ABCG2 Research Articles

Experimental and Computational Methods to Assess Central Nervous System Penetration of Small Molecules.

In CNS drug discovery, the estimation of brain exposure to lead compounds is critical for their optimization. Compounds need to cross the blood-brain barrier (BBB) to reach the pharmacological targets in the CNS. The BBB is a complex system involving passive and active mechanisms of transport and efflux transporters such as P-glycoproteins (P-gp) and breast cancer resistance protein (BCRP), which play an essential role in CNS penetration of small molecules. Several in vivo, in vitro, and in silico methods are available to estimate human brain penetration. Preclinical species are used as in vivo models to understand unbound brain exposure by deriving the Kp,uu parameter and the brain/plasma ratio of exposure corrected with the plasma and brain free fraction. The MDCK-mdr1 (Madin Darby canine kidney cells transfected with the MDR1 gene encoding for the human P-gp) assay is the commonly used in vitro assay to estimate compound permeability and human efflux. The in silico methods to predict brain exposure, such as CNS MPO, CNS BBB scores, and various machine learning models, help save costs and speed up compound discovery and optimization at all stages. These methods enable the screening of virtual compounds, building of a CNS penetrable compounds library, and optimization of lead molecules for CNS penetration. Therefore, it is crucial to understand the reliability and ability of these methods to predict CNS penetration. We review the in silico, in vitro, and in vivo data and their correlation with each other, as well as assess published experimental and computational approaches to predict the BBB penetrability of compounds.

Paynantheine more effectively reverses multidrug resistance in malignant EPG85.257RDB and MCF7MX cells than morphine and speciociliatine.

The possible interactions of morphine, paynantheine and speciociliatine alkaloids with ATP-binding cassette (ABC) transporters was investigated. The compounds were docked against ABCG2 and ABCB1 to predict the binding mode of alkaloids in active binding sites. The cytotoxicity of morphine, paynantheine and speciociliatine for EPG85.257RDB and MCF7MX cells was determined and ABCB1 and ABCG2 gene and protein expression were determined. The binding score of paynantheine to ABCB1 was higher in the docking studies. Paynantheine and speciociliatine had similar binding scores to ABCB1, but higher binding scores to ABCG2 than did morphine. Paynantheine and speciociliatine were more effective against MCF7MX and EPG85.257RDB cells and showed greater cyctotoxicity in the MTT assay. The effect of morphine and paynantheine on the ABCB1 gene and protein expression suggests these compounds can reduce resistance in cancer patients, but that speciociliatine may not be a suitable candidate because of its increased ABCB1 expression while speciociliatine decreased the expression of ABCG2 in MCF7MX cells. This indicates that speciociliatine is a better candidate for reducing drug resistance in this cell line. Structural modification, drug-metabolizing enzymes and differences in the binding sites could cause functional differences between these compounds.

ABCG2 transporter reduces protein aggregation in cigarette smoke condensate-exposed A549 lung cancer cells.

Cigarette smoke-induced protein aggregation damages the lung cells in emphysema and COPD; however, lung cancer cells continue to thrive, evolving to persist in the toxic environment. Here, we showed that upon the cigarette smoke condensate exposure, A549 lung cancer cells exhibit better survival and reduced level of protein aggregation when compared to non-cancerous Beas-2B and H-6053 cells. Our data suggests that upregulation of efflux pumps in cancer cells assists in reducing smoke toxicity. Specifically, we demonstrated that inhibition of the ABCG2 transporter in A549 by febuxostat or its downregulation by shRNA-mediated RNA interference resulted in a significant increase in protein aggregation due to smoke exposure.

Abstract A033: Murine CNS and bone marrow distribution of the aurora A kinase inhibitor alisertib: Pharmacokinetics and exposure at the sites of efficacy and toxicity

Abstract A033: Murine CNS and bone marrow distribution of the aurora A kinase inhibitor alisertib: Pharmacokinetics and exposure at the sites of efficacy and toxicity

Exposure to Low-Frequency Radiation Changes the Expression of Nestin, VEGF, BCRP and Apoptosis Markers During Glioma Treatment Strategy: An In Vitro Study.

