应激压力,导致细胞过氧化损伤。
5. Western blot检测结果表明,正常对照组HL-60细胞中caspases基本以无活性的酶原状态形式存在,裂解活化的caspases(cleaved-caspase 3、cleaved-caspase 9和cleaved-PARP)水平很低,3,6-二羟黄酮作用后,细胞中caspase 3、caspase 9、PARP含量逐渐降低,cleaved-caspase 3、cleaved-caspase 9和cleaved-PARP水平逐渐增高,cleaved-caspases/caspase的比值增高,表明caspases和PARP被裂解活化,凋亡启动和执行的信号转导被显著激活,细胞进入凋亡程序。
6. Western blot检测结果表明,20μM 3,6-二羟黄酮作用于HL-60细胞,细胞内MAPKs信号通路蛋白表达及磷酸化水平发生显著变化,细胞质内磷酸化ERK在处理2 h后显著下降,至24 h恢复到正常水平;JNK与p38MAPK则表现为磷酸化蛋白水平增高,在处理2 h后,磷酸化蛋白含量显著增加,然后缓慢下降,但24 h内仍高于正常水平,提示3,6-二羟黄酮对JNK、p38MAPK信号通路的影响较大。
综上所述,本研究结果表明,3,6-二羟黄酮和2’-羟基二氢黄酮能够有效抑制白血病HL-60细胞增殖,降低HL-60细胞增殖活力,并显著诱导HL-60细胞凋亡;进一步研究表明,3,6-二羟黄酮可显著降低HL-60细胞胞内抗氧化酶系统活性,使细胞过氧化损伤产物显著增加,同时降低线粒体膜电位水平,导致细胞色素C大量释放,激活caspases凋亡启动和执行信号转导,而此过程与MAPKs信号通路的变化可能具有相关性。
Leukemia is a malignant disease of the hematopoietic system. The risk of onset in children and young people is more than the other cancers. It is a significant research topic to seek natural and potent anti-cancer compounds to prevent and cure leukemia. Flavonoids are a large class of polyphenolic compounds, which are ubiquitously present in the plant world and our common diet, such as vegetables, fruits and plant-derived beverages. Several beneficial biological activities have ascribed to flavonoids, including anti-oxidant, anti-inflammatory, anti-cancer and anti-estrogenic properties. Among these pharmacological properties, there has been an increasing scientific interest in the anti-cancer activity of flavonoids due to their potent inhibition effect on the carcinogenesis, proliferation, migration, invasion, angiogenesis and drug resistance of cancer. Our previous study evaluated cytotoxicities of 23 different flavonoids in human cancer cells, and found that 3,6-dihydroxyflavone and 2′-hydroxyflavanone exhibited the most potent cytotoxic effect. This study was based on these researches.
Repaid proliferation is the noticeable characteristic of cancer cells; it is the basic ability for anti-cancer compounds to inhibit proliferation of cancer cells. Cells survival, that is the rate of living cells, reflects the viability of cells. Cells apoptosis appears morphological changes including cell shrinkage and cytoplasmic condensation. The abolition of mitochondria membrane potential and the release of cytochrome-c from mitochondria was the key step for apoptosis originated. Cyto C could activate caspases family in the presence of dATP, and start the apoptosis signal transduction.
The function of antioxidase of cells was important for the regulation of reactive oxygen species (ROS) and the proliferation, apoptosis, inflammatory reaction of cells life activities. The activity of antioxidase in cancer cells was lower than normal cells; ROS could selectively kill the cancer cells. Mitogen-activated protein kinases (MAPK) is an important signal pathway in cells. It has been reported that ROS and oxidative stress could change the MAPK signal pathway and induced apoptosis. Previous study has shown that 3,6-dihydroxyflavone could change the ROS level of HL-60 cells, whether it influence MAPK pathway need further study.
Based on the analysis mentioned above, we selectively study the anti-cancer effect of 3,6-dihydroxyflavone and 2′-hydroxyflavanone on leukemia HL-60 cells. We observed the effect of these two flavonoids on the proliferation and apoptosis of HL-60 cells. By flow cytometric analysis, laser confocal scanning microscopy and western blot, we investigated the changes of mitochondria membrane potential, the release of cytochrome-c, the activation of caspases and MAPK signal pathway under the 3,6-dihydroxyflavone treatment.
