Conjugated Polymer-Ferrocence Nanoparticle as an NIR-II Light Powered Nanoamplifier to Enhance Chemodynamic Therapy.

Chemodynamic therapy (CDT) is a promising therapeutic modality with transition metal ions and endogenous H2O2 as reagents, but its efficiency is impaired by low endogenous H2O2 levels and nonregeneration of metal ions. Most intracellular H2O2 supplement strategies use oxidases and are intensively dependent on oxygen participation. The hypoxia microenvironments of solid tumors weaken their performance. Here, we develop a near-infrared II light powered nanoamplifier to improve the local oxygen level and to enhance CDT. The nanoamplifier CPNP-Fc/Pt consists of ferrocene (Fc)- and cisplatin prodrug (Pt(IV))-modified conjugated polymer nanoparticles (CPNPs). CPNP has a donor-acceptor structure and demonstrates a good photothermal effect under 1064 nm light irradiation, which accelerates blood flow and efficiently elevates the local oxygen content. In response to intracellular glutathione, Pt(II) is released from CPNP-Fc/Pt and triggers enzymatic cascade reactions with nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and superoxide dismutase to convert oxygen into H2O2. The enhanced oxygen level results in efficient intracellular H2O2 supply. Fc is reacted with H2O2 and converted to Fc+ via the Fenton reaction, with the generation of hydroxyl radicals for CDT. Unlike free metal ions, the Fe(III) in Fc+ is reduced to Fe(II) by intracellular NAD(P)H, which achieves the regeneration of Fc. The sufficient intracellular H2O2 supply and efficient Fc regeneration effectively enhance the Fenton reaction and demonstrate good in vivo CDT results with tumor growth suppression. This design offers a promising strategy to enhance CDT efficiency in the hypoxia microenvironment of solid tumors.

Enzymatic/Magnetic Hybrid Micromotors for Synergistic Anticancer Therapy.

Micro/nanomotors (MNMs), which propel by transforming various forms of energy into kinetic energy, have emerged as promising therapeutic nanosystems in biomedical applications. However, most MNMs used for anticancer treatment are only powered by one engine or provide a single therapeutic strategy. Although double-engined micromotors for synergistic anticancer therapy can achieve more flexible movement and efficient treatment efficacy, their design remains challenging. In this study, we used a facile preparation method to develop enzymatic/magnetic micromotors for synergetic cancer treatment via chemotherapy and starvation therapy (ST), and the size of micromotors can be easily regulated during the synthetic process. The enzymatic reaction of glucose oxidase, which served as the chemical engine, led to self-propulsion using glucose as a fuel and ST via a reduction in the energy available to cancer cells. Moreover, the incorporation of Fe3O4 nanoparticles as a magnetic engine enhanced the kinetic power and provided control over the direction of movement. Inherent pH-responsive drug release behavior was observed owing to the acidic decomposition of drug carriers in the intracellular microenvironment of cancer cells. This system displayed enhanced anticancer efficacy owing to the synergetic therapeutic strategies and increased cellular uptake in a targeted area because of the improved motion behavior provided by the double engines. Therefore, the demonstrated micromotors are promising candidates for anticancer biomedical microsystems.

Gene/drug-embedded nanoscale metal azolate framework-7 for the reversal of P-glycoprotein-mediated multidrug resistance.

We report the straightforward synthesis of ATP-responsive nanoscale metal azolate framework-7 (MAF-7) for gene/drug codelivery. The MAF-7 functions as (i) the armour to preserve DNAzymes, (ii) an ATP scavenger to lower the intracellular ATP level, and (iii) a built-in Zn2+ arsenal to initiate the biocatalysis of DNAzymes, ultimately inhibiting P-gp expression to enhance chemotherapy.

Molecular docking and dynamic studies of a potential therapeutic target inhibiting glyoxalase system: Metabolic action of the 3, 3 '- [3- (5-chloro-2-hydroxyphenyl) -3-oxopropane-1, 1-diyl] - Bis-4-hydroxycoumarin leads overexpression of the intracellular level of methylglyoxal and induction of a pro-apoptotic phenomenon in a hepatocellular carcinoma model.

