丹参是一种著名的传统中药，富含丹酚酸和丹参酮。其中，丹参酮的潜在抗肿瘤作用近年来引起众多关注。本研究评价了主要的丹参酮及其衍生物对结肠癌细胞的细胞毒性。结果显示DHTS具有最强的抗结肠癌活性和显著的肿瘤特异性细胞毒性，其细胞毒性主要由于凋亡诱导而不是引起坏死。初步的构效关系分析提示丹参酮母环结构中的A环和B环增加的离域性有助于提高其对结肠癌细胞的细胞毒性，而非二维结构及较小的D环也是进行结构改造的可能方向。 / 基于以上发现，本研究进一步探讨了DHTS的体内外抗肿瘤活性及内在机制。本研究发现DHTS的促凋亡活性并不依赖于p53的表达，而依赖于caspase活性及线粒体介导的细胞质中氧自由基 ROS及钙离子的聚集。DHTS可引起浓度及时间依赖caspase-9/-3/-7的活化而并未显著引起caspase-8的活化，这一现象发生于同样以浓度及时间依赖方式进行的线粒体中cytochrome c及AIF转位之后。在DHTS诱导的结肠癌细胞凋亡中，cytochrome c及caspase介导的凋亡通路及AIF介导的凋亡通路均被激活并显示出两条通路之间的交叉调控。 / 此外，线粒体在DHTS的促凋亡活性中的作用在本研究中被深入探讨。本研究发现线粒体可能是DHTS的一个直接靶点, 而氧化磷酸化复合体III则更可能是其作用位点。DHTS可以引起迅速而明显的线粒体功能障碍，随之引起细胞质中大量的氧自由基及钙离子聚集，诱导凋亡的产生。 / 与体外结果一致，本研究证实了DHTS对免疫缺陷小鼠中的结肠癌移植廇也具有明显的抗肿瘤作用。与溶媒对照组比较，DHTS治疗组中移植廇的增长显著被减缓，在治疗终点时的廇体积与重量也显著被降低。TUNEL检测确认DHTS诱导移植廇中癌细胞的显著凋亡。免疫荧光检测也发现DHTS诱导caspase-3及caspase-7在移植廇中癌细胞的明显活化。 / 综上所述，本研究提供了丹参酮抗结肠癌活性的一些初步构效关系的信息，为提高丹参酮抗结肠癌活性的结构改造提供一定的参考。更重要的是，本研究证明了DHTS的体内外抗结肠癌活性并探讨了其作用机制及可能靶点，为DHTS作为新的应用于抗结肠癌药物或辅助治疗用药提供了临床前研究证据。 / Salvia miltiorrhiza Bunge, also known as Danshen, rich in phenolic acid and tanshinones, has been widely used to treat various kinds of diseases including heart diseases and hepatitis in China with minimal side effects. Among the tanshinones, tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone I are the major bioactive constituents in this herb. In this study, the anti-colon cancer potential of five tanshinones and six derivatives of tanshinone IIA were evaluated in several colon cancer cell lines. It was found that apoptosis but not necrosis contributed significantly to the cytotoxicity of the tanshinones. Dihydrotanshinone I (DHTS) was confirmed to be the most potent and selective anti-cancer compound among the tanshinones tested in this study. Preliminary SAR (structure activity relationship) of tanshinones reveals that the increase of delocalizability of A and B rings in the chemical structure of the tanshinones enhances their cytotoxicity on cancer cells, while compounds with a non-planar and small sized D ring region are better choices for anti-cancer effect. / The underlying mechanisms of the anti-colon cancer activity of DHTS were further studied. It was found that apoptosis induced by DHTS was p53 independent but caspase dependent, which was closely related to intracellular accumulation of ROS (reactive oxidant stress) and calcium mediated by mitochondria. A concentration- and time-dependent activation of caspase-9,-3,-7 but not caspase-8 by DHTS in HCT116 cells was detected after the translocation of cytochrome c and AIF (apoptosis inducing factor) from mitochondria. In this process, the crosstalk between the caspase-dependent and caspase-independent pathways was firstly shown in the apoptotic mechanism of DHTS. To this end, the release of cytochrome c happened first and the translocation of apoptosis inducing factor (AIF) was prevented by a pan caspase inhibitor. In the meantime, the release of cytochrome c and activation of caspase-9 and PARP (poly-ADP-ribose polymerase) cleavage were decreased after AIF knockdown. Especially, mitochondrion was suggested to be the direct target of DHTS and OXPHOS complex III but not OXPHOS complex I was probably the acting site of DHTS. / In accordance with the results obtained in vitro, the potential anti-colon cancer activity of DHTS was also observed in nude mice with xenograft tumors and the compound did not produce any observable systemic toxicity. DHTS efficiently delayed tumor growth by decreasing the tumor size and weight through the induction of apoptosis in cancer cells but not by inhibition of cell proliferation. In the same tissues, a distinct activation of caspase-3 and caspase-7 in tumor cells was also detected by immunofluorescence assay. / Collectively, the present study provides preliminary information about the SAR of the anti-colon cancer activity for tanshinones. It also confirms that DHTS is a promising compound for anti-cancer action both in vitro and in vivo. In addition, this study gives us a better understanding regarding the mechanisms of how DHTS induces apoptosis in cancer cells. All these findings could provide solid pre-clinical evidence to propel the development and application of DHTS and perhaps its derivatives as novel therapeutic or adjuvant agents for the treatment of colon cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Lin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 122-132). / Abstracts also in Chinese. / Wang, Lin.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1077718 |
| Date | January 2013 |
| Contributors | Wang, Lin , active 2014 (author.), Cho, C.-H. (Chi-Hin) (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences, (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource], electronic resource, remote, 1 online resource (iii, xx, 132 leaves) : illustrations (some color), computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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