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1.
Sci Rep ; 14(1): 17334, 2024 Jul 27.
Article in English | MEDLINE | ID: mdl-39068198

ABSTRACT

3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for mechanistic liver homeostasis studies and in vitro to in vivo extrapolation (IVIVE) in drug discovery. However, the kinetics and regulation of drug transporters have not yet been assessed in 3D PHH. Here, we used organic cation transporter 1 (OCT1/SLC22A1) as a model to study both transport kinetics and the long-term regulation of transporter activity via relevant signalling pathways. The kinetics of the OCT1 transporter was studied using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, 3D PHH were treated with xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomic analysis was used to track hepatic phenotypes as well as prototypical changes in other regulated proteins, such as P-glycoprotein and Cytochrome P450 3A4. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14 ± 4.0µM as the mean from three donors. Co-incubation with known OCT1 inhibitors decreased the uptake of ASP+ in the 3D PHH spheroids by 35-52%. The long-term exposure studies showed that OCT1 is relatively stable upon activation of nuclear receptor signalling or exposure to compounds that could induce inflammation, steatosis or liver injury. Our results demonstrate that 3D PHH spheroids express physiologically relevant levels of fully active OCT1 and that its transporter kinetics can be accurately studied in the 3D PHH configuration. We also confirm that OCT1 remains stable and functional during the activation of key metabolic pathways that alter the expression and function of other drug transporters and drug-metabolizing enzymes. These results will expand the range of studies that can be performed using 3D PHH.


Subject(s)
Hepatocytes , Organic Cation Transporter 1 , Spheroids, Cellular , Humans , Hepatocytes/metabolism , Spheroids, Cellular/metabolism , Organic Cation Transporter 1/metabolism , Organic Cation Transporter 1/genetics , Kinetics , Proteomics/methods , Cells, Cultured , Octamer Transcription Factor-1/metabolism , Octamer Transcription Factor-1/genetics , Signal Transduction
2.
Pharmacol Res Perspect ; 12(4): e1238, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38988092

ABSTRACT

Fostemsavir is an approved gp120-directed attachment inhibitor and prodrug for the treatment of human immunodeficiency virus type 1 infection in combination with other antiretrovirals (ARVs) in heavily treatment-experienced adults with multi-drug resistance, intolerance, or safety concerns with their current ARV regimen. Initial in vitro studies indicated that temsavir, the active moiety of fostemsavir, and its metabolites, inhibited organic cation transporter (OCT)1, OCT2, and multidrug and toxin extrusion transporters (MATEs) at tested concentration of 100 uM, although risk assessment based on the current Food and Drug Administration in vitro drug-drug interaction (DDI) guidance using the mechanistic static model did not reveal any clinically relevant inhibition on OCTs and MATEs. However, a DDI risk was flagged with EMA static model predictions. Hence, a physiologically based pharmacokinetic (PBPK) model of fostemsavir/temsavir was developed to further assess the DDI risk potential of OCT and MATEs inhibition by temsavir and predict changes in metformin (a sensitive OCT and MATEs substrate) exposure. No clinically relevant impact on metformin concentrations across a wide range of temsavir concentrations was predicted; therefore, no dose adjustment is recommended for metformin when co-administered with fostemsavir.


Subject(s)
Drug Interactions , Metformin , Organic Cation Transport Proteins , Organic Cation Transporter 2 , Organophosphates , Metformin/pharmacokinetics , Metformin/administration & dosage , Humans , Organic Cation Transport Proteins/metabolism , Organic Cation Transport Proteins/antagonists & inhibitors , Organic Cation Transporter 2/metabolism , Organophosphates/administration & dosage , Organophosphates/pharmacokinetics , Models, Biological , Animals , Organic Cation Transporter 1/metabolism , Anti-HIV Agents/administration & dosage , Anti-HIV Agents/pharmacokinetics , Octamer Transcription Factor-1/metabolism , HIV Infections/drug therapy , HIV Infections/metabolism , Piperazines
3.
Mol Cell ; 84(10): 1932-1947.e10, 2024 May 16.
Article in English | MEDLINE | ID: mdl-38703769

ABSTRACT

Mutations in transporters can impact an individual's response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.


