A 3D printable alloy designed for extreme environments.

Multiprincipal-element alloys are an enabling class of materials owing to their impressive mechanical and oxidation-resistant properties, especially in extreme environments1,2. Here we develop a new oxide-dispersion-strengthened NiCoCr-based alloy using a model-driven alloy design approach and laser-based additive manufacturing. This oxide-dispersion-strengthened alloy, called GRX-810, uses laser powder bed fusion to disperse nanoscale Y2O3 particles throughout the microstructure without the use of resource-intensive processing steps such as mechanical or in situ alloying3,4. We show the successful incorporation and dispersion of nanoscale oxides throughout the GRX-810 build volume via high-resolution characterization of its microstructure. The mechanical results of GRX-810 show a twofold improvement in strength, over 1,000-fold better creep performance and twofold improvement in oxidation resistance compared with the traditional polycrystalline wrought Ni-based alloys used extensively in additive manufacturing at 1,093 °C5,6. The success of this alloy highlights how model-driven alloy designs can provide superior compositions using far fewer resources compared with the 'trial-and-error' methods of the past. These results showcase how future alloy development that leverages dispersion strengthening combined with additive manufacturing processing can accelerate the discovery of revolutionary materials.

Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells.

BACKGROUND: Temozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ. METHODS: Using the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ. RESULTS: Here, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O6-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis. CONCLUSIONS: The understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.

Low-level arsenic causes proteotoxic stress and not oxidative stress.

Prolonged exposure to arsenic has been shown to increase the risk of developing a number of diseases, including cancer and type II diabetes. Arsenic is present throughout the environment in its inorganic forms, and the level of exposure varies greatly by geographical location. The current recommended maximum level of arsenic exposure by the EPA is 10µg/L, but levels>50-1000µg/L have been detected in some parts of Asia, the Middle East, and the Southwestern United States. One of the most important steps in developing treatment options for arsenic-linked pathologies is to understand the cellular pathways affected by low levels of arsenic. Here, we show that acute exposure to non-lethal, low-level arsenite, an environmentally relevant arsenical, inhibits the autophagy pathway. Furthermore, arsenite-induced autophagy inhibition initiates a transient, but moderate ER stress response. Significantly, low-level arsenite exposure does not exhibit an increase in oxidative stress. These findings indicate that compromised autophagy, and not enhanced oxidative stress occurs early during arsenite exposure, and that restoring the autophagy pathway and proper proteostasis could be a viable option for treating arsenic-linked diseases. As such, our study challenges the existing paradigm that oxidative stress is the main underlying cause of pathologies associated with environmental arsenic exposure.

Brusatol overcomes chemoresistance through inhibition of protein translation.

The NRF2 pathway activates a cell survival response when cells are exposed to xenobiotics or are under oxidative stress. Therapeutic activation of NRF2 can also be used prior to insult as a means of disease prevention. However, prolonged expression of NRF2 has been shown to protect cancer cells by inducing the metabolism and efflux of chemotherapeutics, leading to both intrinsic and acquired chemoresistance to cancer drugs. This effect has been termed the "dark side" of NRF2. In an effort to combat this chemoresistance, our group discovered the first NRF2 inhibitor, the natural product brusatol, however the mechanism of inhibition was previously unknown. In this report, we show that brusatol's mode of action is not through direct inhibition of the NRF2 pathway, but through the inhibition of both cap-dependent and cap-independent protein translation, which has an impact on many short-lived proteins, including NRF2. Therefore, there is still a need to develop a new generation of specific NRF2 inhibitors with limited toxicity and off-target effects that could be used as adjuvant therapies to sensitize cancers with high expression of NRF2.

Arsenic Compromises Both p97 and Proteasome Functions.

Exposure to arsenic is a worldwide problem that affects more than 200 million people. The underlying mechanisms of arsenic toxicity have been difficult to ascertain due to arsenic's pleotropic effects. A number of recent investigations have shown that arsenic can compromise protein quality control through the ubiquitin proteasome system (UPS) or the endoplasmic reticulum associated protein degradation (ERAD) pathway. In this article, a link between arsenic and protein quality control is reported. Biochemical and cellular data demonstrate a misregulation of the ATPase cycle of the ATPase associated with various cellular activities (AAA+) chaperone, p97. Interestingly, the loss of p97 activity is due to the increased rate of ATP hydrolysis, which mimics a collection of pathogenic genetic p97 lesions. Cellular studies, using a well characterized reporter of both the proteasome and p97, show the proteasome to also be compromised. This loss of both p97 and proteasome functions can explain the catastrophic protein quality control issues observed in acute, high level arsenic exposures.

