Correction: Reddy et al. Rlip Reduction Induces Oxidative Stress and Mitochondrial Dysfunction in Mutant Tau-Expressed Immortalized Hippocampal Neurons: Mechanistic Insights. Cells 2023, 12, 1646.

The authors wish to make the following changes to their paper [...].

The role of RLIP76 in oxidative stress and mitochondrial dysfunction: Evidence based on autopsy brains from Alzheimer's disease patients.

Several converging lines of evidence from our group support a potential role of RLIP76 (AKA Rlip) in neurodegenerative disorders, including Alzheimer's Disease (AD). However, the role of Rlip in Alzheimer's and other neurodegenerative diseases is not well understood. The purpose of the present study is to determine the role of Rlip in the brains of AD patients and control subjects. To achieve our goals, we used frozen tissues and formalin-fixed paraffin-embedded postmortem brains from AD patients of different Braak stages and age-matched control subjects. Our immunohistology and immunoblotting blotting analysis revealed that expression of Rlip protein gradually and significantly decreased (p = 0.0001) with AD progression, being lowest in Braak stage IV-V. Rlip was colocalized with Amyloid beta (Aß) and phosphorylated tau (p-Tau) as observed by IHC staining and co-immunoprecipitation studies. Lipid peroxidation (4-HNE generation) and H2O2 production were significantly higher (p = 0.004 and 0.0001 respectively) in AD patients compared to controls, and this was accompanied by lower ATP production in AD (p = 0.0009). Oxidative DNA damage was measured by 8-Hydroxyguanosine (8-OHdG) in tissue lysates by ELISA and COMET assay. AD 8-OHdG levels were significantly higher (p = 0.0001) compared to controls. COMET assay was performed in brain cells, isolated from frozen postmortem samples. The control samples showed minimal DNA in comets representing few DNA strand breaks (<20 %), (score-0-1). However, the AD group showed an average of 50 % to 65 % of DNA in comet tails (score-4-5) indicating numerous DNA strand breaks. The difference between the two groups was significant (p = 0.001), as analyzed by Open Comet by ImageJ. Elevated DNA damage was further examined by western blot analysis for phosphorylated histone variant H2AX (γH2AX). Induction of γH2AX was very significant (p < 0.0001) and confirmed the presence of double-strand breaks in DNA. Overall, our results indicate an important role for Rlip in maintaining neuronal health and homeostasis by suppressing cellular oxidative stress and DNA damage. Based on our findings, we cautiously conclude that Rlip is a promising therapeutic target for Alzheimer's disease.

Leveraging cryoablation and checkpoint inhibitors for high-risk triple negative breast cancer.

Breast cancer is the second most common cancer among women in the United States in which the standard of care treatment is surgery with adjunctive therapy. Cryoablation, which destroys the tumor using extremely cold temperatures while preserving the potential tumor antigens, is a promising alternative to surgical resection. It is less invasive, cosmetically appeasing, cost-effective, and capable of contributing to the abscopal effect - the immune response targeting potential distant metastasis. However, to maximize the immunologic benefit of cryoablation in biologically high-risk breast cancers, combination with therapies that enhance immune activation, such as immune checkpoint inhibitors (ICIs) may be necessary. This mini review describes the fundamentals of cryoablation and treatment with ICIs, as well as discuss the caveats in both strategies and current clinical trials aimed to improve this approach to benefit patients.

Rlip Reduction Induces Oxidative Stress and Mitochondrial Dysfunction in Mutant Tau-Expressed Immortalized Hippocampal Neurons: Mechanistic Insights.

