Magnolol derivatives as specific and noncytotoxic inhibitors of breast cancer resistance protein (BCRP/ABCG2).

The breast cancer resistance protein (BCRP/ABCG2) transporter mediates the efflux of numerous antineoplastic drugs, playing a central role in multidrug resistance related to cancer. The absence of successful clinical trials using specific ABCG2 inhibitors reveals the urge to identify new compounds to attend this critical demand. In this work, a series of 13 magnolol derivatives was tested as ABCG2 inhibitors. Only two compounds, derivatives 10 and 11, showed partial and complete ABCG2 inhibitory effect, respectively. This inhibition was selective toward ABCG2, since none of the 13 compounds inhibited neither P-glycoprotein nor MRP1. Both inhibitors (10 and 11) were not transported by ABCG2 and demonstrated a low cytotoxic profile even at high concentrations (up to 100 µM). 11 emerged as the most promising compound of the series, considering the ratio between cytotoxicity (IG50) and ABCG2 inhibition potency (IC50), showing a therapeutic ratio (TR) higher than observed for 10 (10.5 versus 1.6, respectively). This derivative showed a substrate-independent and a mixed type of inhibition. The effect of compound 11 on the ABCG2 ATPase activity and thermostability revealed allosteric protein changes. This compound did not affect the expression levels of ABCG2 and increased the binding of the conformational-sensitive antibody 5D3. A docking study showed that 11 did not share the same binding site with ABCG2 substrate mitoxantrone. Finally, 11 could revert the chemoresistance to SN-38 mediated by ABCG2.

Heteroleptic oxidovanadium(IV)-malate complex improves glucose uptake in HepG2 and enhances insulin action in streptozotocin-induced diabetic rats.

Diabetes mellitus, a complex and heterogeneous disease associated with hyperglycemia, is a leading cause of mortality and reduces life expectancy. Vanadium complexes have been studied for the treatment of diabetes. The effect of complex [VO(bpy)(mal)]·H2O (complex A) was evaluated in a human hepatocarcinoma (HepG2) cell line and in streptozotocin (STZ)-induced diabetic male Wistar rats conditioned in seven groups with different treatments (n = 10 animals per group). Electron paramagnetic resonance and 51V NMR analyses of complex A in high-glucose Dulbecco's Modified Eagle Medium (DMEM) revealed the oxidation and hydrolysis of the oxidovanadium(IV) complex over a period of 24 h at 37 °C to give low-nuclearity vanadates "V1" (H2VO4-), "V2" (H2V2O72-), and "V4" (V4O124-). In HepG2 cells, complex A exhibited low cytotoxic effects at concentrations 2.5 to 7.5 µmol L-1 (IC50 10.53 µmol L-1) and increased glucose uptake (2-NBDG) up to 93%, an effect similar to insulin. In STZ-induced diabetic rats, complex A at 10 and 30 mg kg-1 administered by oral gavage for 12 days did not affect the animals, suggesting low toxicity or metabolic impairment during the experimental period. Compared to insulin treatment alone, complex A (30 mg kg-1) in association with insulin was found to improve glycemia (30.6 ± 6.3 mmol L-1 vs. 21.1 ± 8.6 mmol L-1, respectively; p = 0.002), resulting in approximately 30% additional reduction in glycemia. The insulin-enhancing effect of complex A was associated with low toxicity and was achieved via oral administration, suggesting the potential of complex A as a promising candidate for the adjuvant treatment of diabetes.

