Parthenolide induces rapid thiol oxidation that leads to ferroptosis in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is both a devastating and common disease. Every year in the United States, about 24,500 men and 10,000 women are diagnosed with HCC, and more than half of those diagnosed patients die from this disease. Thus far, conventional therapeutics have not been successful for patients with HCC due to various underlying comorbidities. Poor survival rate and high incidence of recurrence after therapy indicate that the differences between the redox environments of normal surrounding liver and HCC are valuable targets to improve treatment efficacy. Parthenolide (PTL) is a naturally found therapeutic with anti-cancer and anti-inflammatory properties. PTL can alter HCC’s antioxidant environment through thiol modifications leaving tumor cells sensitive to elevated reactive oxygen species (ROS). Investigating the link between altered thiol mechanism and increased sensitivity to iron-mediated lipid peroxidation will allow for improved treatment of HCC. HepG2 (human) and McARH7777 (rat) HCC cells treated with PTL with increasing concentrations decrease cell viability and clonogenic efficiency in vitro. PTL increases glutathione (GSH) oxidation rescued by the addition of a GSH precursor, N-acetylcysteine (NAC). In addition, this elevation in thiol oxidation results in an overall increase in mitochondrial dysfunction. To elucidate if cell death is through lipid peroxidation, using a lipid peroxidation sensor indicated PTL increases lipid oxidation levels after 6 h. Additionally, western blotting reveals glutathione peroxidase 4 (GPx4) protein levels decrease after treatment with PTL suggesting cells are incapable of preventing lipid peroxidation after exposure to PTL. An elevation in lipid peroxidation will lead to a form of cell death known as ferroptosis. To further establish ferroptosis as a critical mechanism of death for HCC in vitro, the addition of ferrostatin-1 combined with PTL demonstrates a partial recovery in a colony survival assay. This study reveals that PTL can induce tumor cell death through elevations in intracellular oxidation, leaving cells sensitive to ferroptosis

The Polyamine Inhibitor Alpha-Difluoromethylornithine Modulates Hippocampus-Dependent Function after Single and Combined Injuries

Exposure to uncontrolled irradiation in a radiologic terrorism scenario, a natural disaster or a nuclear battlefield, will likely be concomitantly superimposed on other types of injury, such as trauma. In the central nervous system, radiation combined injury (RCI) involving irradiation and traumatic brain injury may have a multifaceted character. This may entail cellular and molecular changes that are associated with cognitive performance, including changes in neurogenesis and the expression of the plasticity-related immediate early gene Arc. Because traumatic stimuli initiate a characteristic early increase in polyamine metabolism, we hypothesized that treatment with the polyamine inhibitor alpha-difluoromethylornithine (DFMO) would reduce the adverse effects of single or combined injury on hippocampus structure and function. Hippocampal dependent cognitive impairments were quantified with the Morris water maze and showed that DFMO effectively reversed cognitive impairments after all injuries, particularly traumatic brain injury. Similar results were seen with respect to the expression of Arc protein, but not neurogenesis. Given that polyamines have been found to modulate inflammatory responses in the brain we also assessed the numbers of total and newly born activated microglia, and found reduced numbers of newly born cells. While the mechanisms responsible for the improvement in cognition after DFMO treatment are not yet clear, the present study provides new and compelling data regarding the potential use of DFMO as a potential countermeasure against the adverse effects of single or combined injury

Representative images of Golgi-impregnated cells in the mouse hippocampus.

<p>(A) A dentate gyrus granule neuron and (B) a CA1 pyramidal neuron illustrating basal dendrites in the stratum oriens and apical dendrites in the stratum radiatum. Scale bar = 50 µm.</p

Irradiation affects dendritic spine morphology in the hippocampal DG and CA1 area.

<p>Brain irradiation induced time and region specific changes in the proportion of spine morphological sub types in the DG granule neurons as well as pyramidal neurons from the CA1 region. The open bars represent unirradiated animals and the dark bars represent mice irradiated with 10 Gy of gamma rays. Each bar represents the mean value of 5 animals and error bars are SEM. *represents a p<0.05, and **represents p<0.001.</p

Spatial memory retention in the probe trial following the first day of hidden platform training.

<p>*<i>p</i><0.05 or smaller versus any other quadrant. For details about the overall effect of quadrant and posthoc tests in individual groups, see text. Number of mice per experimental group: Control: n = 7; TBI: n = 8, Irradiation: n = 8, TBI/Irradiation: n = 8; DFMO control: n = 6; DFMO/TBI: n = 6; DFMO/Irradiation: 5; DFMO/TBI/Irradiation: n = 6.</p

Experimental time line.

<p>Two-month-old C57BL/6 mice received whole body irradiation (4 Gy) and immediately after (∼15 min) received either controlled cortical contusion injury or sham craniotomy. Two weeks after injury animals were injected daily for 5 days with BrdU (100 mg/kg). DFMO was made available in the drinking water immediately upon recovery from surgery and for a total of 42 days and was stopped two days before cognitive testing in the Morris water maze. Animals were euthanized 30 minute after the last probe trial.</p

Percentages of hippocampal granule cell expressing Arc protein 30 min after the last probe trail of the Morris water maze.

<p>Two-way ANOVA revealed a significant interaction between drug treatment (DFMO vs vehicle) and injury (trauma, radiation, RCI). Animals treated with vehicle after trauma or RCI showed a significant reduction in the percentage of neurons expressing Arc relative to sham treated animals (#p<0.009, * p<0.02). There was no difference in the percentage of neurons expressing Arc between the different injury groups in the DFMO treated animals. Each treatment group consisted of 8–11 mice. Each bar represents a mean value and error bars are SEM.</p

Total number of BrdU positive cells in the dentate subgranular zone.

<p>There was a significant interaction between drug treatment and injury. RCI animals treated with vehicle showed a significantly higher number of total BrdU compared to sham control animal (*p<0.05). In animals treated with DFMO there was no differences in the total number of BrdU positive cells among the different injury group. Each treatment group consisted of 8–11 mice. Each bar represents a mean value and error bars are SEM.</p

Percentage of mice that spent more time in the target quadrant than any other quadrant.

<p>For details, see text. Number of mice per experimental group: Control: n = 7; TBI: n = 8, Irradiation: n = 8, TBI/Irradiation: n = 8; DFMO control: n = 6; DFMO/TBI: n = 6; DFMO/Irradiation: 5; DFMO/TBI/Irradiation: n = 6.</p

Total number of BrdU/NeuN positive neurons per mm² in the dentate subgranular zone.

<p>Overall, there was no significant interaction between treatment and drug and no effect of the treatment alone or drug alone. In control mice treated with vehicle trauma alone induced no changes while radiation alone decreased the number of BrdU+/NeuN+ cells in the ipsilateral hemisphere. Animals from the RCI group showed an increase in the numbers of newly born neurons compared to radiation alone. Each treatment group consisted of 8–11 mice. Each bar represents a mean value and error bars are SEM.</p