The main results and conclusions were summarized as follows:
1. Cells growth curve showed that the proliferation of normal HL-60 cells was rapid; different dose of 3,6-dihydroxyflavone and 2′-hydroxyflavanone treatment could reduce the proliferation of HL-60 cells differently. The proliferation inhibition effect showed dose-dependent relationship. The treatment of 10μM 3,6-dihydroxyflavone or 20μM 2′-hydroxyflavanone could inhibit proliferation of HL-60 cells, but could not prevent the growth of living cells; 20μM 3,6-dihydroxyflavone could reduce the amount of living cells, showed potent anti-cancer activity. Cells viability assay indicated the similar results that 3,6-dihydroxyflavone and 2′-hydroxyflavanone treatment could dose, time-dependently decrease the cells viability of HL-60 cells, the living cells rate was significantly reduced after the flavonoids treatment.
2. We examined the morphological changes of HL-60 cells after the treatment of 3,6-dihydroxyflavone and 2′-hydroxyflavanone. The normal HL-60 cells showed good growth condition, no cell fraction was found. Treated with 3,6-dihydroxyflavone and 2′-hydroxyflavanone for 24h, dramatic pro-apoptotic morphological changes in comparison with the control were observed, including cell shrinkage and cytoplasmic condensation.
3. The apoptosis analysis by flow cytometric assay showed that 20μM 3,6-dihydroxyflavone or 2′-hydroxyflavanone treatment could significantly induce apoptosis in HL-60 cells. The apoptosis rate was markedly increased after the treatment. The further study showed that after the treatment of 20μM 3,6-dihydroxyflavone, the mitochondria membrane potential was gradually decreased and was significantly lower than the control after 8 h. The level of cytochrome-c in cytosol was significantly increased after treatment. These results indicated that 3,6-dihydroxyflavone treatment could decrease the mitochondria membrane potential and increase the release of cyto C, induce apoptosis.
4. Results of western blot indicated that the caspases in normal HL-60 cells was mainly in inactive form, the level of cleaved-caspases was low. After the treatment of 3,6-dihydroxyflavone, the level of caspases decreased while the level of cleaved-caspases increased, the rate of cleaved-caspase 3/caspase 3, cleaved-caspase 9/caspase 9 and cleaved-PARP/PARP were significantly increased. It indicated that 3,6-dihydroxyflavone actived caspases apoptosis signal transduction.
5. Results of antioxidase activities detection indicated that 3,6-dihydroxy- flavone could significantly decrease the activity of SOD, CAT and GSH-Px. The MDA level was markedly increased. It showed that 3,6-dihydroxyflavone could decrease the antioxidases of HL-60 cells and induce oxidative damage.
6. Our study showed that 3,6-dihydroxyflavone could significantly change the MAPKs signal pathway. The protein level of phosphorylation of ERK in HL-60 cells which were treated for 2h, 8h, 24h by 3,6-dihydroxyflavone was decreased significantly compared with untreated group, the expression of phosphorylation of JNK and p38MAPK increased significantly. It indicated 3,6-dihydroxyflavone influenced more on JNK and p38MAPK signal pathway.
In all, our study indicated potent proliferation inhibition and pro-apoptosis effect of 3,6-dihydroxyflavone or 2′-hydroxyflavanone on leukemia HL-60 cells. Further study showed 3,6-dihydroxyflavone could significantly decrease the antioxidases activities, induce the oxidative damage, cause the abolition of mitochondria membrane potential and the release of cytochrome-c, active caspases apoptosis signal transduction. The anti-cancer mechanism may be associated with the influence on MAPKs signal pathway.