Oxidative stress, mammospheres and Nrf2-new implication for breast cancer therapy?

Mammosphere culture of breast cancer cell lines is an important approach used for enrichment of cancer stem cells (CSCs), which exhibit high tumorigenicity and chemoresistance features. Evidence shows that CSCs maintain lower ROS levels due to elevated expression of ROS-scavenging molecules and antioxidative enzymes, which favors the survival of the CSCs and their chemoresistance. The transcription factor NF-E2-related factor 2 (Nrf2) has emerged as the master regulator of cellular redox homeostasis, by up-regulating antioxidant response element (ARE)-bearing genes products. Although Nrf2 has long-term been regarded as a beneficial defense mechanism, accumulating studies have revealed the "dark side" of Nrf2. High constitutive levels of Nrf2 was observed in many types of tumors and cancer cell lines promoting their resistance to chemotherapeutics. In this study, we report a high expression of Nrf2 and its target genes in mammospheres compared to corresponding adherent cells. In MCF-7 and MDA-MB-231 mammmosphere cells, the Nrf2-mediated cellular protective response is significantly elevated which is associated with increased resistance to taxol and anchorage-independent growth. Brusatol, an inhibitor of the Nrf2 pathway, suppressed the protein level of Nrf2 and its target genes, enhanced intracellular ROS and sensitized mammospheres to taxol, and reduced the anchorage-independent growth. These results suggest that mammospheres rely on abnormal up-regulation of Nrf2 to maintain low intracellular ROS levels. Nrf2 inhibitors, such as brusatol, have the potential to be developed into novel adjuvant chemotherapeutic drug combinations in order to combat refractory tumor initiating CSCs.

Molecular mechanisms of Nrf2 regulation and how these influence chemical modulation for disease intervention.

Nrf2 (nuclear factor erytheroid-derived-2-like 2) transcriptional programmes are activated by a variety of cellular stress conditions to maintain cellular homoeostasis. Under non-stress conditions, Nrf2 is under tight regulation by the ubiquitin proteasome system (UPS). Detailed mechanistic investigations have shown the Kelch-like ECH-associated protein 1 (Keap1)-cullin3 (Cul3)-ring-box1 (Rbx1) E3-ligase to be the primary Nrf2 regulatory system. Recently, both beta-transducin repeat-containing E3 ubiquitin protein ligase (ß-TrCP) and E3 ubiquitin-protein ligase synoviolin (Hrd1) have been identified as novel E3 ubiquitin ligases that negatively regulate Nrf2 through Keap1-independent mechanisms. In addition to UPS-mediated regulation of Nrf2, investigations have revealed a cross-talk between Nrf2 and the autophagic pathway resulting in activation of Nrf2 in a non-canonical manner. In addition to regulation at the protein level, Nrf2 was recently shown to be regulated at the transcriptional level by oncogenic K-rat sarcoma (Ras). A consequence of these differential regulatory mechanisms is the dual role of Nrf2 in cancer: the canonical, protective role and the non-canonical 'dark-side' of Nrf2. Based on the protective role of Nrf2, a vast effort has been dedicated towards identifying novel chemical inducers of Nrf2 for the purpose of chemoprevention. On the other hand, upon malignant transformation, some cancer cells have a constitutively high level of Nrf2 offering a growth advantage, as well as rendering cancer cells resistant to chemotherapeutics. This discovery has led to a new paradigm in cancer treatment; the initially counterintuitive use of Nrf2 inhibitors as adjuvants in chemotherapy. Herein, we will discuss the mechanisms of Nrf2 regulation and how this detailed molecular understanding can be leveraged to develop Nrf2 modulators to prevent diseases, mitigate disease progression or overcome chemoresistance.

Identification of a functional antioxidant response element within the eighth intron of the human ABCC3 gene.