Subject(s)
Molecular Dynamics Simulation , Humans , HEK293 Cells , Structure-Activity Relationship , Mutation, Missense , Pharmacogenetics , Phenotype , Organic Cation Transporter 1/genetics , Organic Cation Transporter 1/metabolism , Mutation , Protein Conformation , Biological Transport , Octamer Transcription Factor-1
4.
BMC Cancer ; 24(1): 554, 2024 May 02.
Article in English | MEDLINE | ID: mdl-38698344

ABSTRACT

BACKGROUND: Prostate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation. METHODS: In this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database. RESULTS: Genome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC. CONCLUSIONS: Our findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management.


Subject(s)
Alcohol Oxidoreductases , Co-Repressor Proteins , Gene Expression Regulation, Neoplastic , Octamer Transcription Factor-1 , Prostatic Neoplasms, Castration-Resistant , Receptors, Androgen , Male , Humans , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/pathology , Prostatic Neoplasms, Castration-Resistant/metabolism , Receptors, Androgen/metabolism , Receptors, Androgen/genetics , Mice , Animals , Octamer Transcription Factor-1/metabolism , Octamer Transcription Factor-1/genetics , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Up-Regulation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Xenograft Model Antitumor Assays , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Tumor Microenvironment , Signal Transduction
5.
Commun Biol ; 7(1): 185, 2024 Feb 15.
Article in English | MEDLINE | ID: mdl-38360857

ABSTRACT

Humoral immunity is vital for host protection, yet aberrant antibody responses can trigger harmful inflammation and immune-related disorders. T follicular helper (Tfh) cells, central to humoral immunity, have garnered significant attention for unraveling immune mechanisms. This study shows the role of B-cell Oct-binding protein 1 (Bob1), a transcriptional coactivator, in Tfh cell regulation. Our investigation, utilizing conditional Bob1-deficient mice, suggests that Bob1 plays a critical role in modulating inducible T-cell costimulator expression and cellular respiration in Tfh cells. This regulation maintains the long-term functionality of Tfh cells, enabling their reactivation from central memory T cells to produce antibodies during recall responses. In a bronchial asthma model induced by house dust mite (HDM) inhalation, Bob1 is observed to enhance HDM-specific antibodies, including IgE, highlighting its pivotal function in Tfh cell regulation. Further exploration of Bob1-dependent mechanisms in Tfh cells holds promise for governing protective immunity and addressing immune-related disorders.


Subject(s)
Immunity, Humoral , Octamer Transcription Factor-1 , T Follicular Helper Cells , Animals , Mice , Antibody Formation , T Follicular Helper Cells/immunology , T-Lymphocytes, Helper-Inducer/immunology , Octamer Transcription Factor-1/genetics , Octamer Transcription Factor-1/metabolism
6.
Int J Biol Sci ; 20(4): 1125-1141, 2024.
Article in English | MEDLINE | ID: mdl-38385081

ABSTRACT

Previous studies have demonstrated that diallyl disulfide (DADS) exhibits potent anti-tumor activity. However, the pharmacological actions of DADS in inhibiting the growth of colorectal cancer (CRC) cells have not been clarified. Herein, we show that DADS treatment impairs the activation of the pentose phosphate pathway (PPP) to decrease PRPP (5-phosphate ribose-1-pyrophosphate) production, enhancing DNA damage and cell apoptosis, and inhibiting the growth of CRC cells. Mechanistically, DADS treatment promoted POU2F1 K48-linked ubiquitination and degradation by attenuating the PI3K/AKT signaling to up-regulate TRIM21 expression in CRC cells. Evidently, TRIM21 interacted with POU2F1, and induced the K272 ubiquitination of POU2F1. The effects of DADS on the enhanced K272 ubiquitination of POU2F1, the PPP flux, PRPP production, DNA damage and cell apoptosis as well as the growth of CRC tumors in vivo were significantly mitigated by TRIM21 silencing or activating the PI3K signaling in CRC cells. Conversely, the effects of DADS were enhanced by TRIM21 over-expression or inhibiting the PI3K/AKT signaling in CRC cells. Collectively, our findings reveal a novel mechanism by which DADS suppresses the growth of CRC by promoting POU2F1 ubiquitination, and may aid in design of novel therapeutic intervention of CRC.


