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13 pages, 8537 KiB

Open AccessArticle

Inhibition of ABCC6 Transporter Modifies Cytoskeleton and Reduces Motility of HepG2 Cells via Purinergic Pathway

by Angela Ostuni, Monica Carmosino, Rocchina Miglionico, Vittorio Abruzzese, Fabio Martinelli, Daniela Russo, Ilaria Laurenzana, Agata Petillo and Faustino Bisaccia

Cells 2020, 9(6), 1410; https://doi.org/10.3390/cells9061410 - 05 Jun 2020

Cited by 16 | Viewed by 2860

Abstract

ABCC6, belonging to sub-family C of ATP-binding cassette transporter, is an ATP-dependent transporter mainly present in the basolateral plasma membrane of hepatic and kidney cells. Although the substrates transported are still uncertain, ABCC6 has been shown to promote ATP release. The extracellular ATP [...] Read more.

ABCC6, belonging to sub-family C of ATP-binding cassette transporter, is an ATP-dependent transporter mainly present in the basolateral plasma membrane of hepatic and kidney cells. Although the substrates transported are still uncertain, ABCC6 has been shown to promote ATP release. The extracellular ATP and its derivatives di- and mono-nucleotides and adenosine by acting on specific receptors activate the so-called purinergic pathway, which in turn controls relevant cellular functions such as cell immunity, inflammation, and cancer. Here, we analyzed the effect of Abcc6 knockdown and probenecid-induced ABCC6 inhibition on cell cycle, cytoskeleton, and motility of HepG2 cells. Gene and protein expression were evaluated by quantitative Reverse Transcription PCR (RT-qPCR) and western blot, respectively. Cellular cycle analysis was evaluated by flow cytometry. Actin cytoskeleton dynamics was evaluated by laser confocal microscopy using fluorophore-conjugated phalloidin. Cell motility was analyzed by in vitro wound-healing migration assay. Cell migration is reduced both in Abcc6 knockdown HepG2 cells and in probenecid treated HepG2 cells by interfering with the extracellular reserve of ATP. Therefore, ABCC6 could contribute to cytoskeleton rearrangements and cell motility through purinergic signaling. Altogether, our findings shed light on a new role of the ABCC6 transporter in HepG2 cells and suggest that its inhibitor/s could be considered potential anti-metastatic drugs. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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20 pages, 1773 KiB

Open AccessArticle

Identification of Novel Rare ABCC1 Transporter Mutations in Tumor Biopsies of Cancer Patients

by Onat Kadioglu, Mohamed Saeed, Markus Munder, Andreas Spuller, Henry Johannes Greten and Thomas Efferth

Cells 2020, 9(2), 299; https://doi.org/10.3390/cells9020299 - 26 Jan 2020

Cited by 1 | Viewed by 3322

Abstract

The efficiency of chemotherapy drugs can be affected by ATP-binding cassette (ABC) transporter expression or by their mutation status. Multidrug resistance is linked with ABC transporter overexpression. In the present study, we performed rare mutation analyses for 12 ABC transporters related to drug [...] Read more.

The efficiency of chemotherapy drugs can be affected by ATP-binding cassette (ABC) transporter expression or by their mutation status. Multidrug resistance is linked with ABC transporter overexpression. In the present study, we performed rare mutation analyses for 12 ABC transporters related to drug resistance (ABCA2, -A3, -B1, -B2, -B5, -C1, -C2, -C3, -C4, -C5, -C6, -G2) in a dataset of 18 cancer patients. We focused on rare mutations resembling tumor heterogeneity of ABC transporters in small tumor subpopulations. Novel rare mutations were found in ABCC1, but not in the other ABC transporters investigated. Diverse ABCC1 mutations were found, including nonsense mutations causing premature stop codons, and compared with the wild-type protein in terms of their protein structure. Nonsense mutations lead to truncated protein structures. Molecular docking and heat map analyses of ABCC1/MRP1 pointed out that Lys498* appeared in a separate cluster branch due to the large deletion, leading to a massive disruption in the protein conformation. The resulting proteins, which are nonfunctional due to nonsense mutations in tumors, offer a promising chemotherapy strategy since tumors with nonsense mutations may be more sensitive to anticancer drugs than wild-type ABCC1-expressing tumors. This could provide a novel tumor-specific toxicity strategy and a way to overcome drug resistance. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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17 pages, 2101 KiB

Open AccessFeature PaperArticle

A Machine Learning-Based Prediction Platform for P-Glycoprotein Modulators and Its Validation by Molecular Docking

by Onat Kadioglu and Thomas Efferth

Cells 2019, 8(10), 1286; https://doi.org/10.3390/cells8101286 - 21 Oct 2019

Cited by 21 | Viewed by 4388

Abstract

P-glycoprotein (P-gp) is an important determinant of multidrug resistance (MDR) because its overexpression is associated with increased efflux of various established chemotherapy drugs in many clinically resistant and refractory tumors. This leads to insufficient therapeutic targeting of tumor populations, representing a major drawback [...] Read more.

