Oxidative stress results in apoptosis of neuronal cells, leading to neurodegenerative disorders. However, the underlying molecular mechanism remains to be elucidated. Here we show that hydrogen peroxide (H2O2), a major oxidant generated when oxidative stress occurs, induced apoptosis of neuronal cells (PC12 cells and primary murine neurons), by inhibiting the mammalian target of rapamycin (mTOR)-mediated phosphorylation of ribosomal p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS), blocked H2O2 inhibition of mTOR signaling. Ectopic expression of wild type (wt) mTOR, constitutively active S6K1 or downregulation of 4E-BP1 partially prevented H2O2 induction of apoptosis. Furthermore, we identified that H2O2 induction of ROS inhibited the upstream kinases, Akt and phosphoinositide-dependent kinase 1 (PDK1), but not the type I insulin-like growth factor receptor (IGFR), and activated the negative regulator, AMP-activated protein kinase α (AMPKα), but not the phosphatase and tensin homolog (PTEN) in the cells. Expression of a dominant negative AMPKα or downregulation of AMPKα1 conferred partial resistance to H2O2 inhibition of phosphorylation of S6K1 and 4E-BP1, as well as cell viability, indicating that H2O2 inhibition of mTOR signaling is at least in part through activation of AMPK. Our findings suggest that AMPK inhibitors may be exploited for prevention of H2O2-induced neurodegenerative diseases.

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd induces neuronal apoptosis in part through activation of the mammalian target of rapamycin (mTOR) pathway. However, the underlying mechanism is unknown. Here we show that Cd induced generation of reactive oxygen species (ROS) by upregulating expression of NADPH oxidase 2 (NOX2) and its regulatory proteins (p22 phox , p67 phox , p40 phox , p47 phox and Rac1) in PC12 and SH-SY5Y cells. Cd induction of ROS contributed to activation of mTOR signaling, as pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, prevented this event. Further studies reveal that Cd induction of ROS increased phosphorylation of type I insulin-like growth factor receptor β subunit (IGFRβ), which was abrogated by NAC. Wortmannin, a phosphoinositide 3′-kinase (PI3K) inhibitor, partially attenuated Cd-induced phosphorylation of Akt, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), as well as apoptosis of the neuronal cells. In addition, overexpression of wild-type phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or pretreatment with aminoimidazole carboxamide ribonucleotide (AICAR), an AMPactivated protein kinase (AMPK) activator, partially prevented Cd-induced ROS and activation of mTOR pathway, as well as cell death. The results indicate that Cd induction of ROS activates mTOR signaling, leading to neuronal cell death, in part by activating the positive regulators IGFR/ PI3K, and by inhibiting the negative regulators PTEN/AMPK. The findings suggest that the inhibitors of PI3K and mTOR, activators of AMPK, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+]i) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+]i elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+]i elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+]i elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+]i, which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Background: Selpercatinib (LOXO-292) and pralsetinib (BLU-667) are highly potent RET-selective protein tyrosine kinase inhibitors (TKIs) for treating advanced RET-altered thyroid cancers and non-small-cell lung cancer (NSCLC). It is critical to analyze RET mutants resistant to these drugs and unravel the molecular basis to improve patient outcomes. Patients and methods: Cell-free DNAs (cfDNAs) were analyzed in a RET-mutant medullary thyroid cancer (MTC) patient and a CCDC6-RET fusion NSCLC patient who had dramatic response to selpercatinib and later developed resistance. Selpercatinib-resistant RET mutants were identified and cross-profiled with pralsetinib in cell cultures. Crystal structures of RET-selpercatinib and RET-pralsetinib complexes were determined based on high-resolution diffraction data collected with synchrotron radiation. Results: RET G810C/S mutations at the solvent front and RET Y806C/N mutation at the hinge region were found in cfDNAs of an MTC patient with RET M918T/V804M/L , who initially responded to selpercatinib and developed resistance. RET G810C mutant was detected in cfDNAs of a CCDC6-RET-fusion NSCLC patient who developed acquired resistance to selpercatinib. Five RET kinase domain mutations at three non-gatekeeper residues were identified from 39 selpercatinib-resistant cell lines. All five selpercatinib-resistant RET mutants were cross-resistant to pralsetinib. X-ray crystal structures of the RETselpercatinib and RET-pralsetinib complexes reveal that, unlike other TKIs, these two RET TKIs anchor one end in the front cleft and wrap around the gate wall to access the back cleft. Conclusions: RET mutations at the solvent front and the hinge are resistant to both drugs. Selpercatinib and pralsetinib use an unconventional mode to bind RET that avoids the interference from gatekeeper mutations but is vulnerable to non-gatekeeper mutations.