Methylglyoxal is a dicarbonyl compound recruited as a potential cytotoxic marker, initially presents in cells and considered as a metabolite of the glycolytic pathway. Our aim is to demonstrate the inhibitory effect of 3, 3'-[3-(5-chloro-2-hydroxyphenyl)-3-oxopropane-1, 1-diyl] Bis (4-hydroxycoumarin) on the glyoxalase system, and indirectly its anticancer activity. The docking of OT-55 was conducted by using Flexible docking protocol, ChiFlex and libdock tools inside the active site of Glo-I indicated that both hydrogen bonding and hydrophobic interactions contributed significantly in establishing potent binding with the active site which is selected as a strong inhibitor with high scoring values and maximum Gibbs free energy. Coumarin-liposome formulation was characterized and evaluated in vivo against chemically induced hepatocarcinoma in Wistar rats. After Diethylnitrosamine (DEN) induction, microscopic assessment was realized; precancerous lesions were developed showing an increase of both tumor-associated lymphocyte and multiple tumor acini supported by the blood investigation. Our finding also suggested a preferential uptake of liposomes respectively in liver, kidney, lung, brain and spleen in the DEN-treated animals. OT-55 has also been shown to inhibit the activity of Glo-I in vitro as well as in DEN-treated rats. An abnormal high level of MGO of up to 50% was recorded followed by a reduction in glucose consumption and lactate dehydrogenase production validated in the positive control. MGO generates apoptosis as depicted by focal hepatic lesions. Also, no deleterious effects in the control group were observed after testing our coumarin but rather a vascular reorganization leading to nodular regenerative hyperplasia. Involved in the detoxification process, liver GSH is restored in intoxicated rats, while no changes are seen between controls. At the endothelial cell, OT-55 appears to modulate the release of NO only in the DEN-treated group. OT-55 would behave both as an anticancer agent but also as an angiogenic factor regarding results obtained.

Chemical characterization (LC-MS-ESI), cytotoxic activity and intracellular localization of PAMAM G4 in leukemia cells.

Generation 4 of polyamidoamine dendrimer (G4-PAMAM) has several biological effects due to its tridimensional globular structure, repetitive branched amides, tertiary amines, and amino-terminal subunit groups liked to a common core. G4-PAMAM is cytotoxic due to its positive charges. However, its cytotoxicity could increase in cancer cells due to the excessive intracellular negative charges in these cells. Furthermore, this work reports G4-PAMAM chemical structural characterization using UHPLC-QTOF-MS/MS (LC-MS) by electrospray ionization to measure its population according to its positive charges. Additionally, the antiproliferative effects and intracellular localization were explored in the HMC-1 and K-562 cell lines by confocal microscopy. The LC-MS results show that G4-PAMAM generated multivalent mass spectrum values, and its protonated terminal amino groups produced numerous positive charges, which allowed us to determine its exact mass despite having a high molecular weight. Additionally, G4-PAMAM showed antiproliferative activity in the HMC-1 tumor cell line after 24 h (IC50 = 16.97 µM), 48 h (IC50 = 7.02 µM) and 72 h (IC50 = 5.98 µM) and in the K-562 cell line after 24 h (IC50 = 15.14 µM), 48 h (IC50 = 14.18 µM) and 72 h (IC50 = 9.91 µM). Finally, our results showed that the G4-PAMAM dendrimers were located in the cytoplasm and nucleus in both tumor cell lines studied.

Engineered synthetic cell penetrating peptide with intracellular anti-inflammatory bioactivity: An in vitro and in vivo study.