Serum 1,5-Anhydroglucitol Concentration as a Biomarker for Type 1 Diabetes in Adults and Children.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease and the leading form of diabetes among young white people. 1,5-Anhydroglucitol (1,5-AG), a nontraditional biomarker of postprandial glycemic control (after 1 - 3 days to 2 weeks), may be useful in T1D screening. We studied serum 1,5-AG concentration as a potential biomarker for T1D screening and diagnosis in adults and children. METHODS: In this case-control study, adults (n = 121; age, 19 - 61 years) and children (n = 19; age, 8 - 14 years) with T1D were matched with healthy subjects (n = 242) according to gender and age. Serum 1,5-AG levels were measured enzymatically (GlycoMark Inc., NY, USA). RESULTS: Patients showed no symptoms of overt kidney disease, assessed by serum creatinine concentrations. The median (25th - 75th percentile) 1,5-AG concentrations for the control group compared with the T1D group were 155 (128 - 183) vs. 21 (14 - 34) µmol/L in adults and 190 (158 - 237) vs. 20 (12 - 30) µmol/L in children (p < 0.001 for both). Receiver operating characteristic curves showed that 1,5-AG cutoffs of ≤ 113 and ≤ 79 µmol/L for adult men and women, respectively, and ≤ 57 µmol/L for children of both genders had > 95% sensitivity and specificity for both groups. CONCLUSIONS: Our results suggest that serum 1,5-AG concentration may be useful as an adjunct measure of hyperglycemia for diagnosing T1D and has the potential to screen for T1D in high-risk subjects.

Characteristics of an Aeromonas trota strain isolated from cerebrospinal fluid.

Aeromonas are ubiquitous in aquatic habitats. However some species can cause infections in humans, but rarely meningitis. Here we describe the isolation and characterization of an Aeromonas strain from cerebrospinal fluid of a meningitis patient. The isolate, identified as A. trota by biochemical and molecular methods, was susceptible to ampicillin but resistant to cephalothin and cefazolin. Genome sequencing revealed virulence factor genes such as type VI secretion system, aerolysin and lateral flagella. The isolate exhibited swarming motility, hemolytic activity and adhesion and cytotoxicity on HeLa cells. This is the first report of A. trota associated with meningitis and its virulence characteristics.

Anticoagulant Choices Affect the Mean Platelet Volume Measurement by Impedance.

BACKGROUND: Mean platelet volume (MPV) is a parameter that evaluates the platelet size. Clinical applications of MPV are limited because of its poor standardization in routine laboratories. This study analyzed the effect of anticoagulants on MPV measurements by impedance technology. METHODS: Blood from 36 healthy volunteers was collected in vacuum tubes filled with K2EDTA and sodium citrate, analyzed immediately (basal) and at 1, 2, and 3 hours after venipuncture. RESULTS: Comparisons between the anticoagulants demonstrated a significant difference (p < 0.05) after 1 hour of exposure with K2EDTA, causing a time-dependent increase on MPV measured. No significant changes in MPV were observed with sodium citrate with 3 hours of exposure (p > 0.05). CONCLUSIONS: The use of sodium citrate is highly indicated for assessment of MPV when the measurement time after blood collection is estimated to be more than 1 hour.

Association of Polymorphism rs17576 of the Metalloproteinase 9 Gene with Gestational Diabetes in Euro-Brazilian Pregnant Women.

BACKGROUND: Metalloproteinase 9 (MMP9) is involved in the degradation of extracellular matrix molecules, and its polymorphism rs17576 (Gln279Arg) has been associated with diabetes. We investigated the association of rs17576 in a case-control study with Euro-Brazilian women with gestational diabetes. METHODS: The study group consisted of a total of 262 Euro-Brazilian pregnant women classified as either healthy (n = 131, control) or with GDM (n = 131). Fluorescent probes with real time PCR (TaqMan system) were applied for genotyping. RESULTS: All groups were in Hardy-Weinberg equilibrium. The minor allele frequencies (G-allele) for rs17567 in healthy and GDM women were 27.1% [95% CI, 22 - 32] and 37.4% [95% CI, 32 - 43], p = 0.011, respectively. Genotypic comparison showed a significant difference (p < 0.05) between the groups. CONCLUSIONS: Polymorphism rs17567 was associated with GDM in the studied population and carriers of the G-allele showed an increased risk for gestational diabetes (Odds ratio 1.61; 95% CI, 1.1 - 2.3).

The Blood Bank for Diabetes Screening: a Feasible Alternative?

