紫草素诱导肝癌细胞凋亡及其机制的初步探讨
摘要
肝癌是最常见的肿瘤之一,往往预后不良,给人体健康造成巨大威胁。尽管人们做了大量监查工作,但是绝大多数肝癌只有在晚期才被诊断出来。在中国,肝癌发病率很高,死亡率居各种肿瘤死亡率前列,已成为威胁我国人们生命健康的主要杀手之一。迄今为止,大部分抗肿瘤药物均有较强的细胞毒性作用,但由于毒副作用大,限制了它的广泛应用和疗效的发挥。因此,寻找毒副作用小、安全有效的天然抗肿瘤药物成为肿瘤治疗研究的热点。紫草素是紫草根部的提取物,为脂溶性萘醌类化合物,具有较强的抗肿瘤作用,有望成为肝癌治疗的有效药物。为寻找具有开发前景的抗肿瘤药物提供理论和实验依据,以肝癌SMMC-7721细胞为研究对象,研究紫草素诱导肝癌SMMC-7721细胞凋亡的作用及对其相关机制进行初步探讨。
1.紫草素体外抑制SMMC-7721细胞增殖试验
以MTT法分别检测紫草素对SMMC-7721细胞和人正常肝细胞HL-7702的细胞增殖的影响。结果显示,紫草素对SMMC-7721细胞具有明显的抑制作用,随紫草素浓度和作用时间的增加,其对肝癌细胞和肝细胞生长的抑制率也明显增加,有明显的时间和浓度依赖性,但对正常肝细胞抑制作用较弱。
2.紫草素诱导肝癌SMMC-7721细胞凋亡的研究
以相差显微镜,共聚显微镜和电镜技术对肝癌SMMC-7721细胞凋亡进行形态学观察;流式细胞仪检测紫草素对肝癌SMMC-7721细胞周期、凋亡率的影响;TUNEL法检测肝癌SMMC-7721细胞凋亡情况;DNA琼脂糖电泳检测肝癌SMMC-7721细胞凋亡的核酸变化。形态学观察显示,肝癌SMMC-7721细胞出现了核固缩、核碎裂、部分细胞变小、折光性差等凋亡特征。流式细胞仪检测肝癌SMMC-7721细胞周期阻滞于G0/G1期,并且随着药物剂量的增大,G0/G1期的比例逐渐增高,而S期细胞的比例下降,G2/M细胞没有比例没有明显变化,细胞凋亡率也呈现明显的浓度时间依赖性。琼脂糖凝胶电泳发现细胞凋亡后,出现了典型的“DNA ladder”条带。
3.紫草素诱导SMMC-7721细胞凋亡机制的初步探讨
以流式细胞仪检测细胞内钙离子浓度、细胞线粒体膜电位的变化,分光光度法检测caspase-3活性的变化,RT-PCR检测凋亡相关基因mRNA水平的表达变化。结果显示,经紫草素作用后,SMMC-7721细胞内钙离子浓度降低,细胞线粒体膜电位不断降低, caspase-3活性升高,bcl-2基因较对照组mRNA水平表达量明显下降,Bax、caspase-3、p53基因较对照组mRNA水平表达量明显升高,bax/bcl-2升高。提示,紫草素可能通过线粒体途径诱导肝癌SMMC-7721细胞凋亡。
Hepatocellular carcinoma (HCC) is the fifth most common tumor worldwide and continues to have a poor prognosis. Despite of surveillance efforts, most tumors are diagnosed at late stages. In China, hepatoma is so high incidence that it has been the main threat of people’s health in our country and has the front rank of the tumor cancers. So far, most antineoplastic drug all have strong cytotoxicity, but the toxic side effects are significant to limit extensive application and efficacy. Therefore, ti is forced on looking for natural anticancer drugs which are insignificant toxicity, safe and effective. Shikonin is extraction of Lithospermum root, which is fat-soluble naphthoquinone compounds with strong antitumor effect. Then shikonin is expected to be an effective drug for treatment of HCC. Hepatoma Cell Line SMMC-7721 as target was used in this study.Test of shikonin induced SMMC-7721 apoptosis and its related mechanisms were carried out in order to provide proof of cancer treatment of shikonin.