Exposure to physical contamination during chemotherapy, including non-ionizing electromagnetic fields, raises concerns about the widespread sources of exposure to this type of radiation. Glioblastoma multiforme (GBM) is an aggressive central nervous system tumor that is hard to treat due to resistance to drugs such as temozolomide (TMZ). Electromagnetic fields (EMF) and haloperidol (HLP) may have anticancer effects. In this study, we investigated the effects of TMZ, HLP, and EMF on GBM cell lines and analyzed the association between non-ionizing radiation and the risk of change in drug performance. Cell viability and reactive oxygen species (ROS) generation were measured by MTT and NBT assay, respectively. Then, the expression levels of breast cancer-resistant protein (BCRP), Bax, Bcl2, Nestin, vascular endothelial growth factor (VEGF) genes, and P53, Bax, and Bcl2 Proteins were evaluated by real-time PCR and western blot. Co-treatment of GBM cells by HLP and TMZ enhanced apoptosis in T-98G and A172 cells by increasing the expression of P53 and Bax and decreasing Bcl-2. Interestingly, exposure of GBM cells to EMF decreased apoptosis in the TMZ+HLP group. In conclusion, EMF reduced the synergistic effect of TMZ and HLP. This hypothesis that patients who are treated for brain tumors and suffer from depression should not be exposed to EMF is proposed in the present study. There appears to be an urgent need to reconsider exposure limits for low-frequency magnetic fields, based on experimental and epidemiological research, the relationship between exposure to non-ionizing radiation and adverse human health effects.

7-Ketocholesterol plays a key role in cholesterol-induced hepatitis via macrophage and neutrophil infiltration

This study sought to explore the role of 7-ketocholesterol (7-KC) in liver damage caused by high cholesterol intake and its potential pathological mechanism in mice. Our in vivo findings indicated that mice fed a high-cholesterol diet had elevated serum levels of 7-KC, accompanied by liver injury and inflammation, similar to human nonalcoholic steatohepatitis. Furthermore, the high-cholesterol diet induced neutrophil infiltration, which played a critical role in liver damage through myeloperoxidase (MPO) activity. Upon stimulation with 7-KC, macrophages exhibited increased expression of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2, as well as ATP-binding cassette transporter A1 (ABCA1) and ABCG1. Hepatocytes, on the other hand, exhibited increased expression of CXCL2 and ABCG1. The infiltration of neutrophils in the liver was primarily caused by CXCL1 and CXCL2, resulting in hepatocyte cell death due to elevated MPO activity. Our data also revealed that the activation of macrophages by 7-KC via ABCA1 or ABCG1 was not associated with lipid accumulation. Collectively, these findings suggest that high cholesterol-induced hepatitis in mice involves, at least partially, the recruitment of neutrophils to the liver by 7-KC-activated macrophages. This is mediated by increased expression of CXCL1 and CXCL2 through ABCA1 or ABCG1, which act as 7-KC efflux transporters. Additionally, hepatocytes contribute to this process by increased expression of CXCL2 through ABCG1. Therefore, our findings suggest that 7-KC may play a role in high cholesterol-induced hepatitis in mice by activating macrophages and hepatocytes, ultimately leading to neutrophil infiltration.

Targeted Delivery of Mesenchymal Stem Cell-Derived Bioinspired Exosome-Mimetic Nanovesicles with Platelet Membrane Fusion for Atherosclerotic Treatment.

Accumulating evidence indicates that mesenchymal stem cells (MSCs)-derived exosomes hold significant potential for the treatment of atherosclerosis. However, large-scale production and organ-specific targeting of exosomes are still challenges for further clinical applications. This study aims to explore the targeted efficiency and therapeutic potential of biomimetic platelet membrane-coated exosome-mimetic nanovesicles (P-ENVs) in atherosclerosis. To produce exosome-mimetic nanovesicles (ENVs), MSCs were successively extruded through polycarbonate porous membranes. P-ENVs were engineered by fusing MSC-derived ENVs with platelet membranes and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. The stability and safety of P-ENVs were also assessed. The targeted efficacy of P-ENVs was evaluated using an in vivo imaging system (IVIS) spectrum imaging system and immunofluorescence. Histological analyses, Oil Red O (ORO) staining, and Western blot were used to investigate the anti-atherosclerotic effectiveness of P-ENVs. Both ENVs and P-ENVs exhibited similar characteristics to exosomes. Subsequent miRNA sequencing of P-ENVs revealed their potential to mitigate atherosclerosis by influencing biological processes related to cholesterol metabolism. In an ApoE-/- mice model, the intravenous administration of P-ENVs exhibited enhanced targeting of atherosclerotic plaques, resulting in a significant reduction in lipid deposition and necrotic core area. Our in vitro experiments showed that P-ENVs promoted cholesterol efflux and reduced total cholesterol content in foam cells. Further analysis revealed that P-ENVs attenuated intracellular cholesterol accumulation by upregulating the expression of the critical cholesterol transporters ABCA1 and ABCG1. This study highlighted the potential of P-ENVs as a novel nano-drug delivery platform for enhancing drug delivery efficiency while concurrently mitigating adverse reactions in atherosclerotic therapy.