RalBP1 (Rlip) is a stress-activated protein that is believed to play a large role in aging and neurodegenerative diseases such as Alzheimer's disease (AD) and other tauopathies. The purpose of our study was to understand the role of Rlip in mutant Tau-expressed immortalized hippocampal HT22 cells. In the current study, we used mutant Tau (mTau)-expressed HT22 neurons and HT22 cells transfected with Rlip-cDNA and/or silenced RNA, and studied the cell survival, mitochondrial respiration, mitochondrial function, immunoblotting, and immunofluorescence analysis of synaptic and mitophagy proteins and the colocalization of Rlip and mTau proteins. We found Rlip protein levels were reduced in mTau-HT22 cells, Rlip silenced HT22 cells, and mTau + Rlip RNA silenced HT22 cells; on the other hand, increased Rlip levels were observed in Rlip cDNA transfected HT22 cells. We found cell survival was decreased in mTau-HT22 cells and RNA-silenced HT22 cells. However, cell survival was increased in Rlip-overexpressed mTau-HT22 cells. A significantly reduced oxygen consumption rate (OCR) was found in mTau-HT22 cells and in RNA-silenced Rlip-HT22 cells, with an even greater reduction in mTau-HT22 + Rlip RNA-silenced HT22 cells. A significantly increased OCR was found in Rlip-overexpressed HT22 cells and in all groups of cells that overexpress Rlip cDNA. Mitochondrial function was defective in mTau-HT22 cells, RNA silenced Rlip in HT22 cells, and was further defective in mTau-HT22 + Rlip RNA-silenced HT22 cells; however, it was rescued in Rlip overexpressed in all groups of HT22 cells. Synaptic and mitophagy proteins were decreased in mTau-HT22 cells, and further reductions were found in RNA-silenced mTau-HT22 cells. However, these were increased in mTau + Rlip-overexpressed HT22 cells. An increased number of mitochondria and decreased mitochondrial length were found in mTau-HT22 cells. These were rescued in Rlip-overexpressed mTau-HT22 cells. These observations strongly suggest that Rlip deficiency causes oxidative stress/mitochondrial dysfunction and Rlip overexpression reverses these defects. Overall, our findings revealed that Rlip is a promising new target for aging, AD, and other tauopathies/neurological diseases.

Rlip overexpression reduces oxidative stress and mitochondrial dysfunction in Alzheimer's disease: Mechanistic insights.

Alzheimer's disease (AD) is a neurodegenerative disease that affects a large proportion of the aging population. RalBP1 (Rlip) is a stress-activated protein that plays a crucial role in oxidative stress and mitochondrial dysfunction in aging and neurodegenerative diseases but its precise role in the progression of AD is unclear. The purpose of our study is to understand the role of Rlip in the progression and pathogenesis of AD in mutant APP/amyloid beta (Aß)-expressed mouse primary hippocampal (HT22) hippocampal neurons. In the current study, we used HT22 neurons that express mAPP, transfected with Rlip-cDNA and/or RNA silenced, and studied cell survival, mitochondrial respiration, mitochondrial function, immunoblotting & immunofluorescence analysis of synaptic and mitophagy protein's and colocalization of Rlip and mutant APP/Aß proteins and mitochondrial length and number. We also assessed Rlip levels in autopsy brains from AD patients and control subjects. We found cell survival was decreased in mAPP-HT22 cells and RNA-silenced HT22 cells. However, cell survival was increased in Rlip-overexpressed mAPP-HT22 cells. Oxygen consumption rate (OCR) was decreased in mAPP-HT22 cells and RNA-silenced Rlip-HT22 cells. OCR was increased in Rlip-overexpressed in mAPP-HT22 cells. Mitochondrial function was defective in mAPP-HT22 cells and RNA silenced Rlip in HT22 cells, however, it was rescued in Rlip overexpressed mAPP-HT22 cells. Synaptic and mitophagy proteins were decreased in mAPP-HT22 cells, further reducing RNA-silenced Rlip-HT22 cells. However, these were increased in mAPP+Rlip-HT22 cells. Colocalization analysis revealed Rlip is colocalized with mAPP/Aß. An increased number of mitochondria and decreased mitochondrial length were found in mAPP-HT22 cells. These were rescued in Rlip overexpressed mAPP-HT22 cells. Reduced Rlip levels were found in autopsy brains from AD patients. These observations strongly suggest that Rlip deficiency causes oxidative stress/mitochondrial dysfunction and Rlip overexpression reduced these defects.

