Anti-metastasis effect of phytochemicals on colorectal cancer cells via heparanase inhibitory activity

肝素酶(Heparanase)為一種葡萄醣醛酸內切酶(endo-beta-D-glucuronidase)，具有切割基底細胞膜與組織硫酸肝素鍵結的能力，可以在原位瘤發生惡化時促進腫瘤細胞轉移，且造成腫瘤細胞周遭微環境變化，對於癌症發生的進程有極大的影響。本研究即利用體外細胞模式，以共培養技術，模擬癌症細胞遭受腫瘤微環境因子影響而發生癌症轉移的現象，並以此平台探討植化素(phytochemicals)之介入對目標蛋白(Heparanase)及下游蛋白Akt, Src, MMPs之作用。本研究第一部分，探討以不同條件PMA誘導THP1細胞分化為polarized M2巨噬細胞的效果，發現以320 nM PMA處理 24小時可表現6.6%的polarized M2巨噬細胞表面抗原，並將此細胞在不同時間點(15-240分鐘)誘導HCT116大腸癌細胞株，發現在誘導時間內皆可促使HCT116細胞發生轉移性，其中又以30分鐘及120分鐘共培養效果最佳。第二部分介入文獻中提出具有抑制癌細胞轉移功效的植化素，在測試其對HCT116細胞毒性及酵素活性之影響後，篩選出五種具有潛力之植化素，進入第三部分的西方點墨法分析。結果顯示，以Quercetin及Resveratrol兩者具有最好的抑制轉移效果。在肝素酶蛋白表現上，以後介入方式施予Quercetin，具有抑制HPA酵素蛋白表現的功效，且有濃度效應，而在p-Src與p-Akt方面，以前介入或後介入方式皆有抑制的效果，而Resveratrol在Heparanase蛋白表現上並無抑制作用，但其可影響Heparanase下游蛋白Akt與Src，使其磷酸化降低，而達到抑制轉移的現象。總括而言，Quercetin為七種植化素中擁有最佳抑制轉移的能力，可以在細胞模式中經由抑制誘發的HCT116肝素酶表現，達到抑制癌症轉移的效果。Heparanase is a type of endo-beta-D-glucuronidase which can cleave heparan sulfate (HS) chains both at the cell surface and in the extracellular matrix. The enzyme is secreted by metastatic tumor cells, destroys surrounding tissue, and alters the microenviroment of the cells. In this study, we used an in vitro cell model which is established by co-cultivation of cancer cells and the tumor microenvironment factors. Then, we investigated the effect of some phytochemicals on the metastasis of the cancer cells. The first part of this study used different concentration of PMA to induce differentiation of THP1 cells to polarized M2 macrophage, and found that 320 nM PMA increase 6.6% polarized M2 cell’s surface antibody. After that, we used the polarized M2 macrophage to induce HCT116 cell at different time points to establish the metastasis mode. We found that the induction occurred at 30 minute and 120 minute resulted in the highest degree of metastasis. In the second part of this study, we first investigated the effects of the some potential phytochemicals on the cell survival rate an