Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain.

Microtubule-severing protein Spastin has been shown to co-localize with actin in migratory glioblastoma cells and is linked to glioblastomas' migration and invasion capacity. However, the effectiveness of Spastin in glioblastoma migration and the molecular mechanism underpinning the orientation of Spastin towards actin filaments remain unknown. Here, we demonstrated that Spastin plays an active role in glioblastoma migration by showing a reduced migratory potential of T98G glioblastoma cells using real-time cell analysis (RTCA) in Spastin-depleted cells. Pull-down assays revealed that a cis-trans isomerase Pin1 interacts with Spastin through binding to the phosphorylated Pin1 recognition motifs in the microtubule-binding domain (MBD), and immunocytochemistry analysis showed that interaction with Pin1 directs Spastin to actin filaments in extended cell regions. Consequently, by utilizing RTCA, we proved that the migration and invasion capacity of T98G glioblastoma cells significantly increased with the overexpression of Spastin, of which the Pin1 recognition motifs in MBD are constitutively phosphorylated, while the overexpression of phospho-mutant form did not have a significant effect on migration and invasion of T98G glioblastoma cells. These findings demonstrate that Pin1 is a novel interaction partner of Spastin, and their interaction drives Spastin to actin filaments, allowing Spastin to contribute to the glioblastomas' migration and invasion abilities.

Salicylidene acylhydrazides attenuate survival of SH-SY5Y neuroblastoma cells through affecting mitotic regulator Speedy/RINGO and ERK/MAPK-PI3K/AKT signaling.

Salicylidene acylhydrazide group synthetic compounds ME0053, ME005 and ME0192 are known for their iron chelating properties and due to these properties they are primarily used for blocking the bacterial type 3 secretory virulence system. On the other side, targeting the metabolic pathways of iron can provide new tools for cancer prognosis and treatment. Therefore, in this study, considering their iron chelating function, the effects of the compounds ME0053, ME0055 and ME0192 were investigated in SH-SY5Y neuroblastoma cell line. Iron chelating compounds are generally known to be effective in tumor development and metastasis by targeting iron in the cell. They can exert this effect through molecules such as cyclin, CDKs, as well as signaling pathways such as PI3K/AKT and ERK/MAPK. For this reason, we analyzed the effect of the iron chelating compounds of ME0053, ME0055 and ME0192 on cell viability and proliferation rate both through ERK/MAPK and PI3K/AKT signal paths, and through the oncogenic Speedy/RINGO protein that is likely to have a regulatory effect on these two signaling pathways. Apoptosis was also investigated by measuring the amount of active caspase-3, an apoptotic marker. Along with the decrease observed in the Speedy/RINGO level, it was observed that the PI3K/AKT and ERK/MAPK signaling were decreased. This suggests that ME0053, ME0055 and ME0192 compounds significantly decrease the Speedy/RINGO expression which has a regulatory effect on the ERK/MAPK and PI3K/AKT signaling. Besides, analyzing active caspase-3 levels showed that the compounds ME0053, ME0055 and ME0192 increased its level by 218%, 60% and 175% in SH-SY5Y cells, respectively. The results of this study will pave the way for better understanding of the regulation of cancer-related ERK/MAPK and PI3K/AKT pathways and the oncogenic Speedy/RINGO which potentially affects these pathways, through synthetic salicylidene acylhydrazides and their therapeutic use in cancer.

Speedy/RINGO protein interacts with ERK/MAPK and PI3K/AKT pathways in SH-SY5Y neuroblastoma cells.

Abnormal activity of ERK/MAPK and PI3K/AKT pathways is one of the most important factors for the development of many cancer types including neuroblastoma cancer. Apart from these two pathways, some cell cycle regulators such as Speedy/RINGO also contribute to neuroblastoma development. There is data reinforcing the possible communication of the components of ERK/MAPK and PI3K/AKT pathways in carcinogenic process. In addition to this, there are studies about the direct/indirect interaction of Speedy/RINGO with these pathways in different cell types other than neuroblastoma. However, there is not any study available showing the interaction of Speedy/RINGO with both pathways in neuroblastoma cells. Therefore, the aim of this study is to determine the possible effect of Speedy/RINGO on PI3K/AKT and ERK/MAPK pathways in SH-SY5Y neuroblastoma cells. For this aim, Speedy/RINGO was silenced by siRNA technique to analyze the effects of direct inhibition of Speedy/RINGO on these pathways. Results showed that Speedy/RINGO silencing caused a significant decrease in MEK1/2 expression and AKT phosphorylation. Afterward, MEK1/2 was inhibited using a specific inhibitor U0126. Data reveal a corresponding decrease in the Speedy/RINGO expression and AKT phosphorylation indicating a reciprocal interaction between ERK/MAPK and Speedy/RINGO. In addition, MTS analysis showed that both ERK/MAPK inhibition and Speedy/RINGO silencing significantly reduced the viability of SH-SY5Y cells. This study provides information about a possible interaction of Speedy/RINGO with PI3K/AKT and ERK/MAPK pathways in SH-SY5Y cells for the first time. It will not only help to better understand the cancer-prone interactions of these pathways but also enable us to identify the appropriate molecular targets for developing efficient treatment strategies.