BACKGROUND: Vascular complications of diabetes mellitus (DM) are associated with 5% of deaths globally every year. Early diagnosis and treatment could reduce this figure. The aim of this project was to investigate the frequency of undiagnosed DM among blood donors and the possibility of blood banks participating in DM screening. METHODS: Of the approximate 5,600 candidates for blood donation who were evaluated, 4,601 were considered suitable. Candidates with any type of DM, hypertension, thyroid disease, and/or continuous use of any drugs were excluded, resulting in the participation of 635 donors aged 18 - 69 years. Glycated hemoglobin (HbA1c) levels were used to classify the donors: HbA1c < 5.7% (low risk of DM), HbA1c 5.7 - 6.4% (pre-diabetes), and HbA1c ≥ 6.5% (diabetes). Another subsample (n = 576) that excluded donors with HbA1c levels < 5.0% or > 6.5% were classified according to the risk of developing DM in 5 years: HbA1c 5 - 5.5% (low risk, < 9%), HbA1c 5.6 - 6.0% (moderate risk, 9 - 25%), and HbA1c 6.1 - 6.5% (high risk, 26 - 50%). RESULTS: Three donors (0.5%) had HbA1c levels suggestive of DM, and 57 donors (9.0%) had levels associated with pre-DM. Regarding the risk of developing DM in 5 years, 111 donors (19.3%) were classified at moderate risk, and 10 donors (1.7%) were classified at high risk. CONCLUSIONS: DM screening in blood banks using HbA1c can identify new cases of DM and individuals at an increased risk of DM. In summary, blood banks could participate in DM screening, benefitting the general public and public health care system in Brazil.

Pre-analytical phase management: a review of the procedures from patient preparation to laboratory analysis.

The pre-analytical phase encompasses all the procedures before the start of laboratory testing. This phase of the testing process is responsible for the majority of the laboratory errors, since the related procedures involve many sorts of non-laboratory professionals working outside the laboratory setting, thus without direct supervision by the laboratory staff. Therefore, either correct organization or management of both personnel and procedures that regard blood specimen collection by venipuncture are of fundamental importance, since the various steps for performing blood collection represent per se sources of laboratory variability. The aim of this (non-systematic) review addressed to healthcare professionals is to highlight the importance of blood specimen management (from patient preparation to laboratory analyses), as a tool to prevent laboratory errors, with the concept that laboratory results from inappropriate blood specimens are inconsistent and do not allow proper treatment nor monitoring of the patient.

The GCKR Gene Polymorphism rs780094 is a Risk Factor for Gestational Diabetes in a Brazilian Population.

BACKGROUND: The glucokinase regulatory protein (GCKR) regulates the activity of the glucokinase (GCK), which plays a key role in glucose homeostasis. Genetic variants in GCK have been associated with diabetes and gestational diabetes (GDM). Due to the relationship between GCKRP and GCK, polymorphisms in GCKR are also candidates for genetic association with GDM. The aim of this study was to evaluate the association between the GCKR rs780094 polymorphism and GDM in a Brazilian population. METHODS: 252 unrelated Euro-Brazilian pregnant women were classified as control (healthy pregnant women, n = 125) and GDM (pregnant women with GDM, n = 127) age-matched groups. Clinical and anthropometric data were obtained from all subjects. The GCKR rs780094 polymorphism was genotyped using fluorescent probes (TaqMan® , code C_2862873_10). RESULTS: Both groups were in Hardy-Weinberg equilibrium. The GCKR rs780094 polymorphism was associated with GDM in codominant and dominant models (P = 0.022 and P = 0.010, respectively). The minor allele (T) frequency for the control group in the study was 38.4% (95% CI: 32-44%), similar to frequencies reported for other Caucasian populations. CONCLUSION: Carriers of the C allele of rs780094 were 1.41 (odds ratio, 95% CI, 0.97-2.03) times more likely to develop GDM.

Functional promoter polymorphisms of the receptor for advanced glycation end products in children and adolescents with type 1 diabetes.