1. Study on hepatoma carcinoma cell proliferation inhibited by shikonin
The growth inhibition of human hepatoma cells lines SMMC-7721 and human normal liver cells HL-7702 treated by various concentration of shikonin after different time were observed with MTT assay.Results showed that shikonin could inhibit the growth of SMMC-7721 cells and HL-7702 cells and it presented time-and concentration-dependent. However, the effect of shikonin on HL-7702 cell was significantly strong than that of HL-7702 cells.
2.Studies on apoptosis of human hepatoma cell induced by shikonin
Motphological changes of SMMC-7721 apoptosis were observed with flouorescance microscop, confocal microscope and electron microscopy. Cell cycle changes and the rates of apoptosis changes were detected by flow cytometry, and the apoptosis of hepatoma cells tunel were detected by DNA agarose gel electrophoresis. Characteristics of typical apoptosis such as nuclear shrinkage, fragmented nuclei et al, could be seen in SMMC-7721 cells treated with shikonin. By flow cytometry, it has been found that cell cycle was inhibited in G0/G1 phase, the ratio of the G0/G1 phase was gradually increased while the proportion of S phase was reduced as the dose of medicine was increased, the proportion of G2/M was not appreciably changing, and the rate of apoptosis showed a clear dose- and time- dependent.“DNA ladder”was found by agarose gel electrophoresis, which was the typical characteristics of cell apoptosis.
3. Preliminary studies on the mechanisms of shikonin-induced human hepatoma cell apoptosis
Concentrations of intracellular calcium and mitochondrial membrane potential changes were determined by flow cytometry, the caspase-3 protein activity was detected with reader, and bcl-2, p53, bax, and caspase-3 gene mRNA levels were detected by semi-quantitative PCR. preliminary studies on the mechanisms of shikonin-induced hepatoma cell line SMMC-7721 apoptosis. The results showed that concentrations of intracellular calcium and mitochondrial membrane potential were continuously reduced, bcl-2 gene had no significant change, while bax, caspase-3, p53 gene expression increased significantly than the control group treaed by shikonin.This shows that mechanism of apoptosis may be way to induce apoptosis through mitochondrial signal pathway.
1.紫草素体外抑制SMMC-7721细胞增殖试验
以MTT法分别检测紫草素对SMMC-7721细胞和人正常肝细胞HL-7702的细胞增殖的影响。结果显示,紫草素对SMMC-7721细胞具有明显的抑制作用,随紫草素浓度和作用时间的增加,其对肝癌细胞和肝细胞生长的抑制率也明显增加,有明显的时间和浓度依赖性,但对正常肝细胞抑制作用较弱。
2.紫草素诱导肝癌SMMC-7721细胞凋亡的研究
以相差显微镜,共聚显微镜和电镜技术对肝癌SMMC-7721细胞凋亡进行形态学观察;流式细胞仪检测紫草素对肝癌SMMC-7721细胞周期、凋亡率的影响;TUNEL法检测肝癌SMMC-7721细胞凋亡情况;DNA琼脂糖电泳检测肝癌SMMC-7721细胞凋亡的核酸变化。形态学观察显示,肝癌SMMC-7721细胞出现了核固缩、核碎裂、部分细胞变小、折光性差等凋亡特征。流式细胞仪检测肝癌SMMC-7721细胞周期阻滞于G0/G1期,并且随着药物剂量的增大,G0/G1期的比例逐渐增高,而S期细胞的比例下降,G2/M细胞没有比例没有明显变化,细胞凋亡率也呈现明显的浓度时间依赖性。琼脂糖凝胶电泳发现细胞凋亡后,出现了典型的“DNA ladder”条带。
3.紫草素诱导SMMC-7721细胞凋亡机制的初步探讨
以流式细胞仪检测细胞内钙离子浓度、细胞线粒体膜电位的变化,分光光度法检测caspase-3活性的变化,RT-PCR检测凋亡相关基因mRNA水平的表达变化。结果显示,经紫草素作用后,SMMC-7721细胞内钙离子浓度降低,细胞线粒体膜电位不断降低, caspase-3活性升高,bcl-2基因较对照组mRNA水平表达量明显下降,Bax、caspase-3、p53基因较对照组mRNA水平表达量明显升高,bax/bcl-2升高。提示,紫草素可能通过线粒体途径诱导肝癌SMMC-7721细胞凋亡。