Effects of proton and oxygen ion irradiation on cardiovascular function and structure in a rabbit model.

PURPOSE: Astronauts on missions beyond low Earth orbit will be exposed to galactic cosmic radiation, and there is concern about potential adverse cardiovascular effects. Most of the research to identify cardiovascular risk of space radiation has been performed in rodent models. To aid in the translation of research results to humans, the current study identified long-term effects of high-energy charged particle irradiation on cardiovascular function and structure in a larger non-rodent animal model. MATERIALS AND METHODS: At the age of 12 months, male New Zealand white rabbits were exposed to whole-body protons (250 MeV) or oxygen ions (16O, 600 MeV/n) at a dose of 0 or 0.5 Gy and were followed for 12 months after irradiation. Ultrasonography was used to measure in vivo cardiac function and blood flow parameters at 10- and 12-months post-irradiation. At 12 months after irradiation, blood cell counts and blood chemistry values were assessed, and cardiac tissue and aorta were collected for histological as well as molecular and biochemical analyses. Plasma was used for metabolomic analysis and to quantify common markers of cardiac injury. RESULTS: A small but significant decrease in the percentage of circulating lymphocytes and an increase in neutrophil percentage was seen 12 months after 0.5 Gy protons, while 16O exposure resulted in an increase in monocyte percentage. Markers of cardiac injury, cardiac troponin I (cTnI) and N-Terminal pro-B-type Natriuretic Peptide were modestly increased in the proton group, and cTnI was also increased after 16O. On the other hand, metabolomics on plasma at 12 months revealed no changes. Both types of irradiation demonstrated alterations in cardiac mitochondrial morphology and an increase in left ventricular protein levels of inflammatory cell marker CD68. However, changes in cardiac function were only mild. CONCLUSION: Low dose charged particle irradiation caused mild long-term changes in inflammatory markers, cardiac function, and structure in the rabbit heart, in line with previous studies in mouse and rat models.

Rlip76: An Unexplored Player in Neurodegeneration and Alzheimer's Disease?

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and is the most common cause of dementia in older people. AD is associated with the loss of synapses, oxidative stress, mitochondrial structural and functional abnormalities, microRNA deregulation, inflammatory responses, neuronal loss, accumulation of amyloid-beta (Aß) and phosphorylated tau (p-tau). AD occurs in two forms: early onset, familial AD and late-onset, sporadic AD. Causal factors are still unknown for a vast majority of AD patients. Genetic polymorphisms are proposed to contribute to late-onset AD via age-dependent increases in oxidative stress and mitochondrial abnormalities. Recent research from our lab revealed that reduced levels of Rlip76 induce oxidative stress, mitochondrial dysfunction and synaptic damage, leading to molecular and behavioral phenotypes resembling late-onset AD. Rlip76 is a multifunctional 76 kDa protein encoded by the RALBP1 gene, located on chromosome 18. Rlip is a stress-protective ATPase of the mercapturic acid pathway that couples clathrin-dependent endocytosis with the efflux of glutathione-electrophile conjugates. Rlip is evolutionarily highly conserved across species and is ubiquitously expressed in all tissues, including AD-affected brain regions, the cerebral cortex and hippocampus, where highly active neuronal metabolisms render the cells highly susceptible to intracellular oxidative damage. In the current article, we summarize molecular and cellular features of Rlip and how depleted Rlip may exacerbate oxidative stress, mitochondrial dysfunction and synaptic damage in AD. We also discuss the possible role of Rlip in aspects of learning and memory via axonal growth, dendritic remodeling, and receptor regulation. We conclude with a discussion of the potential for the contribution of genetic polymorphisms in Rlip to AD progression and the potential for Rlip-based therapies.

Rlip Depletion Alters Oncogene Transcription at Multiple Distinct Regulatory Levels.

