Thioridazine (THD) is a common phenothiazine antipsychotic drug reported to suppress growth in several types of cancer cells. We previously showed that THD acts as an antiglioblastoma and anticancer stem-like cell agent. However, the signaling pathway underlying autophagy and apoptosis induction remains unclear. THD treatment significantly induced autophagy with upregulated AMPK activity and engendered cell death with increased sub-G1 in glioblastoma multiform (GBM) cell lines. Notably, through whole gene expression screening with THD treatment, frizzled (Fzd) proteins, a family of G-protein-coupled receptors, were found, suggesting the participation of Wnt/β-catenin signaling. After THD treatment, Fzd-1 and GSK3β-S9 phosphorylation (inactivated form) was reduced to promote β-catenin degradation, which attenuated P62 inhibition. The autophagy marker LC3-II markedly increased when P62 was released from β-catenin inhibition. Additionally, the P62-dependent caspase-8 activation that induced P53-independent apoptosis was confirmed by inhibiting T-cell factor/β-catenin and autophagy flux. Moreover, treatment with THD combined with temozolomide (TMZ) engendered increased LC3-II expression and caspase-3 activity, indicating promising drug synergism. In conclusion, THD induces autophagy in GBM cells by not only upregulating AMPK activity, but also enhancing P62-mediated autophagy and apoptosis through Wnt/β-catenin signaling. Therefore, THD is a potential alternative therapeutic agent for drug repositioning in GBM.

Two new scalarane sesterterpenoids, 12β-(3′β-hydroxybutanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (1) and 12β-(3′β-hydroxypentanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (2), along with one known tetraprenyltoluquinol-related metabolite (3), were isolated from the sponge Carteriospongia sp. In leukemia Molt 4 cells, 1 at 0.0625 μg/mL (125 nM) triggered mitochondrial membrane potential (MMP) disruption and apoptosis showing more potent effect than 2 and 3. The isolates inhibited topoisomerase IIα expression. The apoptotic-inducing effect of 3 was supported by the in vivo experiment through suppressing the volume of xenograft tumor growth (47.58%) compared with the control. Compound 1 apoptotic mechanism of action in Molt 4 cells was further elucidated through inducing ROS generation, calcium release and ER stress. Using the molecular docking analysis, 1 exhibited more binding affinity to N-terminal ATP-binding pocket of Hsp90 protein than 17-AAG, a standard Hsp90 inhibitor. The expression of Hsp90 client proteins, Akt, p70S6k, NFκB, Raf-1, p-GSK3β, and XIAP, MDM 2 and Rb2, and CDK4 and Cyclin D3, HIF 1 and HSF1 were suppressed by the use of 1. However, the expression of Hsp70, acetylated tubulin, and activated caspase 3 were induced after 1 treatment. Our results suggested that the proapoptotic effect of the isolates is mediated through the inhibition of Hsp90 and topoisomerase activities.

Based on the protein kinase A (PKA)/GSK3β interaction protein (GSKIP)/glycogen synthase kinase 3β (GSK3β) axis, we hypothesized that these might play a role in Tau phosphorylation. Here, we report that the phosphorylation of Tau Ser409 in SHSY5Y cells was increased by overexpression of GSKIP WT more than by PKA- and GSK3β-binding defective mutants (V41/L45 and L130, respectively). We conducted in vitro assays of various kinase combinations to show that a combination of GSK3β with PKA but not Ca2+/calmodulin-dependent protein kinase II (CaMK II) might provide a conformational shelter to harbor Tau Ser409. Cerebrospinal fluid (CSF) was evaluated to extend the clinical significance of Tau phosphorylation status in Alzheimer’s disease (AD), neurological disorders (NAD), and mild cognitive impairment (MCI). We found higher levels of different PKA–Tau phosphorylation sites (Ser214, Ser262, and Ser409) in AD than in NAD, MCI, and normal groups. Moreover, we used the CRISPR/Cas9 system to produce amyloid precursor protein (APPWT/D678H) isogenic mutants. These results demonstrated an enhanced level of phosphorylation by PKA but not by the control. This study is the first to demonstrate a transient increase in phosphor-Tau caused by PKA, but not GSK3β, in the CSF and induced pluripotent stem cells (iPSCs) of AD, implying that both GSKIP and GSK3β function as anchoring proteins to strengthen the cAMP/PKA/Tau axis signaling during AD pathogenesis.

No abstract