【作者】 王雅姝；

【导师】 邓林红；

【作者基本信息】 重庆大学 ， 生物医学工程， 2011， 博士

【摘要】 肝细胞性肝癌(hepatocellular carcinoma,HCC)是当今世界常见的恶性肿瘤之一,其死亡率占世界肿瘤中的第3位,每年全球大约有550,000个新增肝癌病例,超过所有恶性肿瘤的5%,但是目前临床上的治疗结果仍然不容乐观。因此探寻更有效的治疗方法是目前迫在眉睫之事,而传统中药则因其固有的优点无疑是研究肝癌治疗新药物的重要资源。近年来就发现传统中药中的土贝母是一种有效的抗肿瘤药物。土贝母Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae)为葫芦科假贝母属植物,很早就在中国被广泛用于炎症、蛇毒等的治疗,1765年出版的《本草纲目拾遗》中已有记载。土贝母被发现具有较强的抗癌作用后更是受到广泛的关注,并从中成功提取出了有效成分,土贝母苷甲。土贝母苷甲是一种三萜皂苷,已经有实验证明土贝母苷甲可以抑制体外培养的多种人类癌细胞的生长,包括人早幼粒白血病细胞株(HL-60)、子宫颈癌细胞株(HeLa cells)和鼻咽癌细胞株(CNE-2Z)等癌细胞。这些体外研究结果都提示土贝母苷甲有可能是一种有效的抗癌药物成分。然而土贝母苷甲对肝癌的抗肿瘤活性尚未见报道,而且动物模型的药代动力学显示,土贝母苷甲在肝脏中的含量最高,所以土贝母苷甲很有可能对肝癌的治疗效果更佳。因此,有必要研究土贝母苷甲对肝癌的抗肿瘤活性及其相关机制。本课题主要针对体外培养的人肝癌细胞株HepG2细胞,并以正常肝细胞株L-02细胞作为对照,研究了土贝母苷甲对肝癌细胞生长的抑制作用。同时,鉴于肿瘤的发生发展除了因为细胞增殖和分化异常外,细胞凋亡异常也是关键的机制,进一步研究了土贝母苷甲诱导HepG2细胞凋亡的分子生物学机制。另外,本课题还研究了土贝母苷甲对肝癌细胞的迁移、侵袭、细胞骨架刚度等生物力学行为或性质的影响,并对相应的作用机制进行了初步探讨。这些研究为更全面深入地认识土贝母苷甲作用于肝癌细胞,如HepG2细胞的过程和机制奠定了基础,为进一步研究土贝母苷甲用于临床治疗肝癌提供了一定的理论依据。本课题研究取得的主要结果如下:①土贝母苷甲抑制体外培养的肝细胞的生长:采用MTT法试验的结果表明,土贝母苷甲对体外培养的人肝癌细胞(HepG2、HCCLM3)和正常肝细胞(L-02、QSG-7701)的生长均有抑制作用,并呈明显的时间依赖性和剂量依赖性。重要的是,肝癌细胞和正常肝细胞对土贝母苷甲的抑制作用表现出不同的响应,尤其是在10-30μM的浓度范围内,肝癌细胞对于土贝母苷甲作用的敏感性显著地高于正常肝细胞。流式细胞术检测HepG2细胞的细胞周期结果表明土贝母苷甲对肝癌细胞增殖的抑制可能与药物将细胞阻滞于G2/M期有关。②土贝母苷甲诱导肝癌细胞的凋亡:采用相差细胞显微术和Hoechst33258染色剂标记细胞核的实验发现,经过不同浓度的土贝母苷甲处理后,HepG2细胞出现形态皱缩、变圆,细胞核染色质浓缩、断裂等典型的凋亡形态学改变;采用Tubulin-Tracker荧光探针标记的细胞微管骨架也由不加土贝母时明亮的红色荧光、从细胞中心发射状排列分布到出现细胞周围荧光强度减弱、无定形排列、细胞膜易位、细胞形态变圆等显示细胞微管骨架解聚的现象。流式细胞术检测Annexin V/PI双染色法标记细胞的检测结果显示,随着土贝母苷甲浓度的增加,HepG细胞和L-02细胞中早期凋亡和晚期凋亡的细胞数量都相应增加,但是在较低浓度时(e.g.≤15μM)时,早期凋亡的细胞比例与晚期凋亡的细胞比例差别并不明显,当浓度较高(e.g. =30μM)的时候,早期凋亡的细胞比例远远超过晚期凋亡的细胞比例,暗示土贝母苷甲在较高浓度的时候可能更倾向于促进细胞的早期凋亡。另一方面,在同等浓度下,土贝母苷甲对HepG2细胞凋亡的诱导作用要大于对L-02细胞的作用,这与其对细胞增殖的抑制作用结果相一致。采用流式细胞术和Western blotting等的实验结果表明,HepG2细胞在经过土贝母苷甲作用后,其线粒体膜电位下降,细胞色素c从线粒体中释放到细胞浆中,caspase-3和-9的活性增高,抗凋亡蛋白Bcl-2表达降低,而促凋亡蛋白Bax的表达升高,以及Bax和Bcl-2表达的相对比率不断增高等。这些结果说明了线粒体途径参与了土贝母苷甲诱导的HepG2细胞凋亡过程。③土贝母苷甲抑制肝癌细胞迁移与侵袭:肿瘤细胞的转移和侵袭是恶性肿瘤的主要生物学特征,也是直接影响肝癌患者治疗效果和预后的重要原因。本课题的研究显示,土贝母苷甲可以有效抑制HepG2细胞的迁移与侵袭。采用对细胞F-actin骨架的荧光染色和共聚焦显微镜观察显示,经土贝母苷甲作用后的HepG2细胞内F-actin骨架有明显的破坏迹象。明胶酶谱法检测结果则显示经土贝母苷甲作用后的HepG2细胞内的基质金属蛋白酶MMP-2和MMP-9的表达降低。这些结果说明土贝母苷甲可能通过破坏细胞微丝的结构和抑制MMP-2、MMP-9的表达来抑制肝癌细胞的迁移与侵袭。④土贝母苷甲改变肝癌细胞的生物力学性质:根据土贝母苷甲对肝癌细胞F-actin骨架结构的作用,我们推测土贝母苷甲对细胞刚度等生物力学性质也有相应的影响。为了验证这一点,我们采用光学磁微粒扭转系统(OMTC)测量了细胞刚度及其受土贝母苷甲的影响。结果显示,土贝母苷甲确实降低了细胞的刚度,和细胞与加载频率间幂率关系(G～fα)的幂值。此外,HepG2细胞较L-02细胞的本底刚度低,但对土贝母苷甲作用的反应相应更快。这些结果首次揭示了土贝母苷甲对HepG2细胞的F-actin骨架生物力学性质的影响,而细胞骨架生物力学性质对细胞变形、迁移、侵袭、分化等许多重要的生物学行为都起着决定性的作用。综上所述,土贝母苷甲对肝癌(HepG2)细胞生长确实存在显著的抑制作用,而对正常肝细胞(L-02)生长的抑制作用则相对较弱。土贝母苷甲对肝癌(HepG2)细胞生长的抑制作用主要通过由微管和线粒体介导的细胞凋亡机制实现。但另一方面,土贝母苷甲对微丝骨架结构也有破坏作用,从而影响肝癌细胞的刚度和动力学特性,以及相应的肝癌细胞迁移与侵袭能力。所以,土贝母苷甲不仅能抑制肝癌细胞的增殖生长,同是也能抑制肝癌细胞的转移和侵袭。这些研究结果提示土贝母苷甲作为治疗肝癌的中药成分具有一定的潜在价值。更多还原

【Abstract】 Hepatoma is one of the most prevalent malignant tumor types and the third leading cause of cancer-related death worldwide. Each year, approximately 550,000 new cases of hepatoma are reported worldwide, representing more than 5% of all human cancers. Despite the development and use of multimodality therapies including chemotherapy, the clinical outcome of hepatoma treatment remains unsatisfactory, with usually less than 7% of the 5-year overall survival rate. Thus, it is imminent to find more effective methods and/or agents for hepatoma treatment. Traditional Chinese medicinal herbs, owing to its intrinsic advantages, may provide a great source for such search.Indeed, one herb that may have great potential is tubeimoside (TBMS), or the tuber of Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae). In traditional Chinese medicine TBMS has long