Various biomaterials have been used for bone and cartilage regeneration, and inflammation associated with biomaterial implantation is also increased. A 15-mer synthetic anti-inflammatory peptide (SAP15) was designed from human ß-defensin 3 to penetrate cells and induce intracellular downregulation of inflammation. The downregulation of inflammation was achieved by the binding of SAP15 to intracellular histone deacetylase (HDAC5). SAP15-mediated inhibition of inflammation was examined in vitro and in vivo using murine macrophages, human articular chondrocytes, and a collagen-induced arthritis (CIA) rat model. Surface plasmon resonance and immunoprecipitation assays indicated that SAP15 binds to HDAC5. SAP15 inhibited the lipopolysaccharide (LPS)-induced phosphorylation of intracellular HDAC5 and NF-κB p65 in murine macrophages. SAP15 treatment increased aggrecan and type II collagen expression and decreased osteocalcin expression in LPS-induced chondrocytes. Subcutaneous injection of SAP15-loaded sodium hyaluronic acid (HA) solution significantly decreased hind paw swelling, joint inflammation, and serum cytokine levels in CIA rats compared with the effects of sodium HA solution alone. The SAP15-loaded HA group exhibited preservation of cartilage and bone structure in CIA rat joints. Moreover, a more robust anti-inflammatory effect of the SAP15 loaded HA was observed than that of etanercept (an anti-tumor necrosis factor-alpha [TNF-α] antibody)-loaded HA. These findings suggest that SAP15 has an anti-inflammatory effect that is not controlled by sodium HA and is mediated by inhibiting HDAC5, unlike the anti-inflammatory mechanism of etanercept. These results demonstrate that SAP15 is useful as an inflammatory regulator of biomaterials and can be developed as a therapeutic for the treatment of inflammation.

Cell Adhesion-Mediated Piezoelectric Self-Stimulation on Polydopamine-Modified Poly(vinylidene fluoride) Membranes.

Cell adhesion-mediated piezoelectric stimulation provides a noninvasive method for in situ electrical regulation of cell behavior, offering new opportunities for the design of smart materials for tissue engineering and bioelectronic medicines. In particular, the surface potential is mainly dominated by the inherent piezoelectricity of the biomaterial and the dynamic adhesion state of cells. The development of an efficient and optimized material interface would have important implications in cell regulation. Herein, we modified the surface of poled poly(vinylidene fluoride) (PVDF) membranes through polymerization of dopamine and investigated their influence on cell adhesion and electromechanical self-stimulation. Our results demonstrated that mesenchymal stem cells seeded on the poled PVDF membrane exhibited stronger cell spreading and adhesion. Meanwhile, the surface modification through polydopamine significantly improved the hydrophilicity of the samples and contributed to the formation of cell actin bundles and maturation of focal adhesions, which further positively modulated cell piezoelectric self-stimulation and induced intracellular calcium transients. Combining with theoretical simulations, we found that the self-stimulation was enhanced mainly due to the increase of the adhesion site and adhesion force magnitude. These findings provide new insights for probing the cell regulation mechanism on piezoelectric substrates, offering more opportunities for the rational design of piezoelectric biomaterial interfaces for biomedical engineering.

A novel peptide antagonist of the human growth hormone receptor.

Excess circulating human growth hormone (hGH) in vivo is linked to metabolic and growth disorders such as cancer, diabetes, and acromegaly. Consequently, there is considerable interest in developing antagonists of hGH action. Here, we present the design, synthesis, and characterization of a 16-residue peptide (site 1-binding helix [S1H]) that inhibits hGH-mediated STAT5 phosphorylation in cultured cells. S1H was designed as a direct sequence mimetic of the site 1 mini-helix (residues 36-51) of wild-type hGH and acts by inhibiting the interaction of hGH with the human growth hormone receptor (hGHR). In vitro studies indicated that S1H is stable in human serum and can adopt an α-helix in solution. Our results also show that S1H mitigates phosphorylation of STAT5 in cells co-treated with hGH, reducing intracellular STAT5 phosphorylation levels to those observed in untreated controls. Furthermore, S1H was found to attenuate the activity of the hGHR and the human prolactin receptor, suggesting that this peptide acts as an antagonist of both lactogenic and somatotrophic hGH actions. Finally, we used alanine scanning to determine how discrete amino acids within the S1H sequence contribute to its structural organization and biological activity. We observed a strong correlation between helical propensity and inhibitory effect, indicating that S1H-mediated antagonism of the hGHR is largely dependent on the ability for S1H to adopt an α-helix. Taken together, these results show that S1H not only acts as a novel peptide-based antagonist of the hGHR but can also be applied as a chemical tool to study the molecular nature of hGH-hGHR interactions.