RAGE promoter polymorphisms are associated with increases in RAGE expression. A case-control association study was conducted involving a Euro-Brazilian population of children and adolescents with type 1 diabetes (n = 90) and healthy controls (n = 105), which were matched by sex and age. Genotyping by PCR-RFLP the -429T>C (rs1800625), -374T>A (rs1800624), and 63 bp deletion/insertion (-407 to -345 bp) showed no significant differences (P > 0.05) between the groups.

Gestational diabetes mellitus (GDM) decreases butyrylcholinesterase (BChE) activity and changes its relationship with lipids.

Many conditions interfere with butyrylcholinesterase (BChE) activity, e.g., pregnancy or presence of the BCHE gene variant -116A can decrease activity whereas obesity and types I and II diabetes mellitus can increase activity. In this study, we examined BChE activity, -116A and 1615A BCHE gene variants, and anthropometric and biochemical variables associated with diabetes in patients with gestational diabetes mellitus (GDM) and in healthy pregnant women. BChE activity was measured spectrophotometrically using propionylthiocholine as substrate and genotyping of the -116 and 1615 sites of the BCHE gene was done with a TaqMan SNP genotyping assay. Three groups were studied: 150 patients with GDM, 295 healthy pregnant women and 156 non-pregnant healthy women. Mean BChE activity was significantly lower in healthy pregnant women than in women from the general population and was further reduced in GDM patients. BChE activity was significantly reduced in carriers of -116A in GDM patients and healthy pregnant women. Although GDM patients had a significantly higher mean body mass index (BMI) and triglycerides than healthy pregnant women, they had lower mean BChE activity, suggesting that the lowering effect of GDM on BChE activity was stronger than the characteristic enhancing effect of increased BMI and triglycerides.

A Non-Toxic Binuclear Vanadium(IV) Complex as Insulin Adjuvant Improves the Glycemic Control in Streptozotocin-Induced Diabetic Rats.

Diabetes mellitus (DM) complications are a burden to health care systems due to the associated consequences of poor glycemic control and the side effects of insulin therapy. Recently. adjuvant therapies, such as vanadium compounds, have gained attention due to their potential to improve glucose homeostasis in patients with diabetes. In order to determine the anti-diabetic and antioxidant effects of the oxidovanadium(IV) complex (Et3NH)2[{VO(OH}2)(ox)2(µ-ox)] or Vox2), rats with streptozotocin (STZ)-induced diabetes were treated with 30 and 100 mg/kg of Vox2, orally administered for 12 days. Vox2 at 100 mg/kg in association with insulin caused a 3.4 times decrease in blood glucose in STZ rats (424 mg/dL), reaching concentrations similar to those in the normoglycemic animals (126 mg/dL). Compared to insulin alone, the association with Vox2 caused an additional decrease in blood glucose of 39% and 65% at 30 and 100 mg/kg, respectively, and an increased pancreatic GSH levels 2.5 times. Vox2 alone did not cause gastrointestinal discomfort, diarrhea, and hepatic or renal toxicity and was not associated with changes in blood glucose level, lipid profile, or kidney or liver function. Our results highlight the potential of Vox2 in association with insulin in treating diabetes.

Quality impact on diagnostic blood specimen collection using a new device to relieve venipuncture pain.

A new device called Buzzy(®) has been recently presented that combines a cooling ice pack and a vibrating motor in order to relieve the venipuncture pain. The aim of this study was to evaluate the impact of Buzzy(®) use during diagnostic blood specimen collection by venipuncture for routine immunochemistry tests. Blood was collected from 100 volunteers by a single, expert phlebotomist. A vein was located on the left forearm without applying tourniquet, in order to prevent any interference from venous stasis, and blood samples were collected using a 20-G straight needle directly into 5 mL vacuum tubes with clot activator and gel separator. In sequence, external cold and vibration by Buzzy(®) was applied on the right forearm-5 cm above the chosen puncture site-for 1 min before venipuncture and continued until the end of the same procedure already done in the left forearm. The panel of tests included the following: glucose, total cholesterol, HDL-cholesterol, triglycerides, total protein, albumin, c-reactive protein, urea, creatinine, uric acid, alkaline phosphatase, amylase, AST, ALT, g-glutamyltransferase, lactate dehydrogenase, creatine kinase, total bilirubin, phosphorus, calcium, magnesium, iron, sodium, potassium, chloride, lipase, cortisol, insulin, thyroid-stimulating hormone, total triiodothyronine, free triiodothyronine, total thyroxine, free thyroxine and haemolysis index. Clinically significant differences between samples were found only for: total protein, albumin and transferrin. The Buzzy(®) can be used during diagnostic blood specimens collection by venipuncture for the majority of the routine immunochemistry tests. We only suggest avoiding this device during blood collection when protein, albumin and transferrin determinations should be performed.

