An Optimized Liquid Chromatography-Mass Spectrometry Method for Ganglioside Analysis in Cell Lines.

Gangliosides are glycosphingolipids composed of a sialylated glycan head group and a ceramide backbone. These anionic lipids form lipid rafts and play crucial roles in regulating various proteins involved in signal transduction, adhesion, and cell-cell recognition. Neuroblastoma, a pediatric cancer of the sympathetic nervous system, is treated with intensive chemotherapy, radiation, and an antibody targeting the GD2 ganglioside. Gangliosides are critical in neuroblastoma development and serve as therapeutic targets, making it essential to establish a reliable, rapid, and cost-effective method for profiling gangliosides, particularly one capable of isomeric separation of intact species. In this study, liquid chromatography-mass spectrometry (LC-MS) was optimized using standard gangliosides, followed by the optimization of sphingolipid extraction methods from cell lines by comparing Folch and absolute methanol extraction techniques. Percent recovery and the number of identified sphingolipids were used to evaluate the analytical merits of these methods. A standard gangliosides calibration curve demonstrated excellent linearity (R2 = 0.9961-0.9975). The ZIC-HILIC column provided the best separation of ganglioside GD1 isomers with a 25 min runtime. GD1a elutes before GD1b on the ZIC-HILIC column. Absolute methanol yielded better percent recovery (96 ± 7) and identified 121 different sphingolipids, the highest number between the two extraction methods. The optimized method was applied to profile gangliosides in neuroblastoma (COG-N-683), pancreatic cancer (PSN1), breast cancer (MDA-MB-231BR), and brain tumor (CRL-1620) cell lines. The ganglioside profile of the neuroblastoma cell line COG-N-683 showed an inverse relationship between GD1 and GD2. Ceramide, Hex1Cer, GM1, and GM3 were highly abundant in CRL-1620, PSN1, and MDA-MB-231BR, respectively. These results suggest that our method provides a sensitive, reliable, and high-throughput workflow for ganglioside profiling across different cell types.

Targeting Molecular Signaling Pathways and Cytokine Responses to Modulate c-MYC in Acute Myeloid Leukemia.

Overexpression of the MYC oncogene, encoding c-MYC protein, contributes to the pathogenesis and drug resistance of acute myeloid leukemia (AML) and many other hematopoietic malignancies. Although standard chemotherapy has predominated in AML therapy over the past five decades, the clinical outcomes and patient response to treatment remain suboptimal. Deeper insight into the molecular basis of this disease should facilitate the development of novel therapeutics targeting specific molecules and pathways that are dysregulated in AML, including fms-like tyrosine kinase 3 (FLT3) gene mutation and cluster of differentiation 33 (CD33) protein expression. Elevated expression of c-MYC is one of the molecular features of AML that determines the clinical prognosis in patients. Increased expression of c-MYC is also one of the cytogenetic characteristics of drug resistance in AML. However, direct targeting of c-MYC has been challenging due to its lack of binding sites for small molecules. In this review, we focused on the mechanisms involving the bromodomain and extra-terminal (BET) and cyclin-dependent kinase 9 (CDK9) proteins, phosphoinositide-Akt-mammalian target of rapamycin (PI3K/AKT/mTOR) and Janus kinase-signal transduction and activation of transcription (JAK/STAT) pathways, as well as various inflammatory cytokines, as an indirect means of regulating MYC overexpression in AML. Furthermore, we highlight Food and Drug Administration (FDA)-approved drugs for AML, and the results of preclinical and clinical studies on novel agents that have been or are currently being tested for efficacy and tolerability in AML therapy. Overall, this review summarizes our current knowledge of the molecular processes that promote leukemogenesis, as well as the various agents that intervene in specific pathways and directly or indirectly modulate c-MYC to disrupt AML pathogenesis and drug resistance.

Cancer drugs with high repositioning potential for Alzheimer's disease.

