A DNA repair-independent role for alkyladenine DNA glycosylase in alkylation-induced unfolded protein response.

Alkylating agents damage DNA and proteins and are widely used in cancer chemotherapy. While cellular responses to alkylation-induced DNA damage have been explored, knowledge of how alkylation affects global cellular stress responses is sparse. Here, we examined the effects of the alkylating agent methylmethane sulfonate (MMS) on gene expression in mouse liver, using mice deficient in alkyladenine DNA glycosylase (Aag), the enzyme that initiates the repair of alkylated DNA bases. MMS induced a robust transcriptional response in wild-type liver that included markers of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) known to be controlled by XBP1, a key UPR effector. Importantly, this response is significantly reduced in the Aag knockout. To investigate how AAG affects alkylation-induced UPR, the expression of UPR markers after MMS treatment was interrogated in human glioblastoma cells expressing different AAG levels. Alkylation induced the UPR in cells expressing AAG; conversely, AAG knockdown compromised UPR induction and led to a defect in XBP1 activation. To verify the requirements for the DNA repair activity of AAG in this response, AAG knockdown cells were complemented with wild-type Aag or with an Aag variant producing a glycosylase-deficient AAG protein. As expected, the glycosylase-defective Aag does not fully protect AAG knockdown cells against MMS-induced cytotoxicity. Remarkably, however, alkylation-induced XBP1 activation is fully complemented by the catalytically inactive AAG enzyme. This work establishes that, besides its enzymatic activity, AAG has noncanonical functions in alkylation-induced UPR that contribute to cellular responses to alkylation.

Guanosine Neuroprotective Action in Hippocampal Slices Subjected to Oxygen and Glucose Deprivation Restores ATP Levels, Lactate Release and Glutamate Uptake Impairment: Involvement of Nitric Oxide.

Stroke is a major cause of disability and death worldwide. Oxygen and glucose deprivation (OGD) in brain tissue preparations can reproduce several pathological features induced by stroke providing a valuable ex vivo protocol for studying the mechanism of action of neuroprotective agents. Guanosine, an endogenous guanine nucleoside, promotes neuroprotection in vivo and in vitro models of neurotoxicity. We previously showed that guanosine protective effect was mimicked by inhibition of nitric oxide synthases (NOS) activity. This study was designed to investigate the involvement of nitric oxide (NO) in the mechanisms related to the protective role of guanosine in rat hippocampal slices subjected to OGD followed by reoxygenation (OGD/R). Guanosine (100 µM) and the pan-NOS inhibitor, L-NAME (1 mM) afforded protection to hippocampal slices subjected to OGD/R. The presence of NO donors, DETA-NO (800 µM) or SNP (5 µM) increased reactive species production, and abolished the protective effect of guanosine or L-NAME against OGD/R. Guanosine or L-NAME treatment prevented the impaired ATP production, lactate release, and glutamate uptake following OGD/R. The presence of a NO donor also abolished the beneficial effects of guanosine or L-NAME on bioenergetics and glutamate uptake. These results showed, for the first time, that guanosine may regulate cellular bioenergetics in hippocampal slices subjected to OGD/R injury by a mechanism that involves the modulation of NO levels.

New Probucol Analogues Inhibit Ferroptosis, Improve Mitochondrial Parameters, and Induce Glutathione Peroxidase in HT22 Cells.

Probucol, a hypocholesterolemic compound, is neuroprotective in several models of neurodegenerative diseases but has serious adverse effects in vivo. We now describe the design and synthesis of two new probucol analogues that protect against glutamate-induced oxidative cell death, also known as ferroptosis, in cultured mouse hippocampal (HT22) cells and in primary cortical neurons, while probucol did not show any protective effect. Treatment with both compounds did not affect glutathione depletion but still significantly decreased glutamate-induced production of oxidants, mitochondrial superoxide generation, and mitochondrial hyperpolarization in HT22 cells. Both compounds increase glutathione peroxidase (GPx) 1 levels and GPx activity, also exhibiting protection against RSL3, a GPx4 inactivator. These two compounds are therefore potent activators of GPx activity making further studies of their neuroprotective activity in vivo worthwhile.

Piper nigrum ethanolic extract rich in piperamides causes ROS overproduction, oxidative damage in DNA leading to cell cycle arrest and apoptosis in cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ayurvedic and Chinese traditional medicine and tribal people use herbal preparations containing Piper nigrum fruits for the treatment of many health disorders like inflammation, fever, asthma and cancer. In Brazil, traditional maroon culture associates the spice Piper nigrum to health recovery and inflammation attenuation. AIMS OF THE STUDY: The aim of the current work was to evaluate the relationship between reactive oxygen species (ROS) overproduction, DNA fragmentation, cell cycle arrest and apoptosis induced by Piper nigrum ethanolic extract and its antitumor activity. METHODS: The plant was macerated in ethanol. Extract constitution was assessed by TLC, UV-vis and ESI-IT-MS/MS spectrometry. The cytotoxicity, proliferation and intracellular ROS generation was evaluated in MCF-7 cells. DNA damage effects were evaluated through intercalation into CT-DNA, plasmid DNA cleavage and oxidative damage in CT-DNA. Tumor growth inhibition, survival time increase, apoptosis, cell cycle arrest and oxidative stress were assessed in Ehrlich ascites carcinoma-bearing mice. RESULTS: Extraction yielded 64mg/g (36% piperine and 4.2% piperyline). Treatments caused DNA damage and reduced cell viability (EC50=27.1±2.0 and 80.5±6.6µg/ml in MCF-7 and HT-29 cells, respectively), inhibiting cell proliferation by 57% and increased ROS generation in MCF-7 cells (65%). Ehrlich carcinoma was inhibited by the extract, which caused reduction of tumor growth (60%), elevated survival time (76%), cell cycle arrest and induced apoptosis. The treatment with extract increased Bax and p53 and inhibited Bcl-xL and cyclin A expression. It also induced an oxidative stress in vivo verified as enhanced lipid peroxidation and carbonyl proteins content and increased activities of glutathione reductase, superoxide dismutase and catalase. GSH concentration was decreased in tumor tissue from mice. CONCLUSION: The ethanolic extract has cytotoxic and antiproliferative effect on MCF-7 cells and antitumor effect in vivo probably due to ROS overproduction that induced oxidative stress affecting key proteins involved in cell cycle arrest at G1/S and triggering apoptosis. Finally, the overall data from this study are well in line with the traditional claims for the antitumor effect of Piper nigrum fruits.