β-Nitrostyrene 衍生物抑制癌細胞引發之血小板活化與 凝集之機轉探討

Inhibition of tumor cell-induced platelet activation and aggregation by β-nitrostyrenes

魏千凱(Chien-Kai Wei)

指導教授 ： 吳志中

共同指導教授 ： 吳永昌(Yang-Chang Wu)

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2008年)

摘要

過去研究顯示癌細胞進行轉移的能力和其是否可引起血小板凝集具有很大關聯。我們希望從抗血小板藥物中開發可以用來抑制癌細胞轉移的抗癌藥物。在本實驗中我們先嘗試以一具有高度轉移能力的乳癌細胞株MDA-MB-231來誘發血小板凝集。我們發現MDA-MB-231在血漿的幫助下具有引起血小板凝集的能力。在反應前加入thrombin抑制劑hirudin與tissue factor阻斷性抗體均可完全抑制MDA-MB-231引發之血小板凝集。所以MDA-MB-231是透過tissue factor活化凝血因子，促進thrombin產生，進而使血小板凝集。接下來我們以兩個新發現的抗血小板藥物，酪氨酸酶抑制劑3,4-methylene-dioxy-??- nitrostyrene (MNS) 和4-O-benzoyl-3-methoxyl-??- nitrostyrene (BMNS) 來研究它們對MDA-MB-231引起的血小板凝集是否有抑制作用。我們證明MNS與BMNS對MDA-MB-231的促血小板凝集具有濃度相關的抑制能力，其IC50分別為43.7 ?嵱與14.4 ?嵱。我們進一步以流式細胞儀證明MNS與BMNS也可抑制MDA-MB-231促血小板釋出P-selectin，並且抑制血小板附著到MDA-MB-231。此外，MNS與BMNS也會抑制MDA-MB-231促血小板釋出細胞生長因子platelet-derived growth factor (PDGF) 。我們的實驗結果顯示MNS與BMNS具有開發為抗癌細胞轉移藥物的潛力。在藥物作用機轉方面，我們以錐藍排除法 (trypan blue exclusion) 分析MNS與BMNS對MDA-MB-231的細胞毒性。結果顯示，在1小時的藥物處理下，並不會造成明顯的癌細胞死亡。以凝血測試發現，MNS與BMNS並不會影響MDA-MB-231的促凝血活化能力。因此這兩種藥物的抑制作用主要來自於其抗血小板能力。

關鍵字

β-Nitrostyrene ； MDA-MB-231 ； 血小板

並列摘要

Many types of tumor cells have the ability to aggregate blood platelets and, tumor cell-induced platelet aggregation (TCIPA) has been shown to play an important role in cancer metastasis. Therefore, it is possible that blockade of tumor cell-platelet interactions may help to inhibit tumor progression. In the present study, we have tested two novel inhibitors of tyrosine kinases: 3,4-methylene-dioxy-??-nitrostyrene (MNS) and 4-O-benzoyl-3-methoxyl-??- nitrostyrene (BMNS) for their inhibitory effect on TCIPA caused by MDA-MB-231 cells, which are highly metastatic human breast cancer cells. We found that MDA-MB-231 cells caused platelet aggregation in the presence of plasma. Both the blocking antibody to tissue factor (TF) and the thrombin inhibitor hirudin abolished MDA-MB-231 cell-induced platelet aggregation, indicating the involvement of TF-dependent coagulation activation and thrombin generation. We next examined the effect of MNS and BMNS on TCIPA. MNS and BMNS concentration-dependently inhibited TCIPA with IC50 values of 43.7 μM and 14.4 μM, respectively. Furthermore, MNS and BMNS also inhibited tumor cell-induced P-selectin expression in platelets and platelet adhesion to MDA-MB-231 as evidenced by flow cytometry. Furthermore, MNS and BMNS also inhibited tumor cell-induced PDGF release from platelets. Trypan blue exclusion assay confirmed that the inhibitory effects of these two agents were not resulted from their cytotoxicity. MNS and BMNS also did not affect the procoagulant activity of MDA-MB-231 cells. Therefore, in the condition used in our experiments, MNS and BMNS exert inhibition of TCIPA mainly through their antiplatelet activity.

並列關鍵字

β-Nitrostyrene ； MDA-MB-231 ； platelet

參考文獻

Abecassis, J, Beretz, A, Millon-Collard, R, Fricker, JP, Eber, M, Cazenave, JP (1987) In vitro interactions between human breast cancer cells MCF-7 and human blood platelets. Thromb Res 47(6): 693-698.

Alonso-Escolano, D, Strongin, AY, Chung, AW, Deryugina, EI, Radomski, MW (2004) Membrane type-1 matrix metalloproteinase stimulates tumour cell-induced platelet aggregation: role of receptor glycoproteins. Br J Pharmacol 141(2): 241-252.

Amirkhosravi, A, Amaya, M, Siddiqui, F, Biggerstaff, JP, Meyer, TV, Francis, JL (1999) Blockade of GpIIb/IIIa inhibits the release of vascular endothelial growth factor (VEGF) from tumor cell-activated platelets and experimental metastasis. Platelets 10(5): 285-292.

Andersen, H, Greenberg, DL, Fujikawa, K, Xu, W, Chung, DW, Davie, EW (1999) Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity. Proc Natl Acad Sci U S A 96(20): 11189-11193.

Bando, H, Yamashita, T, Tsubura, E (1984) Effects of antiplatelet agents on pulmonary metastases. Gann 75(3): 284-291.