INTRODUCTION: Despite the recent full FDA approval of lecanemab, there is currently no disease modifying therapy (DMT) that can efficiently slow down the progression of Alzheimer's disease (AD) in the general population. This statement emphasizes the need to identify novel DMTs in the shortest time possible to prevent a global epidemic of AD cases as the world population experiences an increase in lifespan. AREAS COVERED: Here, we review several classes of anti-cancer drugs that have been or are being investigated in Phase II/III clinical trials for AD, including immunomodulatory drugs, RXR agonists, sex hormone therapies, tyrosine kinase inhibitors, and monoclonal antibodies. EXPERT OPINION: Given the overall course of brain pathologies during the progression of AD, we express a great enthusiasm for the repositioning of anti-cancer drugs as possible AD DMTs. We anticipate an increasing number of combinatorial therapy strategies to tackle AD symptoms and their underlying pathologies. However, we strongly encourage improvements in clinical trial study designs to better assess target engagement and possible efficacy over sufficient periods of drug exposure.

A Multi-Color Flow Cytometric Assay for Quantifying Dinutuximab Binding to Neuroblastoma Cells in Tumor, Bone Marrow, and Blood.

GD2, a disialoganglioside, is present on the surface of most neuroblastomas, as well as on some other cancers, such as melanoma and osteogenic sarcoma. The anti-GD2 antibody ch14.18 (dinutuximab) has an FDA-registered indication for use as maintenance therapy for high-risk neuroblastoma with cytokines and 13-cis-retinoic acid after myeloablative therapy. Recent studies using immunohistochemistry of tumor or tumor cells in marrow have shown that some neuroblastomas are negative for GD2. Dinutuximab and other anti-GD2 antibodies are increasingly used in combination with cytotoxic chemotherapy for treating relapsed neuroblastoma, so it is important to be able to identify patients with tumor cells with low GD2 expression, as such patients may experience toxicity but not benefit from the antibody therapy. As the most common clinical samples available for relapsed neuroblastoma are bone marrow aspirates, we developed a method to quantify dinutuximab binding density and the frequency of neuroblastoma cells positive for the antibody in bone marrow aspirates. Here, we describe a multi-color flow cytometry assay that employs non-GD2 antibodies to identify neuroblastoma cells in a mixed population (tumor, bone marrow, or blood) and an anti-GD2 antibody to quantify both the frequency and density of GD2 expression on neuroblastoma cells.

Phase I trial of intravenous fenretinide (4-HPR) plus safingol in advanced malignancies.

PURPOSE: Fenretinide (4-HPR) is a synthetic retinoid that induces cytotoxicity through dihydroceramide production. Safingol, a stereochemical-variant dihydroceramide precursor, exhibits synergistic effects when administered with fenretinide in preclinical studies. We conducted a phase 1 dose-escalation clinical trial of this combination. METHODS: Fenretinide was administered as a 600 mg/m2 24-h infusion on Day 1 of a 21-day cycle followed by 900 mg/m2/day on Days 2 and 3. Safingol was concurrently administered as a 48-h infusion on Day 1 and 2 using 3 + 3 dose escalation. Primary endpoints were safety and maximum tolerated dose (MTD). Secondary endpoints included pharmacokinetics and efficacy. RESULTS: A total of 16 patients were enrolled (mean age 63 years, 50% female, median three prior lines of therapy), including 15 patients with refractory solid tumors and one with non-Hodgkin lymphoma. The median number of treatment cycles received was 2 (range 2-6). The most common adverse event (AE) was hypertriglyceridemia (88%; 38% ≥ Grade 3), attributed to the fenretinide intralipid infusion vehicle. Other treatment-related AEs occurring in ≥ 20% of patients included anemia, hypocalcemia, hypoalbuminemia, and hyponatremia. At safingol dose 420 mg/m2, one patient had a dose-limiting toxicity of grade 3 troponinemia and grade 4 myocarditis. Due to limited safingol supply, enrollment was halted at this dose level. Fenretinide and safingol pharmacokinetic profiles resembled those observed in monotherapy trials. Best radiographic response was stable disease (n = 2). CONCLUSION: Combination fenretinide plus safingol commonly causes hypertriglyceridemia and may be associated with cardiac events at higher safingol levels. Minimal activity in refractory solid tumors was observed. TRIAL REGISTRATION NUMBER: NCT01553071 (3.13.2012).

DNA-PKcs as an upstream mediator of OCT4-induced MYC activation in small cell lung cancer.