Subject(s)
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/analogs & derivatives , Allyl Compounds , Colorectal Neoplasms , Disulfides , Humans , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Apoptosis/genetics , Allyl Compounds/pharmacology , Allyl Compounds/therapeutic use , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , DNA Damage , Octamer Transcription Factor-1/genetics
7.
Biomolecules ; 14(1)2024 Jan 17.
Article in English | MEDLINE | ID: mdl-38254723

ABSTRACT

BOB1, a mammalian lymphocyte-specific transcriptional coactivator of the transcription factors OCT1 and OCT2 (OCT1/2), plays important roles in normal immune responses, autoimmunity, and hematologic malignancies. The issue of a DNA sequence preference change imposed by BOB1 was raised more than two decades ago but remains unresolved. In this paper, using the EMSA-SELEX-Seq approach, we have reassessed the intrinsic ability of BOB1 to modulate the specificity of DNA recognition by OCT1 and OCT2. Our results have reaffirmed previous conclusions regarding BOB1 selectivity towards the dimer configuration of OCT1/2. However, they suggest that the monomeric configuration of these factors, assembled on the classical octamer ATGCAAAT and related motifs, are the primary targets of BOB1. Our data further specify the DNA sequence preference imposed by BOB1 and predict the probability of ternary complex formation. These results provide an additional insight into the action of BOB1-an essential immune regulator and a promising molecular target for the treatment of autoimmune diseases and hematologic malignancies.


Subject(s)
Autoimmune Diseases , Hematologic Neoplasms , POU Domain Factors , DNA , Mammals , POU Domain Factors/metabolism , Transcription Factors/genetics , Humans , Octamer Transcription Factor-1/metabolism , Octamer Transcription Factor-2/metabolism
8.
Neurochem Res ; 48(12): 3652-3664, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37592110

ABSTRACT

Evidence demonstrates that DNA methylation is associated with the occurrence and development of various neurological diseases. However, the potential target genes undergoing DNA methylation, as well as their involvement in the chemotherapy drug oxaliplatin-induced neuropathic pain, are still unclear. Here, Lrfn4, which showed hypermethylation in the promoter regions, was screened from the SRA methylation database (PRJNA587622) following oxaliplatin treatment. MeDIP and qPCR assays identified that oxaliplatin treatment increased the methylation in Lrfn4 promoter region and decreased the expression of LRFN4 in the spinal dorsal horn. The assays with gain and loss of LRFN4 function demonstrated that LRFN4 downregulation in spinal dorsal horn contributed to the oxaliplatin-induced mechanical allodynia and cold hyperalgesia. Moreover, oxaliplatin treatment increased the DNA methyltransferases DNMT3a expression and the interaction between DNMT3a and Lrfn4 promoter, while inhibition of DNMT3a prevented the downregulation of LRFN4a induced by oxaliplatin. We also observed that the transcriptional factor POU2F1 can bind to the predicted sites in DNMT3a promoter region, oxaliplatin treatment upregulated the expression of transcriptional factor POU2F1 in dorsal horn neurons. Intrathecal injection of POU2F1 siRNA prevented the DNMT3a upregulation and the LRFN4 downregulation induced by oxaliplatin. Additionally, intrathecal injection of DNMT3a siRNA or POU2F1 siRNA alleviated the mechanical allodynia induced by oxaliplatin. These findings suggested that transcription factor POU2F1 upregulated the expression of DNMT3a, which subsequently decreased LRFN4 expression through hypermethylation modification in spinal dorsal horn, thereby mediating neuropathic pain following oxaliplatin treatment.