P-glycoprotein (P-gp) is an important determinant of multidrug resistance (MDR) because its overexpression is associated with increased efflux of various established chemotherapy drugs in many clinically resistant and refractory tumors. This leads to insufficient therapeutic targeting of tumor populations, representing a major drawback of cancer chemotherapy. Therefore, P-gp is a target for pharmacological inhibitors to overcome MDR. In the present study, we utilized machine learning strategies to establish a model for P-gp modulators to predict whether a given compound would behave as substrate or inhibitor of P-gp. Random forest feature selection algorithm-based leave-one-out random sampling was used. Testing the model with an external validation set revealed high performance scores. A P-gp modulator list of compounds from the ChEMBL database was used to test the performance, and predictions from both substrate and inhibitor classes were selected for the last step of validation with molecular docking. Predicted substrates revealed similar docking poses than that of doxorubicin, and predicted inhibitors revealed similar docking poses than that of the known P-gp inhibitor elacridar, implying the validity of the predictions. We conclude that the machine-learning approach introduced in this investigation may serve as a tool for the rapid detection of P-gp substrates and inhibitors in large chemical libraries. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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12 pages, 2175 KiB

Open AccessArticle

Impact of Q141K on the Transport of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors by ABCG2

by Yutaka Inoue, Takashi Morita, Mari Onozuka, Ken-ichi Saito, Kazumi Sano, Kazuhiko Hanada, Masami Kondo, Yoichi Nakamura, Tohru Kishino, Hiroshi Nakagawa and Yoji Ikegami

Cells 2019, 8(7), 763; https://doi.org/10.3390/cells8070763 - 23 Jul 2019

Cited by 6 | Viewed by 4209

Abstract

The ATP-binding cassette transporter ABCG2 is expressed in various organs, such as the small intestine, liver, and kidney, and influences the pharmacokinetics of drugs that are its substrates. ABCG2 is also expressed by cancer cells and mediates resistance to anticancer agents by promoting [...] Read more.

The ATP-binding cassette transporter ABCG2 is expressed in various organs, such as the small intestine, liver, and kidney, and influences the pharmacokinetics of drugs that are its substrates. ABCG2 is also expressed by cancer cells and mediates resistance to anticancer agents by promoting the efflux of these drugs. In the present study, we investigated the interactions between epidermal growth factor receptor tyrosine kinase inhibitors and ABCG2 by MTT assay, intracellular drug accumulation assay, and FACS. This study showed that four epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) (gefitinib, erlotinib, lapatinib, and afatinib) were transported from tumor cells as substrates of ABCG2. Q141K is a common single-nucleotide polymorphism of ABCG2 in Asians. We demonstrated that the extracellular efflux of gefitinib, erlotinib, and lapatinib was reduced by Q141K, whereas afatinib transport was not affected. In addition, all four EGFR TKIs inhibited the transport of other substrates by both wild-type and variant ABCG2 at 0.1 μM concentrations. Accordingly, epidermal growth factor receptor tyrosine kinase inhibitors may induce interactions with other drugs that are substrates of ABCG2, and single-nucleotide polymorphisms of ABCG2 may influence both the pharmacokinetics and efficacy of these anticancer agents. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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24 pages, 4968 KiB

Open AccessArticle

Functionally Significant Features in the 5′ Untranslated Region of the ABCA1 Gene and Their Comparison in Vertebrates

by Pavel Dvorak, Sarah Leupen and Pavel Soucek

Cells 2019, 8(6), 623; https://doi.org/10.3390/cells8060623 - 21 Jun 2019

Cited by 8 | Viewed by 4690

Abstract

Single nucleotide polymorphisms located in 5′ untranslated regions (5′UTRs) can regulate gene expression and have clinical impact. Recognition of functionally significant sequences within 5′UTRs is crucial in next-generation sequencing applications. Furthermore, information about the behavior of 5′UTRs during gene evolution is scarce. Using [...] Read more.

