摘要
目的川楝素是从我国传统中药楝科植物川楝子和苦楝皮中提取出的一种四环三萜类化合物,具有广泛的药理作用。近几年来研究证明,川楝素在体外可明显抑制多种肿瘤细胞的增殖,但其机制仅有初步探讨。本论文通过体外细胞培养,体内建立荷瘤小鼠模型,全面系统地研究川楝素对实体肿瘤肝癌的抗肿瘤作用,并进一步探讨川楝素的作用机制及毒副作用。同时,考虑到在治疗方法上血液系统恶性肿瘤有别与实体癌,因此,以人慢性髓系白血病K562细胞为研究对象,从增殖抑制、分化、凋亡三方面对川楝素药理作用及其机制进行初步探讨。
方法
1.以人肝癌SMMC-7721(P53﹢)、Hep3B(P53﹣)细胞为实验对象,从以下几个方面研究川楝素在体外对人肝癌细胞生长的抑制作用及机制:细胞计数法和MTT法检测川楝素对SMMC-7721、Hep3B细胞和正常肝细胞IAR-20、LO2生长曲线的影响并计算增殖抑制率;观察SMMC-7721、Hep3B细胞形态学改变;AnnexinV法检测早期凋亡率;比色法检测Caspase-3、8、9相对活性改变;免疫细胞化学法检测凋亡相关蛋白Bcl-2、Bax和Fas的表达变化。
2.建立H22荷瘤小鼠模型,从以下几个方面观察川楝素在体内对小鼠肿瘤生长的抑制作用及机制:量取肿瘤体积,观察肿瘤的生长曲线;剥瘤后称重计算小鼠的肿瘤抑制率;肿瘤组织行病理形态学及HE染色观察;透射电镜观察超微结构改变;观察川楝素对荷瘤小鼠心、肝、脾、肾、胸腺、睾丸组织的影响;免疫组化方法检测肿瘤组织内Bcl-2、Bax和Fas蛋白的表达。
3.以人慢性髓系白血病细胞K562(P53﹣)为研究对象,从以下几个方面研究川楝素在体外对K562细胞增殖、分化、凋亡作用的影响及其机制:MTT法检测川楝素对K562细胞和正常人外周血单个核细胞(PBMCs)增殖的影响;观察K562细胞形态学变化和超微结构的改变;AnnexinV法检测早期凋亡率;DNA ladder分析;联苯胺和POX染色法观察红系和粒系分化情况;比色法检测Caspase-3、8、9相对活性改变;免疫细胞化学法检测凋亡相关蛋白Bcl-2、Bax和Fas的表达。
结果
1.肝癌细胞系体外实验中,0.5μmol/L川楝素可明显抑制肝癌SMMC-7721、Hep3B细胞增殖,且抑制作用呈时间和剂量依赖性,而对正常肝细胞IAR-20、LO2增殖影响较小;川楝素处理后SMMC-7721、Hep3B细胞黏附力降低,部分细胞皱缩变圆;流式细胞术检测到0.5μmol/L川楝素作用SMMC-7721、Hep3B细胞72h的凋亡率分别为21.55%、13.35%,经caspase抑制剂z-VAD-fmk处理后,细胞凋亡率降低,TSN+z-VAD-fmk组与TSN组比较有显著性差异(P< 0.05);川楝素处理SMMC-7721细胞后,Caspase-3、8、9活性均增高,Bcl-2表达减弱,Bax和Fas表达较对照组增强;川楝素处理Hep3B细胞,Caspase-3、Caspase-9活性增高,Caspase-8活性无明显变化,Bcl-2表达减弱,Bax表达增强,而Fas表达无明显变化。
2.动物实验显示,川楝素可显著抑制小鼠体内肿瘤的生长,川楝素低剂量组(0.173mg/kg)、高剂量组(0.69mg/kg)的抑瘤率分别为66.23%、87.01%(P<0.05);肿瘤组织超微结构透射电镜观察可见凋亡小体;HE染色显示小鼠心、肝、脾、肾、睾丸脏器形态正常,而胸腺组织可见胸腺小叶的数量及面积减少甚至消失;免疫组化证实,小鼠肿瘤组织内Bcl-2蛋白表达下调,Bax、Fas蛋白表达上调(P<0.05)。
3.人慢性髓系白血病细胞体外实验中,川楝素对K562细胞有明显增殖抑制作用,呈时间和浓度依赖性,而对PBMC几乎无影响;普通光镜可见K562细胞典型的凋亡形态学改变;超微结构显示凋亡细胞和凋亡小体;10,30,50 nmol/L川楝素处理K562细胞72 h后的早期凋亡率分别为9.66%,26.06%,52.70%(P<0.01);琼脂糖凝胶电泳可见典型“梯状”条带;联苯胺和POX染色结果阴性;激活K562细胞Caspase-3,Caspase-8,Caspase-9活性;凋亡相关蛋白Bcl-xl表达降低,Bax,Fas表达增强(P<0.05)。
结论
1.体内体外实验均表明:川楝素对肝癌细胞有较强的抑制增殖和诱导凋亡作用,涉及线粒体途径的参与。
2.体外实验表明:川楝素对人慢性髓系白血病K562细胞具有抑制增殖,诱导凋亡的药理效应,诱导凋亡的机制涉及线粒体途径和死亡受体途径共同参与;川楝素对K562细胞无诱导其向红系、粒系分化的作用。
3.川楝素对正常人肝细胞LO2、正常大鼠肝细胞IAR-20和正常人外周血单个核细胞几乎无毒性作用。
4.川楝素对P53阳性的SMMC-7721细胞和P53阴性的Hep3B、K562细胞均具有增殖抑制和诱导凋亡的药理效应,可能是一种可通过非P53依赖途径发挥抗癌作用的天然低毒药物。
Objective
