Zhang Hao, Ma Gang, Dong Ming, Zhao Meifen, Shen Xiaodong, Ma Zhigang, Guo Kejian
Department of Surgery, The First Affiliated Hospital of China Medical University, Shenyang, China.
Pancreas. 2006 Jan;32(1):101-9. doi: 10.1097/01.mpa.0000191644.94301.be.
Overexpression of epidermal growth factor (EGF) and EGF receptor has been associated with progression and invasive phenotype in pancreatic cancer. However, the underlying molecular mechanism by which EGF worked in pancreatic cancer cells has been poorly understood. In this study, we determined the effect of EGF on the invasiveness and the related regulatory mechanism in two pancreatic cancer cell lines NOR-P1 and KP4.
Invasion assay was performed to examine the invasiveness of tumor cells induced by EGF, and the expression of matrix metalloproteinase (MMP)-9, MMP-2, and plasminogen activator (uPA) was determined by reverse transcription-polymerase chain reaction and Western blot. Gelatin zymography was used to detect the activities of MMP-9 and MMP-2, and the nuclear factor-kappaB (NF-kappaB) binding activity was determined by electrophoretic mobility shift assay (EMSA).
EGF significantly increased the invasiveness of both cell lines but did not affect cell proliferation or adhesion. Increased invasiveness was associated with the induction of MMP-9 and uPA at both mRNA and protein levels. Furthermore, EGF stimulated NF-kappaB binding activity. Moreover, pretreatment of cells with NF-kappaB inhibitors, pyrrolidine dithiocarbamate or ibuprofen, markedly attenuated EGF-induced NF-kappaB activation. Subsequently, the EGF-induced MMP-9 and uPA expression and MMP-9 activity, as well as cell invasiveness, were also inhibited by these NF-kappaB inhibitors.
Our findings indicated that NF-kappaB-mediated MMP-9 and uPA induction was responsible for EGF-induced invasiveness in these pancreatic cancer cell lines and implicate that such anti-NF-kappaB therapy as the use of NF-kappaB inhibitors may contribute to the reduction of invasiveness of pancreatic cancer.
表皮生长因子（EGF）及其受体的过表达与胰腺癌的进展和侵袭表型相关。然而，EGF在胰腺癌细胞中发挥作用的潜在分子机制尚不清楚。在本研究中，我们确定了EGF对两种胰腺癌细胞系NOR-P1和KP4侵袭性的影响及其相关调控机制。
进行侵袭实验以检测EGF诱导的肿瘤细胞侵袭性，通过逆转录-聚合酶链反应和蛋白质印迹法测定基质金属蛋白酶（MMP）-9、MMP-2和纤溶酶原激活剂（uPA）的表达。用明胶酶谱法检测MMP-9和MMP-2的活性，通过电泳迁移率变动分析（EMSA）测定核因子-κB（NF-κB）的结合活性。
EGF显著增加了两种细胞系的侵袭性，但不影响细胞增殖或黏附。侵袭性增加与mRNA和蛋白质水平上MMP-9和uPA的诱导有关。此外，EGF刺激NF-κB结合活性。而且，用NF-κB抑制剂吡咯烷二硫代氨基甲酸盐或布洛芬预处理细胞，可显著减弱EGF诱导的NF-κB激活。随后，这些NF-κB抑制剂也抑制了EGF诱导的MMP-9和uPA表达、MMP-9活性以及细胞侵袭性。
我们的研究结果表明，NF-κB介导的MMP-9和uPA诱导是EGF诱导这些胰腺癌细胞系侵袭性的原因，这意味着使用NF-κB抑制剂等抗NF-κB治疗可能有助于降低胰腺癌的侵袭性。
