Thrombospondin-1 (TSP-1) Expression and Angiogenesis in Lung Cancer

血管新生 (angiogenesis) 在腫瘤生長 及轉移上扮演重要角色。轉移過程中癌細胞必 需進入血管中，隨血流運行至標的器官，離開 血管並植入組織中，而癌細胞逐漸增生，形成 小腫瘤，再引發局部腫瘤之血管新生；轉移之 癌細胞可再經由相同之方式轉移至其它器 官。 血管新生是一種複雜之過程，包括內皮細 胞分解基底膜、內皮細胞侵入間質、內皮細胞 增生及移行、最後組成管狀之微血管構造。此 過程受到二種分子之調節，即是血管新生因子 及血管新生抑制因子。 血管新生因子包括 bFGF、 aFGF、 VEGF (endothelial cell growth factor)以及其它 之生長因子。 VEGF 之表現在子宮頸癌、乳癌 以及大腸癌之肝轉移被發現與血管新生之程 度呈正相關； VEGF 之單株抗體可抑制腫瘤在 裸鼠上的生長。相反的，血管新生抑制因子則 會抑制血管內皮細胞之增生、移行以及形成微 小血管，並且使內皮細胞維持在不活動之狀 態。血管新生抑制因子可抑制人類絨毛膜癌、 卵巢癌以及子宮內膜癌細胞株之肺部轉移，及 大腸癌細胞株之肝轉移。目前已知有15種自然 的血管新生抑制因子，其中Thrombospondin- 1 (TSP-1)以及angiostatin被認為對 血管內皮細胞有專一性。 TSP-1，一個 450 kD且對鈣敏感之三分子 之醣蛋白，是血小板中α granule 中之主要 成份。它可促進由 thrombin 引發之血小板聚 集。許多其它之細胞及組織亦可分泌TSP-1至 細胞外之基質中 (extracellular matrix)。 TSP-1可調節傷口癒合，蛋白脢活性，以及細 胞之沾連、移動及生長。許多研究顯示，TSP-1 對血管新生有抑制之作用，並且因此也抑制了 腫瘤形成及轉移之能力。TSP-1可抑制兔子眼 角膜之血管新生，也會抑制海棉模式中 granulation tissue 之血管新生。腫瘤細胞 之TSP-1表現量被報告與腫瘤之形成及轉移能 力呈負相關。Weinstat-Sashow DL 等人在裸 鼠之模式中証實將TSP-1之cDNA打入乳癌細胞 株中，可減少接種後腫瘤之大小，以及肺部轉 移之數目，並且使腫瘤中之微小血管密度減 少。Dameron KM 等人也報告腫瘤細胞內TSP-1 之表現受wild-type p53蛋白之調控。 TSP-1在人類肺癌組織中之表現，以及與 腫瘤引發之血管新生之關係仍少被報告。在此 計劃中，我們將用免疫組織化學染色法及實時 間定量反轉錄多聚合脢連鎖反應來評估肺癌 組織中TSP-1蛋白及 mRNA之表現；TSP-1之表 現亦將與腫瘤中之微小血管數做相關性之分 析。 結果顯示，肺癌組織中所表現之TSP-1 mRNA 之量較腫瘤旁正常組織中表現之TSP-1 mRNA 較少，約為腫瘤旁正常組織之0.69 倍。 此結果支持了TSP-1 可能為一血管新生抑制因 子之假說。肺腺癌之TSP-1 mRNA 表現量較鱗 狀上皮細胞癌之TSP-1 mRNA 表現量為多。 另外在免疫組織化學染色中，我們可觀察 到TSP-1 蛋白之表現主要是在肺癌組織之間質 (stromal tissue)中，而肺癌細胞本身則無 TSP-1 蛋白之表現。Angiogenesis play a important role for cancer growth and metastasis. Cancer cell have to enter the systemic circulation to metastasize to targer organs.The degree of angiogenesis induced by tumor was reported to correlate well to the advancement and systemic metastasis in breast cancer, prostate cancer, lung cancer and a variety of malignant tumors. Angiogenesis is a complicate process, which involve degradation of basement menbrane by endothelial cell, invasion of endothelial cell to the stroma, endothelial cell proliferation and migration, and finally organization of proligerated endhothelial cell into capillary structure. This process is under control of local activity of two kind of molecules: angiogenic factor and angiogenesis inhibitors. The angiogenic factors includes bFGF, aFGF, vascular endothelial growth factor (VEGF) and other growth factors. The postive relation of VEGF expression and the angiogenesis had been reported in cervical , liver metastasis of colon cancer and breast cancer. VEGF monoclonal antibody administration also show a dose- and time-dependent inhibition of tumor growth in nude mice. In contrast to angiogenic factors, the angiogenesis inhibitor inhibit the capacity of endothelial cell to proliferate, migration and form microvasculature, and maintain the endothelial cell in quiescent state. Angiogenesis inhibitor has been shown to inhibit the lung metastasis of human choriocarcinoma, obarian cancer and endoterial cancer cell lines, and hepatic metastasis of human colon cancer cell line. There are about 15 naturely occured angiogenesis inhibitors, and among them the thrombospondin -1 (TSP-1) and angiostatin show specificity on endothelial cell. TSP-1, a 450-kD calcium-sentive, disulfied-bonded trimeric glycoprotein, is a major constituent of platelet αgranules, which enhances the platelet aggregation activated by thrombin. TSP-1 is also secreted by a wide variety of tissue where it is incorporated into the extracellular matrix. TSP-1 is one of at least five members of a gene family, and has been reported to modulate the wound healing, protease acitvity, and celluar adherence, motility, and growth. A lot of investigations showed an inhibitory effect of TSP-1 on angiogenesis, which could potentially inhibit both tumorigenesis and metastasis. TSP-1 inhibited angiogenesis in the rat cornea implant and in the granulation tissue invading a sponge implant in vivo, as well as endothelial cell growth, imgration, and cord formation in vitro. Tumor cell TSP-1 production was reported to correlated inversely with the tumorigenecity and metastatic potential in human breast carcinoma, murine melanoma, and human lung carcinoma cell lines. Weinstat-Sashow DL also showed transfection of TSP-1 complementary DNA into breast carcinoma cell line resulted in a dose-dependent inhibition of primary tumor size, spontaneous lung metastasis and capillary density in primary tumor in nude mice model. Dameron KM et al showed TSP-1 producion by tumor cell was regulated by wildtype p53 protein. The expression of TSP-1 in human lung cancer specimen and the relation to the tumor-induced angiogenesis has limitedl been reported. In this study, we assessed the TSP-1 expression in lung cancer specimens by immunohistochemical staining and by real-time quantitative reverse transcription polymerase chain reaction (real- time RT-PCR) using Bactine as an internal control..The TSP-1 expression will be also correlated to the microvessel counts in the lung cacner specimens. The resullts showed that the cancer cell expressed fewer TSP-1 as compared with the surrounding normal tissue (cancer cell TSP-1 mRNA/ normal Tissue TSP-1 mRNA)= 2- 0.52=0.69 Because the angiogenesis increased in cancer tissue, this result support the hypothesis that TSP-1 play a negative regulator of the angiogenesis in lung cancer. The expression of TSP-1 in adenocarcinoma was, however, slightly higher than that in squamous cell carcinoma in this study 3 In immunohistochemical staining, we found that the TSP-1 protein expression was mainly located at the stromal tissue of lung cancer specimen, and the cancer cell itself did not express the TSP-1 protein