Department of Bioenergetics and Physiology of Exercise, Medical University of Gdańsk, Dębinki 1, 80-211, Gdańsk, Poland.
Eur J Nutr. 2012 Oct;51(7):817-25. doi: 10.1007/s00394-011-0260-x. Epub 2011 Oct 22.
P66Shc, an isoform of adaptor proteins, is known to mediate various signals including those leading to apoptosis or cell proliferation. Previously, we have shown that diallyl trisulfide (DATS)-induced prostate cancer cell death was mediated by increased ROS formation. In this study, we investigated the role of p66Shc protein and its serine 36 phosphorylation in DATS induced decrease in prostate cancer cell viability (PC-3).
PC-3 prostate cancer cells were used in this study. Stable cell lines expressing p66ShcS36A or an empty vector have been obtained. Cell viability, concentration of ROS, changes in P-p66Shc and P-Akt and DNA damage were determined.
We observed that DATS treatment increased p66Shc phosphorylation at serine 36. Importantly, the phosphorylation was abolished by JNK inhibitor SP600125. Cells expressing plasmid-encoded variant of p66ShcS36A showed much higher resistance to DATS-induced cells death. In addition to that, we observed that DATS-induced ROS formation was completely abolished in cells expressing the p66ShcS36A variant. Interestingly, SP600125 proved to prevent DATS-induced Akt inactivation. In order to confirm that the observed effect is related to phosphorylation of p66Shc, we performed experiments on a stable cell line expressing p66ShcS36A. In such cells, DATS-induced Akt dephosphorylation was significantly reduced. On the other hand, hydrogen peroxide induced Akt activation in PC-3 cells, which was abrogated in cells expressing p66ShcS36A.
Our results uncover a novel signaling pathway with p66Shc being indispensable for DATS-induced inactivation of Akt due to hypophosphorylation.
衔接蛋白的 P66Shc 同工型已知可介导多种信号，包括导致细胞凋亡或增殖的信号。先前，我们已经表明，二烯丙基三硫化物（DATS）诱导的前列腺癌细胞死亡是由 ROS 形成增加介导的。在这项研究中，我们研究了 p66Shc 蛋白及其丝氨酸 36 磷酸化在 DATS 诱导的前列腺癌细胞活力（PC-3）下降中的作用。
本研究使用 PC-3 前列腺癌细胞。获得了表达 p66ShcS36A 或空载体的稳定细胞系。测定细胞活力、ROS 浓度、p66Shc 和 P-Akt 的变化以及 DNA 损伤。
我们观察到 DATS 处理增加了丝氨酸 36 处的 p66Shc 磷酸化。重要的是，JNK 抑制剂 SP600125 消除了磷酸化。表达质粒编码的 p66ShcS36A 变体的细胞对 DATS 诱导的细胞死亡表现出更高的抗性。除此之外，我们观察到 DATS 诱导的 ROS 形成在表达 p66ShcS36A 变体的细胞中完全被消除。有趣的是，SP600125 被证明可以防止 DATS 诱导的 Akt 失活。为了证实观察到的效应与 p66Shc 的磷酸化有关，我们在表达 p66ShcS36A 的稳定细胞系上进行了实验。在这些细胞中，DATS 诱导的 Akt 去磷酸化明显减少。另一方面，过氧化氢诱导 PC-3 细胞中的 Akt 激活，在表达 p66ShcS36A 的细胞中被阻断。
我们的结果揭示了一种新的信号通路，其中 p66Shc 对于由于低磷酸化导致的 DATS 诱导的 Akt 失活是必不可少的。