Small cell lung cancer (SCLC) is a neuroendocrine tumor noted for the rapid development of both metastases and resistance to chemotherapy. High mutation burden, ubiquitous loss of TP53 and RB1, and a mutually exclusive amplification of MYC gene family members contribute to genomic instability and make the development of new targeted agents a challenge. Previously, we reported a novel OCT4-induced MYC transcriptional activation pathway involving c-MYC, pOCT4S111, and MAPKAPK2 in progressive neuroblastoma, also a neuroendocrine tumor. Using tumor microarray analysis of clinical samples and preclinical models, we now report a correlation in expression between these proteins in SCLC. In correlating c-MYC protein expression with genomic amplification, we determined that some SCLC cell lines exhibited high c-MYC without genomic amplification, implying amplification-independent MYC activation. We then confirmed direct interaction between OCT4 and DNA-PKcs and identified specific OCT4 and DNA-PKcs binding sites. Knock-down of both POU5F1 (encoding OCT4) and PRKDC (encoding DNA-PKcs) resulted in decreased c-MYC expression. Further, we confirmed binding of OCT4 to the promoter/enhancer region of MYC. Together, these data establish the presence of a DNA-PKcs/OCT4/c-MYC pathway in SCLCs. We then disruptively targeted this pathway and demonstrated anticancer activity in SCLC cell lines and xenografts using both DNA-PKcs inhibitors and a protein-protein interaction inhibitor of DNA-PKcs and OCT4. In conclusion, we demonstrate here that DNA-PKcs can mediate high c-MYC expression in SCLCs, and that this pathway may represent a new therapeutic target for SCLCs with high c-MYC expression.

Proposed Mechanism of Long-Term Intraocular Pressure Lowering With the Bimatoprost Implant.

Purpose: The purpose of this study was to evaluate the effects of pharmacologically relevant bimatoprost and bimatoprost free acid (BFA) concentrations on matrix metalloproteinase (MMP) gene expression in cells from human aqueous outflow tissues. Methods: MMP gene expression by human trabecular meshwork (TM), scleral fibroblast (SF), and ciliary muscle (CM) cells exposed to 10 to 1000 µM bimatoprost or 0.1 to 10 µM BFA (intraocular concentrations after intracameral bimatoprost implant and topical bimatoprost dosing, respectively) was measured by polymerase chain reaction array. Results: Bimatoprost dose-dependently upregulated MMP1 and MMP14 mRNA in all cell types and MMP10 and MMP11 mRNA in TM and CM cells; in TM cells from normal eyes, mean MMP1 mRNA levels were 62.9-fold control levels at 1000 µM bimatoprost. BFA upregulated MMP1 mRNA only in TM and SF cells, to two- to three-fold control levels. The largest changes in extracellular matrix (ECM)-related gene expression by TM cells derived from normal (n = 6) or primary open-angle glaucoma (n = 3) eyes occurred with 1000 µM bimatoprost (statistically significant, ≥50% change for 9-11 of 84 genes on the array, versus 1 gene with 10 µM BFA). Conclusions: Bimatoprost and BFA had differential effects on MMP/ECM gene expression. Dramatic upregulation in MMP1 and downregulation of fibronectin, which occurred only with bimatoprost at high concentrations observed in bimatoprost implant-treated eyes, may promote sustained outflow tissue remodeling and long-term intraocular pressure reduction beyond the duration of intraocular drug bioavailability. Variability in bimatoprost-stimulated MMP upregulation among cell strains from different donors may help explain differential long-term responses of patients to bimatoprost implant.

Novel insight into the role of clusterin on intraocular pressure regulation by modifying actin polymerization and extracellular matrix remodeling in the trabecular meshwork.

This study provides comprehensive mechanistic evidence for the role of clusterin, a stress-response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress-related clusterin function loss. Small interfering RNA-mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine-protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and ß5. A complete loss of clusterin as seen in clusterin knockout mice (Clu-/- ) led to significant IOP elevation at postnatal Day 70. Contrarily, constitutive clusterin expression using adenovirus (AdCLU) in HTM cells resulted in the loss of actin polymerization via decreased PRK2, and LIMK1 and negative regulation of integrin αV and ß5. Furthermore, we found that AdCLU treatment in HTM cells significantly decreased the ECM protein expression and distribution by significantly increasing matrix metalloprotease 2 (MMP2) activity and lowering the levels of pro-fibrotic proteins such as transforming growth factor-ß2 (TGFß2), thrombospondin-1 (TSP-1), and plasminogen activator inhibitor-1 (PAI-1). Finally, we found that HTM cells supplemented with recombinant human clusterin attenuated the pro-fibrotic effects of TGFß2. For the first time this study demonstrates the importance of clusterin in the regulation of TM actin cytoskeleton - ECM interactions and the maintenance of IOP, thus making clusterin an interesting target to reverse elevated IOP.

