Towards epigenetic regulation of triple-negative breast cancer via ligand-mediated nanoparticles.

Aims: Develop and analyze triple-negative breast cancer targeted nanoparticles loaded with the demethylating agent decitabine. Materials & methods: The polymers were synthesized by ring-opening polymerization of D,L-lactide and formulated into nanoparticles via emulsion-evaporation method. The nanoparticles were characterized by physicochemical analysis as well as in vitro using breast cancer cell lineages. Results & conclusion: The targeted nanoparticles exhibited a hydrodynamic diameter of 75 ± 12 nm, zeta potential -6.3 ± 0.2 mV and spherical morphology, and displayed greater in vitro accumulation into MDA-MB-231 (triple-negative breast cancer cell-line) compared with MCF7 and HB4A cell lineages as verified by fluorescence confocal microscopy and significant demethylating effects via ADAM33 screening by PCR.

Characterization of Potent ABCG2 Inhibitor Derived from Chromone: From the Mechanism of Inhibition to Human Extracellular Vesicles for Drug Delivery.

Inhibition of ABC transporters is a promising approach to overcome multidrug resistance in cancer. Herein, we report the characterization of a potent ABCG2 inhibitor, namely, chromone 4a (C4a). Molecular docking and in vitro assays using ABCG2 and P-glycoprotein (P-gp) expressing membrane vesicles of insect cells revealed that C4a interacts with both transporters, while showing selectivity toward ABCG2 using cell-based transport assays. C4a inhibited the ABCG2-mediated efflux of different substrates and molecular dynamic simulations demonstrated that C4a binds in the Ko143-binding pocket. Liposomes and extracellular vesicles (EVs) of Giardia intestinalis and human blood were used to successfully bypass the poor water solubility and delivery of C4a as assessed by inhibition of the ABCG2 function. Human blood EVs also promoted delivery of the well-known P-gp inhibitor, elacridar. Here, for the first time, we demonstrated the potential use of plasma circulating EVs for drug delivery of hydrophobic drugs targeting membrane proteins.

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model.

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates. As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 ± 0.6 J/cm2). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug's cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

Targeting breast cancer resistance protein (BCRP/ABCG2): Functional inhibitors and expression modulators.

The primary source of failure of cancer therapies is multidrug resistance (MDR), which can be caused by different mechanisms, including the overexpression of ABC transporters in cancer cells. Among the 48 human ABC proteins, the breast cancer resistance protein (BCRP/ABCG2) has been described as a pivotal player in cancer resistance. The use of functional inhibitors and expression modulators is a promising strategy to overcome the MDR caused by ABCG2. Despite the lack of clinical trials using ABCG2 inhibitors, many compounds have already been discovered. This review presents an overview about various ABCG2 inhibitors that have been identified, discussing some chemical aspects and the main experimental methods used to identify and characterize the mechanisms of new inhibitors. In addition, some biological requirements to pursue preclinical tests are described. Finally, we discuss the potential use of ABCG2 inhibitors in cancer stem cells (CSC) for improving the objective response rate and the mechanism of ABCG2 modulators at transcriptional and protein expression levels.

A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2).

The ABCG2 transporter plays a pivotal role in multidrug resistance, however, no clinical trial using specific ABCG2 inhibitors have been successful. Although ABC transporters actively extrude a wide variety of substrates, photodynamic therapeutic agents with porphyrinic scaffolds are exclusively transported by ABCG2. In this work, we describe for the first time a porphyrin derivative (4B) inhibitor of ABCG2 and capable to overcome multidrug resistance in vitro. The inhibition was time-dependent and 4B was not itself transported by ABCG2. Independently of the substrate, the porphyrin 4B showed an IC50 value of 1.6 µM and a mixed type of inhibition. This compound inhibited the ATPase activity and increased the binding of the conformational-sensitive antibody 5D3. A thermostability assay confirmed allosteric protein changes triggered by the porphyrin. Long-timescale molecular dynamics simulations revealed a different behavior between the ABCG2 porphyrinic substrate pheophorbide a and the porphyrin 4B. Pheophorbide a was able to bind in three different protein sites but 4B showed one binding conformation with a strong ionic interaction with GLU446. The inhibition was selective toward ABCG2, since no inhibition was observed for P-glycoprotein and MRP1. Finally, this compound successfully chemosensitized cells that overexpress ABCG2. These findings reinforce that substrates may be a privileged source of chemical scaffolds for identification of new inhibitors of multidrug resistance-linked ABC transporters.