Dynamic subcellular localization and transcription activity of the SRF cofactor MKL2 in the striatum are regulated by MAPK.

Dopamine type 1 receptor (D1R) signaling activates protein kinase A (PKA), which then activates mitogen-activated protein kinase (MAPK) through Rap1, in striatal medium spiny neurons (MSNs). MAPK plays a pivotal role in reward-related behavior through the activation of certain transcription factors. How D1R signaling regulates behavior through transcription factors remains largely unknown. CREB-binding protein (CBP) promotes transcription through hundreds of different transcription factors and is also important for reward-related behavior. To identify transcription factors regulated by dopamine signaling in MSNs, we performed a phosphoproteomic analysis using affinity beads coated with CBP. We obtained approximately 40 novel candidate proteins in the striatum of the C57BL/6 mouse brain after cocaine administration. Among them, the megakaryoblastic leukemia-2 (MKL2) protein, a transcriptional coactivator of serum response factor (SRF), was our focus. We found that the interaction between CBP and MKL2 was increased by cocaine administration. Additionally, MKL2, CBP and SRF formed a ternary complex in vivo. The C-terminal domain of MKL2 interacted with CBP-KIX and was phosphorylated by MAPK in COS7 cells. The activation of PKA-MAPK signaling induced the nuclear localization of MKL2 and increased SRF-dependent transcriptional activity in neurons. These results demonstrate that dopamine signaling regulates the interaction of MKL2 with CBP in a phosphorylation-dependent manner and thereby controls SRF-dependent gene expression. Cover Image for this issue: https://doi.org/10.1111/jnc.15067.

Improved photothermal therapy of brain cancer cells and photogeneration of reactive oxygen species by biotin conjugated gold photoactive nanoparticles.

Herein, we report on the design and development of functionalized acrylic polymeric nanoparticles with Spiropyrans (SPs) and imidazole moieties via superficial polymerizations. Then, Au3+ ions were immobilized and reduced on their surface to obtain photoresponsive gold-decorated polymer nanoparticles(Au-NPs). The synthesized Au-NPs were surface adapted with biotin as specific targeting tumor penetration cells and enhance the intercellular uptake through the endocytosis. FT-IR (Fourier-transform Infrared Spectroscopy), UV-Vis (Ultra Violet-Visible Spectrophotometer), EDS (Energy Dispersive X-Ray Spectroscopy), SEM (Scanning Electron Microscope) and HR-TEM (High-resolution transmission electron microscopy) descriptions were engaged to illustrate their spectral analysis and morphological examinations of Bt@Au-NPs. Fluorescence microscopy images of cellular uptake descriptions and ICP-MS (Inductively coupled plasma mass spectrometry) investigation established the cell lines labeling ability and enhanced targetting efficacy of biotin-conjugated Au-NPs (Bt@Au-NPs) toward C6 glioma cells (brain cancer cells) with 72.5% cellular uptake relative to 30.2% for non-conjugated lone. These were further established through intracellular ROS examinations and in vitro cytotoxicity investigation on the C6 glioma cell line. The solid surface plasmon absorptions of the Au-NPs and Bt@Au-NPs providing raised photothermal therapy under UV irradiation. The synthesized multifunctional Bt@Au-NPs with an inclusive combination of potential resources presented encouraging nanoprobe with targeting capability, improved photodynamic and photothermal cancer therapy.

Self-assembled Camptothecin derivatives - Curcuminoids conjugate for combinatorial chemo-photodynamic therapy to enhance anti-tumor efficacy.