been widely used for treatment of illness such as inflammation and snake venoms, and was listed in the Supplement to the Compendium of Materia Medica published in early 1765. In the 1980’s TBMS was first reported to show potent anti-tumor activity. Such anti-tumor activity in part motivated the successful isolation of tubeimoside I (TBMS I), a triterpenoid saponin. And subsequent studies confirmed that TBMS I can indeed inhibit growth of cultured cancer cells of several human cancer cell lines including human promyelocytic leukemia (HL-60), nasopharyngeal carcinoma CNE-2Z cell line (CNE-2Z) and HeLa cells. These studies appear to suggest that TBMS I may be a potential candidate as a novel anti-tumor drug, However, TBMS I so far has not been well studied for its anti-tumor activity against hepatoma, even though that TBMS I is known to preferentially distribute in the liver during in vivo metabolism, and thus might better target liver cancer, or hepatoma.Therefore, we evaluated TBMS I for its cytotoxicity to cultured human hepatoma cells or normal liver cells, from HepG2 or L-02 cell lines, respectively. Because the genesis and progression of cancer are known to involve not only abnormal proliferation and differentiation, but also abnormal apoptosis of the cells, we thus investigated apoptosis-associated molecular events as potential mechanisms responsible for the cytotoxic effects of TBMS I on HepG2 cells. Furthermore, we evaluated the effects of TBMS I on migration, invasion, F-actin structuer, and cytoskeletal stiffness of the cultured HepG2 cells. All together, this study may serve as a foundation for more comprehensive understanding of the pharmacology of TBMS I with HepG2 cells, and provide a scientific basis for further development of TBMS I as a novel drug for treating hepatoma in clinical practice. The main findings of this study are as follows:①TBMS I inhibited growth of cultured cells. The results from MTT assay demonstrated that TBMS I inhibited growth of HepG2 cells, HCCLM3 cells, L-02 cells and QSG-7701 cells in a typical concentration- and time-dependent manner. However, as compared to the normal human liver cells, hepatoma cells appeared to be more sensitive to TBMS I, particularly at the dose range of 10-30μM. Cell cycle analysis by flow cytometry demonstrated that exposure to TBMS I inhibited HepG2 cell proliferation via G2/M phase arrest in a dose-dependent manner.②TBMS I induced apoptosis in HepG2 cells. When HepG2 cells were examined for cell and nucleus morphology by phase contrast microscopy and Hoechst 33258 staining, it was found that after exposure to TBMS I induced cell shrinkage and even detachment, nuclear condensation and fragmentation. The cytoplasmic microtubules labeled with Tubulin-Tracker fluorescent probe also changed from bright fluorescence and appeared radiating from center to periphery of the cells without TBMS I to an apparent decrease in the fluorescence intensity, membrane translocation, and amorphous appearance, all suggesting tubulin depolymerization. Analysis of the cells double stained with Annexin V/PI by flow cytometry demonstrated that the percentage of the early apoptotic cells induced by TBMS I exposure seemed indifferent from that of the late ones when TBMS I concentration was low (e.g.