Sunman Jeffrey A, Foster Michael S, Folse Stacey L, May Sheldon W, Matesic Diane F
Department of Pharmaceutical Sciences, Southern School of Pharmacy, Mercer University, Atlanta, Georgia 30341, USA.
Mol Carcinog. 2004 Dec;41(4):231-46. doi: 10.1002/mc.20060.
Recent studies have shown that the proliferation of some tumor cells is dependent on autocrine growth loops that require amidated autocrine growth factors. Peptidylglycine alpha-monooxygenase (PAM) is required for amidation of these growth factors and, therefore, this enzyme is an attractive target for anti-tumor compounds. 4-Phenyl-3-butenoic acid (PBA) is an irreversible turnover-dependent inhibitor of PAM in vitro and has been shown to decrease lung cancer cell proliferation by inhibiting the synthesis of amidated growth factors. We show here that PBA (0.1 mg/mL) inhibits the growth of Ras-transformed epithelial cells (WB-Ras) but has little effect on the proliferation of normal epithelial cells (WB-Neo). The methyl ester derivative of PBA (PBA-Me) at 10-fold lower concentration also exhibits a selective inhibition of Ras-transformed cell growth compared to normal epithelial cell growth. In addition, PBA produces a significant upregulation of gap junctional communication between WB-Ras cells following 2-5 day treatments, with a corresponding increase in the degree of connexin 43 phosphorylation and an increase in the number of connexin 43-containing plasma membrane gap junction plaques. Western blot analyses indicate no effect of PBA on the proportion of p21 Ras in the membrane versus cytosolic fractions or on p44/42 MAP kinase phosphorylation. Furthermore, the cell morphology of PBA-treated WB-Ras cells is altered, so as to more closely resemble that of non-transformed WB-Neo cells. PAM activity was assayed in both WB-Ras and WB-Neo cells, and we demonstrate that PBA at long treatment times (4 days) inhibits PAM activity in both cell types at concentrations that produce selective growth inhibition of WB-Ras cells. Shorter PBA treatment times (24 h), however, inhibit PAM activity in WB-Ras but not WB-Neo cells, an effect that was mimicked by PBA-Me. Taken together, these results clearly demonstrate that PBA returns Ras-transformed cells to a more normal phenotype, a finding consistent with the known increased dominance of the Ras signaling pathway in transformed epithelial cells.
最近的研究表明，一些肿瘤细胞的增殖依赖于自分泌生长环，这些生长环需要酰胺化的自分泌生长因子。肽基甘氨酸α-单加氧酶（PAM）是这些生长因子酰胺化所必需的，因此，这种酶是抗肿瘤化合物的一个有吸引力的靶点。4-苯基-3-丁烯酸（PBA）在体外是一种不可逆的、依赖周转的PAM抑制剂，并且已被证明通过抑制酰胺化生长因子的合成来降低肺癌细胞的增殖。我们在此表明，PBA（0.1mg/mL）抑制Ras转化的上皮细胞（WB-Ras）的生长，但对正常上皮细胞（WB-Neo）的增殖影响很小。与正常上皮细胞生长相比，PBA的甲酯衍生物（PBA-Me）在浓度低10倍时也表现出对Ras转化细胞生长的选择性抑制。此外，PBA在处理2-5天后使WB-Ras细胞之间的间隙连接通讯显著上调，同时连接蛋白43的磷酸化程度相应增加，且含连接蛋白43的质膜间隙连接斑块数量增加。蛋白质印迹分析表明，PBA对膜组分与胞质组分中p21 Ras的比例或对p44/42丝裂原活化蛋白激酶的磷酸化没有影响。此外，经PBA处理的WB-Ras细胞的形态发生改变，从而更类似于未转化的WB-Neo细胞。在WB-Ras和WB-Neo细胞中均检测了PAM活性，并且我们证明，长时间处理（4天）时，PBA在产生对WB-Ras细胞选择性生长抑制的浓度下，抑制两种细胞类型中的PAM活性。然而，较短的PBA处理时间（24小时）抑制WB-Ras细胞中的PAM活性，但不抑制WB-Neo细胞中的PAM活性，PBA-Me也模拟了这种效应。综上所述，这些结果清楚地表明，PBA使Ras转化细胞恢复到更正常的表型，这一发现与已知的Ras信号通路在转化上皮细胞中优势增加一致。