Polyoxovanadates as new P-glycoprotein inhibitors: insights into the mechanism of inhibition.

A promising strategy to overcome multidrug resistance is the use of inhibitors of ABC drug transporters. For this reason, we evaluated the polyoxovanadates (POVs) [V10 O28 ]6- (V10 ), [H6 V14 O38 (PO4 )]5- (V14 ), [V15 O36 Cl]6- (V15 ) and [V18 O42 I]7- (V18 ) as inhibitors of three major multidrug resistance-linked ABC transporters: P-glycoprotein (P-gp), ABCG2 and MRP1. All of the POVs selectively inhibited P-gp. V10 and V18 were the two most promising compounds, with IC50 values of transport inhibition of 25.4 and 22.7 µm, respectively. Both compounds inhibited P-gp ATPase activity, with the same IC50 value of 1.26 µm. V10 and V18 triggered different conformational changes in the P-gp protein with time-dependent inhibition, which was confirmed using the synthesized salt of V10 with rhodamine B, RhoB-V10 . The hydrophilic nature of POVs supports the hypothesis that these compounds target an unusual ligand-binding site, opening new possibilities in the development of potent modulators of ABC transporters.

Uso da citometria de fluxo no diagnóstico sorológico da COVID-19 / The use of flow cytometry for the serological diagnosis of COVID-19

A pandemia da COVID-19 tem tido um impacto devastador em todo o mundo e levou ao rápido desenvolvimento de testes diagnósticos. Diferentes tecnologias vêm sendo utilizadas para a detecção de imunoglobulinas frente à infecção por SARS-CoV-2. Ensaios imunoenzimáticos (ELISA), quimioluminescentes e imunocromatográficos estão disponíveis e, no geral, apresentam poder diagnóstico limitado, principalmente para a detecção de IgA. A citometria de fluxo tem surgido como alternativa para o desenvolvimento de métodos sensíveis e específicos para a COVID-19 aplicados para diagnóstico, triagem e estratificação da doença. A citometria de fluxo é uma tecnologia óptica baseada em laser que detecta características físico-químicas de células ou partículas em um fluido heterogêneo. O artigo explora a citometria de fluxo para o diagnóstico da COVID-19 em duas estratégias para a detecção de anticorpos no soro ou plasma, uma utilizando antígenos virais expressos na superfície de células de mamíferos e outra com estes elementos imobilizados em microesferas (beads). A possibilidade de detecção rápida de múltiplos anticorpos simultaneamente, com pequeno volume de amostra e elevada sensibilidade e especificidade, torna a citometria de fluxo uma metodologia promissora para o laboratório clínico, como ferramenta de referência para auxiliar na contenção do processo pandêmico da COVID-19 e futuros eventos similares.

The COVID-19 pandemic has had a devastating impact around the world and has led to the rapid development of diagnostic tests. Different technologies have been used to detect immunoglobulins produced against SARS-CoV-2 infection. Immunoenzymatic (ELISA), chemiluminescent and immunochromatographic assays are available and, in general, they have limited diagnostic accuracy, especially for the detection of IgA. Flow cytometry has emerged as an alternative for the development of sensitive and specific methods for COVID-19 applied for diagnosis, screening and stratification of the disease. Flow cytometry is a laser-based optical technology that detects physicochemical characteristics of cells or particles in a heterogeneous fluid. The article explores flow cytometry for the diagnosis of COVID-19 in two strategies for detecting antibodies in serum or plasma, the first one using viral antigens expressed on the surface of mammalian cells and the other one with these elements immobilized on microspheres (beads). The possibility of rapid detection of multiple antibodies simultaneously, with a small sample volume and high sensitivity and specificity, makes flow cytometry a promising methodology for the clinical laboratory, as a reference tool to help stop the COVID-19 pandemic process and similar future events.

