Daveri Elena, Valacchi Giuseppe, Romagnoli Roberta, Maellaro Emilia, Maioli Emanuela
Department of Life Sciences, University of Siena, Via Aldo Moro 7, 53100 Siena, Italy.
Department of Biology and Evolution, University of Ferrara, Via Luigi Borsari 46, 44100 Ferrara, Italy.
Evid Based Complement Alternat Med. 2015;2015:545838. doi: 10.1155/2015/545838. Epub 2015 May 28.
Melanoma is the most aggressive and chemoresistant form of skin cancer. Mutated, constitutively active B-RAF is believed to play a crucial role, although the selective B-RAF inhibition has shown poor clinical success, since phenomena of resistance usually occur, likely arising from additional genetic aberrations, such as loss of function of p53 and PTEN, overexpression of cyclin D1, hyperactivation of NF-κB, and downregulation of p21/Cip1. Since all of them are present in the Sk-Mel-28 melanoma cells, this cell line could be an ideal, albeit hard to study, model to develop new therapeutic strategies. In the current study, we tested the cytostatic action of Rottlerin on Sk-Mel-28 melanoma cells, on the basis of the known Rottlerin effects on the main proliferative signaling pathways. We presented evidence that the drug inhibits cell growth by an Akt- and p21/Cip1-independent mechanism, involving the dual inhibition of ERK and NF-κB and downregulation of cyclin D1. In addition, we found that Rottlerin increases ERK phosphorylation, but, surprisingly, this resulted in decreased ERK activity. Pull-down experiments, using Rottlerin-CNBr-conjugated Sepharose beads, revealed that Rottlerin binds to ERK, independently from its phosphorylation status. This direct interaction could in part explain the paradoxical blockage of ERK downstream signaling and growth arrest. We would like to dedicate this paper to the memory of our friend and colleague, prematurely deceased, Claudia Torricelli, who actively contributed to this project.
黑色素瘤是皮肤癌中最具侵袭性且对化疗耐药的类型。尽管选择性B-RAF抑制在临床上效果不佳，因为通常会出现耐药现象，可能源于其他基因异常，如p53和PTEN功能丧失、细胞周期蛋白D1过表达、NF-κB过度激活以及p21/Cip1下调，但突变的、组成型激活的B-RAF被认为起着关键作用。由于所有这些异常都存在于Sk-Mel-28黑色素瘤细胞中，该细胞系可能是开发新治疗策略的理想模型，尽管难以研究。在本研究中，基于已知的rottlerin对主要增殖信号通路的作用，我们测试了rottlerin对Sk-Mel-28黑色素瘤细胞的细胞生长抑制作用。我们提供的证据表明，该药物通过一种不依赖Akt和p21/Cip1的机制抑制细胞生长，该机制涉及对ERK和NF-κB的双重抑制以及细胞周期蛋白D1的下调。此外，我们发现rottlerin增加了ERK磷酸化，但令人惊讶的是，这导致ERK活性降低。使用与溴化氰偶联的rottlerin琼脂糖珠进行的下拉实验表明，rottlerin与ERK结合，与其磷酸化状态无关。这种直接相互作用可能部分解释了ERK下游信号传导的矛盾性阻断和生长停滞。我们谨以此文纪念我们过早离世的朋友和同事克劳迪娅·托里切利，她为这个项目做出了积极贡献。
