Nishida Shozo, Yoshioka Shohei, Kinoshita-Kimoto Saori, Kotani Michiyo, Tsubaki Masanobu, Fujii Yoshiki, Tomura Takanori T, Irimajiri Kiyohiro
Department of Pharmacotheraphy, Kinki University, School of Pharmaceutical Sciences, 3-4-1 Kowakae, Higashiosaka, Osaka 577-8502, Japan.
Life Sci. 2003 Dec 26;74(6):781-92. doi: 10.1016/j.lfs.2003.07.012.
Tumor necrosis factor alpha (TNF-alpha) modulates various events through several different pathways. Many tumor cells are resistant to this cytokine. Pretreatment of these cells with actinomycin D enhances TNF-alpha-induced apoptosis. In the present study, we investigated the mechanism of this enhancement and whether or not the apoptosis of TNF-alpha-resistant cancer cells can be induced by the inhibition of Protein kinase C (PKC). When TNF-alpha was added after inhibition of PKC by H7, apoptosis was observed, and companied with the activation of nuclear factor kappa B (NF-kappaB). After the inhibition of protein kinase B (Akt) by LY294002 or p38 mitogen-activated protein kinase (p38MAPK) by SB203580, the addition of TNF-alpha did not cause apoptosis. However, after the inhibition of MAPK/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) with U0126, apoptosis was observed when TNF-alpha was added. In the Western blotting analysis, phosphorylation of MEK1/2 occurred at 60 minutes after the addition of TNF-alpha. However, it was noted that after pretreatment with H7, a significant decrease in phosphorylated MEK1/2 was observed. The present findings suggest that MEK1/2 plays an important role in TNF-alpha-resistance in TNF-alpha-resistant B16 melanoma BL6 cells. Furthermore, it was found that MEK1/2 is more important than NF-kappaB, Akt, and p38MAPK in anti-apoptotic PKC signaling and that TNF-alpha-resistance can be overcome by inhibiting MEK1/2. These results suggest the possibility of development of a new anticancer drug treatment.
肿瘤坏死因子α（TNF-α）通过多种不同途径调节各种事件。许多肿瘤细胞对这种细胞因子具有抗性。用放线菌素D预处理这些细胞可增强TNF-α诱导的细胞凋亡。在本研究中，我们研究了这种增强的机制，以及抑制蛋白激酶C（PKC）是否能诱导TNF-α抗性癌细胞的凋亡。当用H7抑制PKC后加入TNF-α时，观察到细胞凋亡，并伴有核因子κB（NF-κB）的激活。用LY294002抑制蛋白激酶B（Akt）或用SB203580抑制p38丝裂原活化蛋白激酶（p38MAPK）后，加入TNF-α不会导致细胞凋亡。然而，用U0126抑制丝裂原活化蛋白激酶/细胞外信号调节激酶激酶1/2（MEK1/2）后，加入TNF-α时观察到细胞凋亡。在蛋白质印迹分析中，加入TNF-α后60分钟出现MEK1/2的磷酸化。然而，值得注意的是，用H7预处理后，观察到磷酸化的MEK1/2显著减少。本研究结果表明，MEK1/2在TNF-α抗性B16黑色素瘤BL6细胞的TNF-α抗性中起重要作用。此外，发现在抗凋亡PKC信号传导中，MEK1/2比NF-κB、Akt和p38MAPK更重要，并且抑制MEK1/2可以克服TNF-α抗性。这些结果提示了开发新型抗癌药物治疗方法的可能性。
