Graduate Institute of Pharmacy, School of Pharmacy, Taipei Medical University, Taipei, Taiwan.
J Cell Physiol. 2010 Nov;225(2):472-81. doi: 10.1002/jcp.22226.
An increase in MMP-9 gene expression and enzyme activity with stimulating the migration of GBM8401 glioma cells via wound healing assay by 12-O-tetradecanoylphorbol-13-acetate (TPA) was detected in glioblastoma cells GBM8401. TPA-induced translocation of protein kinase C (PKC)alpha from the cytosol to membranes, and migration of GBM8401 elicited by TPA was suppressed by adding the PKCalpha inhibitors, GF109203X and H7. Activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) by TPA was identified, and TPA-induced migration and MMP-9 activity was significantly blocked by ERK inhibitor PD98059 and U0126, but not JNK inhibitor SP600125. Activation of NF-kappaB protein p65 nuclear translocation and IkappaBalpha protein phosphorylation with increased NF-kappaB-directed luciferase activity by TPA were observed, and these were blocked by the PD98059 and IkB inhibitor BAY117082 accompanied by reducing migration and MMP-9 activity induced by TPA in GBM8401 cells. Transfection of GBM8401 cells with PKCalpha siRNA specifically reduced PKCalpha protein expression with blocking TPA-induced MMP-9 activation and migration. Additionally, suppression of TPA-induced PKCalpha/ERK/NK-kappaB activation, migration, and MMP-9 activation by flavonoids including kaempferol (Kae; 3,5,7,4'-tetrahydroxyflavone), luteolin (Lut; 5,7,3'4'-tetrahydroxyflavone), and wogonin (Wog; 5,7-dihydroxy-8-methoxyflavone) was demonstrated, and structure-activity relationship (SAR) studies showed that hydroxyl (OH) groups at C4' and C8 are critical for flavonoids' action against MMP-9 enzyme activation and migration/invasion of glioblastoma cells elicited by TPA. Application of flavonoids to prevent the migration/invasion of glioblastoma cells through blocking PKCalpha/ERK/NF-kappaB activation is first demonstrated herein.
通过用 12-O-十四烷酰佛波醇-13-乙酸酯（TPA）刺激 GBM8401 神经胶质瘤细胞的划痕愈合试验，检测到 MMP-9 基因表达和酶活性在神经胶质瘤细胞 GBM8401 中的增加。TPA 诱导蛋白激酶 C（PKC）alpha 从细胞质向膜易位，并且 TPA 诱导的 GBM8401 迁移被添加 PKCalpha 抑制剂 GF109203X 和 H7 抑制。鉴定了 TPA 对细胞外信号调节激酶（ERK）和 c-Jun-N-末端激酶（JNK）的激活，并且 ERK 抑制剂 PD98059 和 U0126 显著阻断了 TPA 诱导的迁移和 MMP-9 活性，但 JNK 抑制剂 SP600125 则不然。观察到 TPA 诱导的 NF-kappaB 蛋白 p65 核易位和 IkappaBalpha 蛋白磷酸化，以及 NF-kappaB 定向荧光素酶活性增加，并通过 PD98059 和 IkB 抑制剂 BAY117082 阻断了这些，同时减少了 TPA 在 GBM8401 细胞中诱导的迁移和 MMP-9 活性。用 PKCalpha siRNA 转染 GBM8401 细胞可特异性降低 PKCalpha 蛋白表达，阻断 TPA 诱导的 MMP-9 激活和迁移。此外，抑制 TPA 诱导的 PKCalpha/ERK/NK-kappaB 激活、迁移和 MMP-9 激活被黄酮类化合物如山奈酚（Kae；3,5,7,4'-四羟基黄酮）、木犀草素（Lut；5,7,3'4'-四羟基黄酮）和高良姜素（Wog；5,7-二羟基-8-甲氧基黄酮）所证明，结构活性关系（SAR）研究表明，C4'和 C8 上的羟基（OH）基团对于黄酮类化合物抑制 TPA 诱导的 MMP-9 酶激活和神经胶质瘤细胞的迁移/侵袭至关重要。本文首次证明了应用黄酮类化合物通过阻断 PKCalpha/ERK/NF-kappaB 激活来防止神经胶质瘤细胞的迁移/侵袭。
