Fifty Years of Diazeniumdiolate Research: A Tribute to Dr. Larry K. Keefer.

The pioneering studies of Dr. Larry Keefer and colleagues with diazeniumdiolates or NONOates as a platform have unraveled the chemical biology of many nitric oxides and have led to the design of a variety of promising therapeutic agents in oncology, gastroenterology, antimicrobials, wound healing, and the like. This dedication to Dr. Larry Keefer briefly highlights some of his studies using the diazeniumdiolate platform in the cancer arena.

Half-Sandwich Type Platinum-Group Metal Complexes of C-Glucosaminyl Azines: Synthesis and Antineoplastic and Antimicrobial Activities.

While platinum-based compounds such as cisplatin form the backbone of chemotherapy, the use of these compounds is limited by resistance and toxicity, driving the development of novel complexes with cytostatic properties. In this study, we synthesized a set of half-sandwich complexes of platinum-group metal ions (Ru(II), Os(II), Ir(III) and Rh(III)) with an N,N-bidentate ligand comprising a C-glucosaminyl group and a heterocycle, such as pyridine, pyridazine, pyrimidine, pyrazine or quinoline. The sugar-containing ligands themselves are unknown compounds and were obtained by nucleophilic additions of lithiated heterocycles to O-perbenzylated 2-nitro-glucal. Reduction of the adducts and, where necessary, subsequent protecting group manipulations furnished the above C-glucosaminyl heterocycles in their O-perbenzylated, O-perbenzoylated and O-unprotected forms. The derived complexes were tested on A2780 ovarian cancer cells. Pyridine, pyrazine and pyridazine-containing complexes proved to be cytostatic and cytotoxic on A2780 cells, while pyrimidine and quinoline derivatives were inactive. The best complexes contained pyridine as the heterocycle. The metal ion with polyhapto arene/arenyl moiety also impacted on the biological activity of the complexes. Ruthenium complexes with p-cymene and iridium complexes with Cp* had the best performance in ovarian cancer cells, followed by osmium complexes with p-cymene and rhodium complexes with Cp*. Finally, the chemical nature of the protective groups on the hydroxyl groups of the carbohydrate moiety were also key determinants of bioactivity; in particular, O-benzyl groups were superior to O-benzoyl groups. The IC50 values of the complexes were in the low micromolar range, and, importantly, the complexes were less active against primary, untransformed human dermal fibroblasts; however, the anticipated therapeutic window is narrow. The bioactive complexes exerted cytostasis on a set of carcinomas such as cell models of glioblastoma, as well as breast and pancreatic cancers. Furthermore, the same complexes exhibited bacteriostatic properties against multiresistant Gram-positive Staphylococcus aureus and Enterococcus clinical isolates in the low micromolar range.

Pyridine Scaffolds, Phenols and Derivatives of Azo Moiety: Current Therapeutic Perspectives.

Synthetic heterocyclic compounds have incredible potential against different diseases; pyridines, phenolic compounds and the derivatives of azo moiety have shown excellent antimicrobial, antiviral, antidiabetic, anti-melanogenic, anti-ulcer, anticancer, anti-mycobacterial, anti-inflammatory, DNA binding and chemosensing activities. In the present review, the above-mentioned activities of the nitrogen-containing heterocyclic compounds (pyridines), hydroxyl (phenols) and azo derivatives are discussed with reference to the minimum inhibitory concentration and structure-activity relationship, which clearly indicate that the presence of nitrogen in the phenyl ring; in addition, the hydroxyl substituent and the incorporation of a diazo group is crucial for the improved efficacies of the compounds in probing different diseases. The comparison was made with the reported drugs and new synthetic derivatives that showed recent therapeutic perspectives made in the last five years.

Hypoxia-Overcoming Breast-Conserving Treatment by Magnetothermodynamic Implant for a Localized Free-Radical Burst Combined with Hyperthermia.