Subject(s)
DNA Methylation , Neuralgia , Down-Regulation , Hyperalgesia/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Neuralgia/chemically induced , Neuralgia/drug therapy , Neuralgia/metabolism , Octamer Transcription Factor-1/metabolism , Oxaliplatin/adverse effects , RNA, Small Interfering/therapeutic use , Spinal Cord Dorsal Horn/metabolism , Animals , Rats
9.
Arterioscler Thromb Vasc Biol ; 43(8): 1429-1440, 2023 08.
Article in English | MEDLINE | ID: mdl-37381986

ABSTRACT

BACKGROUND: Increasing evidence suggests that superoxide ions produced by NOX (nicotinamide adenine dinucleotide phosphate oxidases) mediate vascular effects of Ang II (angiotensin II) evoked by atherogenic diets. Here, we analyzed the mechanism by which NOX2 contributes to Ang II-induced ET-1 (endothelin 1) production in human microvascular endothelial cells. METHODS: The effects of high-fat diet were compared between WT (wild type) and Nox2 (mouse NOX2 gene)-deficient mice. ET-1 production and NOX2 expression by human microvascular endothelial cells in vitro were analyzed by ELISA, reverse transcription quantitative polymerase chain reaction, electrophoretic mobility shift assay, promoter deletions, RNA interference, and pharmacological inhibition. Production of superoxide anions was visualized by fluorescent cell labeling. RESULTS: Feeding mice high-fat diet for 10 weeks increased cardiac expression and plasma levels of Ang II and ET-1 in WT but not in Nox2-deficient animals. Exposure of human microvascular endothelial cells to Ang II resulted in increased ET-1 production, which could be blocked by silencing NOX2 (human NOX2 gene). Ang II promoted NOX2 expression through induction of the Oct-1 (human/mouse octamer binding transcription factor 1 protein) and activation of the NOX2 promoter region containing Oct-1-binding sites. Stimulation of NOX2 expression by Ang II was associated with increased production of superoxide anions. Inhibition of Oct-1 by small interfering RNA reduced Ang II-induced NOX2 expression and superoxide anion production, and neutralization of superoxide by SOD (superoxide dismutase) abolished Ang II-stimulated ET1 (human ET-1 gene) promoter activity, ET1 mRNA expression, and ET-1 release. CONCLUSIONS: Ang II may promote ET-1 production in the endothelium in response to atherogenic diets through a mechanism that involves the transcription factor Oct-1 and the increased formation of superoxide anions by NOX2.


Subject(s)
Endothelial Cells , Superoxides , Mice , Animals , Humans , Superoxides/metabolism , Endothelial Cells/metabolism , Octamer Transcription Factor-1 , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Angiotensin II/pharmacology , Angiotensin II/metabolism , Reactive Oxygen Species/metabolism
10.
Exp Cell Res ; 420(1): 113335, 2022 11 01.
Article in English | MEDLINE | ID: mdl-36084669

ABSTRACT

Multitargeted kinase inhibitors (MKIs) including sorafenib and lenvatinib, are applied for first-line treatment for inoperable hepatocellular carcinoma (HCC) patients, but the therapeutic effect is limited because of drug resistance. Therefore, we sought potential biomarkers to indicate sorafenib and lenvatinib resistance in HCC. In this article, we report a novel long non-coding RNA (lncRNA), AC026401.3, in promoting sorafenib and lenvatinib resistance of HCC cells. AC026401.3 is upregulated in HCC tissues and is positively relevant to HCC patients with large tumor size, cancer recurrence, advanced TNM stage, and poor prognosis. AC026401.3 knockdown or knockout enhances the sensitivity of HCC cells to sorafenib and lenvatinib, respectively. Moreover, AC026401.3 upregulates the expression of the transcription factor E2F2. Mechanistically, AC026401.3 interacts with OCT1 and promotes the recruitment of OCT1 to the promoter region of E2F2, intensifying sorafenib and lenvatinib resistance in HCC by activating the transcription of E2F2. In conclusion, our results reveal that lncRNA AC026401.3 is a risk factor for HCC patients by enhancing sorafenib and lenvatinib resistance of HCC cells, and targeting the AC026401.3-OCT1-E2F2 signaling axis would be a promising strategy for HCC therapeutics.