Roles of breast cancer resistance protein and organic anion transporting polypeptide 2B1 in gastrointestinal toxicity induced by SN-38 under inflammatory conditions

Drug transporters are among the factors that determine the pharmacokinetic profiles after drug administration. In this study, we investigated the roles of drug transporters involved in transport of SN-38, which is an active metabolite of irinotecan, in the intestine under inflammatory conditions in vitro and determined their functional consequences. The expression alterations of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 2B1 were determined at the mRNA and protein levels, and the subsequent functional alterations were evaluated via an accumulation study with the representative transporter substrates [prazosin and dibromofluorescein (DBF)] and SN-38. We also determined the cytotoxicity of SN-38 under inflammatory conditions. Decreased BCRP expression and increased OATP2B1 expression were observed under inflammatory conditions in vitro, which led to altered accumulation profiles of prazosin, DBF, and SN-38, and the subsequent cytotoxic profiles of SN-38. Treatment with rifampin or novobiocin supported the significant roles of BCRP and OATP2B1 in the transport and cytotoxic profile of SN-38. Collectively, these results suggest that BCRP and OATP2B1 are involved in the increased cytotoxicity of SN-38 under inflammatory conditions in vitro. Further comprehensive research is warranted to completely understand SN-38-induced gastrointestinal cytotoxicity and aid in the successful treatment of cancer with irinotecan.

NAT10-mediated mRNA N4-acetylcytidine modification of MDR1 and BCRP promotes breast cancer progression.

N-acetyltransferase 10 (NAT10) serves as a critical enzyme in mediating the N4-acetylcytidine (ac4C) that ensures RNA stability and effective translation processes. The role of NAT10 in driving the advancement of breast cancer remains uninvestigated. We observed an increase in NAT10 expression, both at mRNA level through the analysis of the Cancer Genome Atlas (TCGA) database and at the protein level of tumor tissues from breast cancer patients. We determined that a heightened expression of NAT10 served as a predictor of an unfavorable clinical outcome. By screening the Cancer Cell Line Encyclopedia (CCLE) cell bank, this expression pattern of NAT10 was consistency found across almost all the classic breast cancer cell lines. Functionally, interference of NAT10 expression exerts an inhibitory effect on proliferation and invasion of breast cancer cells. By using ac4C RNA immunoprecipitation (ac4c-RIP) and acRIP-qPCR assays, we identified a reduction of ac4C enrichment within the ATP binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP), consequent to NAT10 suppression. Expressions of MDR1 and BCRP exhibited a positive correlation with NAT10 expression in tumor tissues, and the inhibition of NAT10 in breast cancer cells resulted in a decrease of MDR1 and BCRP expression. Therefore, the overexpressing of MDR1 and BCRP could partially rescue the adverse consequences of NAT10 depletion. In addition, we found that, remodelin, a NAT10 inhibitor, reinstated the susceptibility of capecitabine-resistant breast cancer cells to the chemotherapy, both in vitro and in vivo. The results of our study demonstrated the essential role of NAT10-mediated ac4c-modification in breast cancer progression and provide a novel strategy for overcoming chemoresistance challenges.

Novel Screening System for Biliary Excretion of Drugs Using Human Cholangiocyte Organoid Monolayers with Directional Drug Transport.

It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes. The purpose of this study was to culture the organoids in monolayers on inserts to investigate the biliary excretory capacity of drugs. Cholangiocyte organoids prepared from hepatocytes had significantly higher mRNA expression of CK19, a bile duct epithelial marker, compared to hepatocytes. The organoids also expressed mRNA for efflux transporters involved in biliary excretion of drugs, P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). The subcellular localization of each protein was observed. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane side. Transport studies showed that the permeation rate from the B side to the A side was faster than from the A side to the B side for the substrates of each efflux transporter, but this directionality disappeared in the presence of inhibitor of each transporter. In conclusion, the cholangiocyte organoid monolayer system has the potential to quantitatively evaluate the biliary excretion of drugs. The results of the present study represent an unprecedented system using human cholangiocyte organoids, which may be useful as a screening model to directly quantify the contribution of biliary excretion to the clearance of drugs.

