β1,4-N-acetylgalactosaminyltransferase-III在大腸癌細胞中扮演的角色

Abstract

醣基化作用(Glycosylation)在蛋白質的後轉譯修飾作用中扮演相當重要的角色。這些醣基化作用所產生的醣類結構經由改變細胞膜上蛋白質的結構而影響細胞的行為，包括訊息傳遞以及細胞和細胞間的黏附作用等。近年來對癌症的研究發現，蛋白質及脂質上的異常醣基化作用往往造成細胞的癌化，例如:形成T-antigen影響癌細胞的轉移、侵襲和生長等能力。這些醣類結構由單醣所構成，其改變往往藉由去醣基酵素(glycosidase)和醣轉移酵素(glycosyltransferase)的合作形成多種醣類結構。 最近才被發現的β1,4-N-acetylgalactosaminyltransferase-III (β4GalNAc-T3)是一種醣轉移酵素(glycosyltransferase)，它能使GalNAc結合在帶有GlcNAc的醣類上形成N,N’-diacetylgalactosediamine (GalNAcβ1-4GlcNAc)的結構。這種結構在O-linked及N-linked的醣類結構上都曾被發現。另外，之前的研究指出，β4GalNAc-T3的mRNA在胃以及大腸都有大量的表現，但是其功能還不十分了解。因此，在本篇研究中，我們利用HCT116大腸癌細胞株進行β4GalNAc-T3的基因轉殖，建立能表現β4GalNAc-T3的穩定細胞株，觀察β4GalNAc-T3的過量表現在大腸癌細胞中扮演的角色。 我們發現，β4GalNAc-T3的細胞株較早伸出pseudopodium，形成尖細紡錘狀。我們利用螢光染色檢測CD29, FAK [pY397], actin以及 FAK這些與細胞黏附分子有關的蛋白質在兩株細胞中的表現，發現β4GalNAc-T3造成細胞骨架的重新分布。另外，將兩株細胞分別植入SCID和NOD-SCID品系的老鼠，發現帶有β4GalNAc-T3基因的腫瘤在體積和重量上都比較大。此外我們看見有許多血管分布在腫瘤表面，為進一步瞭解β4GalNAc-T3是否能引起血管新生因子的釋放進而影響血管新生，利用組織免疫染色檢測CD31/PECAM-1分布情況並計算血管密度，發現並沒有顯著差異。同時，待兩株細胞株長滿後收集細胞培養液加入初級培養的人類臍靜脈內皮細胞並進行MTT細胞生長檢測。實驗結果發現，兩株細胞的培養液對於內皮細胞的生長也沒有明顯差異，這表示β4GalNAc-T3造成腫瘤生長較大可能不是藉由血管新生造成。 然而，兩株細胞的MTT生長檢測以及trypan blue染色顯示β4GalNAc-T3基因直接影響細胞生長的速率，促使細胞生長較mock細胞株快速。爲了解β4GalNAc-T3造成細胞生長快速的機制，利用西方免疫轉漬法，發現磷酸化的ERK在β4GalNAc-T3的穩定細胞株中表現量比mock上升；當以ERK上游MEK的抑制劑，PD98059處理細胞後，磷酸化的ERK在β4GalNAc-T3的細胞株中表現量下降，進一步利用MTT生長檢測觀察經PD98059處理後細胞的生長狀況，發現β4GalNAc-T3的細胞株其生長速率亦隨之下降。定量即時PCR顯示E-cadherin以及 β-catenin的mRNA在β4GalNAc-T3的細胞株表現量較mock高。這些結果暗示著β4GalNAc-T3可能藉由MEK/ERK MAP kinase這條路徑來調控癌細胞的生長快速；此外，E-cadherin以及 β-catenin的活化也可能參與β4GalNAc-T3調控細胞生長速率的機制。