Tumor-Associated Macrophages as Multifaceted Regulators of Breast Tumor Growth.

Breast cancer is the most commonly occurring cancer in women of Western countries and is the leading cause of cancer-related mortality. The breast tumor microenvironment contains immune cells, fibroblasts, adipocytes, mesenchymal stem cells, and extracellular matrix. Among these cells, macrophages or tumor-associated macrophages (TAMs) are the major components of the breast cancer microenvironment. TAMs facilitate metastasis of the breast tumor and are responsible for poor clinical outcomes. High TAM density was also found liable for the poor prognosis of breast cancer. These observations make altering TAM function a potential therapeutic target to treat breast cancer. The present review summarizes the origin of TAMs, mechanisms of macrophage recruitment and polarization in the tumor, and the contributions of TAMs in tumor progression. We have also discussed our current knowledge about TAM-targeted therapies and the roles of miRNAs and exosomes in re-educating TAM function.

PPE decontamination to overcome PPE shortage in rural area during pandemic.

Despite remarkable developments in healthcare, the world was not ready to stop the spread of the novel COVID-19 pandemic almost a century after the great influenza pandemic. The explosive increase in the number of patients stalled the healthcare system, and the first and apparent issue was the shortage of personal protective equipment (PPE). Our group established a system using a hydrogen peroxide vaporization method to decontaminate and reuse N95 respirators for healthcare workers. The system decontaminated over 12,000 units of PPE to cover institutions in West Texas. This service provided support at the most needed time during the pandemic.

Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and OCT4-MK2 interactions.

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 (MK2) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the Ser111 and the Ser93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds ("hits") that inhibited the luminescence reaction between DNA-PKcs and OCT4, and 65 hits inhibiting the MK2-OCT4 interaction. Using custom antibodies specific for pOCT4S93 and pOCT4S111 , the "hits" were validated for their effect on OCT4 phosphorylation and activation. Using a two-step method for validation, we identified two candidate compounds from the DNA-PKcs assay and three from the MK2 assay. All five compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found five compounds that may function as lead compounds for further development.

A phase I study of intravenous fenretinide (4-HPR) for patients with malignant solid tumors.

BACKGROUND: Fenretinide is a synthetic retinoid that can induce cytotoxicity by several mechanisms. Achieving effective systemic exposure with oral formulations has been challenging. An intravenous lipid emulsion fenretinide formulation was developed to overcome this barrier. We conducted a study to establish the maximum tolerated dose (MTD), preliminary efficacy, and pharmacokinetics of intravenous lipid emulsion fenretinide in patients with advanced solid tumors. METHODS: Twenty-three patients with advanced solid tumors refractory to standard treatments received fenretinide as a continuous infusion for five consecutive days in 21-day cycles. Five different dose cohorts were evaluated between doses of 905 mg/m2 and 1414 mg/m2 per day using a 3 + 3 dose escalation design. A priming dose of 600 mg/m2 on day 1 was introduced in an attempt to address the asymptomatic serum triglyceride elevations related to the lipid emulsion. RESULTS: The treatment-related adverse events occurring in ≥ 20% of patients were anemia, hypertriglyceridemia, fatigue, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) increase, thrombocytopenia, bilirubin increase, and dry skin. Five evaluable patients had stable disease as best response, and no patients had objective responses. Plasma steady-state concentrations of the active metabolite were significantly higher than with previous capsule formulations. CONCLUSION: Fenretinide emulsion intravenous infusion had a manageable safety profile and achieved higher plasma steady-state concentrations of the active metabolite compared to previous capsule formulations. Single-agent activity was minimal but combinatorial approaches are under evaluation.

Vorinostat and fenretinide synergize in preclinical models of T-cell lymphoid malignancies.