Rlip76 (Rlip) is a multifunctional membrane protein that facilitates the high metabolic rates of cancer cells through the efflux of toxic metabolites and other functions. Rlip inhibition or depletion results in broad-spectrum anti-cancer effects in vitro and in vivo. Rlip depletion effectively suppresses malignancy and causes global reversion of characteristic CpG island methylomic and transcriptomic aberrations in the p53-null mouse model of spontaneous carcinogenesis through incompletely defined signaling and transcriptomic mechanisms. The methylome and transcriptome are normally regulated by the concerted actions of several mechanisms that include chromatin remodeling, promoter methylation, transcription factor interactions, and miRNAs. The present studies investigated the interaction of Rlip depletion or inhibition with the promoter methylation and transcription of selected cancer-related genes identified as being affected by Rlip depletion in our previous studies. We constructed novel promoter CpG island/luciferase reporter plasmids that respond only to CpG methylation and transcription factors. We found that Rlip depletion regulated expression by a transcription factor-based mechanism that functioned independently of promoter CpG methylation, lipid peroxidation, and p53 status.

Rlip Protein: A Potential Target for COVID-19.

On January 30, 2020, the COVID-19 epidemic was declared an international public health emergency by the World Health Organization. Given the growing impact of the pandemic, there is great interest in finding potential targets for treating infected or hospitalized COVID-19 patients. Therapeutic studies have been conducted on pre-existing drugs, which vary by country, including anti-malarial agents, antiviral agents, and convalescent plasma. However, many of these agents are ineffective at reducing mortality or only shorten the severity or duration of COVID-19 illness in hospitalized patients. As such, other alternatives for treating COVID-19 are being investigated. One such target of interest has been clathrin-dependent endocytosis (CDE). Clathrin-dependent endocytosis is the most commonly observed mechanism of viral entry into cells. However, there have been no published studies to date on CDE inhibition strategies against COVID-19. One such target is Rlip or RLIP76 (human gene RALBP1, 18p11.22). Among its many functions, Rlip is a stress-protective, Ral-regulated ATPase of the mercapturic acid pathway that transports glutathione-electrophile conjugates of electrophilic toxins, which are precursors of mercapturic acid that precedes de-glutamylation by gamma-glutamyl transferase. Rlip is also regulated by several G-proteins that coordinate movement of cells, organelles, membranes, cytoskeleton, macromolecules, and other small molecules. Previous studies have link Rlip in the pathogenesis of several viral illness. In this paper, we want to propose that RLIP76 (Rlip or RALBP1) may be a novel target for treating SARS-CoV-2 viral infections.

Anticancer Activity of Ω-6 Fatty Acids through Increased 4-HNE in Breast Cancer Cells.

Her2-amplified breast cancers resistant to available Her2-targeted therapeutics continue to be a challenge in breast cancer therapy. Dox is the mainstay of chemotherapy of all types of breast cancer, but its usefulness is limited by cumulative cardiotoxicity. Because oxidative stress caused by dox generates the pro-apoptotic Ω-6 PUFA metabolite 4-hydroxynonenal (4-HNE), we surmised that Ω-6 PUFAs would increase the effectiveness of dox chemotherapy. Since the mercapturic acid pathway enzyme RALBP1 (also known as RLIP76 or Rlip) that limits cellular accumulation of 4-HNE also mediates dox resistance, the combination of Ω-6 PUFAs and Rlip depletion could synergistically improve the efficacy of dox. Thus, we studied the effects of the Ω-6 PUFA arachidonic acid (AA) and Rlip knockdown on the antineoplastic activity of dox towards Her2-amplified breast cancer cell lines SK-BR-3, which is sensitive to Her2 inhibitors, and AU565, which is resistant. AA increased lipid peroxidation, 4-HNE generation, apoptosis, cellular dox concentration and dox cytotoxicity in both cell lines while sparing cultured immortalized cardiomyocyte cells. The known functions of Rlip including clathrin-dependent endocytosis and dox efflux were inhibited by AA. Our results support a model in which 4-HNE generated by AA overwhelms the capacity of Rlip to defend against apoptosis caused by dox or 4-HNE. We propose that Ω-6 PUFA supplementation could improve the efficacy of dox or Rlip inhibitors for treating Her2-amplified breast cancer.