Hepatocellular carcinoma (HCC) is the fifth most common tumor worldwide and continues to have a poor prognosis. Despite of surveillance efforts, most tumors are diagnosed at late stages. In China, hepatoma is so high incidence that it has been the main threat of people’s health in our country and has the front rank of the tumor cancers. So far, most antineoplastic drug all have strong cytotoxicity, but the toxic side effects are significant to limit extensive application and efficacy. Therefore, ti is forced on looking for natural anticancer drugs which are insignificant toxicity, safe and effective. Shikonin is extraction of Lithospermum root, which is fat-soluble naphthoquinone compounds with strong antitumor effect. Then shikonin is expected to be an effective drug for treatment of HCC. Hepatoma Cell Line SMMC-7721 as target was used in this study.Test of shikonin induced SMMC-7721 apoptosis and its related mechanisms were carried out in order to provide proof of cancer treatment of shikonin.
1. Study on hepatoma carcinoma cell proliferation inhibited by shikonin
The growth inhibition of human hepatoma cells lines SMMC-7721 and human normal liver cells HL-7702 treated by various concentration of shikonin after different time were observed with MTT assay.Results showed that shikonin could inhibit the growth of SMMC-7721 cells and HL-7702 cells and it presented time-and concentration-dependent. However, the effect of shikonin on HL-7702 cell was significantly strong than that of HL-7702 cells.
2.Studies on apoptosis of human hepatoma cell induced by shikonin
Motphological changes of SMMC-7721 apoptosis were observed with flouorescance microscop, confocal microscope and electron microscopy. Cell cycle changes and the rates of apoptosis changes were detected by flow cytometry, and the apoptosis of hepatoma cells tunel were detected by DNA agarose gel electrophoresis. Characteristics of typical apoptosis such as nuclear shrinkage, fragmented nuclei et al, could be seen in SMMC-7721 cells treated with shikonin. By flow cytometry, it has been found that cell cycle was inhibited in G0/G1 phase, the ratio of the G0/G1 phase was gradually increased while the proportion of S phase was reduced as the dose of medicine was increased, the proportion of G2/M was not appreciably changing, and the rate of apoptosis showed a clear dose- and time- dependent.“DNA ladder”was found by agarose gel electrophoresis, which was the typical characteristics of cell apoptosis.
3. Preliminary studies on the mechanisms of shikonin-induced human hepatoma cell apoptosis
Concentrations of intracellular calcium and mitochondrial membrane potential changes were determined by flow cytometry, the caspase-3 protein activity was detected with reader, and bcl-2, p53, bax, and caspase-3 gene mRNA levels were detected by semi-quantitative PCR. preliminary studies on the mechanisms of shikonin-induced hepatoma cell line SMMC-7721 apoptosis. The results showed that concentrations of intracellular calcium and mitochondrial membrane potential were continuously reduced, bcl-2 gene had no significant change, while bax, caspase-3, p53 gene expression increased significantly than the control group treaed by shikonin.This shows that mechanism of apoptosis may be way to induce apoptosis through mitochondrial signal pathway.
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