Camptothecin (CPT), an alkaloid, was first discovered from plants and has potent anti-tumor activity. Since then, CPT analogs (namely Irinotecan and Topotecan) have been approved by the FDA for cancer treatments. Curcumin, on the other hand, is a widely used photosensitizer in photodynamic therapy (PDT) treatment. In our previous work, we have reported a straightforward strategy to construct a drug self-delivery system in which two-molecular species Irinotecan and Curcumin can self-assembly into a complex of ion pairs, namely ICN, through intermolecular non-covalent interactions. We found that ICN has slightly better chemotherapy efficacy than its individual components with much fewer side effects. In this paper, we aim to combine the chemotherapy and the PDT of ICN to further improve its anti-tumor performance. The efficient cellular uptake of ICNs was observed by confocal microscopy. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay was used to detect the generation of singlet oxygen species. We found that the cell viability was 9% with both chemotherapy and PDT, and 31% with chemotherapy alone for the case with an ICN concentration of 10 µM, which demonstrated that the anti-tumor efficacy against the HT-29 cancer cell line was enhanced substantially with the combination therapy strategy. The study with an in vivo mouse model has further verified that the chemo-PDT dual therapy can inhibit tumor growth by 84% and 18.8% comparing with the control group and the chemotherapy group, respectively. Our results demonstrated that the new strategy using self-assembly and carrier-free nanoparticles with their chemo-PDT dual therapy may provide new opportunities to develop future combinatorial therapy methods in treating cancer.

Aggregated LDL turn human macrophages into foam cells and induce mitochondrial dysfunction without triggering oxidative or endoplasmic reticulum stress.

Uptake of modified lipoproteins by macrophages turns them into foam cells, the hallmark of the atherosclerotic plaque. The initiation and progression of atherosclerosis have been associated with mitochondrial dysfunction. It is known that aggregated low-density lipoproteins (agLDL) induce massive cholesterol accumulation in macrophages in contrast with native LDL (nLDL) and oxidized LDL (oxLDL). In the present study we aimed to assess the effect of agLDL on the mitochondria and ER function in macrophage-derived foam cells, in an attempt to estimate the potential of these cells, known constituents of early fatty streaks, to generate atheroma in the absence of oxidative stress. Results show that agLDL induce excessive accumulation of free (FC) and esterified cholesterol in THP-1 macrophages and determine mitochondrial dysfunction expressed as decreased mitochondrial membrane potential and diminished intracellular ATP levels, without generating mitochondrial reactive oxygen species (ROS) production. AgLDL did not stimulate intracellular ROS (superoxide anion or hydrogen peroxide) production, and did not trigger endoplasmic reticulum stress (ERS) or apoptosis. In contrast to agLDL, oxLDL did not modify FC levels, but stimulated the accumulation of 7-ketocholesterol in the cells, generating oxidative stress which is associated with an increased mitochondrial dysfunction, ERS and apoptosis. Taken together, our results reveal that agLDL induce foam cells formation and mild mitochondrial dysfunction in human macrophages without triggering oxidative or ERS. These data could partially explain the early formation of fatty streaks in the intima of human arteries by interaction of monocyte-derived macrophages with non-oxidatively aggregated LDL generating foam cells, which cannot evolve into atherosclerotic plaques in the absence of the oxidative stress.

Dual-targeted 5-aminolevulinic acid derivatives with glutathione depletion function for enhanced photodynamic therapy.

Photodynamic therapy (PDT) is a promising tumor therapy which utilizes reactive oxygen species (ROSs) to cause tumor cells death. 5-aminolevulinic acid (ALA) and two of its esters are FDA-approved photosensitizers. However, their clinical application suffers from their instability and lack of tumor selectivity. In addition, the overexpression of glutathione (GSH) in some tumor cells reduces the PDT efficiency due to the ROS-scavenging ability of GSH. In this work, we present three multifunctional ALA derivates with the characteristics of dual-targeting and GSH depletion to improve the therapeutic effect of ALA-based PDT. The general structure of these compounds consists of an ALA methyl ester (ALA-OMe) moiety that can metabolize to photosensitive protoporphyin IX (PpIX) inside the cells, a biotin group for targeting biotin receptor-positive tumor cells and a disulfide bond-based self-immolative linker which can be activated by GSH to liberate ALA-OMe. Simultaneously, the reaction between the disulfide bond and GSH also depletes intracellular GSH, causing tumor cells more vulnerable to ROSs. All three compounds exhibited high stability under physiological conditions. In vitro experiments demonstrated that the more lipophilic compounds 1 and 2 were much more efficient in inducing PpIX production in biotin receptor-overexpressed HeLa cells as compared with their parent compound (ALA-OMe). And the PpIX generation induced by compounds 1 and 2 was positively correlated with the overexpression of biotin receptor and GSH level in tumor cells. More importantly, the GSH depletion ability of them significantly increased their phototoxicity. Furthermore, in comparison with ALA-OMe, compound 2 showed much higher in vivo efficiency in PpIX production. All the results demonstrate that the combination strategy of dual-targeting and GSH depletion can be used to concurrently enhance the tumor-specificity and anti-tumor efficiency of ALA-based PDT. And this strategy may be used for designing other ALA-based photosensitizers with higher tumor-specificity and better therapeutic effects.