The ATP-binding cassette (ABC) family of transporters, including ABCC3, is a large family of efflux pumps that plays a pivotal role in the elimination of xenobiotics from the body. ABCC3 has been reported to be induced during hepatic stress conditions and through the progression of some forms of cancer. Several lines of evidence have implicated the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in this induction. However, although rodent models have been investigated, a functional antioxidant response element (ARE) in the human ABCC3 gene has not been identified. The purpose of this study was to identify and characterize the ARE(s) responsible for mediating the Nrf2-dependent induction of the human ABCC3 gene. A high-throughput chromatin immunoprecipitation-sequencing analysis performed in A549 cells revealed a specific interaction between Nrf2 and the eighth intron of the human ABCC3 gene rather than the more prototypical flanking region of the gene. Subsequent in silico analysis of the intron identified two putative ARE elements that contained the core consensus ARE sequence commonly found in several Nrf2-responsive genes. Functional characterization of these two AREs using luciferase-reporter constructs with ARE mutant constructs revealed that one of these putative AREs is functionally active. Finally, DNA pull-down assays confirmed specific binding of these intronic AREs by Nrf2 in vitro. Our findings identify a functional Nrf2 response element within the eighth intron of the ABCC3 gene, which may provide mechanistic insight into the induction of ABCC3 during antioxidant response stimuli.

Plant extracts of the family Lauraceae: a potential resource for chemopreventive agents that activate the nuclear factor-erythroid 2-related factor 2/antioxidant response element pathway.

Cells and tissues counteract insults from exogenous or endogenous carcinogens through the expression of genes encoding antioxidants and phase II detoxifying enzymes regulated by antioxidant response element promoter regions. Nuclear factor-erythroid 2-related factor 2 plays a key role in regulating the antioxidant response elements-target gene expression. Hence, the Nrf2/ARE pathway represents a vital cellular defense mechanism against damage caused by oxidative stress and xenobiotics, and is recognized as a potential molecular target for discovering chemopreventive agents. Using a stable antioxidant response element luciferase reporter cell line derived from human breast cancer MDA-MB-231 cells combined with a 96-well high-throughput screening system, we have identified a series of plant extracts from the family Lauraceae that harbor Nrf2-inducing effects. These extracts, including Litsea garrettii (ZK-08), Cinnamomum chartophyllum (ZK-02), C. mollifolium (ZK-04), C. camphora var. linaloolifera (ZK-05), and C. burmannii (ZK-10), promoted nuclear translocation of Nrf2, enhanced protein expression of Nrf2 and its target genes, and augmented intracellular glutathione levels. Cytoprotective activity of these extracts against two electrophilic toxicants, sodium arsenite and H2O2, was investigated. Treatment of human bronchial epithelial cells with extracts of ZK-02, ZK-05, and ZK-10 significantly improved cell survival in response to sodium arsenite and H2O2, while ZK-08 showed a protective effect against only H2O2. Importantly, their protective effects against insults from both sodium arsenite and H2O2 were Nrf2-dependent. Therefore, our data provide evidence that the selected plants from the family Lauraceae are potential sources for chemopreventive agents targeting the Nrf2/ARE pathway.

Pacritinib is a potent ACVR1 inhibitor with significant anemia benefit in patients with myelofibrosis.

In patients with cytopenic myelofibrosis, treatment with the JAK2/IRAK1 inhibitor pacritinib was associated with anemia benefit in the phase 3 PERSIST-2 study. The impact of pacritinib on transfusion independence (TI) has not been previously described, nor has the mechanism by which pacritinib improves anemia been elucidated. Because it has been previously postulated that inhibition of activin A receptor, type 1 (ACVR1)/activin receptor-like kinase-2 improves anemia in patients with myelofibrosis via suppression of hepcidin production, we assessed the relative inhibitory potency of pacritinib compared with other JAK2 inhibitors against ACVR1. Pacritinib inhibited ACVR1 with greater potency (half-maximal inhibitory concentration [IC50] = 16.7 nM; Cmax:IC50 = 12.7) than momelotinib (IC50 = 52.5 nM; Cmax:IC50 = 3.2), fedratinib (IC50 = 273 nM; Cmax:IC50 = 1.0), or ruxolitinib (IC50 > 1000; Cmax:IC50 < 0.01). Pacritinib's inhibitory activity against ACVR1 was corroborated via inhibition of downstream SMAD signaling in conjunction with marked suppression of hepcidin production. Among patients on PERSIST-2 who were not transfusion independent at baseline based on Gale criteria, a significantly greater proportion achieved TI on pacritinib compared with those treated on best available therapy (37% vs 7%, P = .001), and significantly more had a ≥50% reduction in transfusion burden (49% vs 9%, P < .0001). These data indicate that the anemia benefit of the JAK2/IRAK1 inhibitor pacritinib may be a function of potent ACVR1 inhibition.