Tetrahydroquinoline/4,5-Dihydroisoxazole Molecular Hybrids as Inhibitors of Breast Cancer Resistance Protein (BCRP/ABCG2).

Multidrug resistance (MDR) is one of the major factors in the failure of many chemotherapy approaches. In cancer cells, MDR is mainly associated with the expression of ABC transporters such as P-glycoprotein, MRP1 and ABCG2. Despite major efforts to develop new selective and potent inhibitors of ABC drug transporters, no ABCG2-specific inhibitors for clinical use are yet available. Here, we report the evaluation of sixteen tetrahydroquinoline/4,5-dihydroisoxazole derivatives as a new class of ABCG2 inhibitors. The affinity of the five best inhibitors was further investigated by the vanadate-sensitive ATPase assay. Molecular modelling data, proposing a potential binding mode, suggest that they can inhibit the ABCG2 activity by binding on site S1, previously reported as inhibitors binding region, as well targeting site S2, a selective region for substrates, and by specifically interacting with residues Asn436, Gln398, and Leu555. Altogether, this study provided new insights into THQ/4,5-dihydroisoxazole molecular hybrids, generating great potential for the development of novel most potent ABCG2 inhibitors.

Mechanistic basis of breast cancer resistance protein inhibition by new indeno[1,2-b]indoles.

The ATP-binding cassette transporter ABCG2 mediates the efflux of several chemotherapeutic drugs, contributing to the development of multidrug resistance (MDR) in many cancers. The most promising strategy to overcome ABCG2-mediated MDR is the use of specific inhibitors. Despite many efforts, the identification of new potent and specific ABCG2 inhibitors remains urgent. In this study, a structural optimization of indeno[1,2-b]indole was performed and a new generation of 18 compounds was synthesized and tested as ABCG2 inhibitors. Most compounds showed ABCG2 inhibition with IC50 values below 0.5 µM. The ratio between cytotoxicity (IG50) and ABCG2 inhibition potency (IC50) was used to identify the best inhibitors. In addition, it was observed that some indeno[1,2-b]indole derivatives produced complete inhibition, while others only partially inhibited the transport function of ABCG2. All indeno[1,2-b]indole derivatives are not transported by ABCG2, and even the partial inhibitors are able to fully chemosensitize cancer cells overexpressing ABCG2. The high affinity of these indeno[1,2-b]indole derivatives was confirmed by the strong stimulatory effect on ABCG2 ATPase activity. These compounds did not affect the binding of conformation-sensitive antibody 5D3 binding, but stabilized the protein structure, as revealed by the thermostabilization assay. Finally, a docking study showed the indeno[1,2-b]indole derivatives share the same binding site as the substrate estrone-3-sulfate.

Componentes químicos del aceite esencial de Ocotea paranaensis (Lauraceae) y sus propiedades antioxidantes, anticancer y antimicrobianas / Chemical constituents of Ocotea paranaensis (Lauraceae) essential oil and their antioxidant, anticancer and antimicrobial properties

La composición química del aceite esencial obtenido de las ramas de Ocotea paranaensis se estudió por cromatografía de gases/espectrometría de masas (CG/MS). Se identificaron veintisiete compuestos, que comprenden el 94,82% de los componentes totales. El aceite se caracterizó por una concentración relativamente alta de sesquiterpenos (62,96%), sesquiterpenos oxigenados (33,33%) y diterpeno (3,70%). En cuanto a los compuestos principales, se destacaron Z-nerolidol (19,16%), germacreno D (12,92%) y α-bulnesene (8,47%), que correspondieron al 40,55% de las sustancias encontradas. El aceite esencial analizado de Ocotea paranaensis tiene una buena acción reductora de fosfomolibdeno y es moderadamente tóxico para la Artemia salina (LC50 = 147,91 µg/mL). Mostró potencial hemolítico y actividad moderada contra Staphylococcus aureus (concentración inhibitoria mínima MIC = 250 µg/mL) y Pseudomonas aeruginosa (MIC = 500 µg/mL). No se observaron resultados satisfactorios de citotoxicidad en el linaje H460 y HeLa.

