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16 pages, 1930 KiB

Open AccessArticle

Protein Abundance of Drug Metabolizing Enzymes in Human Hepatitis C Livers

by Marek Drozdzik, Joanna Lapczuk-Romanska, Christoph Wenzel, Lukasz Skalski, Sylwia Szeląg-Pieniek, Mariola Post, Arkadiusz Parus, Marta Syczewska, Mateusz Kurzawski and Stefan Oswald

Int. J. Mol. Sci. 2023, 24(5), 4543; https://doi.org/10.3390/ijms24054543 - 25 Feb 2023

Cited by 4 | Viewed by 1606

Abstract

Hepatic drug metabolizing enzymes (DMEs), whose activity may be affected by liver diseases, are major determinants of drug pharmacokinetics. Hepatitis C liver samples in different functional states, i.e., the Child–Pugh class A (n = 30), B (n = 21) and C [...] Read more.

Hepatic drug metabolizing enzymes (DMEs), whose activity may be affected by liver diseases, are major determinants of drug pharmacokinetics. Hepatitis C liver samples in different functional states, i.e., the Child–Pugh class A (n = 30), B (n = 21) and C (n = 7) were analyzed for protein abundances (LC-MS/MS) and mRNA levels (qRT-PCR) of 9 CYPs and 4 UGTs enzymes. The protein levels of CYP1A1, CYP2B6, CYP2C8, CYP2C9, and CYP2D6 were not affected by the disease. In the Child–Pugh class A livers, a significant up-regulation of UGT1A1 (to 163% of the controls) was observed. The Child–Pugh class B was associated with down-regulation of the protein abundance of CYP2C19 (to 38% of the controls), CYP2E1 (to 54%), CYP3A4 (to 33%), UGT1A3 (to 69%), and UGT2B7 (to 56%). In the Child–Pugh class C livers, CYP1A2 was found to be reduced (to 52%). A significant trend in down-regulation of the protein abundance was documented for CYP1A2, CYP2C9, CYP3A4, CYP2E1, UGT2B7, and UGT2B15. The results of the study demonstrate that DMEs protein abundances in the liver are affected by hepatitis C virus infection and depend on the severity of the disease. Full article

(This article belongs to the Special Issue Channels and Transporters in Cells and Tissues 3.0)

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12 pages, 2264 KiB

Open AccessArticle

Selective Fluorescent Probes for High-Throughput Functional Diagnostics of the Human Multidrug Transporter P-Glycoprotein (ABCB1)

by Edit Szabó, Anna Kulin, Bálint Jezsó, Nóra Kucsma, Balázs Sarkadi and György Várady

Int. J. Mol. Sci. 2022, 23(18), 10599; https://doi.org/10.3390/ijms231810599 - 13 Sep 2022

Viewed by 1373

Abstract

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer [...] Read more.

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells. Full article

(This article belongs to the Special Issue Channels and Transporters in Cells and Tissues 3.0)

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12 pages, 2578 KiB

Open AccessArticle

Albumin Stimulates Epithelial Na⁺ Transport and Barrier Integrity by Activating the PI3K/AKT/SGK1 Pathway

by Mandy Laube and Ulrich H. Thome

Int. J. Mol. Sci. 2022, 23(15), 8823; https://doi.org/10.3390/ijms23158823 - 8 Aug 2022

Viewed by 1861

Abstract

Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na⁺ transport drives alveolar fluid clearance (AFC), enabling [...] Read more.

Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na⁺ transport drives alveolar fluid clearance (AFC), enabling air breathing. Whether or not albumin affects AFC and Na⁺ transport is yet unknown. We therefore determined the acute and chronic effects of albumin on Na⁺ transport in fetal distal lung epithelial (FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial Na⁺ transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of Na⁺ transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the chronic effects. The elevated Na⁺ transport was caused by an increased maximal ENaC activity, while Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the stimulating effect of BSA. BSA therefore enhanced epithelial Na⁺ transport and barrier integrity by activating the PI3K/AKT/SGK1 pathway. Full article

(This article belongs to the Special Issue Channels and Transporters in Cells and Tissues 3.0)

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13 pages, 1427 KiB

Open AccessArticle

Deubiquitinating Enzyme USP7 Is Required for Self-Renewal and Multipotency of Human Bone Marrow-Derived Mesenchymal Stromal Cells

by You Ji Kim, Kwang Hwan Park, Kyoung-Mi Lee, Yong-Min Chun and Jin Woo Lee

Int. J. Mol. Sci. 2022, 23(15), 8674; https://doi.org/10.3390/ijms23158674 - 4 Aug 2022

Cited by 5 | Viewed by 1740

Abstract

Ubiquitin-specific protease 7 (USP7) is highly expressed in a variety of malignant tumors. However, the role of USP7 in regulating self-renewal and differentiation of human bone marrow derived mesenchymal stromal cells (hBMSCs) remains unknown. Herein, we report that USP7 regulates self-renewal of hBMSCs [...] Read more.