Developments in Blood-Brain Barrier Penetrance and Drug Repurposing for Improved Treatment of Glioblastoma.

Glioblastoma (GBM) is one of the most common, deadly, and difficult-to-treat adult brain tumors. Surgical removal of the tumor, followed by radiotherapy (RT) and temozolomide (TMZ) administration, is the current treatment modality, but this regimen only modestly improves overall patient survival. Invasion of cells into the surrounding healthy brain tissue prevents complete surgical resection and complicates treatment strategies with the goal of preserving neurological function. Despite significant efforts to increase our understanding of GBM, there have been relatively few therapeutic advances since 2005 and even fewer treatments designed to effectively treat recurrent tumors that are resistant to therapy. Thus, while there is a pressing need to move new treatments into the clinic, emerging evidence suggests that key features unique to GBM location and biology, the blood-brain barrier (BBB) and intratumoral molecular heterogeneity, respectively, stand as critical unresolved hurdles to effective therapy. Notably, genomic analyses of GBM tissues has led to the identification of numerous gene alterations that govern cell growth, invasion and survival signaling pathways; however, the drugs that show pre-clinical potential against signaling pathways mediated by these gene alterations cannot achieve effective concentrations at the tumor site. As a result, identifying BBB-penetrating drugs and utilizing new and safer methods to enhance drug delivery past the BBB has become an area of intensive research. Repurposing and combining FDA-approved drugs with evidence of penetration into the central nervous system (CNS) has also seen new interest for the treatment of both primary and recurrent GBM. In this review, we discuss emerging methods to strategically enhance drug delivery to GBM and repurpose currently-approved and previously-studied drugs using rational combination strategies.

The TNF receptor family member Fn14 is highly expressed in recurrent glioblastoma and in GBM patient-derived xenografts with acquired temozolomide resistance.

Background: Glioblastoma (GBM) is a difficult to treat brain cancer that nearly uniformly recurs, and recurrent tumors are largely therapy resistant. Our prior work has demonstrated an important role for the tumor necrosis factor-like weak inducer of apoptosis (TWEAK) receptor fibroblast growth factor-inducible 14 (Fn14) in GBM pathobiology. In this study, we investigated Fn14 expression in recurrent GBM and in the setting of temozolomide (TMZ) resistance. Methods: Fn14 mRNA expression levels in nonneoplastic brain, primary (newly diagnosed) GBM, and recurrent GBM (post-chemotherapy and radiation) specimens were obtained from The Cancer Genome Atlas data portal. Immunohistochemistry was performed using nonneoplastic brain, patient-matched primary and recurrent GBM, and gliosarcoma (GSM) specimens to examine Fn14 protein levels. Western blot analysis was used to compare Fn14 expression in parental TMZ-sensitive or matched TMZ-resistant patient-derived xenografts (PDXs) established from primary or recurrent tumor samples. The migratory capacity of control and Fn14-depleted TMZ-resistant GBM cells was assessed using the transwell migration assay. Results: We found that Fn14 is more highly expressed in recurrent GBM tumors than their matched primary GBM counterparts. Fn14 expression is also significantly elevated in GSM tumors. GBM PDX cells with acquired TMZ resistance have higher Fn14 levels and greater migratory capacity than their corresponding parental TMZ-sensitive cells, and the migratory difference is due, at least in part, to Fn14 expression in the TMZ-resistant cells. Conclusions: This study demonstrates that the Fn14 gene is highly expressed in recurrent GBM, GSM, and TMZ-resistant GBM PDX tumors. These findings suggest that Fn14 may be a valuable therapeutic target or drug delivery portal for treatment of recurrent GBM and GSM patients.

Reduced Nrf2 expression mediates the decline in neural stem cell function during a critical middle-age period.