Foretinib, a c-MET receptor tyrosine kinase inhibitor, tackles multidrug resistance in cancer cells by inhibiting ABCB1 and ABCG2 transporters

BackgroundABC transporter-mediated multidrug resistance (MDR) remains a major obstacle for cancer pharmacological treatment. Some tyrosine kinase inhibitors (TKIs) have been shown to reverse MDR. The present study was designed to evaluate for the first time whether foretinib, a multitargeted TKI, can circumvent ABCB1 and ABCG2-mediated MDR in treatment-resistant cancer models. MethodsAccumulation of fluorescent substrates of ABCB1 and ABCG2 in ABCB1-overexpressing MES-SA/DX5 and ABCG2-overexpressing MCF-7/MX and their parenteral cells was evaluated by flow cytometry. The growth inhibitory activity of single and combination therapy of foretinib and chemotherapeutic drugs on MDR cells was examined by MTT assay. Analysis of combined interaction effects was performed using CalcuSyn software. ResultsIt was firstly proved that foretinib increased the intracellular accumulation of rhodamine 123 and mitoxantrone in MES-SA/DX5 and MCF-7/MX cancer cells, with accumulation ratios of 12 and 2.2 at 25 μM concentration, respectively. However, it did not affect the accumulation of fluorescent substrates in the parental cells. Moreover, foretinib synergistically improved the cytotoxic effects of doxorubicin and mitoxantrone. The means of combination index (CI) values at fraction affected (Fa) values of 0.5, 0.75, and 0.9 were 0.64 ± 0.08 and 0.47 ± 0.09, in MES-SA/DX5 and MCF-7/MX cancer cells, respectively. In silico analysis also suggested that the drug-binding domain of ABCB1 and ABCG2 transporters could be considered as potential target for foretinib. ConclusionOverall, our results suggest that foretinib can target MDR-linked ABCB1 and ABCG2 transporters in clinical cancer therapy.

Cadmium transport by mammalian ATP-binding cassette transporters.

Cellular responses to toxic metals depend on metal accessibility to intracellular targets, reaching interaction sites, and the intracellular metal concentration, which is mainly determined by uptake pathways, binding/sequestration and efflux pathways. ATP-binding cassette (ABC) transporters are ubiquitous in the human body-usually in epithelia-and are responsible for the transfer of indispensable physiological substrates(e.g. lipids and heme), protection against potentially toxic substances, maintenance of fluid composition, and excretion of metabolic waste products. Derailed regulation and gene variants of ABC transporters culminate in a wide array of pathophysiological disease states, such as oncogenic multidrug resistance or cystic fibrosis. Cadmium (Cd) has no known physiological role in mammalians and poses a health risk due to its release into the environment as a result of industrial activities, and eventually passes into the food chain. Epithelial cells, especially within the liver, lungs, gastrointestinal tract and kidneys, are particularly susceptible to the multifaceted effects of Cd because of the plethora of uptake pathways available. Pertinent to their broad substrate spectra, ABC transporters represent a major cellular efflux pathway for Cd and Cd complexes. In this review, we summarize current knowledge concerning transport of Cd and its complexes (mainly Cd bound to glutathione) by the ABC transporters ABCB1 (P-glycoprotein, MDR1), ABCB6, ABCC1 (multidrug resistance related protein 1, MRP1), ABCC7 (cystic fibrosis transmembrane regulator, CFTR), and ABCG2 (breast cancer related protein, BCRP). Potential detoxification strategies underlying ABC transporter-mediated efflux of Cd and Cd complexesare discussed.