≤15μM), but at high concentrations of TBMS I (e.g. =30μM), the exposure induced much greater percentage of the early apoptotic cells as compared to the late apoptotic cells. This suggests that exposure to TBMS I at high concentrations may predominately promote early apoptosis in HepG2 cells. TBMS I induced a similar trend of apoptotic process in L-02 cells, but with less total apoptotic cells, which was consistent with the results of proliferation inhibition.The mitochondrial apoptotic pathway has been described as an important signaling pathway of apoptosis. In the present study, we examined whether this pathway was involved in the TBMS I-induced apoptosis. Results obtained from flow cytometry and Western blotting demonstrated that exposure of HepG2 cells to TBMS I resulted in loss of mitochondrial membrane integrity, release of cytochrome c from mitochondria to the cytoplasm, marked activation of caspase-9, and -3, decrease in Bcl-2 expression and increase in Bax expression that led to increase of Bax/Bcl-1 ratio. All these results show that mitochondrial apoptotic pathways participate in TBMS I-induced apoptosis. ③TBMS I effectively inhibited migration and invasion of HepG2 cells. Cell migration and invasion are major biological behaviors that characterize tumore malignancy, and important determinanats of therapeutic efficacy and prognosis in clinical treatment of hepatoma patients. In this study, we used Transwell assay to evaluate the effect of TBMS I on the migration and invasion of HepG2 cells. The results showed that as the cells were treated with increasing concentration of TBMS I, both the migration and invasion of HepG2 cells were increasingly inhibited. In parallel, the cells treated with TBMS I show destruction and desolution of the F-actin cytoskeleton labeled with FITC-Phalloidin, and also decreased expression of matrix metalloproteinase (MMP)-2 and -9 as measured by Gelatin zymography. These results suggest that F-actin cytoskeleton and MMPs signal pathways may be alternative targets of TBMS I through which TBMS I may regulate cytoskeleton dynamics and cell-matrix interactions, then inhibit migration and invasion of the cells.④TBMS I affected biomechanical properties of HepG2 cells. Since TBMS I induced destruction and desolution of F-actin cytoskeleton, we proposed that TBMS I may affect the biomechanical properties of the HepG2 cells. To test this idea, we measured the stiffness of HepG2 cells before and after exposure to TBMS I using optical magnetic twisting cytometry (OMTC). The results showed that TBMS I indeed affected the stiffness of HepG2 cells. Exposure of the HepG2 cells to increasing concentration of TBMS I resulted in decreases in both cell stiffness (G’) and the value of exponent (α) for the power-law between the cell stiffness and the loading frequency (G~fα). In addition, while the baseline stiffness of HepG2 cells was lower than that of L-02 cells, the response of HepG2 cells to TBMS I was faster than than of L-02 cells. This was the first time that biomechanical properties of HepG2 cells were studied with regards TBMS I.Taken together, we conclude that TBMS I was a potent agent to suppress the growth of hepatoma cells in vitro. And the growth inhibition was in large part mediated via apoptosis-associated mitochondrial dysfunction and microtubule pathways. On the other hand, TBMS I also showed destruction and desolution of the F-actin cytoskeleton, then affected the stiffness and further inhibited migration and invasion of HepG2 cells. So TBMS I not only inhibited the growth of hepatoma cells but also inhibited the migration and invasion, together with its preferential distribution in the liver and its origin of natural medicinal plant, suggest that TBMS I may be a preferred drug candidate for treating liver cancer.更多还原