Effect of light emitting diode photobiomodulation on murine macrophage function after Bothrops envenomation.

Several reports have suggested that photobiomodulation, owing to its analgesic, anti-inflammatory, and healing effects, may be an effective therapeutic option for local effects of snakebites when the availability and accessibility of conventional serum therapy are inefficient and far from medical care centers. Although there have been studies that demonstrate the application of photobiomodulation in the treatment of local adverse events due to snakebites from snakes of the genus Bothrops, its role in the activation of leukocytes, particularly macrophages, has not been evaluated. Here, we assessed the effect of light-emitting diode (LED) treatment on macrophage activation induced by B. jararacussu venom (BjV). LED treatment caused an increase in the viability of macrophages incubated with BjV. This treatment reduced reactive oxygen species (ROS) and nitric oxide (NO) production by macrophages after incubation with BjV. However, LED treatment did not interfere with IL-1ß and IL-10 production by macrophages after incubation with BjV. In conclusion, this study showed that LED treatment has the potential to be used in combination with conventional serum therapy to prevent or minimize the progression of local to severe symptoms after Bothrops envenomation.

Astaxanthin protects mesenchymal stem cells from oxidative stress by direct scavenging of free radicals and modulation of cell signaling.

Recent evidence has shown that mesenchymal stem cells (MSCs) play vital roles in cell therapy of ischemia/hypoxia damaged tissues. However, after the transplantation, they might undergo apoptosis due to oxidative stress. Thus, some strategies have been developed to support stem cells in harsh conditions, including pre-treatment of the cells with antioxidants. Of various antioxidants, in this study, astaxanthin (ATX) was used to protect adipose-derived MSCs against oxidative stress. The MSCs were exposed to different doses of hydrogen peroxide, and then the expression of key genes involved in the redox signaling pathway was studied, including nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NADPH quinine oxidoreductase 1 (NQO1). The balance of intracellular reactive oxygen species was detected with the H2DCFDA molecular probe. Additionally, for the detection of apoptosis and protective effect of ATX, the DAPI/Phallacidin and annexin V cell staining were performed. The results of cellular studies revealed that ATX reduced the H2O2-induced cell apoptosis and oxidative stress. Furthermore, after the induction of oxidative stress, the cells' native antioxidants (HO-1 and NQO1) were overexpressed but they were modulated with ATX treatments (p < 0.023). Based on our findings, ATX could increase the expression of Nrf2 as a key transcription factor of antioxidant enzymes (p < 0.05). These findings support the notion that ATX can act as an effective antioxidant in the pre-treatment of MSCs before cell therapy. Thus, to enhance the viability of stem cells during the transplantation in harsh conditions, the concurrent use of ATX in cell therapy modalities is proposed.

Monensin-Induced Increase in Intracellular Na+ Induces Changes in Na+ and Ca2+ Currents and Regulates Na+-K+ and Na+-Ca2+ Transport in Cardiomyocytes.

BACKGROUND/AIMS: Monensin, an Na ionophore, increases intracellular Na ([Na]i). Alteration of [Na]i influences ion transport through the sarcolemmal membrane. So far, the effects of monensin on ventricular myocytes have not been examined in detail. The main objective of this study was to elucidate the mechanism via which monensin-evoked increases in [Na]i affect the membrane potential and currents in ventricular myocytes of guinea pigs. METHODS: Membrane potentials and currents were measured using the whole-cell patch-clamp technique in single myocytes. The concentration of intracellular Ca ([Ca]i) was evaluated by measuring fluorescence intensity of Fluo-4. RESULTS: Monensin (10-5M) shortened the action potential duration (APD) and reduced the amplitude of the plateau phase. In addition, monensin decreased the sodium current (INa) and shifted the inactivation curve to the hyperpolarized direction. Moreover, it decreased the L-type calcium current (ICa). However, this effect was attenuated by increasing the buffering capacity of [Ca]i. The Na-Ca exchange current (INa-Ca) was activated particularly in the reverse mode. Na-K pump current (INa-K) was also activated. Notably, the inward rectifying K current (IK1) was not affected, and the change in the delayed outward K current (IK) was not evident. CONCLUSION: These results suggest that the monensin-induced shortened APD and reduced amplitude of the plateau phase are primarily due to the decrease in the ICa, the activation of the reverse mode of INa-Ca, and the increased INa-K, and second due to the decreased INa. The IK and the IK1 may not be associated with the abovementioned changes induced by monensin. The elevation of [Na]i can exert multiple influences on electrophysiological phenomena in cardiac myocytes.

Specifically Eliminating Tumor-Associated Macrophages with an Extra- and Intracellular Stepwise-Responsive Nanocarrier for Inhibiting Metastasis.

Metastasis is the primary cause of death for most cancer patients, in which tumor-associated macrophages (TAMs) are involved through several mechanisms. While hitherto there is still a lack of study on exclusive elimination of TAMs to inhibit metastasis due to the difficulties in specific targeting of TAMs, we construct an extra- and intracellular stepwise-responsive delivery system p-(aminomethyl)benzoic acid (PAMB)/doxorubicin (DOX) to achieve specific TAM depletion for the first time, thereby preventing tumor metastasis. Once accumulated into the tumor, PAMB/DOX would stepwise responsively (hypoxia and reactive oxygen species (ROS) responsively) disintegrate to expose the TAM-targeting ligand and release DOX sequentially, which depletes TAMs effectively in vivo. Owing to the inhibition of extracellular matrix (ECM) degradation, neovascularization, and tumor invasion contributed by TAM depletion, lung metastasis was successfully inhibited. Furthermore, PAMB/DOX showed efficient inhibition against tumor growth as well as spontaneous metastasis formation when combined with additional chemotherapy, representing a safe and efficient nanoplatform to modulate the adverse tumor microenvironment via TAM elimination.

Chronic IL-1 exposure drives LNCaP cells to evolve androgen and AR independence.

Chronic inflammation promotes prostate cancer (PCa) initiation and progression. We previously reported that acute intereluekin-1 (IL-1) exposure represses androgen receptor (AR) accumulation and activity, providing a possible mechanism for IL-1-mediated development of androgen- and AR-independent PCa. Given that acute inflammation is quickly resolved, and chronic inflammation is, instead, co-opted by cancer cells to promote tumorigenicity, we set out to determine if chronic IL-1 exposure leads to similar repression of AR and AR activity observed for acute IL-1 exposure and to determine if chronic IL-1 exposure selects for androgen- and AR-independent PCa cells. We generated isogenic sublines from LNCaP cells chronically exposed to IL-1α or IL-1ß. Cells were treated with IL-1α, IL-1ß, TNFα or HS-5 bone marrow stromal cells conditioned medium to assess cell viability in the presence of cytotoxic inflammatory cytokines. Cell viability was also assessed following serum starvation, AR siRNA silencing and enzalutamide treatment. Finally, RNA sequencing was performed for the IL-1 sublines. MTT, RT-qPCR and western blot analysis show that the sublines evolved resistance to inflammation-induced cytotoxicity and intracellular signaling and evolved reduced sensitivity to siRNA-mediated loss of AR, serum deprivation and enzalutamide. Differential gene expression reveals that canonical AR signaling is aberrant in the IL-1 sublines, where the cells show constitutive PSA repression and basally high KLK2 and NKX3.1 mRNA levels and bioinformatics analysis predicts that pro-survival and pro-tumorigenic pathways are activated in the sublines. Our data provide evidence that chronic IL-1 exposure promotes PCa cell androgen and AR independence and, thus, supports CRPCa development.

