Meneses Ana Maria, Medina Rodolfo A, Kato Sumie, Pinto Mauricio, Jaque Maria Paz, Lizama Isabel, García María de los Angeles, Nualart Francisco, Owen Gareth I
Laboratorio de Biología Celular y Molecular, MIFAB, Universidad Nacional Andrés Bello, Santiago, Chile.
J Cell Physiol. 2008 Jan;214(1):110-6. doi: 10.1002/jcp.21166.
Increased glucose uptake as a principal energy source is a requirement for the continued survival of tumour cells. Facilitative glucose transporter-1 (GLUT1) and -3 (GLUT3) have been previously shown to be present and regulated in breast cancer cells and are associated with poor patient prognosis. In cancer cells, the cAMP secondary messenger pathway is known to potentiate described glucose transporter activators and regulate cell fate. However, no regulation of the glucose transporters in breast cancer cells by cAMP has previously been examined. Herein, we determined in the well-characterized breast cancer cell line ZR-75, if the cAMP analogue 8-br-cAMP was capable of regulating GLUT1 and GLUT3 expression and thus glucose uptake. We demonstrated that 8-br-cAMP transiently up-regulates GLUT3 mRNA levels. The use of actinomycin-D and the cloning of 1,200 bp upstream of the human GLUT3 promoter demonstrated that this regulation was transcriptional. Immunocytochemistry and Western blotting confirmed that the increase in mRNA was reflected by an increase in protein levels. No notable regulation of GLUT1 in the presence of 8-br-cAMP was detected. Finally, we determined using the non-metabolizable glucose analogue 2-DOG if this up-regulation in GLUT3 increased glucose uptake. We observed the presence of two uptake components, one corresponding to the Km of GLUT1/4 and the other to GLUT3. A doubling in the uptake velocity was observed only at the Km corresponding to GLUT3. In conclusion, we demonstrate and characterize for the first time, an up-regulation of GLUT3 mRNA, protein and glucose uptake by the cAMP pathway in breast cancer cells.
增加葡萄糖摄取作为主要能量来源是肿瘤细胞持续存活的必要条件。促进性葡萄糖转运蛋白1（GLUT1）和 -3（GLUT3）先前已被证明存在于乳腺癌细胞中并受其调控，且与患者预后不良相关。在癌细胞中，已知cAMP第二信使途径可增强所述葡萄糖转运蛋白激活剂并调节细胞命运。然而，此前尚未研究过cAMP对乳腺癌细胞中葡萄糖转运蛋白的调控作用。在此，我们在特征明确的乳腺癌细胞系ZR - 75中确定cAMP类似物8 - br - cAMP是否能够调节GLUT1和GLUT3的表达，从而调节葡萄糖摄取。我们证明8 - br - cAMP可短暂上调GLUT3 mRNA水平。使用放线菌素 - D以及克隆人GLUT3启动子上游1200 bp的序列表明这种调控是转录水平的。免疫细胞化学和蛋白质印迹证实mRNA的增加反映在蛋白质水平的增加上。在8 - br - cAMP存在的情况下未检测到GLUT1有明显调控。最后，我们使用不可代谢的葡萄糖类似物2 - DOG确定GLUT3的这种上调是否增加了葡萄糖摄取。我们观察到存在两种摄取成分，一种对应于GLUT1/4的Km值，另一种对应于GLUT3的Km值。仅在对应于GLUT3的Km值处观察到摄取速度加倍。总之，我们首次证明并表征了cAMP途径在乳腺癌细胞中对GLUT3 mRNA、蛋白质和葡萄糖摄取的上调作用。