肿瘤细胞最显著的特点是不受控制的快速增殖特性,能够有效抑制肿瘤细胞的增殖是抗肿瘤作用的最基本表现。细胞存活率,即活细胞的比例,反映出该群细胞的整体增殖活力状况,细胞存活率越高反映出该群细胞的增殖活性越好,反之则表明该群细胞中的死亡细胞、凋亡细胞比例高,细胞整体增殖活性低。细胞凋亡是以细胞核浓缩、染色体DNA被以核小体为单位切成梯状片段、细胞缩小,最终形成细胞凋亡小体等形态变化为特征。研究表明,细胞凋亡发生的关键环节在于线粒体膜功能发生改变,内膜跨膜电位消失和线粒体内蛋白酶活化物(如细胞色素C)的释放,激发各种凋亡相关的代谢变化,从而启动凋亡程序。线粒体跨膜电位消失与细胞色素C从线粒体的释放被认为是细胞凋亡启动的关键步骤:释放到细胞浆的细胞色素C在dATP存在的条件下激活caspase家族,从而启动和执行凋亡信号转导。
细胞中抗氧化酶系统的功能对于调控活性氧(ROS)以及调节包括增殖、凋亡、炎症反应的细胞生命活动具有极其重要的作用。肿瘤细胞内的抗氧化酶活性都显著低于正常细胞,肿瘤细胞清除ROS和生成GSH的能力较低,而肿瘤细胞内的ROS水平往往高于正常细胞,降低细胞内抗氧化酶活性能够选择性杀伤肿瘤细胞,这可能是抗肿瘤药物发挥效应的重要机制。促分裂素原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号通路是真核生物信号传递网络中的重要途径之一,在基因表达调控和细胞质功能活动中发挥关键作用。近年来一些研究表明,ROS和氧化应激可介导MAPKs信号途径的改变,诱导细胞凋亡,其中JNK/SAPK和p38MAPK两条途径在氧化应激的研究中引起广泛关注。前期研究表明3,6-二羟黄酮可显著影响HL-60细胞胞内ROS水平,3,6-二羟黄酮是否因此影响MAPK信号通路,值得探讨。
基于以上分析,本课题选用前期药效实验筛选出的具有较强抗肿瘤效应的3,6-二羟黄酮和2’-羟基二氢黄酮,观察两种植物黄酮化合物对白血病HL-60细胞增殖和凋亡的影响,利用流式细胞分析、激光共聚焦扫描显微镜、Western blot等分子生物学检测分析技术,检测3,6-二羟黄酮对HL-60细胞线粒体膜电位水平降低、细胞色素C释放以及caspase家族激活这一凋亡启动和执行信号转导的影响,同时探讨3,6-二羟黄酮诱导HL-60细胞凋亡过程中,对细胞抗氧化酶活性和MAPKs信号通路的影响。
本研究主要实验结果和结论如下:
1.细胞生长曲线结果显示,不同剂量的3,6-二羟黄酮和2’-羟基二氢黄酮作用于HL-60细胞,细胞增殖能力产生不同程度降低,显示出浓度-依赖性和时间-依赖性效应关系;10μM浓度的3,6-二羟黄酮和20μM的2’-羟基二氢黄酮能够有效抑制HL-60细胞的快速增殖,20μM以上浓度的3,6-二羟黄酮则可有效阻止HL-60细胞的增殖,显示出杀灭HL-60细胞的抗肿瘤活性。与此一致,细胞活力分析结果表明,3,6-二羟黄酮和2’-羟基二氢黄酮能够剂量、时间-依赖性的降低HL-60细胞的细胞活力。以上结果表明,3,6-二羟黄酮和2’-羟基二氢黄酮能够显著降低HL-60细胞的细胞活力,抑制其增殖。
2.细胞形态学观察可见,正常HL-60细胞生长状态良好,形态完整,细胞呈珍珠(圆球)形,悬浮生长,细胞排列紧密,大小均匀,组织完整,未见破裂细胞和细胞碎片;3,6-二羟黄酮和2’-羟基二氢黄酮处理24h后,HL-60细胞形态随处理剂量的增加逐渐呈现出显著异常,表现为细胞排列稀疏、紊乱,大小不均匀,形态异常,大量细胞皱缩变圆、胞质凝缩或肿大破裂,肿胀细胞、破裂细胞和细胞碎片逐渐增多,这些形态变化都是凋亡细胞或死亡细胞的表现特征。这表明,3,6-二羟黄酮和2’-羟基二氢黄酮作用HL-60细胞后,细胞中死亡细胞、凋亡细胞和细胞碎片大量增加。
3.流式细胞凋亡分析结果表明,20μM的3,6-二羟黄酮和2’-羟基二氢黄酮处理,能够显著诱导HL-60细胞发生凋亡,凋亡细胞比例显著增高,显示出良好的抗肿瘤效应。进一步研究表明,20μM的3,6-二羟黄酮作用于HL-60细胞,细胞线粒体膜电位水平随作用时间增加而逐渐降低,并在作用8 h后显著低于正常对照组水平;Western blot方法检测胞浆蛋白中细胞色素C的含量,结果表明,20μM的3,6-二羟黄酮作用后2 h即见细胞胞浆中的细胞色素C含量显著升高,并随作用时间的增加逐渐增高;这表明3,6-二羟黄酮可诱导HL-60细胞线粒体膜电位显著降低,并导致线粒体中的细胞色素C大量释放至胞浆,启动细胞凋亡。
4.细胞抗氧化酶活性检测结果显示,3,6-二羟黄酮可显著降低HL-60细胞胞内抗氧化酶SOD、CAT、GSH-Px的活性,并显著降低SOD的表达,细胞中脂质过氧化物MDA水平显著升高。表明3,6-二羟黄酮可通过降低胞内抗氧化酶活性增加HL-60细胞内的氧化应激压力,导致细胞过氧化损伤。
5. Western blot检测结果表明,正常对照组HL-60细胞中caspases基本以无活性的酶原状态形式存在,裂解活化的caspases(cleaved-caspase 3、cleaved-caspase 9和cleaved-PARP)水平很低,3,6-二羟黄酮作用后,细胞中caspase 3、caspase 9、PARP含量逐渐降低,cleaved-caspase 3、cleaved-caspase 9和cleaved-PARP水平逐渐增高,cleaved-caspases/caspase的比值增高,表明caspases和PARP被裂解活化,凋亡启动和执行的信号转导被显著激活,细胞进入凋亡程序。
6. Western blot检测结果表明,20μM 3,6-二羟黄酮作用于HL-60细胞,细胞内MAPKs信号通路蛋白表达及磷酸化水平发生显著变化,细胞质内磷酸化ERK在处理2 h后显著下降,至24 h恢复到正常水平;JNK与p38MAPK则表现为磷酸化蛋白水平增高,在处理2 h后,磷酸化蛋白含量显著增加,然后缓慢下降,但24 h内仍高于正常水平,提示3,6-二羟黄酮对JNK、p38MAPK信号通路的影响较大。