RALBP1 in Oxidative Stress and Mitochondrial Dysfunction in Alzheimer's Disease.

The purpose of our study is to understand the role of the RALBP1 gene in oxidative stress (OS), mitochondrial dysfunction and cognition in Alzheimer's disease (AD) pathogenesis. The RALPB1 gene encodes the 76 kDa protein RLIP76 (Rlip). Rlip functions as a stress-responsive/protective transporter of glutathione conjugates (GS-E) and xenobiotic toxins. We hypothesized that Rlip may play an important role in maintaining cognitive function. The aim of this study is to determine whether Rlip deficiency in mice is associated with AD-like cognitive and mitochondrial dysfunction. Brain tissue obtained from cohorts of wildtype (WT) and Rlip+/- mice were analyzed for OS markers, expression of genes that regulate mitochondrial fission/fusion, and synaptic integrity. We also examined mitochondrial ultrastructure in brains obtained from these mice and further analyzed the impact of Rlip deficiency on gene networks of AD, aging, stress response, mitochondrial function, and CREB signaling. Our studies revealed a significant increase in the levels of OS markers and alterations in the expression of genes and proteins involved in mitochondrial biogenesis, dynamics and synapses in brain tissues from these mice. Furthermore, we compared the cognitive function of WT and Rlip+/- mice. Behavioral, basic motor and sensory function tests in Rlip+/- mice revealed cognitive decline, similar to AD. Gene network analysis indicated dysregulation of stress-activated gene expression, mitochondrial function and CREB signaling genes in the Rlip+/- mouse brain. Our results suggest that Rlip deficiency-associated increases in OS and mitochondrial dysfunction could contribute to the development or progression of OS-related AD processes.

Dietary supplementation with sulforaphane ameliorates skin aging through activation of the Keap1-Nrf2 pathway.

Visible impairments in skin appearance, as well as a subtle decline in its functionality at the molecular level, are hallmarks of skin aging. Activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-pathway, which is important in controlling inflammation and oxidative stress that occur during aging, can be triggered by sulforaphane (SFN), an isothiocyanate found in plants from the Brassicaceae family. This study aimed to assess the effects of SFN intake on age-related skin alterations. Male C57BL6 young (2 months) and old (21 months) mice were treated for 3 months with SFN diet (442.5 mg per kg) or control diet. The antioxidant capacities of the skin were increased in old SFN-treated animals as measured by mRNA levels of Nrf2 (P<.001) and its target genes NQO1 (P<.001) and HO1 (P<.01). Protein expression for Nrf2 was also increased in old SFN fed animals (P<.01), but not the protein expression of NQO1 or HO1. Additionally, ROS and MMP9 protein levels were significantly decreased (P<.05) in old SFN fed animals. Histopathological analysis confirmed that there was no difference in epidermal thickness in old, when compared to young, SFN treated animals, while the dermal layer thickness was lower in old vs. young, treated animals (P<.05). Moreover, collagen deposition was improved with SFN treatment in young (P<.05) and structurally significantly improved in the old mice (P<.001). SFN dietary supplementation therefore ameliorates skin aging through activation of the Nrf2-pathway.

Haploinsufficiency Interactions between RALBP1 and p53 in ERBB2 and PyVT Models of Mouse Mammary Carcinogenesis.