T-cell lymphoid malignancies (TCLMs) are in need of novel and more effective therapies. The histone deacetylase (HDAC) inhibitors and the synthetic cytotoxic retinoid fenretinide have achieved durable clinical responses in T-cell lymphomas as single agents, and patients who failed prior HDAC inhibitor treatment have responded to fenretinide. We have previously shown fenretinide synergized with the class I HDAC inhibitor romidepsin in preclinical models of TCLMs. There exist some key differences between HDAC inhibitors. Therefore, we determined if the pan-HDAC inhibitor vorinostat synergizes with fenretinide. We demonstrated cytotoxic synergy between vorinostat and fenretinide in nine TCLM cell lines at clinically achievable concentrations that lacked cytotoxicity for non-malignant cells (fibroblasts and blood mononuclear cells). In vivo, vorinostat + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous TCLM xenograft models than other groups. Fenretinide + vorinostat increased reactive oxygen species (ROS, measured by 2',7'-dichlorodihydrofluorescein diacetate dye), resulting in increased apoptosis (via transferase dUTP nick end labeling assay) and histone acetylation (by immunoblotting). The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + vorinostat was abrogated by the antioxidant vitamin C. Like romidepsin, vorinostat combined with fenretinide achieved synergistic cytotoxic activity and increased histone acetylation in preclinical models of TCLMs, but not in non-malignant cells. As vorinostat is an oral agent and not a P-glycoprotein substrate it may have advantages in such combination therapy. These data support conducting a clinical trial of vorinostat combined with fenretinide in relapsed and refractory TCLMs.

Mithramycin induces promoter reprogramming and differentiation of rhabdoid tumor.

Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is the known oncogenic driver, there are limited effective therapeutic options for these patients. Here we use unbiased screening of cell line panels to identify a heightened sensitivity of rhabdoid tumor to mithramycin and the second-generation analogue EC8042. The sensitivity of MMA and EC8042 was superior to traditional DNA damaging agents and linked to the causative mutation of the tumor, SMARCB1 deletion. Mithramycin blocks SMARCB1-deficient SWI/SNF activity and displaces the complex from chromatin to cause an increase in H3K27me3. This triggers chromatin remodeling and enrichment of H3K27ac at chromHMM-defined promoters to restore cellular differentiation. These effects occurred at concentrations not associated with DNA damage and were not due to global chromatin remodeling or widespread gene expression changes. Importantly, a single 3-day infusion of EC8042 caused dramatic regressions of RT xenografts, recapitulated the increase in H3K27me3, and cellular differentiation described in vitro to completely cure three out of eight mice.

Antineoplastic Agents Targeting Sphingolipid Pathways.

Emerging studies in the enigmatic area of bioactive lipids have made many exciting new discoveries in recent years. Once thought to play a strictly structural role in cellular function, it has since been determined that sphingolipids and their metabolites perform a vast variety of cellular functions beyond what was previously believed. Of utmost importance is their role in cellular signaling, for it is now well understood that select sphingolipids serve as bioactive molecules that play critical roles in both cancer cell death and survival, as well as other cellular responses such as chronic inflammation, protection from intestinal pathogens, and intrinsic protection from intestinal contents, each of which are associated with oncogenesis. Importantly, it has been demonstrated time and time again that many different tumors display dysregulation of sphingolipid metabolism, and the exact profile of said dysregulation has been proven to be useful in determining not only the presence of a tumor, but also the susceptibility to various chemotherapeutic drugs, as well as the metastasizing characteristics of the malignancies. Since these discoveries surfaced it has become apparent that the understanding of sphingolipid metabolism and profile will likely become of great importance in the clinic for both chemotherapy and diagnostics of cancer. The goal of this paper is to provide a comprehensive review of the current state of chemotherapeutic agents that target sphingolipid metabolism that are undergoing clinical trials. Additionally, we will formulate questions involving the use of sphingolipid metabolism as chemotherapeutic targets in need of further research.

MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma.