综上所述,本研究结果表明,3,6-二羟黄酮和2’-羟基二氢黄酮能够有效抑制白血病HL-60细胞增殖,降低HL-60细胞增殖活力,并显著诱导HL-60细胞凋亡;进一步研究表明,3,6-二羟黄酮可显著降低HL-60细胞胞内抗氧化酶系统活性,使细胞过氧化损伤产物显著增加,同时降低线粒体膜电位水平,导致细胞色素C大量释放,激活caspases凋亡启动和执行信号转导,而此过程与MAPKs信号通路的变化可能具有相关性。
Leukemia is a malignant disease of the hematopoietic system. The risk of onset in children and young people is more than the other cancers. It is a significant research topic to seek natural and potent anti-cancer compounds to prevent and cure leukemia. Flavonoids are a large class of polyphenolic compounds, which are ubiquitously present in the plant world and our common diet, such as vegetables, fruits and plant-derived beverages. Several beneficial biological activities have ascribed to flavonoids, including anti-oxidant, anti-inflammatory, anti-cancer and anti-estrogenic properties. Among these pharmacological properties, there has been an increasing scientific interest in the anti-cancer activity of flavonoids due to their potent inhibition effect on the carcinogenesis, proliferation, migration, invasion, angiogenesis and drug resistance of cancer. Our previous study evaluated cytotoxicities of 23 different flavonoids in human cancer cells, and found that 3,6-dihydroxyflavone and 2′-hydroxyflavanone exhibited the most potent cytotoxic effect. This study was based on these researches.
Repaid proliferation is the noticeable characteristic of cancer cells; it is the basic ability for anti-cancer compounds to inhibit proliferation of cancer cells. Cells survival, that is the rate of living cells, reflects the viability of cells. Cells apoptosis appears morphological changes including cell shrinkage and cytoplasmic condensation. The abolition of mitochondria membrane potential and the release of cytochrome-c from mitochondria was the key step for apoptosis originated. Cyto C could activate caspases family in the presence of dATP, and start the apoptosis signal transduction.
The function of antioxidase of cells was important for the regulation of reactive oxygen species (ROS) and the proliferation, apoptosis, inflammatory reaction of cells life activities. The activity of antioxidase in cancer cells was lower than normal cells; ROS could selectively kill the cancer cells. Mitogen-activated protein kinases (MAPK) is an important signal pathway in cells. It has been reported that ROS and oxidative stress could change the MAPK signal pathway and induced apoptosis. Previous study has shown that 3,6-dihydroxyflavone could change the ROS level of HL-60 cells, whether it influence MAPK pathway need further study.