Ubiquitin-specific protease 7 (USP7) is highly expressed in a variety of malignant tumors. However, the role of USP7 in regulating self-renewal and differentiation of human bone marrow derived mesenchymal stromal cells (hBMSCs) remains unknown. Herein, we report that USP7 regulates self-renewal of hBMSCs and is required during the early stages of osteogenic, adipogenic, and chondrogenic differentiation of hBMSCs. USP7, a deubiquitinating enzyme (DUB), was found to be downregulated during hBMSC differentiation. Furthermore, USP7 is an upstream regulator of the self-renewal regulating proteins SOX2 and NANOG in hBMSCs. Moreover, we observed that SOX2 and NANOG are poly-ubiquitinated and their expression is downregulated in USP7-deficient hBMSCs. Overall, this study showed that USP7 is required for maintaining self-renewal and multipotency in cultured hBMSCs. Targeting USP7 might be a novel strategy to preserve the self-renewal capacity of hBMSCs intended for stem cell therapy. Full article

(This article belongs to the Special Issue Channels and Transporters in Cells and Tissues 3.0)

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13 pages, 3397 KiB

Open AccessArticle

Lysosomal Exocytosis of Olivacine on the Way to Explain Drug Resistance in Cancer Cells

by Benita Wiatrak, Tomasz Gębarowski, Eddie Czwojdziński, Kazimierz Gąsiorowski and Beata Tylińska

Int. J. Mol. Sci. 2022, 23(11), 6119; https://doi.org/10.3390/ijms23116119 - 30 May 2022

Cited by 1 | Viewed by 1659

Abstract

Ellipticine is an indole alkaloid with proven antitumor activity against various tumors in vitro and a diverse mechanism of action, which includes topoisomerase II inhibition, intercalation, and cell cycle impact. Olivacine—ellipticine’s isomer—shows similar properties. The objectives of this work were as follows: (a) [...] Read more.

Ellipticine is an indole alkaloid with proven antitumor activity against various tumors in vitro and a diverse mechanism of action, which includes topoisomerase II inhibition, intercalation, and cell cycle impact. Olivacine—ellipticine’s isomer—shows similar properties. The objectives of this work were as follows: (a) to find a new path of olivacine synthesis, (b) to study the cytotoxic properties of olivacine and ellipticine in comparison to doxorubicin as well as their impact on the cell cycle, and (c) to investigate the cellular pharmacokinetics of the tested compounds to understand drug resistance in cancer cells better. SRB and MTT assays were used to study the anticancer activity of olivacine and ellipticine in vitro. Both compounds showed a cytotoxic effect on various cell lines, most notably on the doxorubicin-resistant LoVo/DX model, with olivacine’s cytotoxicity approximately three times higher than doxorubicin. Olivacine proved to be less effective against cancer cells and less cytotoxic to normal cells than ellipticine. Olivacine proved to have fluorescent properties. Microscopic observation of cells treated with olivacine showed the difference in sensitivity depending on the cell line, with A549 cells visibly affected by a much lower concentration of olivacine than normal NHDF cells. An increased percentage of cells in G0/G1 was observed after treatment with olivacine and ellipticine, suggesting an impact on cell cycle progression, potentially via higher p53 protein expression, which blocks the transition from G0/G1 to the S phase. Ellipticine induced apoptosis at a concentration as low as 1 μM. It has been proved that the tested compounds (ellipticine and olivacine) undergo lysosomal exocytosis. Reducing exocytosis is possible through the use of compounds that inhibit the activity of the proton pump. Olivacine and ellipticine exhibited diverse cytotoxicity against a panel of cancer cells. Analysis of the lysosomal exocytosis of olivacine and ellipticine shows the need to look for derivatives with comparable anticancer activity but reduced weak base character. Full article

(This article belongs to the Special Issue Channels and Transporters in Cells and Tissues 3.0)

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17 pages, 2787 KiB

Open AccessArticle

Differential Serotonin Uptake Mechanisms at the Human Maternal–Fetal Interface

by Petra Baković, Maja Kesić, Maja Perić, Ivona Bečeheli, Marina Horvatiček, Meekha George, Lipa Čičin-Šain, Gernot Desoye, Christian Wadsack, Ute Panzenboeck and Jasminka Štefulj

Int. J. Mol. Sci. 2021, 22(15), 7807; https://doi.org/10.3390/ijms22157807 - 21 Jul 2021

Cited by 11 | Viewed by 3214

Abstract

Serotonin (5-HT) plays an extensive role during pregnancy in regulating both the placental physiology and embryonic/fetal development. The uptake of 5-HT into cells is central to the control of local concentrations of 5-HT near its molecular targets. Here, we investigated the mechanisms of [...] Read more.