Although it is known that the regenerative function of neural stem/progenitor cells (NSPCs) declines with age, causal mechanisms underlying this phenomenon are not understood. Here, we systematically analyze subventricular zone (SVZ) NSPCs, in various groups of rats across the aging spectrum, using in vitro and in vivo histological and behavioral techniques. These studies indicate that although NSPC function continuously declines with advancing age, there is a critical time period during middle age (13-15 months) when a striking reduction in NSPC survival and regeneration (proliferation and neuronal differentiation) occurs. The studies also indicate that this specific temporal pattern of NSPC deterioration is functionally relevant at a behavioral level and correlates with the decreasing expression of the redox-sensitive transcription factor, Nrf2, in the NSPCs. When Nrf2 expression was suppressed in 'young' NSPCs, using short interfering RNAs, the survival and regeneration of the NSPCs was significantly compromised and mirrored 'old' NSPCs. Conversely, Nrf2 overexpression in 'old' NSPCs rendered them similar to 'young' NSPCs, and they showed increased survival and regeneration. Furthermore, examination of newborn Nrf2 knockout (Nrf2 -/-) mice revealed a lower number of SVZ NSPCs in these animals, when compared to wild-type controls. In addition, the proliferative and neurogenic potential of the NSPCs was also compromised in the Nrf2-/- mice. These results identify a novel regulatory role for Nrf2 in NSPC function during aging and have important implications for developing NSPC-based strategies to support healthy aging and to treat age-related neurodegenerative disorders.

p62 links autophagy and Nrf2 signaling.

The Nrf2-Keap1-ARE pathway is a redox and xenobiotic sensitive signaling axis that functions to protect cells against oxidative stress, environmental toxicants, and harmful chemicals through the induction of cytoprotective genes. To enforce strict regulation, cells invest a great deal of energy into the maintenance of the Nrf2 pathway to ensure rapid induction upon cellular insult and rapid return to basal levels once the insult is mitigated. Because of the protective role of Nrf2 transcriptional programs, controlled activation of the pathway has been recognized as a means for chemoprevention. On the other hand, constitutive activation of Nrf2, due to somatic mutations of genes that control Nrf2 degradation, promotes carcinogenesis and imparts chemoresistance to cancer cells. Autophagy, a bulk protein degradation process, is another tightly regulated complex cellular process that functions as a cellular quality control system to remove damaged proteins or organelles. Low cellular nutrient levels can also activate autophagy, which acts to restore metabolic homeostasis through the degradation of macromolecules to provide nutrients. Recently, these two cellular pathways were shown to intersect through the direct interaction between p62 (an autophagy adaptor protein) and Keap1 (the Nrf2 substrate adaptor for the Cul3 E3 ubiquitin ligase). Dysregulation of autophagy was shown to result in prolonged Nrf2 activation in a p62-dependent manner. In this review, we will discuss the progress that has been made in dissecting the intersection of these two pathways and the potential tumor-promoting role of prolonged Nrf2 activation.

Pharmacological evidence for an abstinence-induced switch in 5-HT1B receptor modulation of cocaine self-administration and cocaine-seeking behavior.

Studies examining serotonin-1B (5-HT1B) receptor manipulations on cocaine self-administration and cocaine-seeking behavior initially seemed discrepant. However, we recently suggested based on viral-mediated 5-HT1B-receptor gene transfer that the discrepancies are likely due to differences in the length of abstinence from cocaine prior to testing. To further validate our findings pharmacologically, we examined the effects of the selective 5-HT1B receptor agonist CP 94,253 (5.6 mg/kg, s.c.) on cocaine self-administration during maintenance and after a period of protracted abstinence with or without daily extinction training. We also examined agonist effects on cocaine-seeking behavior at different time points during abstinence. During maintenance, CP 94,253 shifted the cocaine self-administration dose-effect function on an FR5 schedule of reinforcement to the left, whereas following 21 days of abstinence CP 94,253 downshifted the function and also decreased responding on a progressive ratio schedule of reinforcement regardless of extinction history. CP 94,253 also attenuated cue-elicited and cocaine-primed drug-seeking behavior following 5 days, but not 1 day, of forced abstinence. The attenuating effects of CP 94,253 on the descending limb of the cocaine dose-effect function were blocked by the selective 5-HT1B receptor antagonist SB 224289 (5 mg/kg, i.p.) at both time points, indicating 5-HT1B receptor mediation. The results support a switch in 5-HT1B receptor modulation of cocaine reinforcement from facilitatory during self-administration maintenance to inhibitory during protracted abstinence. These findings suggest that the 5-HT1B receptor may be a novel target for developing medication for treating cocaine dependence.