Specific Inhibition of Tumor Growth by T Cell Receptor-Drug Conjugates Targeting Intracellular Cancer-Testis Antigen NY-ESO-1/LAGE-1.

Despite the significant therapeutic advances in T-cell immunotherapy, many malignancies remain unresponsive, which might be because of the negative regulation of T cells by the tumor microenvironment (TME). T cells discriminate tumor cells and normal cells through T-cell receptors (TCRs); therefore, we generated a novel type of TCR-drug conjugates (TDCs) by referring antibody-drug conjugations (ADCs) to overcome the effects of the TME on T cells while preserving the specificity of TCR for tumor recognition. We selected HLA-A2/NY-ESO-1157-165 (peptide NY-ESO-1157-165 in complex with human leukocyte antigen serotype HLA-A*02:01) as the antigen and the antigen-specific TCR (1G4113) as the carrier. By sortase A-mediated ligation, we obtained three NY-TCR-vcMMAEs and further studied their properties, antitumor activity, and toxicity in vitro and in vivo. We found that all the NY-TCR-vcMMAEs had high endocytosis efficiency and specifically killed HLA-A2/NY-ESO-1157-165 positive tumor cells. In xenograft models, one of the TDCs, NY-TCR-2M, was effectively and specifically distributed into tumor tissues and inhibited tumor growth without inducing obvious toxicity. Our results demonstrated that TCRs can be carriers of toxic payloads and that the TDCs thus formed can specifically inhibit tumor growth, neglecting the immune microenvironment.

The Phosphodiesterase Inhibitor Ensifentrine Reduces Production of Proinflammatory Mediators in Well Differentiated Bronchial Epithelial Cells by Inhibiting PDE4.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel that impair airway salt and fluid secretion. Excessive release of proinflammatory cytokines and chemokines by CF bronchial epithelium during airway infection leads to chronic inflammation and a slow decline in lung function; thus, there is much interest in finding safe and effective treatments that reduce inflammation in CF. We showed previously that the cyclic nucleotide phosphodiesterase (PDE) inhibitor ensifentrine (RPL554; Verona Pharma) stimulates the channel function of CFTR mutants with abnormal gating and also those with defective trafficking that are partially rescued using a clinically approved corrector drug. PDE inhibitors also have known anti-inflammatory effects; therefore, we examined whether ensifentrine alters the production of proinflammatory cytokines in CF bronchial epithelial cells. Ensifentrine reduced the production of monocyte chemoattractant protein-1 and granulocyte monocyte colony-stimulating factor (GM-CSF) during challenge with interleukin-1ß Comparing the effect of ensifentrine with milrinone and roflumilast, selective PDE3 and PDE4 inhibitors, respectively, demonstrated that the anti-inflammatory effect of ensifentrine was mainly due to inhibition of PDE4. Beneficial modulation of GM-CSF was further enhanced when ensifentrine was combined with low concentrations of the ß 2-adrenergic agonist isoproterenol or the corticosteroid dexamethasone. The results indicate that ensifentrine may have beneficial anti-inflammatory effects in CF airways particularly when used in combination with ß 2-adrenergic agonists or corticosteroids. SIGNIFICANCE STATEMENT: Airway inflammation that is disproportionate to the burden of chronic airway infection causes much of the pathology in the cystic fibrosis (CF) lung. We show here that ensifentrine beneficially modulates the release of proinflammatory factors in well differentiated CF bronchial epithelial cells that is further enhanced when combined with ß2-adrenergic agonists or low-concentration corticosteroids. The results encourage further clinical testing of ensifentrine, alone and in combination with ß2-adrenergic agonists or low-concentration corticosteroids, as a novel anti-inflammatory therapy for CF.