从血小板-肿瘤细胞相互作用探讨丹酚酸B对卵巢癌增殖的抑制作用及其作用机制

Platelets have a well-studied role in coagulation and in thromboembolism. The latter was recognized to be a feature of occult cancer more than 100 years ago. It has been increasingly appreciated that, as a component of the tumor microenvironment, platelets also have important functions in the mechanisms involved in carcinogenesis, tumor growth, tumor angiogenesis, tumor-related host organ inflammation and immune responses, and tumor metastasis, and in the modulation of tumor therapy. Therapeutic alteration of platelet numbers and function has increasingly gained attention for cancer prevention, survival prolongation and possibly for therapy. ..As one of the most commonly used traditional Chinese medicines promoting blood circulation to remove blood stasis, Salvia miltiorrhiza (Danshen, DAN) can inhibit activation and aggregation of platelets. In addition, this Chinese Medicine was also reported significant inhibitive effects on the proliferation of cancer cells. Furthermore, it was demonstrated that the anti-proliferative properties of Salvia miltiorrhiza are associated with its cytotoxic effects. Based on the interaction between platelets and cancer cells, our previous studies found that incubation of platelets with SKOV3 cells, a kind of ovarian cancer cell line, increase cancer cell proliferation, and this proliferative effect of platelets were reduced by DAN. Moreover, the proliferation of SKOV3 cells was also simulated by supernatants of platelets activated by thrombin, and DAN blocked significant cell growth induced by platelets. It was also showed the same effects on proliferation of SKOV3 cell when these tumor cells were incubated with platelets. Furthermore, the effects of several constituents from DAN on the inhibition of platelet-tumor cell interaction were tested. It was found that salvianolic acid B (SAB) also showed inhibitory effects on platelet-tumor cell interaction. Upon these evidences, we here hypothesize that SAB shows significant anti-tumor properties, and the mechanisms of action may be associated with suppression of interaction between platelets and ovarian cancer cells. The specific aim is investigation of the inhibitory effects of SAB on the interaction between platelets and cancer cells, and elucidation of the underlying mechanisms of this interaction. To test these hypotheses, we will: (1) evaluate the effects of SAB on the proliferation of ovarian cancer cells induced by platelets in vitro and also in vivo; (2) analyze the mechanisms of inhibitory activities of SAB on interaction between platelets and ovarian cancer cells. These investigations will provide new knowledge on anti-tumor properties of traditional Chinese medicines promoting blood circulation to remove blood stasis.

丹参具有抗肿瘤增殖活性，其机制研究主要围绕细胞毒作用展开，但这不能完全体现其活血化瘀特点。鉴于血小板异常在肿瘤血瘀证中的作用，前期从血小板与卵巢癌细胞相互作用入手考察了丹参的抗肿瘤增殖作用。结果表明，丹参提取物可抑制血小板诱导的卵巢癌细胞增殖。进一步研究发现，其活性成分丹酚酸B也有此活性，而且不仅可抑制血小板颗粒直接诱导的细胞增殖，还可干扰血小板通过分泌作用诱导的细胞增殖，其机制分别与下调NF-κB和TGF-β1信号通路活性有关。据此，本研究将在“活血化瘀”理论指导下，从血小板与癌细胞相互作用入手，探讨丹酚酸B的抗肿瘤作用及其机制。首先，建立多种血小板-癌细胞相互作用的动物和细胞模型，从二者的直接和间接作用入手考察丹酚酸B的体、内外抗卵巢癌作用；同时，我们还将从TGF-β1、NF-κB信号转导出发研究其分子机制，为探索丹参活血化瘀功效及其抗癌作用间的内在联系、阐明其作用特点提供证据。

丹参具有抗肿瘤增殖和抗肿瘤转移活性。现有研究表明其抗癌作用主要体现在对癌细胞的直接杀灭上。然而仅从细胞毒作用探讨丹参对癌细胞增殖的机制不能体现其化散瘀滞的作用特点。前期研究证实丹参提取物不仅可抑制血小板与癌细胞的直接作用，还可干扰它们间的间接作用，本研究在此基础上探讨丹参的活性成分丹酚酸B抑制癌细胞增殖的作用及机制。 .首先，我们考察了血小板对肿瘤细胞的影响，并观察了丹酚酸B的作用。结果发现，一方面，血小板上清液能够促进卵巢癌ID8细胞增殖，丹酚酸B可显著抑制ID8细胞的增殖，其作用机制与干扰PI3K/Akt信号通路有关。另一方面，血小板颗粒也能促进小鼠卵巢癌ID8细胞的增殖，且丹酚酸B可显著抑制这种由血小板颗粒引起的细胞增殖。丹酚酸B的这种对卵巢癌ID8细胞的抑制作用可能是通过下调NF-κB信号通路的p-IκBα/IκBα、p-p65/p65的表达有关。另外，本研究还发现血小板颗粒能显著促进肿瘤细胞迁移、侵袭与黏附等细胞的转移能力，丹酚酸B可抑制血小板诱导体系下肿瘤细胞的迁移、侵袭与黏附能力，其抗转移作用的机制与其EMT逆转作用有关。.其次，我们还考察了肿瘤细胞对血小板活性的影响，同时观察了丹酚酸B的作用。结果发现，肿瘤细胞培养上清可显著促进血小板的分泌、聚集和粘附功能，丹酚酸B对上述作用则表现出明显的抑制活性，其机制可能与其抑制血小板中PI3K/Akt信号通路的转导相关。另外，本研究还发现，乳腺癌细胞分泌的外泌体可显著抑制血小板的聚集，而丹酚酸B则可显著刺激乳腺癌细胞分泌外泌体，其机制可能与Rab27A 和 Rab27B 等调控外泌体释放的 蛋白及其效应蛋白Slp4、Slac2b的活性有关。.本研究从血小板和肿瘤细胞的相互作用的角度，对丹参重要活性成分丹酚酸B的抗肿瘤作用及其机制开展了系统研究，并对其可能的作用机制进行了初步探索，研究结果将对阐明丹参在治疗肿瘤血瘀证中的作用特点提供数据支持，也可进一步丰富人们对“肿瘤血瘀证” 、“活血化瘀”的认识，为活血化瘀药的临床使用提供实验室支持。