IGFBP-3透過調控TSP-1的表現以抑制腫瘤的血管新生

Abstract

類胰島素生長因子結合蛋白質-3 (IGFBP-3) 是個已知的抑癌基因。在人類卵巢類子宮內膜癌細胞株-OVTW59，具高度侵襲轉移能力的P4分株的IGFBP-3基因表現量明顯比原始的細胞株低 (Oncogene 2008; 27: 2137-47)。我們將帶有人類IGFBP-3基因的質體 (pKG3226-hIGFBP-3) 與空的質體轉殖到P4細胞株中，利用cDNA微陣列基因表達分析技術分析兩個轉殖細胞株的基因表現差異，我們發現，血小板反應蛋白質-1 (TSP - 1)，一個內源性的血管生成抑製蛋白，與IGFBP-3有類似的基因表達模式。為了分析IGFBP-3與TSP-1之間的關係，我們建立一個利用四環黴素誘導 IGFBP-3的表達質體pBIG2i-hIGFBP-3轉殖到P4細胞株中。經由四環黴素刺激兩個小時後，IGFBP-3的表現量提高四倍，同時TSP-1的表現量也跟著增加。在人類的臍靜脈內皮細胞 (Human umbilical vein endothelial cell, HUVECs) 的管狀結構形成實驗中發現，IGFBP-3的表現會抑制管狀結構的形成；加入拮抗TSP-1抗體後，HUVECs的管狀結構形成能力會恢復。在雞胚絨毛尿囊膜 (chick embryo chorioallantoic membrane, CAM)試驗中，增加IGFBP-3的表現會抑制尿囊膜毛細血管的形成。在動物實驗中，利用四環黴素刺激IGFBP-3表達後5天，腫瘤的生長明顯被抑制，腫瘤的被抑制生長狀態持續了約 10天。在腫瘤切片免疫染色上發現，在生長抑制期間，除了腫瘤內微血管的密度減少，同時細胞凋亡增加。這些研究結果顯示，IGFBP-3可以透過調控TSP-1的表現，以抑制腫瘤血管的生成，使得腫瘤的生長受到抑制。這些實驗結果暗示， IGFBP-3及TSP-1可做為卵巢類子宮內膜癌之標的治療。

Insulin-like growth factor binding protein 3 (IGFBP-3) is known as an antiproliferative and an invasion-suppressor protein. In human ovarian endometrioid carcinoma (OEC) OVTW59, highly invasive subline P4 showed lower IGFBP-3 gene expression than the original P0 cell line (Oncogene 2008; 27:2137-47). By cDNA microarray analysis of differential gene expression between IGFBP-3 re-expression in P4-pKG3226-hIGFBP-3 transfectant and the control line, thrombospondin-1 (TSP-1) was identified to be significantly co-expression with IGFBP-3. TSP-1 is a known endogenous inhibitor of angiogenesis. We further established a tetracycline-inducible IGFBP-3 expressing OEC cell line using pBIG2i plasmid. This cell line showed 4-fold increase in IGFBP-3 expression 2 hours after tetracycline treatment in associated with a paraellel increase in TSP-1. In human umbilical vein endothelial cell (HUVECs) tube formation assay, IGFBP-3 expression was associated with a decrease in capillary tube formation. This reaction was reversed after anti-TSP-1 treatment. By chick embryo chorioallantoic membrane (CAM) assay, IGFBP-3 expression showed a significant decrease in capillary formation. Heterotransplantation of IGFBP-3-transfectants showed significant decrease in tumor growth 5 days after IGFBP-3 induction. The dormancy state of tumor growth persisted for about 10 days. Xenograft tumors in this period showed a decrease in micro vessel density and an increase in tumor apoptosis. In conclusion, we have identified a novel association between IGFBP-3 and TSP-1. IGFBP-3 could acts through the regulation of TSP-1 to decrease angiogenesis formation, and resulted in the dormancy state of cancer growth. By impairing the processes of angiogenesis, IGFBP-3 could then play as a tumor suppressor gene in ovarian cancer. Both IGFBP-3 and TSP-1 may serve for the molecular targeted therapy in patients with ovarian endometroid cancer.