For the purpose of improving the quality of life and minimizing the psychological morbidity of a mastectomy, breast-conserving treatment (BCT) has become the more preferable choice in breast cancer patients. Meanwhile, tumor hypoxia has been increasingly recognized as a major deleterious factor in cancer therapies. In the current study, a novel, effective, and noninvasive magnetothermodynamic strategy based on an oxygen-independent free-radical burst for hypoxia-overcoming BCT is proposed. Radical precursor (AIPH) and iron oxide nanoparticles (IONPs) are coincorporated within the alginate (ALG) hydrogel, which is formed in situ within the tumor tissue by leveraging the cross-linking effect induced by the local physiological Ca2+ with ALG solution. Inductive heating is mediated by IONPs under AMF exposure, and consequently, regardless of the tumor hypoxia condition, a local free-radical burst is achieved by thermal decomposition of AIPH via AMF responsivity. The combination of magnetic hyperthermia and oxygen-irrelevant free-radical production effectively enhances the in vitro cytotoxic effect and also remarkably inhibits tumor proliferation. This study provides a valuable protocol for an hypoxia-overcoming strategy and also an alternative formulation candidate for noninvasive BCT.

Tumor-Penetrable Nitric Oxide-Releasing Nanoparticles Potentiate Local Antimelanoma Therapy.

Although nitric oxide (NO) has been emerging as a novel local anticancer agent because of its potent cytotoxic effects and lack of off-target side effects, its clinical applications remain a challenge because of the short effective diffusion distance of NO that limits its anticancer activity. In this study, we synthesized albumin-coated poly(lactic-co-glycolic acid) (PLGA)-conjugated linear polyethylenimine diazeniumdiolate (LP/NO) nanoparticles (Alb-PLP/NO NPs) that possess tumor-penetrating and NO-releasing properties for an effective local treatment of melanoma. Sufficient NO-loading and prolonged NO-releasing characteristics of Alb-PLP/NO NPs were acquired through PLGA-conjugated LP/NO copolymer (PLP/NO) synthesis, followed by nanoparticle fabrication. In addition, tumor penetration ability was rendered by the electrostatic adsorption of the albumin on the surface of the nanoparticles. The Alb-PLP/NO NPs showed enhanced intracellular NO delivery efficiency and cytotoxicity to B16F10 murine melanoma cells. In B16F10-tumor-bearing mice, the Alb-PLP/NO NPs showed improved extracellular matrix penetration and spatial distribution in the tumor tissue after intratumoral injection, resulting in enhanced antitumor activity. Taken together, the results suggest that Alb-PLP/NO NPs represent a promising new modality for the local treatment of melanoma.

O2-(2,4-dinitrophenyl) diazeniumdiolate derivative induces G2/M arrest via PTEN-mediated inhibition of PI3K/Akt pathway in hepatocellular carcinoma cells.

OBJECTIVES: The study aimed to investigate whether G2/M arrest caused by O2-(2,4-dinitrophenyl) diazeniumdiolate derivative (JS-K) was related to PTEN-mediated inhibition of PI3K/Akt pathway in hepatocellular carcinoma cells. METHODS: The cell apoptosis was detected by DAPI staining and Annexin V-FITC/PI dual staining. The cell cycle was analysed by PI staining. The expressions of cell cycle-related proteins, PTEN and PI3K/AKT pathway were measured by Western blot. The rat model of primary hepatic carcinoma was established with diethylnitrosamine to verify the antitumour effects of JS-K. KEY FINDINGS: The morphological features of apoptosis were obviously reversed when the cells were pre-treated with bpv(pic), followed by treatment with JS-K. JS-K mediated G2/M arrest and down-regulated expressions of cyclin B1. Meanwhile, it up-regulated the expression of p-Cdk1, p-Chk2 and p-CDC25C while down-regulated that of Cdk1 and CDC25C. Furthermore, JS-K also enhanced the expressions of p21 and p27, PTEN and p53 while decreased the expressions of p-PTEN, PI3K and p-AKT. However, bpv(pic) and Carboxy-PTIO could reverse JS-K-induced G2/M cell arrest and PTEN-mediated inhibition of the PI3K/AKT pathway. The same results were also testified in the rat model of primary hepatic carcinoma. CONCLUSIONS: JS-K caused G2/M arrest through PTEN-mediated inhibition of the PI3K/AKT pathway involving Chk2/CDC25C/Cdk1 checkpoint.

Activation of aryl hydrocarbon receptor (AhR) in mesenchymal stem cells modulates macrophage polarization in asthma.

Macrophage polarization has been demonstrated to exert a vital role on asthma pathogenesis. Mesenchymal stem cells (MSC) have the capacity to modulate macrophage differentiation from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype. However, the impact of MSC-macrophage interactions on asthma development and underlying mechanisms responsible for this interaction remain largely unknown. The aim of this study was to investigate the role of AhR expressed on MSC in macrophage polarization in a cockroach extract (CRE)-induced asthma mouse model. The studies here revealed that MSC polarized macrophages from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype in this model. The mRNA levels of interleukin (IL)-6, IL-1ß, and NOS2 as M1 markers were significantly decreased while those of select M2 markers such as Arg-1, FIZZ1, and YM-1 were significantly enhanced. It was also observed that aryl hydrocarbon receptor (AhR) signaling was significantly increased during asthma pathogenesis as demonstrated by enhanced mRNA expression of AhR, CYP1a1, and CYP1b1. It was also seen that the elevated AhR signaling was able to attenuate the onset of asthma. Use of an AhR antagonist (CH223191) resulted in significant inhibition of the AhR signaling and increases in M2 marker expression, but led to elevation of expression of M1 markers in the CRE-induced asthma model. Taken together, the current study showed that MSC can modulate macrophage polarization, in part, via activation of AhR signaling during CRE-induced asthma.

Localized Free Radicals Burst Triggered by NIR-II Light for Augmented Low-Temperature Photothermal Therapy.

As a novel treatment modality of tumors, hypothermal hyperthermia employed relatively lower temperature (<45 °C) to damage cancer cells with mild toxicity to normal tissues. However, beyond that inducible heat resistance of tumor cells, the discounted therapeutic effect of low temperature hyperthermia was also ascribed to poor penetration of exogenous light stimulation and low accumulation of photothermal agents in tumor sites. Herein, we constructed a multifunctional in situ hydrogel of sodium alginate (ALG) via Ca2+ coordinated with ALG to encapsulate the photothermal agent of Ink and azo initiator of 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (AIPH) for effective tumor treatment. The designed ALG hydrogel was used to improve the therapeutic effect by increased accumulation of Ink and AIPH and avoid potential side-effects caused by the unexpected spread to the surrounding normal tissues. After injection, local low temperature stimulation was generated with near-infrared-II irradiation by a 1064 nm laser, triggering rapid decomposition of AIPH to produce alkyl radicals. The synergistic low temperature photothermal therapy and cytotoxic-free radicals enhanced the apoptosis of tumor cells via physical heat damage and lipid peroxidation. Thus, remarkable inhibition of tumor growth was observed in a subcutaneous colorectal cancer with negligible side effects. Furthermore, the formulation could also exert strong photoacoustic signals, which were utilized to monitor the stability of the composite hydrogel.

Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders.

HIV-associated neurocognitive disorders (HAND) have been linked to dysregulation of glutamate metabolism in the central nervous system (CNS) culminating in elevated extracellular glutamate and disrupted glutamatergic neurotransmission. Increased glutamate synthesis via upregulation of glutaminase (GLS) activity in brain immune cells has been identified as one potential source of excess glutamate in HAND. However, direct evidence for this hypothesis in an animal model is lacking, and the viability of GLS as a drug target has not been explored. In this brief report, we demonstrate that GLS inhibition with the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) can reverse cognitive impairment in the EcoHIV-infected mouse model of HAND. However, due to peripheral toxicity DON is not amenable to clinical use in a chronic disease such as HAND. We thus tested JHU083, a novel, brain penetrant DON prodrug predicted to exhibit improved tolerability. Systemic administration of JHU083 reversed cognitive impairment in EcoHIV-infected mice similarly to DON, and simultaneously normalized EcoHIV-induced increases in cerebrospinal fluid (CSF) glutamate and GLS activity in microglia-enriched brain CD11b + cells without observed toxicity. These studies support the mechanistic involvement of elevated microglial GLS activity in HAND pathogenesis, and identify JHU083 as a potential treatment option. Graphical Abstract Please provide Graphical Abstract caption.Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders .

Azodyes as markers for tumor hypoxia imaging and therapy: An up-to-date review.

Tumor hypoxia is the low tissue oxygen levels seen characteristically in rapidly proliferating and expanding neoplasms. It affects both malignant tumor cells and its microenvironment, resulting in dysfunctional neovascularization. This leads to epithelial-to-mesenchymal transition phenotype, facilitating tumor progression through cell mobility, invasion, and metastasis. The hypoxic condition in solid tumors is thus an indicator of the process of cancer progression towards an aggressive malignant phenotype with an enhanced possibility of metastasis and resistance to treatment. Advancements in the detection of tumor hypoxia and its utilization as a treatment modality in solid tumors are highly imperative. The use of fluorescent probes is an evolving field for detecting hypoxic tumors in biological systems. The present review is an attempt to provide a contextual knowledge on the prominent role of tumor hypoxia in cancer progression and dissemination. The use of azodyes in detecting tumor hypoxia aiding in cancer diagnosis through fluorescence off-on imaging and azodye-based hypoxia selective pro-drugs for assisting cancer therapy are presented. The limitations of fluorescence based hypoxia imaging and further investigations desired for clinical usage of azodye based hypoxic probes for fluorescence imaging are also considered.

The Small Molecule Inhibitor QLT-0267 Decreases the Production of Fibrin-Induced Inflammatory Cytokines and Prevents Post-Surgical Peritoneal Adhesions.

Peritoneal adhesions develop after abdominal surgery, trauma or intraperitoneal infections, and have important consequences. The deposition of peritoneal fibrin is a common pathophysiological pathway for the formation of adhesions. Here, we aimed to examine the effects of fibrin-induced cytokine production on peritoneal mesothelial cells (PMCs), and to block the effects of fibrin using an integrin-linked kinase (ILK) inhibitor, QLT-0267. PMCs were cultured from the enzymatic disaggregation of rat omentum. After the PMCs were covered with fibrin, the expression of IL-1ß, IL-6, TNFα and VEGF-A increased. This increase in cytokine production was attenuated by QLT-0267, which acted via the inhibition of both the ILK and focal adhesion kinase (FAK) pathways, and subsequently via the GSK-3ß pathway. We found that QLT-0267 decreased both the severity of peritoneal adhesion and the serum levels of IL-6 in our post-surgical adhesion mouse model. In conclusion, our study provides novel evidence that fibrin-induced cytokine production may involve in the mechanism of peritoneal adhesion formation. Furthermore, the use of the small molecule inhibitor QLT-0267 is a new strategy in preventing peritoneal adhesion in patients undergoing abdominal surgery.

Identification of a Small Molecule That Selectively Inhibits ERG-Positive Cancer Cell Growth.

Oncogenic activation of the ETS-related gene (ERG) by recurrent gene fusions (predominantly TMPRSS2-ERG) is one of the most validated and prevalent genomic alterations present in early stages of prostate cancer. In this study, we screened small-molecule libraries for inhibition of ERG protein in TMPRSS2-ERG harboring VCaP prostate cancer cells using an In-Cell Western Assay with the highly specific ERG-MAb (9FY). Among a subset of promising candidates, 1-[2-Thiazolylazo]-2-naphthol (NSC139021, hereafter ERGi-USU) was identified and further characterized. ERGi-USU selectively inhibited growth of ERG-positive cancer cell lines with minimal effect on normal prostate or endothelial cells or ERG-negative tumor cell lines. Combination of ERGi-USU with enzalutamide showed additive effects in inhibiting growth of VCaP cells. A screen of kinases revealed that ERGi-USU directly bound the ribosomal biogenesis regulator atypical kinase RIOK2 and induced ribosomal stress signature. In vivo, ERGi-USU treatment inhibited growth of ERG-positive VCaP tumor xenografts with no apparent toxicity. Structure-activity-based derivatives of ERGi-USU recapitulated the ERG-selective activity of the parental compound. Taken together, ERGi-USU acts as a highly selective inhibitor for the growth of ERG-positive cancer cells and has potential for further development of ERG-targeted therapy of prostate cancer and other malignancies.Significance: A highly selective small-molecule inhibitor of ERG, a critical driver of early stages of prostate cancer, will be imperative for prostate cancer therapy. Cancer Res; 78(13); 3659-71. ©2018 AACR.

Encapsulation of N-Diazeniumdiolates within Liposomes for Enhanced Nitric Oxide Donor Stability and Delivery.

The rapid decomposition of nitric oxide (NO) donors in aqueous environments remains a limitation for applications requiring extended NO release. Herein, we report the synthesis of dipalmitoylphosphatidylcholine-based liposomes capable of extended NO release using low molecular weight NO donors and a reverse-phase evaporation technique. The encapsulation of the NO donors within the liposomes enabled both prolonged NO release and enhanced storage compared to free NO donors alone. The NO-releasing liposomes also demonstrated enhanced efficacy against human pancreatic cancer cells. These NO-release vehicles represent attractive anticancer therapeutics due to their potential to store the majority of their NO payload until reaching cancerous tissue at which time the lower pH inherent to such environments will trigger an avalanche of NO.

Stromal characterization and comparison of odontogenic cysts and odontogenic tumors using picrosirius red stain and polarizing microscopy: A retrospective and histochemical study.

INTRODUCTION: Odontogenic lesions represent a range of conditions, the features of which probably depend on the stage of induction towards tooth formation reached prior to neoplastic or hamartomatous proliferation. It has been also suggested that inductive changes may allow progression from one type of odontogenic tumor to another. The epithelium also plays an important role in the pathogenesis of these lesions; even stroma is likely to play an equally important role in the pathogenesis and biological behavior. So, this study was performed to investigate, compare, and correlate different types of collagen fibers in odontogenic cysts and odontogenic tumors. MATERIALS AND METHODS: Thirty each pre-diagnosed odontogenic cysts and tumors were histochemically analyzed using a special stain (Picrosirius red stain) and polarizing microscopy. RESULTS: Seven cases (99%) of inflammatory cysts exhibited predominantly greenish-yellow birefringence indicating procollagen, intermediate, or pathologic collagen fibers suggestive of loosely packed collagen fibers. Predominant yellowish-orange birefringence exhibited by 21 cases (99%) of developmental cysts was comparable to the yellowish-orange and orangish-red to red birefringence exhibited by odontogenic tumors suggesting tightly packed fibers. CONCLUSIONS: The Picrosirius red stain in conjunction with polarizing microscopy serves as a specific and sensitive tool in characterizing collagen fibers in odontogenic cysts and odontogenic tumor.

Effect of a Pluronic(®) P123 formulation on the nitric oxide-generating drug JS-K.

PURPOSE: O(2)-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] or JS-K is a nitric oxide-producing prodrug of the arylated diazeniumdiolate class with promising anti-tumor activity. JS-K has challenging solubility and stability properties. We aimed to characterize and compare Pluronic(®) P123-formulated JS-K (P123/JS-K) with free JS-K. METHODS: We determined micelle size, shape, and critical micelle concentration of Pluronic(®) P123. Efficacy was evaluated in vitro using HL-60 and U937 cells and in vivo in a xenograft in NOD/SCID IL2Rγ (null) mice using HL-60 cells. We compared JS-K and P123/JS-K stability in different media. We also compared plasma protein binding of JS-K and P123/JS-K. We determined the binding and Stern Volmer constants, and thermodynamic parameters. RESULTS: Spherical P123/JS-K micelles were smaller than blank P123. P123/JS-K formulation was more stable in buffered saline, whole blood, plasma and RPMI media as compared to free JS-K. P123 affected the protein binding properties of JS-K. In vitro it was as efficacious as JS-K alone when tested in HL-60 and U937 cells and in vivo greater tumor regression was observed for P123/JS-K treated NOD/SCID IL2Rγ (null) mice when compared to free JS-K-treated NOD/SCID IL2Rγ (null) mice. CONCLUSIONS: Pluronic(®) P123 solubilizes, stabilizes and affects the protein binding characteristics of JS-K. P123/JS-K showed more in vivo anti-tumor activity than free JS-K.

Complex formation, thermal behavior and stability competition between Cu(II) ion and Cu(0) nanoparticles with some new azo dyes. Antioxidant and in vitro cytotoxic activity.

Four triazole and thiadiazole-based azo chromophores namely [(E)-4-((1H-1,2,4-triazol-3-yl)diazenyl)benzene-1,3-diol.(HL(1)), (E)-4-((5-(methylthio)-1H-1,2,4-triazol-3-yl)diazenyl)benzene-1,3-diol.(HL(2)), (E)-4-((1,3,4-thiadiazol-2-yl)diazenyl)benzene-1,3-diol.(HL(3)) and (E)-4-((5-mercapto-1,3,4-thiadiazol-2-yl)diazenyl)benzene-1,3-diol.(HL(4))] were synthesized and characterized by elemental analyses, IR, UV-Vis as well as mass spectroscopy. Cu(II) complexes of the investigated azo dyes have been synthesized and characterized by elemental analyses, IR, electronic and ESR spectra, magnetic susceptibility and thermogravimetric analyses. The bond lengths and bond angles have been calculated to confirm the geometry of the ligands and their Cu(II) complexes. The mode of interaction of the azodyes to copper nanoparticles was described as coordination mode of charged dye molecules on the colloidal Cu(0) surface through anchoring OH(-) group. The apparent association constants of the colloidal copper nanoparticles azodye complexes in solution were evaluated using the spectral method and compared with the formation constant of the Cu(II) azo complexes. The antitumor and antioxidant activities of the synthesized azo dyes and their Cu(II) azo complexes have been evaluated.

Evaluation of responsive gene expression as a sensitive and specific biomarker in patients with ulcerative colitis.

BACKGROUND: Clinical trials in ulcerative colitis (UC) rely on certain parameters to evaluate responses that are highly subjective or of low sensitivity. Here, using a select group of genes, we tested the accuracy of gene expression analysis as a biomarker of clinical, endoscopic, and histologic improvements. METHODS: Intestinal biopsies were obtained from UC patients included in two cohorts. Cohort 1 was used to select for genes whose expression was modulated in active (vs. inactive) UC. Cohort 2 included patients recruited in a phase II study receiving placebo, mesalazine, or dersalazine sodium for 4 weeks. The expression of 44 genes identified in Cohort 1 was assessed at weeks 0 and 4, and was then correlated with biomarkers, as well as with clinical, endoscopic, and histologic scores. RESULTS: Significant changes in the expression of 31 of the 44 genes tested were detected in Cohort 2 at week 4. Gene expression (ΔCt) significantly correlated with the total Mayo score, C-reactive protein (CRP), and fecal calprotectin. The number of genes significantly regulated at week 4 was highly associated with histologic and endoscopic responses. Logistic regression analysis identified four separate genes (IFITM1, ITGB2, IL1R2, IL2RA) whose relative change was independently associated with endoscopic remission with high specificity and sensitivity. CONCLUSIONS: Change in the expression of a select set of genes can serve as an early biomarker, one with high specificity and sensitivity to clinical, endoscopic, and histologic responses. This could represent a new tool for identifying early response to treatment in mild to moderately active UC patients.