The chemical composition of the essential oil obtained from the branches of Ocotea paranaensis was studied by gas chromatography/mass spectrometry (GC/MS). Twenty-seven compounds, comprising 94.82% of the total components, were identified. The oil showed relatively high concentration of sesquiterpenes (62.96%), oxygenated sesquiterpenes (33.33%), and diterpene (3.70%). Regarding the major compounds, Z-nerolidol (19.16%), germacrene D (12.92%) and α-bulnesene (8.47%) could be highlighted, which corresponded to 40.55% of the substances that were found. The essential oil from Ocotea paranaensis has phosphomolybdenum reducing action and is moderately toxic to the Artemia salina (LC50 = 147.91 µg/mL). It showed haemolytic potential and moderate activity against Staphylococcus aureus, (minimum inhibitory concentration MIC = 250 µg/mL) and Pseudomonas aeruginosa (MIC = 500 µg/mL). No satisfactory cytotoxicity results were observed in lineage H460 and HeLa.

Photodynamic effect of meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins on HaCaT keratinocytes.

Sixteen porphyrins, including neutral, anionic and cationic meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins were herein evaluated in terms of their photosensitizing properties against HaCaT keratinocytes. After an initial screening, the cationic porphyrins were studied in more details, by both determining their log POW and performing PDT assays in lower porphyrin concentrations. Porphyrins presenting two or more adjacent positively charged groups, directly linked to the macrocycle meso positions, appeared to be the most effective photosensitizers. The present study also included the dicationic 5,10-diphenyl-15,20-di(1-methylpyridinium-4-yl)porphyrin (14b), which has previously shown promising results on a psoriasis-like in vivo model. Overall results indicated that the beneficial effect related to porphyrins on psoriasis can be related to the decreasing of keratinocyte viability. Furthermore, some of the cationic porphyrins studied appeared as candidates to be utilized as photosensitizers for psoriasis treatment.

Antioxidant properties of glutamine and its role in VEGF-Akt pathways in portal hypertension gastropathy.

AIM: To investigate the effects of glutamine on oxidative/nitrosative stress and the vascular endothelial growth factor (VEGF)-Akt-endothelial nitric oxide synthase (eNOS) signaling pathway in an experimental model of portal hypertension induced by partial portal vein ligation (PPVL). METHODS: Portal hypertension was induced by PPVL. The PPVL model consists of a partial obstruction of the portal vein, performed using a 20 G blunt needle as a guide, which is gently removed after the procedure. PPVL model was performed for 14 d beginning treatment with glutamine on the seventh day. On the fifteenth day, the mesenteric vein pressure was checked and the stomach was removed to test immunoreactivity and oxidative stress markers. We evaluated the expression and the immunoreactivity of proteins involved in the VEGF-Akt-eNOS pathway by Western blotting and immunohistochemical analysis. Oxidative stress was measured by quantification of the cytosolic concentration of thiobarbituric acid reactive substances (TBARS) as well as the levels of total glutathione (GSH), superoxide dismutase (SOD) activity, nitric oxide (NO) production and nitrotyrosine immunoreactivity. RESULTS: All data are presented as the mean ± SE. The production of TBARS and NO was significantly increased in PPVL animals. A reduction of SOD activity was detected in PPVL + G group. In the immunohistochemical analyses of nitrotyrosine, Akt and eNOS, the PPVL group exhibited significant increases, whereas decreases were observed in the PPVL + G group, but no difference in VEGF was detected between these groups. Western blotting analysis detected increased expression of phosphatidylinositol-3-kinase (PI3K), P-Akt and eNOS in the PPVL group compared with the PPVL + G group, which was not observed for the expression of VEGF when comparing these groups. Glutamine administration markedly alleviated oxidative/nitrosative stress, normalized SOD activity, increased levels of total GSH and blocked NO overproduction as well as the formation of peroxynitrite. CONCLUSION: Glutamine treatment demonstrated to reduce oxidative damage but does not reduce angiogenesis induced by PH in gastric tissue, demonstrating a beneficial role for the PI3K-Akt-eNOS pathway.