Overexpression of SCN5A overcomes ABC transporter-mediated multidrug resistance in acute myeloid leukemia through promoting apoptosis

ABSTRACT Background This study aimed to explore the effect and mechanism of SCN5A overcoming ATP-binding cassette (ABC) transporter-mediated multidrug resistance (MDR) in acute myeloid leukemia (AML) through promoting apoptosis. Research design and methods The tissues derived from AML patients were divided into Sensitive group and Resistance group according to the presence of drug-resistance. Human AML cell line HL-60 and drug-resistant strain HL-60/ADR were divided into HL-60/ADR-vector group, HL-60/ADR-SCN5A group, HL-60-vector group and HL-60-SCN5A group. RT-qPCR was used to detect the mRNA expression level of SCN5A; MTT assay to assess the survival rate and proliferation level of cells; flow cytometry to determine the apoptosis level; and western blot to check the levels of SCN5A, P-glycoprotein (P-gp), MDR protein 1 (MRP1), MDR gene 1 (MDR1), breast cancer resistance protein (BCRP), Bcl-2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl-2) proteins in cells. Results SCN5A expressed lowly in drug-resistant AML tissues and cells. Up-regulation of SCN5A inhibited MDR in HL-60 cells, enhanced the chemosensitivity of HL-60/ADR, and increased the apoptosis levels of HL-60 and HL-60/ADR cells. However, over-expression of SCN5A inhibited the expression of MDR-related proteins. Conclusions SCN5A may overcome ABC transporter-mediated MDR in AML through enhancing the apoptosis and inhibiting the expression of MDR proteins.

A Pilot Study To Assess the Suitability of Riboflavin As a Surrogate Marker of Breast Cancer Resistance Protein in Healthy Participants.

We recently showed that riboflavin is a selected substrate of breast cancer resistance protein (BCRP) over P-glycoprotein (P-gp) and demonstrated its prediction performance in preclinical drug-drug interaction (DDI) studies. The aim of this study was to investigate the suitability of riboflavin to assess BCRP inhibition in humans. First, we assessed the substrate potential of riboflavin toward other major drug transporters using established transfected cell systems. Riboflavin is a substrate for organic anion transporter (OAT)1, OAT3, and multidrug and toxin extrusion protein (MATE)2-K, with uptake ratios ranging from 2.69 to 11.6, but riboflavin is not a substrate of organic anion-transporting polypeptide (OATP)1B1, OATP1B3, organic cation transporter (OCT)2, and MATE1. The effects of BMS-986371, a potent in vitro inhibitor of BCRP (IC 50 0.40 μM), on the pharmacokinetics of riboflavin, isobutyryl carnitine, and arginine were then examined in healthy male adults (N = 14 or 16) after oral administration of methotrexate (MTX) (7.5 mg) and enteric-coated (EC) sulfasalazine (SSZ) (1000 mg) alone or in combination with BMS-986371 (150 mg). Oral administration of BMS-986371 increased the area under the plasma concentration-time curves (AUCs) of rosuvastatin and immediate-release (IR) SSZ to 1.38- and 1.51-fold, respectively, and significantly increased AUC(0-4h), AUC(0-24h), and C max of riboflavin by 1.25-, 1.14-, and 1.11-fold (P-values of 0.003, 0.009, and 0.025, respectively) compared with the MTX/SSZ EC alone group. In contrast, BMS-986371 did not significantly influence the AUC(0-24h) and C max values of isobutyryl carnitine and arginine (0.96- to 1.07-fold, respectively; P > 0.05). Overall, these data indicate that plasma riboflavin is a promising biomarker of BCRP that may offer a possibility to assess drug candidate as a BCRP modulator in early drug development. SIGNIFICANCE STATEMENT: Endogenous compounds that serve as biomarkers for clinical inhibition of breast cancer resistance protein (BCRP) are not currently available. This study provides the initial evidence that riboflavin is a promising BCRP biomarker in humans. For the first time, the value of leveraging the substrate of BCRP with acceptable prediction performance in clinical studies is shown. Additional clinical investigations with known BCRP inhibitors are needed to fully validate and showcase the utility of this biomarker.

Improving brain penetration using elacridara P-glycoprotein and BCRP inhibitor

The central nervous system (CNS) is a site for a myriad of disorders and diseases, such as schizophrenia, Alzheimers disease, and Parkinsons disease. Many medications targeting these illnesses remain challenged due to efflux transporters forming a blood-brain barrier (BBB). To combat such challenges, elacridar shown promise at inhibiting such transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), therefore improving brain penetration. However, as an early clinical candidate, many remain unknown about elacridars pharmacokinetic (PK) properties in the human body. This paper aims to obtain a better understanding of elacridars pharmacokinetic profile and predict the optimal dose and dose interval for its application in the clinic. To begin with, a series of basic PK models were created using fundamental PK equations and the models were fit to elacridar clinical data to obtain elacridar-specific PK parameters. Next, elacridar preclinical PK as well as in vitro inhibition assay were used to determine the therapeutic window. Our final, multiple-dose extravascular PK model reveals that the most convenient and effective dose regimen is 900 mg BID (bis in die; twice per day). With this elacridar PK model, researchers can leverage elacridar human PK to improve clinical outcomes.

