NEK6 Regulates Redox Balance and DNA Damage Response in DU-145 Prostate Cancer Cells

NEK6 is a central kinase in developing castration-resistant prostate cancer (CRPC). However, the pathways regulated by NEK6 in CRPC are still unclear. Cancer cells have high reactive oxygen species (ROS) levels and easily adapt to this circumstance and avoid cell death by increasing antioxidant defenses. We knocked out the NEK6 gene and evaluated the redox state and DNA damage response in DU-145 cells. The knockout of NEK6 decreases the clonogenic capacity, proliferation, cell viability, and mitochondrial activity. Targeting the NEK6 gene increases the level of intracellular ROS; decreases the expression of antioxidant defenses (SOD1, SOD2, and PRDX3); increases JNK phosphorylation, a stress-responsive kinase; and increases DNA damage markers (p-ATM and γH2AX). The exogenous overexpression of NEK6 also increases the expression of these same antioxidant defenses and decreases γH2AX. The depletion of NEK6 also induces cell death by apoptosis and reduces the antiapoptotic Bcl-2 protein. NEK6-lacking cells have more sensitivity to cisplatin. Additionally, NEK6 regulates the nuclear localization of NF-κB2, suggesting NEK6 may regulate NF-κB2 activity. Therefore, NEK6 alters the redox balance, regulates the expression of antioxidant proteins and DNA damage, and its absence induces the death of DU-145 cells. NEK6 inhibition may be a new strategy for CRPC therapy

Antioxidant and Neuroprotective Effects of the First Tryptophyllin Found in Snake Venom (Bothrops moojeni)

In this study, we report the isolation, characterization, and synthesis of the peptide BmT-2 belonging to the tryptophyllins family, isolated from the venom of the snake Bothrops moojeni. This is the first time a tryptophyllin is identified in snake venom. We tested whether BmT-2 had cytotoxic effects and antioxidant activity in a set of experiments that included both in vitro and cell-based assays. BmT-2 presented a radical scavenging activity toward ABTS• and AAPH-derived radicals. BmT-2 protected fluorescein, DNA molecules, and human red blood cells (RBCs) from free radicals generated by the thermal decomposition of AAPH. The novel tryptophyllin was not toxic in cell viability tests, where it (up to 0.4 mg/mL) did not cause hemolysis of human RBCs and did not cause significant loss of cell viability, showing a CC50 > 1.5 mM for cytotoxic effects against SK-N-BE(2) neuroblastoma cells. BmT-2 prevented the arsenite-induced upregulation of Nrf2 in Neuro-2a neuroblasts and the phorbol myristate acetate-induced overgeneration of reactive oxygen species and reactive nitrogen species in SK-N-BE(2) neuroblastoma cells. Electronic structure calculations and full atomistic reactive molecular dynamics simulations revealed the relevant contribution of aromatic residues in BmT-2 to its antioxidant properties. Our study presents a novel peptide classified into the family of the tryptophyllins, which has been reported exclusively in amphibians. Despite the promising results on its antioxidant activity and low cytotoxicity, the mechanisms of action of BmT-2 still need to be further elucidated

Increased mTOR Signaling and Impaired Autophagic Flux Are Hallmarks of SARS-CoV-2 Infection

The COVID-19 (Coronavirus Disease 2019), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), severely affects mainly individuals with pre-existing comorbidities. Here our aim was to correlate the mTOR (mammalian/mechanistic Target of Rapamycin) and autophagy pathways with the disease severity. Through western blotting and RNA analysis, we found increased mTOR signaling and suppression of genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2 as well as in transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients. Immunofluorescence co-localization assays also indicated that SARS-CoV-2 colocalizes within autophagosomes but not with a lysosomal marker. Our findings indicate that SARS-CoV-2 can benefit from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling