Dually crosslinked degradable polyionic micelles for sustained glucose-responsive insulin release.

Glucose -sensitive delivery systems hold great promise as a therapeutic approach for high-incidence diabetes owing to their ability to release insulin whenever elevated glycemia is detected. However, they are unstable in a hyperglycemic environment, which leads to short-term sustained insulin release. Herein, we designed dually crosslinked insulin polyionic micelles (DCM@insulin) based on triblock polymers of o-glycol and phenylboronic acid-functionalized poly(ethylene glycol)-poly(dimethylamino carbonate)-poly(dimethylamino-trimethylene carbonate) (mPEG-P(AC-co-MPD)-PDMAC and mPEG-P(AC-co-MAPBA)-PDMATC, respectively) for sustained glucose-responsive insulin release. DCM@insulin with a phenylboronic acid ester structure (first crosslinking structure) enhanced glycemic responsiveness by regulating insulin release in a hyperglycemic environment. Additionally, the UV-crosslinking structure (second crosslinking structure) formed by the residual double bonds in AC units endowed DCM@insulin with the ability to effectively protect the loaded insulin against protease degradation and avoid burst release under multiple insulin release. The in vivo findings demonstrated that DCM@insulin effectively maintained glycemic levels (BGLs) within the normal range for 6 h in comparison to single-crosslinked micelles (SCM@insulin). Therefore, the glucose-responsive and dually crosslinked polyionic micelle system exhibits potential as a viable option for the treatment of diabetes.

Novel orally bioavailable piperidine derivatives as extracellular arginase inhibitors developed by a ring expansion.

Arginase is a multifaced enzyme that plays an important role in health and disease being regarded as a therapeutic target for the treatment of various pathological states such as malignancies, asthma, and cardiovascular disease. The discovery of boronic acid-based arginase inhibitors in 1997 revolutionized attempts of medicinal chemistry focused on development of drugs targeting arginase. Unfortunately, these very polar compounds had limitations such as analysis and purification without chromophores, synthetically challenging space, and poor oral bioavailability. Herein, we present a novel class of boronic acid-based arginase inhibitors which are piperidine derivatives exhibiting a different pharmacological profile compared to our drug candidate in cancer immunotherapy -OATD-02 - dual ARG1/2 inhibitor with high intracellular activity. Compounds from this new series show low intracellular activity, hence they can inhibit mainly extracellular arginase, providing different therapeutic space compared to a dual intracellular ARG1/2 inhibitor. The disclosed series showed good inhibitory potential towards arginase enzyme in vitro (IC50 up to 160 nM), favorable pharmacokinetics in animal models, and encouraging preliminary in vitro and in vivo tolerability. Compounds from the new series have moderate-to-high oral bioavailability (up to 66 %) and moderate clearance in vivo. Herein we describe the development and optimization of the synthesis of the new class of boronic acid-based arginase inhibitors via a ring expansion approach starting from the inexpensive chirality source (d-hydroxyproline). This upgraded methodology facilitated a gram-scale delivery of the final compound and eliminated the need for costly and time-consuming chiral resolution.

A Landmark Paper That Introduced Proteasome Inhibition in Myeloma.

The ongoing therapeutic revolution in multiple myeloma care can be traced to the turn of the millennium with the unanticipated discovery in 1999 that the cereblon binding small molecule thalidomide had profound clinical effectiveness and, simultaneously, the emergence of a new class of targeted therapies inhibiting the proteasome, both of which ultimately target ubiquitinated protein degradation. These contemporaneous discoveries forever changed the landscape of multiple myeloma care, substantially extending survival. Foreshadowing this seismic change, Nobel Prize winning work on the proteasome ubiquitin pathway had stimulated the development of highly specific proteasome inhibitor small molecules, particularly PS-341 (later named bortezomib). An abundance of the proteasome in hematologic malignancies had been recognized and thus PS-341 was logically being explored in relevant preclinical models. Concurrent with phase I trials, which were soon to prove the significant clinical relevance of preclinical models, the laboratory of Dr. Kenneth Anderson and colleagues at Dana-Farber, in partnership with Dr. Julian Adams and scientists at ProScript (later Millennium Pharmaceuticals) first demonstrated that the proteasome inhibitor PS-341 inhibited growth, induced apoptosis, and overcame drug resistance in human multiple myeloma cells. This landmark paper in Cancer Research set the stage for a paradigm shift in how multiple myeloma was managed across all stages of the disease, which changed the lives of patients worldwide. See related article by Hideshima and colleagues, Cancer Res 2001;61:3071-6.

Molecular docking, synthesis, anticancer activity, and metabolomics study of boronic acid ester-containing fingolimod derivatives.

In recent years, drugs that contain boronic acid groups, such as ixazomib (Ninlaro™) and bortezomib (Velcade™), have been used in the treatment of bone marrow cancer. The activity of compounds has been found to increase with the addition of boron atoms to the structure. In addition to these compounds, studies have found that fingolimod (FTY720) is more effective against breast cancer than cisplatin. Therefore, in this study, the first examples of boron-containing derivatives of fingolimod were designed and synthesized; in addition, their structures were confirmed by spectroscopic techniques. The synthesized boron-containing drug candidates were found to significantly inhibit cell proliferation and induce apoptosis-mediated cell death in HT-29 (colorectal cells), SaOs-2 (osteosarcoma cells), and U87-MG (glioblastoma cells). Moreover, we revealed that the anticancer effects of boron-containing fingolimod compounds were found to be significantly enhanced over boron-free control groups and, strikingly, over the widely used anticancer drug 5-fluorouracil. The metabolomic analysis confirmed that administration of the boron-containing drug candidates induces significant changes in the metabolite profiles in HT-29, SaOs-2, and U87-MG cells. Altogether, our results showed that boron-containing fingolimod compounds can be further examined to reveal their potential as anticancer drug candidates.

High-content microscopy reveals a morphological signature of bortezomib resistance.

Drug resistance is a challenge in anticancer therapy. In many cases, cancers can be resistant to the drug prior to exposure, that is, possess intrinsic drug resistance. However, we lack target-independent methods to anticipate resistance in cancer cell lines or characterize intrinsic drug resistance without a priori knowledge of its cause. We hypothesized that cell morphology could provide an unbiased readout of drug resistance. To test this hypothesis, we used HCT116 cells, a mismatch repair-deficient cancer cell line, to isolate clones that were resistant or sensitive to bortezomib, a well-characterized proteasome inhibitor and anticancer drug to which many cancer cells possess intrinsic resistance. We then expanded these clones and measured high-dimensional single-cell morphology profiles using Cell Painting, a high-content microscopy assay. Our imaging- and computation-based profiling pipeline identified morphological features that differed between resistant and sensitive cells. We used these features to generate a morphological signature of bortezomib resistance. We then employed this morphological signature to analyze a set of HCT116 clones (five resistant and five sensitive) that had not been included in the signature training dataset, and correctly predicted sensitivity to bortezomib in seven cases, in the absence of drug treatment. This signature predicted bortezomib resistance better than resistance to other drugs targeting the ubiquitin-proteasome system, indicating specificity for mechanisms of resistance to bortezomib. Our results establish a proof-of-concept framework for the unbiased analysis of drug resistance using high-content microscopy of cancer cells, in the absence of drug treatment.

Organoboronic acids/esters as effective drug and prodrug candidates in cancer treatments: challenge and hope.

Boronic acids/esters have recently emerged in the field of medicinal and pharmaceutical research due to their exceptional oxophilicity, low toxicity, and unique structure. They are known as potent enzyme inhibitors, cancer therapy capture agents, and can mimic certain types of antibodies to fight infections. They have been designed and developed into drugs, and this approach has emerged in the last 20 years. Five boronic acid drugs have been approved by the FDA and Health Canada, two of which are used to treat cancer, specifically multiple myeloma. The purpose of this review is to investigate boronic acid/ester derivatives as potential pharmaceutical agents as well as the mechanism of action. It will concentrate on six types of cancer: multiple myeloma, prostate cancer, breast cancer, lung cancer, cervical cancer, and colon cancer. Some newly developed boron-containing compounds have already demonstrated highly promising activities, but further investigation is required before final conclusions can be drawn.

Effects of invivo CXCR4 blockade and proteasome inhibition on bone marrow plasma cells in HLA-sensitized kidney transplant candidates.

To date, plasma cell (PC)-targeted therapies have been limited by suboptimal PC depletion and antibody rebound. We hypothesized this is partly because of PC residence in protective bone marrow (BM) microenvironments. The purpose of this proof-of-concept study was to examine the effects of the CXCR4 antagonist, plerixafor, on PC BM residence; its safety profile (alone and in combination with a proteasome inhibitor, bortezomib); and the transcriptional effect on BMPCs in HLA-sensitized kidney transplant candidates. Participants were enrolled into 3 groups: group A (n = 4), plerixafor monotherapy; and groups B (n = 4) and C (n = 4), plerixafor and bortezomib combinations. CD34+ stem cell and PC levels increased in the blood after plerixafor treatment. PC recovery from BM aspirates varied depending on the dose of plerixafor and bortezomib. Single-cell RNA sequencing on BMPCs from 3 group C participants pretreatment and posttreatment revealed multiple populations of PCs, with a posttreatment enrichment of oxidative phosphorylation, proteasome assembly, cytoplasmic translation, and autophagy-related genes. Murine studies demonstrated dually inhibiting the proteasome and autophagy resulted in greater BMPC death than did monotherapies. In conclusion, this pilot study revealed anticipated effects of combined plerixafor and bortezomib on BMPCs, an acceptable safety profile, and suggests the potential for autophagy inhibitors in desensitization regimens.

Entinostat-Bortezomib Hybrids against Multiple Myeloma.

Although proteasome inhibitors have emerged as the therapeutic backbone of multiple myeloma treatment, patients often relapse and become drug refractory. The combination between proteasome and histone deacetylase inhibitors has shown to be more efficient compared to monotherapy by enhancing the anti-myeloma activity and improving the patient's lifetime expectancy. Hybrid molecules, combining two drugs/pharmacophores in a single molecular entity, offer improved effectiveness by modulating more than one target and circumventing differences in the pharmacokinetic and pharmacodynamic profiles, which are the main disadvantages of combination therapy. Therefore, eleven histone deacetylase-proteasome inhibitor hybrids were synthesized, combining pharmacophores of entinostat and bortezomib. Compound 3 displayed the strongest antiproliferative activity with an IC50 value of 9.5 nM in the multiple myeloma cells RPMI 8226, 157.7 nM in the same cell line resistant to bortezomib, and 13.1 nM in a 3D spheroid model containing multiple myeloma and mesenchymal stem cells. Moreover, the compound inhibited 33% of histone deacetylase activity when RPMI 8226 cells were treated for 8 h at 10 µM. It also inhibited the proteasome activity with an IC50 value of 23.6 nM.

Discovery of non-boronic acid Arginase 1 inhibitors through virtual screening and biophysical methods.

Inhibiting Arginase 1 (ARG1), a metalloenzyme that hydrolyzes l-arginine in the urea cycle, has been demonstrated as a promising therapeutic avenue in immuno-oncology through the restoration of suppressed immune response in several types of cancers. Most of the currently reported small molecule inhibitors are boronic acid based. Herein, we report the discovery of non-boronic acid ARG1 inhibitors through virtual screening. Biophysical and biochemical methods were used to experimentally profile the hits while X-ray crystallography confirmed a class of trisubstituted pyrrolidine derivatives as optimizable alternatives for the development of novel classes of immuno-oncology agents targeting this enzyme.

Boronic Acid: A Novel Pharmacophore Targeting Src Homology 2 (SH2) Domain of STAT3.

SH2 domains have been recognized as promising targets for various human diseases. However, targeting SH2 domains with phosphopeptides or small-molecule inhibitors derived from bioisosteres of the phosphate group is still challenging. Identifying novel bioisosteres of the phosphate group to achieve favorable in vivo potency is urgently needed. Here, we report the feasibility of targeting the STAT3-SH2 domain with a boronic acid group and the identification of a highly potent inhibitor compound 7 by replacing the carboxylic acid of compound 4 with a boronic acid. Compound 7 shows higher binding affinity, better cellular potency, more favorable PK profiles, and higher in vivo antitumor activity than 4. The stronger anticancer effect of 7 partially stems from its covalent binding mode with the SH2 domain, verified by the washout experiments. The relatively high level of sequence conservation among SH2 domains makes the results presented here of general significance.

Arsenic Prodrug-Mediated Tumor Microenvironment Modulation Platform for Synergetic Glioblastoma Therapy.

Glioblastoma (GBM) has a distinct internal environment characterized by high levels of glutathione (GSH) and low oxygen partial pressure, which significantly restrict most drugs' effectiveness. Arsenic-based drugs are emerging candidates for treating solid tumors; however, relatively high doses in solo systems and inconsistent complementary systems severely damage the normal tissues. We proposed a novel covalently conjugated strategy for arsenic-based therapy via arsenic-boronic acid complex formation. The boronic acid was modified on silver (AgL) to capture AsV under an alkaline condition named arsenate plasmonic complex (APC) with a distinct Raman response. The APC can precisely release the captured AsV in lysosomal acidic pH that specifically targets TME to initiate a multimodal therapeutic effect such as GSH depletion and reactive oxygen species generation. In addition, GSH activation leads to subconverted AsV into AsIII, which further facilitated glutathione peroxidase (GPx) and superoxide dismutase inhibition, whereas the tumor selective etching of the silver core triggered by endogenous H2O2 that can oxidize to generate highly toxic Ag ions produces and supplies O2 to help the alleviated hypoxia. Both in vitro and in vivo data verify the APC-based chemotherapy paving the way for efficient nanomedicine-enabled boronate affinity-based arsenic chemotherapeutics for on demand site-specific cancer combination treatment of GBM tumors.

ISG20L2 suppresses bortezomib antimyeloma activity by attenuating bortezomib binding to PSMB5.

The proteasome inhibitors (PIs) bortezomib and carfilzomib, which target proteasome 20S subunit beta 5 (PSMB5) in cells, are widely used in multiple myeloma (MM) treatment. In this study, we demonstrated the role of interferon-stimulated 20 kDa exonuclease-like 2 (ISG20L2) in MM PI resistance. Gain- and loss-of-function studies showed that ISG20L2 suppressed MM cell sensitivity to PIs in vitro and in vivo. Patients with ISG20L2lo MM had a better response to PIs and a longer overall survival than patients with ISG20L2hi MM. Biotinylated bortezomib pull-down assays showed that ISG20L2 competed with PSMB5 in binding to bortezomib. The surface plasmon resonance assay confirmed the direct binding of bortezomib to ISG20L2. In ISG20L2hi MM cells, ISG20L2 attenuated the binding of bortezomib to PSMB5, resulting in lower inhibition of proteasome activity and therefore less bortezomib-induced cell death. Overall, we identified a potentially novel mechanism by which ISG20L2 conferred bortezomib resistance on MM. The expression of ISG20L2 correlated with MM PI responses and patient treatment outcomes.

Metformin Induces Resistance of Cancer Cells to the Proteasome Inhibitor Bortezomib.

The anti-diabetic drug metformin is currently tested for the treatment of hematological and solid cancers. Proteasome inhibitors, e.g., Bortezomib, are approved for the treatment of multiple myeloma and mantle cell lymphoma but are also studied for lung cancer therapy. We here analyzed the interaction of the two drugs in two cell lines, namely the mantle cell lymphoma Jeko-1 and the non-small-cell lung cancer (NSCLC) H1299 cells, using proliferation and survival assays, native-gel analysis for proteasome activity and assembly, and expression analysis of proteasome assembly factors. Our results demonstrate that metformin treatment induces resistance of cancer cells to the proteasome inhibitor Bortezomib by impairing the activity and assembly of the 26S proteasome complexes. These effects of metformin on proteasome inhibitor sensitivity in cancer cells are of potential relevance for patients that receive proteasome inhibitor therapy.

Bortezomib inhibits the proteasome, leading to cell death via apoptosis in feline injection site sarcoma cells in vitro.

OBJECTIVE: To determine the in vitro effects of the proteasome inhibitor bortezomib in feline injection site sarcoma (FISS) cell lines. SAMPLE: In vitro cultures of the FISS cell lines Ela-1, Hamilton, and Kaiser. PROCEDURES: Cells were treated with increasing doses of bortezomib or vehicle alone (dimethyl sulfoxide) and evaluated for cell viability via an adenosine triphosphate concentration assay, proteasome activity via a commercially available proteasome assay, accumulation of ubiquitinated proteins via Western blot, and apoptosis via flow cytometry. RESULTS: All 3 cell lines were sensitive to bortezomib with a 50% inhibitory concentration after 48 hours of treatment at 17.46 nM (95% CI, 15.47 to 19.72 nM) for Ela-1, 19.48 nM (95% CI, 16.52 to 23.00 nM) for Hamilton, and 21.38 nM (95% CI, 19.24 to 23.78 nM) for Kaiser. In the Ela-1 cell line, 20 nM bortezomib inhibited 20S proteasome activity by 90.9% compared with the vehicle-only control. In the Kaiser cell line, 20 nM bortezomib decreased 20S proteasome activity by 70%, compared with the untreated vehicle-only control. Last, treatment with bortezomib (25 and 40 nM) resulted in statistically significant decreases in viable cells accompanied by a statistically significant increase in apoptotic cells. CLINICAL RELEVANCE: Treatment options for FISS, especially nonresectable FISS, are currently very limited. These results support further investigation of bortezomib either alone or in combination with other treatments in such cases.

A Hyaluronan and Proteoglycan Link Protein 1 Matrikine: Role of Matrix Metalloproteinase 2 in Multiple Myeloma NF-κB Activation and Drug Resistance.

The NF-κB signaling pathway plays key roles in inflammation and the pathogenesis of many solid and hematologic malignancies, including multiple myeloma, a malignancy of the plasma cells. While proteasome inhibitors, such as bortezomib, employed in multiple myeloma treatments may inhibit NF-κB signaling pathways, multiple myeloma cells often become drug resistant in part due to non-cell autonomous mechanism(s) from the multiple myeloma tumor microenvironment. We previously found that fragments of, but not full-length, hyaluronan and proteoglycan link protein 1 (HAPLN1), produced by multiple myeloma bone marrow stromal cells (BMSC), activate an atypical bortezomib-resistant NF-κB pathway in multiple myeloma cells. In our current study, we found that multiple myeloma cells promote HAPLN1 expression and matrix metalloproteinase 2 (MMP2) activity in cocultured BMSCs and MMP2 activity is higher in BMSCs established from multiple myeloma patients' BM aspirates relative to normal equivalents. Moreover, MMP2 cleaves HAPLN1 into forms similar in size to those previously observed in patients with multiple myeloma with progressive disease. Both HAPLN1 and MMP2 in BMSCs were required to enhance NF-κB activation and resistance to bortezomib-induced cell death in cocultured multiple myeloma cells. We propose that MMP2-processing of HAPLN1 produces a matrikine that induces NF-κB activation and promotes bortezomib resistance in multiple myeloma cells. IMPLICATIONS: HAPLN1 and MMP2 produced by BMSCs obtained from patients with multiple myeloma promote NF-κB activity and resistance to bortezomib toxicity in multiple myeloma cells, uncovering their potential as biomarkers or therapeutic targets to address bortezomib resistance in patients with multiple myeloma.