Despite the improvement in clinical outcome with 13-cis-retinoic acid (13-cisRA) + anti-GD2 antibody + cytokine immunotherapy given in first response ~40% of high-risk neuroblastoma patients die of recurrent disease. MYCN genomic amplification is a biomarker of aggressive tumors in the childhood cancer neuroblastoma. MYCN expression is downregulated by 13-cisRA, a differentiating agent that is a component of neuroblastoma therapy. Although MYC amplification is rare in neuroblastoma at diagnosis, we report transcriptional activation of MYC medicated by the transcription factor OCT4, functionally replacing MYCN in 13-cisRA-resistant progressive disease neuroblastoma in large panels of patient-derived cell lines and xenograft models. We identified novel OCT4-binding sites in the MYC promoter/enhancer region that regulated MYC expression via phosphorylation by MAPKAPK2 (MK2). OCT4 phosphorylation at the S111 residue by MK2 was upstream of MYC transcriptional activation. Expression of OCT4, MK2, and c-MYC was higher in progressive disease relative to pre-therapy neuroblastomas and was associated with inferior patient survival. OCT4 or MK2 knockdown decreased c-MYC expression and restored the sensitivity to 13-cisRA. In conclusion, we demonstrated that high c-MYC expression independent of genomic amplification is associated with disease progression in neuroblastoma. MK2-mediated OCT4 transcriptional activation is a novel mechanism for activating the MYC oncogene in progressive disease neuroblastoma that provides a therapeutic target.

TGFß2-Hepcidin Feed-Forward Loop in the Trabecular Meshwork Implicates Iron in Glaucomatous Pathology.

Purpose: Elevated levels of transforming-growth-factor (TGF)-ß2 in the trabecular meshwork (TM) and aqueous humor are associated with primary open-angle glaucoma (POAG). The underlying mechanism includes alteration of extracellular matrix homeostasis through Smad-dependent and independent signaling. Smad4, an essential co-Smad, upregulates hepcidin, the master regulator of iron homeostasis. Here, we explored whether TGF-ß2 upregulates hepcidin, implicating iron in the pathogenesis of POAG. Methods: Primary human TM cells and human and bovine ex vivo anterior segment organ cultures were exposed to bioactive TGF-ß2, hepcidin, heparin (a hepcidin antagonist), or N-acetyl carnosine (an antioxidant), and the change in the expression of hepcidin, ferroportin, ferritin, and TGF-ß2 was evaluated by semiquantitative RT-PCR, Western blotting, and immunohistochemistry. Increase in reactive oxygen species (ROS) was quantified with dihydroethidium, an ROS-sensitive dye. Results: Primary human TM cells and bovine TM tissue synthesize hepcidin locally, which is upregulated by bioactive TGF-ß2. Hepcidin downregulates ferroportin, its downstream target, increasing ferritin and iron-catalyzed ROS. This causes reciprocal upregulation of TGF-ß2 at the transcriptional and translational levels. Heparin downregulates hepcidin, and reduces TGF-ß2-mediated increase in ferritin and ROS. Notably, both heparin and N-acetyl carnosine reduce TGF-ß2-mediated reciprocal upregulation of TGF-ß2. Conclusions: The above observations suggest that TGF-ß2 and hepcidin form a self-sustained feed-forward loop through iron-catalyzed ROS. This loop is partially disrupted by a hepcidin antagonist and an anti-oxidant, implicating iron and ROS in TGF-ß2-mediated POAG. We propose that modification of currently available hepcidin antagonists for ocular use may prove beneficial for the therapeutic management of TGF-ß2-associated POAG.

Role of OCT4 in cancer stem-like cells and chemotherapy resistance.

Cancer stem-like cells (CSCs) contribute to the tumorigenicity, progression, and chemoresistance of cancers. It is not known whether CSCs arise from normal stem cells or if they arise from differentiated cancer cells by acquiring self-renewal features. These CSCs share stem cell markers that normal stem cells express. There is a rising interest in octamer-binding transcription factor 4 (OCT4), one of the stem cell factors that are essential in embryogenesis and pluripotency. OCT4 is also overexpressed in CSCs of various cancers. Although the majority of the studies in CSCs reported a positive association between the expression of OCT4 and chemoresistance and an inverse correlation between OCT4 and clinical prognosis, there are studies rebuking these findings, possibly due to the sparsity of stem cells within tumors and the heterogeneity of tumors. In addition, post-translational modification of OCT4 affects its activity and warrants further investigation for its association with chemoresistance and prognosis.

Deep neuromuscular blockade during spinal surgery reduces intra-operative blood loss: A randomised clinical trial.