Colorectal cancer (CRC) is one of the most common tumors and it is a major cause of cancer death worldwide. Colorectal carcinogenesis is a complex multi-step process involving progressive disruption of intestinal epithelial-cell proliferation, apoptosis, differentiation, and survival mechanism. In recent studies, malignant transformation often accompanies aberrant glycosylation changes of glycoproteins and glycolipids. These aberrant glycans can affect a variety of normal cellular functions, such as cell signaling and cell-cell adhesion, and thereby may affect tumor progression. The glycans, which are composed of various monosaccharides, are enzymatically synthesized by glycosyltransferases. β1,4-N-acetylgalactosaminyltransferase-Ⅲ (β4GalNAc-T3), which was recently cloned and identified, is a glycosyltransferase and highly expressed in the stomach, colon and testis. β4GalNAc-T3 can transfer GalNAc residues to GlcNAc and effectively synthesize, GalNAcβ1-4GlcNAc, at non-reducing termini of various acceptors. The N,N’-diacetylgalactosediamine structures (GalNAcβ1-4GlcNAc) are present on not only N-glycans but also O-glycans. In our study, we used HCT116 colorectal carcinoma cells transfected with β4GalNAc-T3 as a model to investigate the role of β4GalNAc-T3 in colon cancer cell growth in vitro and in vivo. The mock and β4GalNAc-T3 stable clones of HCT116 colorectal carcinoma cells were previously established in our lab. The morphological observation of mock and β4GalNAc-T3 stable clones by a phase-contrast microscope for 24 hours showed that β4GalNAc-T3 stably transfected cells exhibited fibroblastoid morphology with trailing pseudopodia. The immunocytochemical analysis of CD29, FAK [pY397], phalloidin and FAK, which are involved in cell adhesion complex, exhibited that CD29 and FAK protein levels did not change. However, the FAK [pY397] and actin protein levels were greater in β4GalNAc-T3 than in mock transfectants. These data suggest that β4GalNAc-T3 may interfere in the cytoskeleton dynamics. The mock and β4GalNAc-T3 xenograft in SCID and NOD-SCID mice showed that β4GalNAc-T3 enhanced the tumor growth in vivo. Since many blood vessels were located near the surface of excised tumors, the tumors were subjected to immunohistochemistry by CD31/PECAM-1 antibody and the microvessel density was identified. In addition, primary human umbilical vein endothelial cells (HUVECs) were subjected to MTT proliferation assay with conditioned media obtained from mock and β4GalNAc-T3 stable clones. The microvessel density in β4GalNAc-T3 xenograft was similar with those in mock xenograft. Simultaneously, HUVEC cells cultured in the conditioned medium obtained from mock and β4GalNAc-T3 stable clones did not show a significant change in the proliferation rate. These results suggested that β4GalNAc-T3 on enhanced tumor growth does not result from tumor angiogenesis. Nevertheless, the effect of β4GalNAc-T3 gene on HCT116 cell proliferation by using MTT assay and trypan blue exclusion assay displayed that β4GalNAc-T3 promotes cell growth in vitro. To elucidate the mechanism by which β4GalNAc-T3 increases cell proliferation, Western blotting was performed with anti-phospho-ERK1/2, anti-phospho-p38, and anti-phospho-JNK. The expression of phospho-ERK1/2 in β4GalNAc-T3 stable clones was increased as about two times as those in mock. This data indicated that overexpression of β4GalNAc-T3 results in upregulation of phosphorylation level of ERK1/2. When treating with MEK inhibitor, PD98059, the phospho-ERK1/2 was decreased. Simultaneously, the proliferation rate of HCT116/β4GalNAc-T3 stable clones was decreased. We also observed that mRNA expression of MMP-7, TIMP-1, and β-catenin was upregulated in β4GalNAc-T3 stable clones. These results suggest that β4GalNAc-T3 may promotes tumor cell proliferation via activation of MEK/ERK MAP kinase cascades and also be influenced by MMP-7, TIMP-1, and β-catenin.