Dietary Flavonoids with Catechol Moiety Inhibit Anticancer Action of Bortezomib: What about the other Boronic Acid-based Drugs?

Approval of the first boronic acid group-containing drug, bortezomib, in 2003 for the treatment of multiple myeloma sparked an increased interest of medicinal chemists in boronic acidbased therapeutics. As a result, another boronic acid moiety-harboring medication, ixazomib, was approved in 2015 as a second-generation proteasome inhibitor for multiple myeloma; and dutogliptin is under clinical investigation in combination therapy against myocardial infarction. Moreover, a large number of novel agents with boronic acid elements in their structure are currently in intensive preclinical studies, allowing us to suppose that at least some of them will enter clinical trials in the near future. On the other hand, only some years after bortezomib approval, direct interactions between its boronic acid group and catechol moiety of green tea catechins as well as some other common dietary flavonoids like quercetin and myricetin were discovered, leading to the formation of stable cyclic boronate esters and abolishing the anticancer activities. Although highly relevant, to date, no reports on possible co-effects of catechol group-containing flavonoids with new-generation boronic acidbased drugs can be found. However, this issue cannot be ignored, especially considering the abundance of catechol moiety-harboring flavonoids in both plant-derived food items as well as over-thecounter dietary supplements and herbal products. Therefore, in parallel with the intensified development of boronic acid-based drugs, their possible interactions with catechol groups of plant-derived flavonoids must also be clarified to provide dietary recommendations to patients for maximizing therapeutic benefits. If concurrently consumed flavonoids can indeed antagonize drug efficacy, it may pose a real risk to clinical outcomes.

Palladium catalyzed migratory heck coupling of arteannuin B and boronic acids: An approach towards the synthesis of antiproliferative agents in breast and lung cancer cells.

We have recently highlighting the role of spiroisoxazoline arteannuin B derivatives in mediating proinflammatory cytokines like IL-6, TNfα and NO in vitro. In the present study, a series of new ß-arylated arteannuin B analogues were synthesized through coupling with arylboroic acids and evaluated for their in vitro cytotoxic activity in a panel of six cancer cell lines. The binding efficiency was verified by docking of the original ligand within the active site of ATPase domain of GRP78 (PDB ID: 3LDL) at a resolution of 2.30 Å with the score energy of -8.07 kcal/mol. Among the new compounds 3a, 3b, 3d, 3i, 3j and 3n displayed potent cytotoxic potential with an IC50 from 2 to 18 µM and compound 3i was proven to be the most potent cytotoxic and anti-proliferative compound of all the six distinct cell lines. Compound 3i exhibited promising apoptosis inducing potential in breast cancer cells and stalled their wound healing properties and was effective in blocking the migration of cancer cells.

Synthesis, equilibrium, and biological study of a C-7 glucose boronic acid derivative as a potential candidate for boron neutron capture therapy.

The synthesis of d-glucoheptose derivative containing a boronic moiety is described herein. Starting from benzyl 6,7-dideoxy-2,3,4-tri-O-benzyl-ß-d-gluco-ept-6-enopyranoside, the introduction of the boronic acid was performed through a metathesis reaction by using MIDA vinyl boronic acid and the 2nd generation Grubbs catalyst. Hydrogenation led to the final product in only two reaction steps. This new sugar-containing boronic acid in the skeleton could mimic carbohydrate behavior and follow the glucose uptake in living cells. The in vitro toxicity tests performed in fibroblasts and glioma tumor cell lines showed minimal toxicity. Boron uptake measured using ICP-MS was minimal in fibroblasts, while in glioma cells showed a value of 6 ng of total boron accumulation per mg of cells, implying that compound 1a is able to accumulate selectively in the tumor tissues compared to normal.