BACKGROUND: Spinal surgery is usually performed in the prone position using a posterior approach. However, the prone position may cause venous engorgement in the back and thus increase surgical bleeding with interruption of surgery. The prone position also affects cardiac output since large vessels are compressed decreasing venous return to the heart. OBJECTIVE: We hypothesised that deep neuromuscular blockade would be associated with less surgical bleeding during spinal surgery in the prone position. DESIGN: Randomised, single blinded trial. SETTING: University teaching hospital. PARTICIPANTS: Eighty-eight patients in two groups. INTERVENTIONS: Patients were randomly assigned to moderate neuromuscular blockade or deep neuromuscular blockade. In the moderate neuromuscular blockade group, administration of rocuronium was adjusted such that the train-of-four count was one to two. In the deep neuromuscular blockade group, rocuronium administration was adjusted such that the train-of-four count was zero with a posttetanic count 2 or less. MAIN OUTCOME MEASURES: The primary outcome was the volume of intra-operative surgical bleeding. The surgeon's satisfaction with operating conditions, haemodynamic and respiratory status, and postoperative pain scores were evaluated. RESULTS: The median [IQR] volume of intra-operative surgical bleeding was significantly less in the deep neuromuscular blockade group than in the moderate neuromuscular blockade group; 300 ml [200 to 494] vs. 415 ml [240 to 601]; difference: 117 ml (95% CI, 9 to 244; P = 0.044). The mean ±â€ŠSD surgeon's satisfaction with the intra-operative surgical conditions was greater in the deep neuromuscular blockade group than in the moderate neuromuscular blockade group; 3.5 ±â€Š1.0 vs. 2.9 ±â€Š0.9 (P = 0.004). In intergroup comparisons of respiratory variables, peak inspiratory pressure was lower in the deep neuromuscular blockade group overall (P < 0.001). The median [IQR] postoperative pain score was lower in the deep neuromuscular blockade group than the moderate neuromuscular blockade group; 50 [36 to 60] vs. 60 [50 to 70], (P = 0.023). CONCLUSION: Deep neuromuscular blockade reduced intra-operative surgical bleeding in patients undergoing spinal surgery. This may be related to greater relaxation in the back muscles and lower intra-operative peak inspiratory pressure when compared with moderate neuromuscular blockade. TRIAL REGISTRATION: KCT0001264 (http://cris.nih.go.kr).

Fenretinide via NOXA Induction, Enhanced Activity of the BCL-2 Inhibitor Venetoclax in High BCL-2-Expressing Neuroblastoma Preclinical Models.

Recurrent high-risk neuroblastoma is a childhood cancer that often fails to respond to therapy. Fenretinide (4-HPR) is a cytotoxic retinoid with clinical activity in recurrent neuroblastoma and venetoclax (ABT-199) is a selective inhibitor of the antiapoptotic protein B-cell lymphoma-2 (BCL-2). We evaluated activity of 4-HPR + ABT-199 in preclinical models of neuroblastoma. Patient-derived cell lines and xenografts from progressive neuroblastoma were tested. Cytotoxicity was evaluated by DIMSCAN, apoptosis by flow cytometry, and gene expression by RNA sequencing, quantitative RT-PCR, and immunoblotting. 4-HPR + ABT-199 was highly synergistic against high BCL-2-expressing neuroblastoma cell lines and significantly improved event-free survival of mice carrying high BCL-2-expressing patient-derived xenografts (PDX). In 10 matched-pair cell lines [established at diagnosis (DX) and progressive disease (PD) from the same patients], BCL-2 expression in the DX and PD lines was comparable, suggesting that BCL-2 expression at diagnosis may provide a biomarker for neuroblastomas likely to respond to 4-HPR + ABT-199. In a pair of DX (COG-N-603x) and PD (COG-N-623x) PDXs established from the same patient, COG-N-623x was less responsive to cyclophosphamide + topotecan than COG-N-603x, but both DX and PD PDXs were responsive to 4-HPR + ABT-199. Synergy of 4-HPR + ABT-199 was mediated by induction of NOXA via 4-HPR stimulation of reactive oxygen species that induced expression of ATF4 and ATF3, transcription factors for NOXA. Thus, fenretinide + venetoclax is a synergistic combination that warrants clinical testing in high BCL-2-expressing neuroblastoma.