Toosendanin(TSN), a triterpenoid derivative isolated from Melia toosendan Sieb.et Zucc, possesses different pharmacological effects in human and important values in agriculture. As indicated by previous reports, TSN could confer anti-proliferative and pro-apoptotic effects on human cancer cell lines, But the mechanism by which TSN induced apoptosis remains poorly clarified.we investigated the inhibitory effects of TSN in vitro and in vivo with cell cultivating method, H22 xenograft tumor models estabilishing in BALB/C, and explored the probable mechanisms of TSN in solid carcinoma. And to further explore the mechanisms and side effects of TSN. As the difference methods in the treatment of hematologic malignancies and solid cancers, Taking the human chronic myeloid leukemia (CML) K562 cells as our experimental object, we observed on the proliferation, differentiation and apoptosis of K562 cells treated with TSN and discussed the probable mechanisms of anti-tumor effects.
Methods
1. Taking the SMMC-7721 and Hep3B cells as our experimental object, we observed the SMMC-7721 and Hep3B cells’proliferation inhibion treated with TSN and discussed the probable mechanisms of anti-tumor effects in vitro from the following aspects: the inhibitory rate of cell proliferation and cell growth curve were detected using MTT assay and cell counting method in SMMC-7721、Hep3B、IAR-20、LO2 cells; the morphlolgical changes of cells was observed by the optical microscope; cell apoptosis at the early stage was detected with AnnexinV-FITC/PI assay; The activities of Caspase-8, Caspase-9 and Caspase-3 were analyzed by spectrophotometry; the expression of Bcl-2、Bax and Fas were detected using immunohistochemistry assay.
2. H22 xenograft tumor models were established in BALB/C. The tumor suppression effects of TSN was studied in vivo from the following aspects: the xenograft tumor were excised and weighted to calculate the tumor growth inhibitory rate; the tumor volume were measured; sections from BALB/C xenograft tumor was subjected to HE staining to reveal the changes of histomorphologism; transmission electron microscopy was used to test the ultramicro-structure alternations of BALB/C xenograft tumor cells, Pathological features of heart, liver, spleen, kidney, thymus and testis of cancer bearing mice were observed; the expression of Bcl-2、Bax and Fas were detected using immunohistochemistry assay.