Local dose-dense chemotherapy for triple-negative breast cancer via minimally invasive implantation of 3D printed devices

Dose-dense chemotherapy is the preferred first-line therapy for triple-negative breast cancer (TNBC), a highly aggressive disease with a poor prognosis. This treatment uses the same drug doses as conventional chemotherapy but with shorter dosing intervals, allowing for promising clinical outcomes with intensive treatment. However, the frequent systemic administration used for this treatment results in systemic toxicity and low patient compliance, limiting therapeutic efficacy and clinical benefit. Here, we report local dose-dense chemotherapy to treat TNBC by implanting 3D printed devices with time-programmed pulsatile release profiles. The implantable device can control the time between drug releases based on its internal microstructure design, which can be used to control dose density. The device is made of biodegradable materials for clinical convenience and designed for minimally invasive implantation via a trocar. Dose density variation of local chemotherapy using programmable release enhances anti-cancer effects in vitro and in vivo. Under the same dose density conditions, device-based chemotherapy shows a higher anti-cancer effect and less toxic response than intratumoral injection. We demonstrate local chemotherapy utilizing the implantable device that simulates the drug dose, number of releases, and treatment duration of the dose-dense AC (doxorubicin and cyclophosphamide) regimen preferred for TNBC treatment. Dose density modulation inhibits tumor growth, metastasis, and the expression of drug resistance-related proteins, including p-glycoprotein and breast cancer resistance protein. To the best of our knowledge, local dose-dense chemotherapy has not been reported, and our strategy can be expected to be utilized as a novel alternative to conventional therapies and improve anti-cancer efficiency.

Cocaine regulates antiretroviral therapy CNS access through pregnane-x receptor-mediated drug transporter and metabolizing enzyme modulation at the blood brain barrier

BackgroundAppropriate interactions between antiretroviral therapies (ART) and drug transporters and metabolizing enzymes at the blood brain barrier (BBB) are critical to ensure adequate dosing of the brain to achieve HIV suppression. These proteins are modulated by demographic and lifestyle factors, including substance use. While understudied, illicit substances share drug transport and metabolism pathways with ART, increasing the potential for adverse drug:drug interactions. This is particularly important when considering the brain as it is relatively undertreated compared to peripheral organs and is vulnerable to substance use-mediated damage.MethodsWe used an in vitro model of the human BBB to determine the extravasation of three first-line ART drugs, emtricitabine (FTC), tenofovir (TFV), and dolutegravir (DTG), in the presence and absence of cocaine, which served as our illicit substance model. The impact of cocaine on BBB integrity and permeability, drug transporters, metabolizing enzymes, and their master transcriptional regulators were evaluated to determine the mechanisms by which substance use impacted ART central nervous system (CNS) availability.ResultsWe determined that cocaine had a selective impact on ART extravasation, where it increased FTC’s ability to cross the BBB while decreasing TFV. DTG concentrations that passed the BBB were below quantifiable limits. Interestingly, the potent neuroinflammatory modulator, lipopolysaccharide, had no effect on ART transport, suggesting a specificity for cocaine. Unexpectedly, cocaine did not breach the BBB, as permeability to albumin and 4 kDa FITC-dextran, as well as tight junction proteins and adhesion molecules remained unchanged. Rather, cocaine selectively decreased the pregnane-x receptor (PXR), but not constitutive androstane receptor (CAR). Consequently, drug transporter expression and activity decreased in endothelial cells of the BBB, including p-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 4 (MRP4). Further, cytochrome P450 3A4 (CYP3A4) enzymatic activity increased following cocaine treatment that coincided with decreased expression. Finally, cocaine modulated adenylate kinases that are required to facilitate biotransformation of ART prodrugs to their phosphorylated, pharmacologically active counterparts.ConclusionOur findings indicate that additional considerations are needed in CNS HIV treatment strategies for people who use cocaine, as it may limit ART efficacy through regulation of drug transport and metabolizing pathways at the BBB.