Serotonin (5-HT) plays an extensive role during pregnancy in regulating both the placental physiology and embryonic/fetal development. The uptake of 5-HT into cells is central to the control of local concentrations of 5-HT near its molecular targets. Here, we investigated the mechanisms of 5-HT uptake into human primary placental cells and cord blood platelets, all isolated immediately after birth. Trophoblasts and cord blood platelets showed 5-HT uptake with similar Michaelis constant (Km) values (~0.6 μM), typical of the high-affinity serotonin transporter (SERT). The uptake of 5-HT into trophoblasts was efficiently inhibited by various SERT-targeting drugs. In contrast, the uptake of 5-HT into feto-placental endothelial cells was not inhibited by a SERT blocker and showed a Km value (~782 μM) in the low-affinity range. Consistent with this, SERT mRNAs were abundant in term trophoblasts but sparse in feto-placental endothelial cells, whereas the opposite was found for the low-affinity plasma membrane monoamine transporter (PMAT) mRNAs. Organic cation transporter (OCT) 1, 2, and 3 mRNAs were absent or sparse in both cell types. In summary, the results demonstrate, for the first time, the presence of functional 5-HT uptake systems in feto-placental endothelial cells and fetal platelets, cells that are in direct contact with fetal blood plasma. The data also highlight the sensitivity to various psychotropic drugs of 5-HT transport into trophoblasts facing the maternal blood. The multiple, high-, and low-affinity systems present for the cellular uptake of 5-HT underscore the importance of 5-HT homeostasis at the maternal–fetal interface. Full article

(This article belongs to the Special Issue Channels and Transporters in Cells and Tissues 3.0)

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Review

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9 pages, 835 KiB

Open AccessReview

Membrane Fusion and SNAREs: Interaction with Ras Proteins

by Azzurra Margiotta

Int. J. Mol. Sci. 2022, 23(15), 8067; https://doi.org/10.3390/ijms23158067 - 22 Jul 2022

Cited by 10 | Viewed by 2747

Abstract

The superfamily of Ras proteins comprises different molecules belonging to the GTPase family. They normally cycle between an active state bound to GTP which activates effectors while the protein is membrane-associated, and an inactive GDP-bound state. They regulate the intracellular trafficking and other [...] Read more.

The superfamily of Ras proteins comprises different molecules belonging to the GTPase family. They normally cycle between an active state bound to GTP which activates effectors while the protein is membrane-associated, and an inactive GDP-bound state. They regulate the intracellular trafficking and other cellular processes. The family of Rab proteins includes several members and they have been found, among other Ras proteins, to be fundamental for important biological processes, such as endocytosis and exocytosis. SNARE proteins control the fusion of vesicles by forming quaternary complexes which are divided into two small groups on the two different compartments. Generally, the association of three SNARE proteins on the donor compartment with the one on the target compartment determines the formation of the SNARE complex, the opening of the fusion pore and the formation of one single bigger vesicle. Interestingly, novel interactions between other molecules involved in intracellular trafficking, endosomal fusion and maturation have recently been found, such as the interaction between invariant chain and the Qb SNARE vti1b, and more functional connections between Rab proteins and SNAREs are supposed to be fundamental for the regulation of membrane fusion. Full article

(This article belongs to the Special Issue Channels and Transporters in Cells and Tissues 3.0)

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19 pages, 2214 KiB

Open AccessReview

Role of K⁺ and Ca²⁺-Permeable Channels in Osteoblast Functions

by Hiroaki Kito and Susumu Ohya

Int. J. Mol. Sci. 2021, 22(19), 10459; https://doi.org/10.3390/ijms221910459 - 28 Sep 2021

Cited by 14 | Viewed by 3238

Abstract

Bone-forming cells or osteoblasts play an important role in bone modeling and remodeling processes. Osteoblast differentiation or osteoblastogenesis is orchestrated by multiple intracellular signaling pathways (e.g., bone morphogenetic proteins (BMP) and Wnt signaling pathways) and is modulated by the extracellular environment (e.g., parathyroid [...] Read more.

Bone-forming cells or osteoblasts play an important role in bone modeling and remodeling processes. Osteoblast differentiation or osteoblastogenesis is orchestrated by multiple intracellular signaling pathways (e.g., bone morphogenetic proteins (BMP) and Wnt signaling pathways) and is modulated by the extracellular environment (e.g., parathyroid hormone (PTH), vitamin D, transforming growth factor β (TGF-β), and integrins). The regulation of bone homeostasis depends on the proper differentiation and function of osteoblast lineage cells from osteogenic precursors to osteocytes. Intracellular Ca²⁺ signaling relies on the control of numerous processes in osteoblast lineage cells, including cell growth, differentiation, migration, and gene expression. In addition, hyperpolarization via the activation of K⁺ channels indirectly promotes Ca²⁺ signaling in osteoblast lineage cells. An improved understanding of the fundamental physiological and pathophysiological processes in bone homeostasis requires detailed investigations of osteoblast lineage cells. This review summarizes the current knowledge on the functional impacts of K⁺ channels and Ca²⁺-permeable channels, which critically regulate Ca²⁺ signaling in osteoblast lineage cells to maintain bone homeostasis. Full article

(This article belongs to the Special Issue Channels and Transporters in Cells and Tissues 3.0)

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