3. Taking K562 cells as our experimental object, we observed the K562 cell’s proliferation inhibion, differentiation, apoptosis treated with TSN and discussed the probable mechanisms of anti-tumor effects in vitro from the following aspects: The growth inhibition rate of K562 cells and Peripheral Blood Mononuclear Cell (PBMC) was measured by MTT; Morphology of K562 cells was detected by wright’s stain; The ultrastructure changes of K562 cells were analyzed by transmission electron microscope; DNA fragmentation and the percentage of apoptotic cells were examined, respectively; benzidine staining and POX staining was detected. The activities of Caspase-8, Caspase-9 and Caspase-3 were analyzed by spectrophotometry; the expression of Bcl-2、Bax and Fas were detected using immunohistochemistry assay.
Results
1. TSN presented the striking proliferation inhibition, as well as apoptosis induction potency on SMMC-7721 and Hep3B cells in vitro in a time-and dose-dependent manner(P<0.05); the number of TSN-treated cells was significantly reduced accompanied by decreased adhesion ability,a fraction of cells was shrunk and round, without toxicity to IAR-20 and LO2 cells; Flow cytometry indicated the apoptosis rate of SMMC-7721 cells and Hep3B cells was 21.55% and 13.35%, respectively, the apoptosis of these cells induced by TSN could be partially blocked by z-VAD-fmk(P < 0.05); The activities of caspase 3, 8 and 9 in SMMC-7721 cells were markedly enhanced accompanied by decreased expression of Bcl-2 and increased expression of Bax as well as Fas, In Hep3B cells treated with 0.5μM TSN for 72 h, the activities of caspase 3 and 9 were markedly increased, but the activity of caspase 8 was not profoundly changed. Additionally, the expression of Bcl-2 was decreased accompanied by increased expression of Bax and no evident changes were found in the expression of Fas.
2. The experiment in H22 tumor-bearing BALB/C documented the TSN had obvious effects on tumor inhibition. Its inhibitory rate were 66.23%、87.01% in the BALB/C administrated TSN with low dose (0.173 mg/kg)、high dose (0.69 mg/kg) , respectively. The ultrastructure alterantions of tumor cells in BALB/C xenograft tumor treated with TSN exhibited apoptotic body appearance; HE staining of heart, liver, spleen, kidney, thymus as well as testis indicated no significant difference in the morphology of these tissues was noted between the control group and TSN group, but the number and area of thymus lobules in TSN group were decreased or even loss; The expression of Bcl-2 protein was down-regulated while expression of Bax and Fas protein was up-regulated in tumor tissue treated with TSN compared with the control groups.
3. TSN was a potent inhibitor of proliferation of K562 cells, without toxicity to PBMC. It is shown that the inhibitory efficacy of TSN in K562 cell depended on the reaction time and dosage; The morphological features of apoptosis were observed by light microscopy and transmission electron microscopy in cell shrinkage, even appearance of apoptosis body. After toosendanin treatment at 10 , 30 and 50 nmol/L for 72 h, the apoptosis rate of K562 cells was 9.66%, 26.06% and 52.70%, respectively(P<0.01); A typical ladder pattern was identified in DNA electrophoresis; benzidine staining and POX staining could not singnifcantly change compared with the control groups; The enzyme activity changes of Caspase-3、Caspase-8 and Caspase-9 were detected. Immunocytochemistry technique showed that toosendanin could significantly down-regulate Bcl-xl protein expression and up-regulate Bax and Fas proteins expression (P<0.05)
Conclusions
1. Results indicated TSN in vivo and in vitro could inhibit proliferation and induce apoptosis in hepatocellular carcinoma cells. the toosendanin-induced apoptosis involved the mitochondrial pathway.
2. TSN in vitro inhibit proliferation and induce apoptosis of K562 cells by mitochondrion and death-receptor dual pathways.but K562 cells can be induced in vitro to erythroid differentiation or granulocytic differentiation.
3. TSN in vitro on human normal liver cell line L02, normal rat hepatocytes IAR-20 and PBMCs were almost no toxic effects.
4. TSN could inhibit proliferation and induce apoptosis both in P53 positive SMMC-7721 cells and and P53 negative Hep3B、K562 cells, TSN can induce human cancer cell lines apoptosis via P53-independent mechanisms and play as a natural low toxicity anti-cancer drugs.
引文
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