We recently reported that loss of one or both alleles of Ralbp1, which encodes the stress-protective protein RLIP76 (Rlip), exerts a strong dominant negative effect on both the inherent cancer susceptibility and the chemically inducible cancer susceptibility of mice lacking one or both alleles of the tumor suppressor p53. In this paper, we examined whether congenital Rlip deficiency could prevent genetically-driven breast cancer in two transgenic mouse models: the MMTV-PyVT model, which expresses the polyomavirus middle T antigen (PyVT) under control of the mouse mammary tumor virus promoter (MMTV) and the MMTV-Erbb2 model which expresses MMTV-driven erythroblastic leukemia viral oncogene homolog 2 (Erbb2, HER2/Neu) and frequently acquires p53 mutations. We found that loss of either one or two Rlip alleles had a suppressive effect on carcinogenesis in Erbb2 over-expressing mice. Interestingly, Rlip deficiency did not affect tumor growth but significantly reduced the lung metastatic burden of breast cancer in the viral PyVT model, which does not depend on either Ras or loss of p53. Furthermore, spontaneous tumors of MMTV-PyVT/Rlip+/+ mice showed no regression following Rlip knockdown. Finally, mice lacking one or both Rlip alleles differentially expressed markers for apoptotic signaling, proliferation, angiogenesis, and cell cycling in PyVT and Erbb2 breast tumors. Our results support the efficacy of Rlip depletion in suppressing p53 inactivated cancers, and our findings may yield novel methods for prevention or treatment of cancer in patients with HER2 mutations or tumor HER2 expression.

Sulforaphane prevents age-associated cardiac and muscular dysfunction through Nrf2 signaling.

Age-associated mitochondrial dysfunction and oxidative damage are primary causes for multiple health problems including sarcopenia and cardiovascular disease (CVD). Though the role of Nrf2, a transcription factor that regulates cytoprotective gene expression, in myopathy remains poorly defined, it has shown beneficial properties in both sarcopenia and CVD. Sulforaphane (SFN), a natural compound Nrf2-related activator of cytoprotective genes, provides protection in several disease states including CVD and is in various stages of clinical trials, from cancer prevention to reducing insulin resistance. This study aimed to determine whether SFN may prevent age-related loss of function in the heart and skeletal muscle. Cohorts of 2-month-old and 21- to 22-month-old mice were administered regular rodent diet or diet supplemented with SFN for 12 weeks. At the completion of the study, skeletal muscle and heart function, mitochondrial function, and Nrf2 activity were measured. Our studies revealed a significant drop in Nrf2 activity and mitochondrial functions, together with a loss of skeletal muscle and cardiac function in the old control mice compared to the younger age group. In the old mice, SFN restored Nrf2 activity, mitochondrial function, cardiac function, exercise capacity, glucose tolerance, and activation/differentiation of skeletal muscle satellite cells. Our results suggest that the age-associated decline in Nrf2 signaling activity and the associated mitochondrial dysfunction might be implicated in the development of age-related disease processes. Therefore, the restoration of Nrf2 activity and endogenous cytoprotective mechanisms by SFN may be a safe and effective strategy to protect against muscle and heart dysfunction due to aging.

Multi-Omic Analysis Reveals Different Effects of Sulforaphane on the Microbiome and Metabolome in Old Compared to Young Mice.

Dietary factors modulate interactions between the microbiome, metabolome, and immune system. Sulforaphane (SFN) exerts effects on aging, cancer prevention and reducing insulin resistance. This study investigated effects of SFN on the gut microbiome and metabolome in old mouse model compared with young mice. Young (6-8 weeks) and old (21-22 months) male C57BL/6J mice were provided regular rodent chow ± SFN for 2 months. We collected fecal samples before and after SFN administration and profiled the microbiome and metabolome. Multi-omics datasets were analyzed individually and integrated to investigate the relationship between SFN diet, the gut microbiome, and metabolome. The SFN diet restored the gut microbiome in old mice to mimic that in young mice, enriching bacteria known to be associated with an improved intestinal barrier function and the production of anti-inflammatory compounds. The tricarboxylic acid cycle decreased and amino acid metabolism-related pathways increased. Integration of multi-omic datasets revealed SFN diet-induced metabolite biomarkers in old mice associated principally with the genera, Oscillospira, Ruminococcus, and Allobaculum. Collectively, our results support a hypothesis that SFN diet exerts anti-aging effects in part by influencing the gut microbiome and metabolome. Modulating the gut microbiome by SFN may have the potential to promote healthier aging.

Effects of single-dose protons or oxygen ions on function and structure of the cardiovascular system in male Long Evans rats.