Cell division cycle 25 A (Cdc25A), a dual-specificity protein phosphatase, is one of the most crucial cell cycle regulators, which removes the inhibitory phosphorylation in cyclin-dependent kinases (CDKs), such as CDK2, CDK4, and CDK6, and positively regulates the activities of CDKs that lead to cell cycle progression. In addition, Cdc25A also acts as a regulator of apoptosis. Overexpression of Cdc25A promotes tumorigenesis, and is frequently observed in various types of cancer. Here we briefly summarize current understanding of the role of Cdc25A in cell proliferation and apoptosis, as well as the impact of overexpression of Cdc25A on tumorigenesis.

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Ciclopirox olamine (CPX) is a synthetic antifungal agent clinically used to treat mycoses of the skin and nails. Here, we show that CPX inhibited tumor growth in human breast cancer MDA-MB-231 xenografts. To unveil the underlying mechanism, we further studied the antitumor activity of CPX in cell culture. The results indicate that CPX inhibited cell proliferation and induced apoptosis in human rhabdomyosarcoma (Rh30), breast carcinoma (MDA-MB231) and colon adenocarcinoma (HT-29) cells in a concentration-dependent manner. By cell cycle analysis, CPX induced accumulation of cells in G 1 /G 0 phase of the cell cycle. Concurrently, CPX downregulated cellular protein expression of cyclins (A, B1, D1 and E) and cyclin-dependent kinases (CDK2 and CDK4) and upregulated expression of the CDK inhibitor p21Cip1 , leading to hypophosphorylation of retinoblastoma protein. CPX also downregulated protein expression of Bcl-xL and survivin and enhanced cleavages of Bcl-2. Z-VAD-FMK, a pan-caspase inhibitor, partially prevented CPX-induced cell death, suggesting that CPX-induced apoptosis of cancer cells is mediated at least in part through caspase-dependent mechanism. The results indicate that CPX is a potential antitumor agent.Ciclopirox olamine (CPX) (also called Batrafen, Loprox, Penlac and Stieprox), the ethanolamine salt of 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone, is a synthetic antifungal agent used to treat mycoses of the skin and nails for more than 20 years. 1-3Studies have shown that CPX has a very broad spectrum of action against dermatophytes, yeast, filamentous fungi and bacteria. 4,5 The mechanisms of these actions of CPX seem diverse, involving disruption of membrane function in fungi or targeting different metabolic (respiratory) and energy-producing processes in bacteria.1,2 In the yeast Saccharomyces cerevisiae, CPX may also exert its effect by disrupting DNA repair, cell division signals and structures (mitotic spindles) as well as some elements of intracellular transport.6 Apart from its antimycotic and antibacterial activities, CPX arrests the cell cycle at G 1 phase in mammalian cells 7,8 and G 2 /M phase in the yeast S. cerevisiae. 9 CPX also prevents the death of tropic factor-deprived PC12 cells and postmitotic sympathetic neurons by blocking the cell cycle progression 7 or the death of cerebellar granule neurons in low K þ -containing medium, 10 but it induces an active cell death in S. cerevisiae. 9In addition, CPX is a well-known iron chelator, inhibiting the iron-containing enzymes, such as catalase and peroxidase.11 Most recent studies have revealed that the chelation of intracellular iron and the inhibition of the iron-dependent enzyme ribonucleotide reductase were associated with CPXinduced cell death.12 It appeared that CPX induced cell death in primary human acute myeloid leukemia (AML) cells and inhibited engraftment of primary AML cells in NOD/SCID mouse models without gross organ toxicity or loss of body weight.12 Previous safety and toxicity studies of CPX also demonstrated that a 4-w...

The canonical Wnt/β-catenin signaling pathway plays a central role in development and cancer. The p21-activated kinase 4 (PAK4) involves in a wide range of cellular processes, including cytoskeletal reorganization, cell proliferation, gene transcription and oncogenic transformation. However, the cross talk between the Wnt and PAK4 signaling pathways is poorly understood. Here, we show that PAK4 is a nucleo-cytoplasmic shuttling protein, containing three nuclear export signals (NESs) and two nuclear localization signals (NLSs). PAK4 is exported by the chromosome region maintenance-1 (CRM-1)-dependent pathway and is imported into the nucleus in an importin α5-dependent manner. PAK4 interacts with and phosphorylates β-catenin on Ser675, which promotes the TCF/LEF transcriptional activity and stabilizes β-catenin through inhibition of its degradation. Moreover, nuclear import of PAK4 accompanies with the nuclear import of β-catenin and increased TCF/LEF transcriptional activity. We further demonstrated that PAK4 associates with the TCF/LEF transcriptional complex by ChIP assays. These findings uncover a novel role for PAK4 in modulating intracellular translocation and signaling of β-catenin.