3-Pyridinylboronic Acid Ameliorates Rotenone-Induced Oxidative Stress Through Nrf2 Target Genes in Zebrafish Embryos.

Parkinson's disease (PD) is one of the most common forms of neurodegenerative diseases and research on potential therapeutic agents for PD continues. Rotenone is a neurotoxin that can pass the blood-brain barrier and is used to generate PD models in experimental animals. Boron is a microelement necessary for neural activity in the brain. Antioxidant, non-cytotoxic, anti-genotoxic, anti-carcinogenic effects of boric acid, the salt compound of boron has been reported before. Boronic acids have been approved for treatment by FDA and are included in drug discovery studies and pyridine boronic acids are a subclass of heterocyclic boronic acids used in drug design and discovery as substituted pyridines based on crystal engineering principles. The aim of our study was to determine the effect of 3-pyridinylboronic acid in rotenone-exposed zebrafish embryos, focusing on oxidant-antioxidant parameters and gene expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) target genes gclm, gclc, hmox1a, nqo1, and PD related genes, brain-derived neurotrophic factor, dj1, and tnfα. Zebrafish embryos were exposed to Rotenone (10 µg/l); Low Dose 3-Pyridinylboronic acid (100 µM); High Dose 3-Pyridinylboronic acid (200 µM); Rotenone + Low Dose-3-Pyridinylboronic acid (10 µg/l + 100 µM); Rotenone + High Dose-3-Pyridinylboronic acid (10 µg/l + 200 µM) in well plates for 96 h post-fertilization (hpf). Our study showed for the first time that 3-pyridinylboronic acid, as a novel sub-class of the heterocyclic boronic acid compound, improved locomotor activities, ameliorated oxidant-antioxidant status by decreasing LPO and NO levels, and normalized the expressions of bdnf, dj1, tnf⍺ and Nrf2 target genes hmox1a and nqo1 in rotenone exposed zebrafish embryos. On the other hand, it caused the deterioration of the oxidant-antioxidant balance in the control group through increased lipid peroxidation, nitric oxide levels, and decreased antioxidant enzymes. We believe that these results should be interpreted in the context of the dose-toxicity and benefit-harm relationship of the effects of 3-pyridinylboronic.

The Inhibitory Effect of Blue Light on Phagocytic Activity by ARPE-19 Cells.

Chronic exposure of the retina to short wavelength visible light is a risk factor in pathogenesis of age-related macular degeneration. The proper functioning and survival of photoreceptors depends on efficient phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelium. The purpose of this study was to analyze the phagocytic activity of blue light-treated ARPE-19 cells, and to examine whether the observed effects could be related to altered levels of POS phagocytosis receptor proteins and/or to oxidation of cellular proteins and lipids. POS phagocytosis was measured by flow cytometry. Phagocytosis receptor proteins αv and ß5 integrin subunits and Mer tyrosine kinase (MerTK) were quantified by western blotting. The intact functional heterodimer αvß5 was quantified by immunoprecipitation followed by immunoblotting. Cellular protein and lipid hydroperoxides were analyzed by coumarin boronic acid probe and iodometric assay, respectively. Cell irradiation induced reversible inhibition of specific phagocytosis and transient reductions in phagocytosis receptor proteins. Full recovery of functional heterodimer was apparent. Significant photooxidation of cellular proteins and lipids was observed. The results indicate that transient inhibition of specific phagocytosis by blue light could be related to the reduction in phagocytosis receptor proteins. Such changes may arise from oxidative modifications of cell phagocytic machinery components.