PURPOSE: Studies are required to determine whether exposures to radiation encountered during manned missions in deep space may have adverse effects on the cardiovascular system. Most of the prior studies on effects of simulated space radiation on the heart and vasculature have been performed in mouse models. To provide data from a second animal species, two studies were performed to assess effects of high-energy charged particle radiation on the heart and abdominal aorta in a rat model. MATERIALS AND METHODS: In study A, male Long Evans rats were exposed to whole-body protons (250 MeV, 0.5 Gy) or oxygen ions (16O, 600 MeV/n, 0.5 Gy), and ultrasonography was used to measure in vivo cardiac function and blood flow parameters at 3, 5, 9 and 12 months after radiation, followed by tissue collection at 12 months. In study B, male Long Evans rats were exposed to 16O (1 GeV/n, 0.01-0.25 Gy), and hearts collected at 6 to 7 and 12 months for histology and western-blots. RESULTS: Both protons (250 MeV) and 16O (600 MeV/n) caused a decrease in left ventricular posterior wall thickness at 3-5 months, but did not change echocardiographic measures of cardiac function. In Pulsed-wave Doppler assessment of the abdominal aorta, an increase was seen in mean velocity, peak velocity, and velocity time integral at 12 months after 16O (600 MeV/n), suggesting a change in vascular function. There were no significant changes in histopathology or histological quantification of total collagens in heart or aorta. On the other hand, an increase was seen in a 75 kDa peptide of collagen type III in the left ventricle of rats exposed to protons (250 MeV) and 16O (600 MeV/n and 1 GeV/n), suggesting that radiation caused remodeling of existing collagens in the heart. 16O (600 MeV/n and 1 GeV/n) caused increases in left ventricular protein levels of immune cell markers CD2, CD4, CD8, and CD68. CONCLUSION: A single low dose of whole body protons or 16O in male Long Evans rats did not change cardiac function or induce gross pathological changes in the heart or aorta, but induced mild changes in vascular function and remodeling of existing collagens in the heart. Altogether, studies in prior mouse models and the current work in rats indicate minor changes in cardiac function and structure after a low dose of single-ion radiation.

Rlip Depletion Suppresses Growth of Breast Cancer.

RLIP76 (RAL-binding protein-1, Rlip) is a stress-protective mercapturic-acid-pathway transporter protein that also plays a key role in regulating clathrin-dependent endocytosis as a Ral effector. Targeted inhibition or depletion of Rlip causes regression of xenografts of many cancers and is capable of abrogating tumor formation in p53-null mice. This is associated with the reversion of the abnormal methylomic profile of p53-null mice to wild-type. In a query of The Cancer Genome Atlas (TCGA) databases, we found that Rlip expression was associated with poor survival and with significant differences in the frequencies of PIK3CA mutation, MYC amplification, and CDKN2A/B deletion, which were the most commonly mutated, amplified, and deleted genes, respectively, among TCGA breast cancer patients. We conducted the present study to further examine the effects of Rlip inhibition and to evaluate the in vitro and in vivo efficacy in breast cancer. Using immunogold electron microscopy, we found that plasma-membrane Rlip was accessible to cell-surface antibodies in the MCF7 (ER+) breast cancer cell line. Rlip depletion resulted in decreased survival of MCF7 and MDA-MB-231 cells and increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity and DNA laddering, indicating apoptotic cell death. Additionally, in vitro knockdown of Rlip inhibited EGF endocytosis and WNT/MAPK signaling. Xenograft studies in nude mice showed regression of breast cancer via antisense-mediated depletion of Rlip mRNA as well as by anti-Rlip antibody. Finally, knockdown of Rlip by antisense locked nucleic acid oligonucleotides increased markers for apoptotic signaling and decreased markers for proliferation, angiogenesis, and cell cycling in MCF7 and MDA-MB-231luc xenografts. Our findings validate Rlip as an attractive target in breast cancer.

Topical 2'-Hydroxyflavanone for Cutaneous Melanoma.