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.

Structural and molecular characterization of lopinavir and ivermectin as breast cancer resistance protein (BCRP/ABCG2) inhibitors.

A current clinical challenge in cancer is multidrug resistance (MDR) mediated by ABC transporters. Breast cancer resistance protein (BCRP) or ABCG2 transporter is one of the most important ABC transporters implicated in MDR and the use of inhibitors is a promising approach to overcome the resistance in cancer. This study aimed to characterize the molecular mechanism of ABCG2 inhibitors identified by a repurposing drug strategy using antiviral, anti-inflammatory and antiparasitic agents. Lopinavir and ivermectin can be considered as pan-inhibitors of ABC transporters, since both compounds inhibited ABCG2, P-glycoprotein and MRP1. They inhibited ABCG2 activity showing IC50 values of 25.5 and 23.4 µM, respectively. These drugs were highly cytotoxic and not transported by ABCG2. Additionally, these drugs increased the 5D3 antibody binding and did not affect the mRNA and protein expression levels. Cell-based analysis of the type of inhibition suggested a non-competitive inhibition, which was further corroborated by in silico approaches of molecular docking and molecular dynamics simulations. These results showed an overlap of the lopinavir and ivermectin binding sites on ABCG2, mainly interacting with E446 residue. However, the substrate mitoxantrone occupies a different site, binding to the F436 region, closer to the L554/L555 plug. In conclusion, these results revealed the mechanistic basis of lopinavir and ivermectin interaction with ABCG2. See also the Graphical abstract(Fig. 1).

Apolipoprotein M gene polymorphisms in childhood-onset type 1 diabetes in southern Brazil.

Type 1 diabetes mellitus (T1DM), associated with autoimmune destruction of pancreatic ß cells, is observed in children and adolescents. OBJECTIVE: We investigated the potential association of the apolipoprotein M (APOM) polymorphisms rs707921, rs805264, rs805296, rs805297, and rs9404941 in childhood-onset T1DM (n = 144) and compared them to those in healthy (mostly Euro-Brazilian) children (n = 168). METHODS: This project was approved by the Ethics Committee of the Federal University of Parana (CAAE 24676613.6.0000.0102). Genotyping was performed using PCR-restriction fragment length polymorphisms (rs805296 and rs9404941) and TaqMan probes (rs707921, rs805264, and rs805297). RESULTS: All polymorphisms were in Hardy-Weinberg equilibrium. In the codominant model, no significant differences (P > 0.05) were observed in genotype and allele frequencies between healthy controls and children with T1DM. The minor allele frequencies (95% CI) for healthy subjects were rs707921 (A, 10.7%; 7-14%), rs805264 (A, 6.5%; 4-9%), rs805296 (C, 3.6%; 2-6%), rs805297 (A, 22.6%; 22-31%), and rs9404941 (C, 2.7%; 1-4%). The frequencies of the rs805297 A allele and rs805296 C allele were similar to those of other Caucasian populations; both the rs707921 and rs805264 A alleles were similar to American and Latin American populations, whereas that of the rs9404941 C allele was lower than that observed in the Caucasian and Asian populations. CONCLUSIONS: Haplotype analysis suggests that rs805297-C, rs9404941-T, rs805296-T, rs805264-G, and rs707921-C conferred risk (OR: 4.25; 95% CI: 1.81-10.1) to childhood-onset T1DM in the Euro-Brazilian population.

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.

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.

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.