Establishment of a genotyping method for the junior blood group and identification of a rare blood type with partial DVI.3 and Jr(a-)

To develop a genotyping method for the Junior blood type and report on a rare blood type with Jr(a-). Healthy O-type RhD+ volunteer donors of the Shenzhen Blood Center from January to May 2021 (n = 1 568) and a pedigree with difficult cross-matching (n = 3) were selected as the study subjects. Serological methods were used for proband's blood type identification, unexpected antibody identification, and antibody titer determination. Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used for typing the proband's RhD gene. ABCG2 gene coding region sequencing and a PCR-SSP genotyping method were established for determining the genotypes of the proband and his family members and screening of Jra antigen-negative rare blood type among the 1 568 blood donors. The proband's ABO and RhD blood types were respectively determined as B and partial D (RHDDVI.3/RHD01N.01), Junior blood type Jra antigen was negative, and plasma had contained anti-D and anti-Jra. Sequencing of the ABCG2 gene revealed that the proband's genotype was ABGG201N.01/ABGG201N.01 [homozygous c.376C>T (p.Gln126X) variants], which is the most common Jr(a-) blood type allele in the Asian population. Screening of the voluntary blood donors has detected no Jr(a-) rare blood type. Statistical analysis of the heterozygotes suggested that the allelic frequency for ABCG2*01N.01 (c.376T) was 0.45%, and the frequency of Jr(a-) rare blood type with this molecular background was about 0.2‰. A very rare case of partial DVI.3 type and Jr(a-) rare blood type has been identified. And a method for identifying the Junior blood type through sequencing the coding regions of the ABCG2 gene and PCR-SSP has been established.

Decreased NMIIA heavy chain phosphorylation at S1943 promotes mitoxantrone resistance by upregulating BCRP and N-cadherin expression in breast cancer cells.

Mitoxantrone (MX) is an effective treatment for breast cancer; however, high efflux of MX that is accomplished by breast cancer resistance protein (BCRP) leads to acquired multidrug resistance (MDR), reducing MX's therapeutic efficacy in breast cancer. Non-muscle myosin IIA (NMIIA) and its heavy phosphorylation at S1943 have been revealed to play key roles in tumor metastasis and progression, including in breast cancer; however, their molecular function in BCRP-mediated MDR in breast cancer remains unknown. In this study, we revealed that the expression of NMIIA heavy chain phosphorylation at S1943 was downregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and stable expression of NMIIA-S1943A mutant increased BCRP expression and promoted the resistance of MCF-7/MX cells to MX. Meanwhile, NMIIA S1943 phosphorylation induced by epidermal growth factor (EGF) was accompanied by the downregulation of BCRP in MCF-7/MX cells. Furthermore, stable expression of NMIIA-S1943A in MCF-7/MX cells resulted in upregulation of N-cadherin and the accumulation of β-catenin on the cell surface, which inhibited the nucleus translocation of β-catenin and Wnt/β-catenin-based proliferative signaling. EGF stimulation of MCF-7/MX cells showed the downregulation of N-cadherin and β-catenin. Our results suggest that decreased NMIIA heavy phosphorylation at S1943 increases BCRP expression and promotes MX resistance in breast cancer cells via upregulating N-cadherin expression.

ATP-Binding Cassette Subfamily G Member 2 in Acute Myeloid Leukemia: A New Molecular Target?

Despite the progress in the knowledge of disease pathogenesis and the identification of many molecular markers as potential targets of new therapies, the cure of acute myeloid leukemia remains challenging. Disease recurrence after an initial response and the development of resistance to old and new therapies account for the poor survival rate and still make allogeneic stem cell transplantation the only curative option. Multidrug resistance (MDR) is a multifactorial phenomenon resulting from host-related characteristics and leukemia factors. Among these, the overexpression of membrane drug transporter proteins belonging to the ABC (ATP-Binding Cassette)-protein superfamily, which diverts drugs from their cellular targets, plays an important role. Moreover, a better understanding of leukemia biology has highlighted that, at least in cancer, ABC protein's role goes beyond simple drug transport and affects many other cell functions. In this paper, we summarized the current knowledge of ABCG2 (formerly Breast Cancer Resistance Protein, BCRP) in acute myeloid leukemia and discuss the potential ways to overcome its efflux function and to revert its ability to confer stemness to leukemia cells, favoring the persistence of leukemia progenitors in the bone marrow niche and justifying relapse also after therapy intensification with allogeneic stem cell transplantation.