Single nucleotide polymorphisms located in 5′ untranslated regions (5′UTRs) can regulate gene expression and have clinical impact. Recognition of functionally significant sequences within 5′UTRs is crucial in next-generation sequencing applications. Furthermore, information about the behavior of 5′UTRs during gene evolution is scarce. Using the example of the ATP-binding cassette transporter A1 (ABCA1) gene (Tangier disease), we describe our algorithm for functionally significant sequence finding. 5′UTR features (upstream start and stop codons, open reading frames (ORFs), GC content, motifs, and secondary structures) were studied using freely available bioinformatics tools in 55 vertebrate orthologous genes obtained from Ensembl and UCSC. The most conserved sequences were suggested as hot spots. Exon and intron enhancers and silencers (sc35, ighg2 cgamma2, ctnt, gh-1, and fibronectin eda exon), transcription factors (TFIIA, TATA, NFAT1, NFAT4, and HOXA13), some of them cancer related, and microRNA (hsa-miR-4474-3p) were localized to these regions. An upstream ORF, overlapping with the main ORF in primates and possibly coding for a small bioactive peptide, was also detected. Moreover, we showed several features of 5′UTRs, such as GC content variation, hairpin structure conservation or 5′UTR segmentation, which are interesting from a phylogenetic point of view and can stimulate further evolutionary oriented research. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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21 pages, 4266 KiB

Open AccessArticle

TNF-α Modulates P-Glycoprotein Expression and Contributes to Cellular Proliferation via Extracellular Vesicles

by Tandressa S. Berguetti, Lucas S. P. Quintaes, Thais Hancio Pereira, Marcela C. Robaina, André L. S. Cruz, Raquel C. Maia and Paloma Silva de Souza

Cells 2019, 8(5), 500; https://doi.org/10.3390/cells8050500 - 24 May 2019

Cited by 14 | Viewed by 5820

Abstract

P-glycoprotein (Pgp/ABCB1) overexpression is associated with multidrug resistance (MDR) phenotype and, consequently, failure in cancer chemotherapy. However, molecules involved in cell death deregulation may also support MDR. Tumor necrosis factor-alpha (TNF-α) is an important cytokine that may trigger either death or tumor growth. [...] Read more.

P-glycoprotein (Pgp/ABCB1) overexpression is associated with multidrug resistance (MDR) phenotype and, consequently, failure in cancer chemotherapy. However, molecules involved in cell death deregulation may also support MDR. Tumor necrosis factor-alpha (TNF-α) is an important cytokine that may trigger either death or tumor growth. Here, we examined the role of cancer cells in self-maintenance and promotion of cellular malignancy through the transport of Pgp and TNF-α molecules by extracellular vesicles (membrane microparticles (MP)). By using a classical MDR model in vitro, we identified a positive correlation between endogenous TNF-α and Pgp, which possibly favored a non-cytotoxic effect of recombinant TNF-α (rTNF-α). We also found a positive feedback involving rTNF-α incubation and TNF-α regulation. On the other hand, rTNF-α induced a reduction in Pgp expression levels and contributed to a reduced Pgp efflux function. Our results also showed that parental and MDR cells spontaneously released MP containing endogenous TNF-α and Pgp. However, these MP were unable to transfer their content to non-cancer recipient cells. Nevertheless, MP released from parental and MDR cells elevated the proliferation index of non-tumor cells. Collectively, our results suggest that Pgp and endogenous TNF-α positively regulate cancer cell malignancy and contribute to changes in normal cell behavior through MP. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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18 pages, 1891 KiB

Open AccessArticle

Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort

by Yu Toyoda, Andrea Mančíková, Vladimír Krylov, Keito Morimoto, Kateřina Pavelcová, Jana Bohatá, Karel Pavelka, Markéta Pavlíková, Hiroshi Suzuki, Hirotaka Matsuo, Tappei Takada and Blanka Stiburkova

Cells 2019, 8(4), 363; https://doi.org/10.3390/cells8040363 - 18 Apr 2019

Cited by 44 | Viewed by 6916

Abstract

ATP-binding cassette subfamily G member 2 (ABCG2) is a physiologically important urate transporter. Accumulating evidence demonstrates that congenital dysfunction of ABCG2 is an important genetic risk factor in gout and hyperuricemia; recent studies suggest the clinical significance of both common and rare variants [...] Read more.

ATP-binding cassette subfamily G member 2 (ABCG2) is a physiologically important urate transporter. Accumulating evidence demonstrates that congenital dysfunction of ABCG2 is an important genetic risk factor in gout and hyperuricemia; recent studies suggest the clinical significance of both common and rare variants of ABCG2. However, the effects of rare variants of ABCG2 on the risk of such diseases are not fully understood. Here, using a cohort of 250 Czech individuals of European descent (68 primary hyperuricemia patients and 182 primary gout patients), we examined exonic non-synonymous variants of ABCG2. Based on the results of direct sequencing and database information, we experimentally characterized nine rare variants of ABCG2: R147W (rs372192400), T153M (rs753759474), F373C (rs752626614), T421A (rs199854112), T434M (rs769734146), S476P (not annotated), S572R (rs200894058), D620N (rs34783571), and a three-base deletion K360del (rs750972998). Functional analyses of these rare variants revealed a deficiency in the plasma membrane localization of R147W and S572R, lower levels of cellular proteins of T153M and F373C, and null urate uptake function of T434M and S476P. Accordingly, we newly identified six rare variants of ABCG2 that showed lower or null function. Our findings contribute to deepening the understanding of ABCG2-related gout/hyperuricemia risk and the biochemical characteristics of the ABCG2 protein. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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11 pages, 1588 KiB

Open AccessArticle

A Human ABC Transporter ABCC4 Gene SNP (rs11568658, 559 G > T, G187W) Reduces ABCC4-Dependent Drug Resistance

by Megumi Tsukamoto, Miho Yamashita, Tsuyoshi Nishi and Hiroshi Nakagawa

Cells 2019, 8(1), 39; https://doi.org/10.3390/cells8010039 - 10 Jan 2019

Cited by 16 | Viewed by 3693

Abstract

Broad-spectrum drug resistance is a major obstacle in cancer treatment, which is often caused by overexpression of ABC transporters the levels of which vary between individuals due to single-nucleotide polymorphisms (SNPs) in their genes. In the present study, we focused on the human [...] Read more.

Broad-spectrum drug resistance is a major obstacle in cancer treatment, which is often caused by overexpression of ABC transporters the levels of which vary between individuals due to single-nucleotide polymorphisms (SNPs) in their genes. In the present study, we focused on the human ABC transporter ABCC4 and one major non-synonymous SNP variant of the ABCC4 gene in the Japanese population (rs11568658, 559 G > T, G187W) whose allele frequency is 12.5%. Cells expressing ABCC4 (G187W) were established using the Flp-In™ system based on Flp recombinase-mediated transfection to quantitatively evaluate the impacts of this non-synonymous SNP on drug resistance profiles of the cells. Cells expressing ABCC4 (WT) or (G187W) showed comparable ABCC4 mRNA levels. 3-(4,5-Dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay indicated that the EC₅₀ value of the anticancer drug, SN-38, against cells expressing ABCC4 (G187W) was 1.84-fold lower than that against cells expressing ABCC4 (WT). Both azathioprine and 6-mercaptopurine showed comparable EC₅₀ values against cells expressing ABCC4 (G187W) and those expressing ABCC4 (WT). These results indicate that the substitution of Gly at position 187 of ABCC4 to Trp resulted in reduced SN-38 resistance. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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Review

Jump to: Research

15 pages, 1555 KiB

Open AccessReview

Cellular Processing of the ABCG2 Transporter—Potential Effects on Gout and Drug Metabolism

by Orsolya Mózner, Zsuzsa Bartos, Boglárka Zámbó, László Homolya, Tamás Hegedűs and Balázs Sarkadi

Cells 2019, 8(10), 1215; https://doi.org/10.3390/cells8101215 - 08 Oct 2019

Cited by 18 | Viewed by 5401

Abstract

The human ABCG2 is an important plasma membrane multidrug transporter, involved in uric acid secretion, modulation of absorption of drugs, and in drug resistance of cancer cells. Variants of the ABCG2 transporter, affecting cellular processing and trafficking, have been shown to cause gout [...] Read more.

The human ABCG2 is an important plasma membrane multidrug transporter, involved in uric acid secretion, modulation of absorption of drugs, and in drug resistance of cancer cells. Variants of the ABCG2 transporter, affecting cellular processing and trafficking, have been shown to cause gout and increased drug toxicity. In this paper, we overview the key cellular pathways involved in the processing and trafficking of large membrane proteins, focusing on ABC transporters. We discuss the information available for disease-causing polymorphic variants and selected mutations of ABCG2, causing increased degradation and impaired travelling of the transporter to the plasma membrane. In addition, we provide a detailed in silico analysis of an as yet unrecognized loop region of the ABCG2 protein, in which a recently discovered mutation may actually promote ABCG2 membrane expression. We suggest that post-translational modifications in this unstructured loop at the cytoplasmic surface of the protein may have special influence on ABCG2 processing and trafficking. Full article

(This article belongs to the Special Issue ABC Transporters: From Basic Functions to Diseases)

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