Daveri E, Maellaro E, Valacchi G, Ietta F, Muscettola M, Maioli E
Department of Life Sciences, University of Siena, via Aldo Moro 7, 53100 Siena, Italy.
Department of Molecular and Developmental Medicine, University of Siena, Via Aldo Moro 7, 53100 Siena, Italy.
Cancer Lett. 2016 Sep 28;380(1):106-13. doi: 10.1016/j.canlet.2016.06.018. Epub 2016 Jun 22.
Earlier studies demonstrated that Rottlerin exerts a time- and dose-dependent antiproliferative effect on SK-Mel-28 melanoma cells during 24 h of treatment, but cytotoxicity due to cell death began only after a 48 h exposure. In the current study, in order to identify the type of cell death in this cell line, which is notoriously refractory to most anticancer therapies, and to clarify the underlying mechanisms of this delayed outcome, we searched for apoptotic, necrotic/necroptotic and autophagic traits in Rottlerin-exposed cells. Although SK-Mel-28 cells are both apoptosis and autophagy competent, Western blotting analysis, caspase activity assay, nuclear imaging and the effects of autophagy, apoptosis and necroptosis inhibitors, indicated that Rottlerin cytotoxicity was due to none of the aforementioned death mechanisms. Nevertheless, in growth arrested cells, the death did occur after a prolonged treatment and most likely ensued from the observed blockage of protein synthesis that reached levels expected to be incompatible with cell survival. From a mechanistic point of view, we ascribed this effect to the documented inhibition of mTORC1 activity; mTORC1 inhibition on the one hand led to a not deadly, rather protective autophagic response but, on the other hand caused a near complete arrest of protein synthesis. Interestingly, no cytotoxicity was found towards normal skin fibroblasts, which only resulted mildly growth arrested by the drug.
早期研究表明，在24小时的治疗过程中，rottlerin对SK-Mel-28黑色素瘤细胞具有时间和剂量依赖性的抗增殖作用，但细胞死亡导致的细胞毒性仅在暴露48小时后才开始。在本研究中，为了确定这种对大多数抗癌疗法具有 notoriously refractory（此处可能有误，推测是“众所周知难以治疗”之类的意思）的细胞系中的细胞死亡类型，并阐明这种延迟结果的潜在机制，我们在rottlerin处理的细胞中寻找凋亡、坏死/坏死性凋亡和自噬特征。尽管SK-Mel-28细胞具有凋亡和自噬能力，但蛋白质印迹分析、半胱天冬酶活性测定、核成像以及自噬、凋亡和坏死性凋亡抑制剂的作用表明，rottlerin的细胞毒性并非由上述任何一种死亡机制引起。然而，在生长停滞的细胞中，长时间处理后确实发生了死亡，最有可能是由于观察到的蛋白质合成受阻，其水平达到了预期与细胞存活不相容的程度。从机制角度来看，我们将这种效应归因于已记录的mTORC1活性抑制；mTORC1抑制一方面导致了一种并非致命而是具有保护作用的自噬反应，但另一方面导致了蛋白质合成几乎完全停滞。有趣的是，未发现对正常皮肤成纤维细胞有细胞毒性，药物仅使其轻度生长停滞。