Based on the analysis mentioned above, we selectively study the anti-cancer effect of 3,6-dihydroxyflavone and 2′-hydroxyflavanone on leukemia HL-60 cells. We observed the effect of these two flavonoids on the proliferation and apoptosis of HL-60 cells. By flow cytometric analysis, laser confocal scanning microscopy and western blot, we investigated the changes of mitochondria membrane potential, the release of cytochrome-c, the activation of caspases and MAPK signal pathway under the 3,6-dihydroxyflavone treatment.
The main results and conclusions were summarized as follows:
1. Cells growth curve showed that the proliferation of normal HL-60 cells was rapid; different dose of 3,6-dihydroxyflavone and 2′-hydroxyflavanone treatment could reduce the proliferation of HL-60 cells differently. The proliferation inhibition effect showed dose-dependent relationship. The treatment of 10μM 3,6-dihydroxyflavone or 20μM 2′-hydroxyflavanone could inhibit proliferation of HL-60 cells, but could not prevent the growth of living cells; 20μM 3,6-dihydroxyflavone could reduce the amount of living cells, showed potent anti-cancer activity. Cells viability assay indicated the similar results that 3,6-dihydroxyflavone and 2′-hydroxyflavanone treatment could dose, time-dependently decrease the cells viability of HL-60 cells, the living cells rate was significantly reduced after the flavonoids treatment.
2. We examined the morphological changes of HL-60 cells after the treatment of 3,6-dihydroxyflavone and 2′-hydroxyflavanone. The normal HL-60 cells showed good growth condition, no cell fraction was found. Treated with 3,6-dihydroxyflavone and 2′-hydroxyflavanone for 24h, dramatic pro-apoptotic morphological changes in comparison with the control were observed, including cell shrinkage and cytoplasmic condensation.
3. The apoptosis analysis by flow cytometric assay showed that 20μM 3,6-dihydroxyflavone or 2′-hydroxyflavanone treatment could significantly induce apoptosis in HL-60 cells. The apoptosis rate was markedly increased after the treatment. The further study showed that after the treatment of 20μM 3,6-dihydroxyflavone, the mitochondria membrane potential was gradually decreased and was significantly lower than the control after 8 h. The level of cytochrome-c in cytosol was significantly increased after treatment. These results indicated that 3,6-dihydroxyflavone treatment could decrease the mitochondria membrane potential and increase the release of cyto C, induce apoptosis.
4. Results of western blot indicated that the caspases in normal HL-60 cells was mainly in inactive form, the level of cleaved-caspases was low. After the treatment of 3,6-dihydroxyflavone, the level of caspases decreased while the level of cleaved-caspases increased, the rate of cleaved-caspase 3/caspase 3, cleaved-caspase 9/caspase 9 and cleaved-PARP/PARP were significantly increased. It indicated that 3,6-dihydroxyflavone actived caspases apoptosis signal transduction.
5. Results of antioxidase activities detection indicated that 3,6-dihydroxy- flavone could significantly decrease the activity of SOD, CAT and GSH-Px. The MDA level was markedly increased. It showed that 3,6-dihydroxyflavone could decrease the antioxidases of HL-60 cells and induce oxidative damage.
6. Our study showed that 3,6-dihydroxyflavone could significantly change the MAPKs signal pathway. The protein level of phosphorylation of ERK in HL-60 cells which were treated for 2h, 8h, 24h by 3,6-dihydroxyflavone was decreased significantly compared with untreated group, the expression of phosphorylation of JNK and p38MAPK increased significantly. It indicated 3,6-dihydroxyflavone influenced more on JNK and p38MAPK signal pathway.
In all, our study indicated potent proliferation inhibition and pro-apoptosis effect of 3,6-dihydroxyflavone or 2′-hydroxyflavanone on leukemia HL-60 cells. Further study showed 3,6-dihydroxyflavone could significantly decrease the antioxidases activities, induce the oxidative damage, cause the abolition of mitochondria membrane potential and the release of cytochrome-c, active caspases apoptosis signal transduction. The anti-cancer mechanism may be associated with the influence on MAPKs signal pathway.
引文
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