2'-hydroxyflavanone (2HF) is a dietary flavonoid with anticancer activity towardsmultiple cancers. Here, we report that topically applied 2HF inhibits the growth of intradermalimplants of melanoma in immunocompetent mice. 2HF induced apoptosis and inhibited the growthof the human SK-MEL-24 as well as murine B16-F0 and B16-F10 melanoma cell lines in vitro.Apoptosis was associated with depletion of caspase-3, caspase-9, and PARP1 in B16-F0 and SKMEL-24 cells. Caspase-9 and MEKK-15 were undetected even in untreated B16-F10 cells. Signalingproteins TNFα, and phospho-PDGFR-ß were depleted in all three cell lines; MEKK-15 was depletedby 2HF in SK-MEL-24 cells. 2HF enhanced sunitinib (an MEK and PDGFR-ß inhibitor) and AZD2461 (a PARP1 inhibitor) cytotoxicity. 2HF also depleted the Ral-regulated, stress-responsive,antiapoptotic endocytic protein RLIP76 (RALBP1), the inhibition of which has previously beenshown to inhibit B16-F0 melanoma growth in vivo. Functional inhibition of RLIP76 was evidentfrom inhibition of epidermal growth factor (EGF) endocytosis by 2HF. We found that topicallyapplied 2HF-Pluronic Lecithin Organogel (PLO) gel inhibited B16-F0 and B16-F10 tumorsimplanted in mice and caused no overt toxicity despite significant systemic absorption. 2HFtreatment reduced phospho-AKT, vimentin, fibronectin, CDK4, cyclinB1, and BCL2, whereas itincreased BIM and phospho-AMPK in excised tumors. Several cancer signals are controlled byendocytosis, a process strongly inhibited by RLIP76 depletion. We conclude that 2HF-PLO gel maybe useful for topical therapy of cutaneous metastases of melanoma and could enhance theantineoplastic effects of sunitinib and PARP1 inhibitors. The mechanism of action of 2HF inmelanoma overlaps with RLI76 inhibitors.

Effects of low-dose oxygen ions and protons on cardiac function and structure in male C57BL/6J mice.

PURPOSE: Astronauts traveling beyond low-Earth orbit will be exposed to high linear-energy transfer charged particles. Because there is concern about the adverse effects of space radiation on the cardiovascular system, this study assessed cardiac function and structure and immune cell infiltration in a mouse model of charged-particle irradiation. MATERIALS AND METHODS: Male C57BL/6 J mice were exposed to oxygen ions (16O, 600 MeV/n at 0.25-0.26 Gy/min to a total dose of 0, 0.05, 0.1, 0.25, or 1 Gy), protons (150 MeV, 0.35-0.55 Gy/min to 0, 0.5, or 1 Gy), or protons (150 MeV, 0.5 Gy) followed by 16O (600 MeV/n, 0.1 Gy). Separate groups of mice received 137Cs γ-rays (1 Gy/min to 0, 0.5, 1, or 3 Gy) as a reference. Cardiac function and blood velocity were measured with ultrasonography at 3, 5, 7, and 9 months after irradiation. At 2 weeks, 3 months, and 9 months, cardiac tissue was collected to assess apoptosis, tissue remodeling, and markers of immune cells. RESULTS: Ejection fraction and fractional shortening decreased at 3 and 7 months after 16O. These parameters did not change in mice exposed to γ-rays, protons, or protons followed by 16O. Each of the radiation exposures caused only small increases in cleaved caspase-3 and numbers of apoptotic nuclei. Changes in the levels of α-smooth muscle cell actin and a 75-kDa peptide of collagen type III in the left ventricle suggested tissue remodeling, but there was no significant change in total collagen deposition at 2 weeks, 3 months, and 9 months. Increases in protein amounts of cluster of differentiation (CD)2, CD68, and CD45 as measured with immunoblots at 2 weeks, 3 months, and 9 months after exposure to protons or 16O alone suggested immune cell infiltration. For type III collagen, CD2 and CD68, the efficacy in inducing protein abundance of CD2, CD68, and CD45 was 16O > protons > γ-rays > protons followed by 16O. CONCLUSIONS: Low-dose, high-energy charged-particle irradiation caused mild changes in cardiac function and tissue remodeling in the mouse.