Subject(s)
Antineoplastic Agents , Carcinoma, Hepatocellular , Liver Neoplasms , Octamer Transcription Factor-1/metabolism , RNA, Long Noncoding , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , E2F2 Transcription Factor , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Neoplasm Recurrence, Local/drug therapy , Phenylurea Compounds/pharmacology , Quinolines , RNA, Long Noncoding/genetics , Sorafenib/pharmacology , Sorafenib/therapeutic use , Transcription Factors
11.
Int J Mol Sci ; 23(15)2022 Jul 29.
Article in English | MEDLINE | ID: mdl-35955563

ABSTRACT

Many organic cations (OCs) may be transported through membranes by a genetically still uncharacterized proton-organic cation (H + OC) antiporter. Here, we characterized an extended substrate spectrum of this antiporter. We studied the uptake of 72 drugs in hCMEC/D3 cells as a model of the human blood-brain barrier. All 72 drugs were tested with exchange transport assays and the transport of 26 of the drugs was studied in more detail concerning concentration-dependent uptake and susceptibility to specific inhibitors. According to exchange transport assays, 37 (51%) drugs were good substrates of the H + OC antiporter. From 26 drugs characterized in more detail, 23 were consistently identified as substrates of the H + OC antiporter in six different assays and transport kinetic constants could be identified with intrinsic clearances between 0.2 (ephedrine) and 201 (imipramine) mL × minute-1 × g protein-1. Excellent substrates of the H + OC antiporter were no substrates of organic cation transporter OCT1 and vice versa. Good substrates of the H + OC antiporter were more hydrophobic and had a lower topological polar surface area than non-substrates or OCT1 substrates. These data and further research on the H + OC antiporter may result in a better understanding of pharmacokinetics, drug-drug interactions and variations in pharmacokinetics.


Subject(s)
Antiporters , Octamer Transcription Factor-1/metabolism , Organic Cation Transporter 1 , Antiporters/genetics , Antiporters/metabolism , Biological Transport , Brain/metabolism , Cations , Humans , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolism , Organic Cation Transporter 1/metabolism , Protons
12.
Int J Mol Sci ; 23(15)2022 Jul 30.
Article in English | MEDLINE | ID: mdl-35955590

ABSTRACT

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are associated with obesity. They are accompanied by increased levels of free cholesterol in the liver. Most free cholesterol resides within the plasma membrane. We assessed the impact of adding or removing free cholesterol on the function and localization of two hepatocellular uptake transporters: the Na+/taurocholate cotransporting polypeptide (NTCP) and the organic cation transporter 1 (OCT1). We used a cholesterol-MCD complex (cholesterol) to add cholesterol and methyl-ß-cyclodextrin (MCD) to remove cholesterol. Our results demonstrate that adding cholesterol decreases NTCP capacity from 132 ± 20 to 69 ± 37 µL/mg/min and OCT1 capacity from 209 ± 66 to 125 ± 26 µL/mg/min. Removing cholesterol increased NTCP and OCT1 capacity to 224 ± 65 and 279 ± 20 µL/mg/min, respectively. In addition, adding cholesterol increased the localization of NTCP within lipid rafts, while adding or removing cholesterol increased OCT1 localization in lipid rafts. These results demonstrate that increased cholesterol levels can impair NTCP and OCT1 function, suggesting that the free cholesterol content of the liver can alter bile acid and drug uptake into the liver. This could explain the increased plasma bile acid levels in NAFLD and NASH patients and potentially lead to altered drug disposition.


Subject(s)
Non-alcoholic Fatty Liver Disease , Octamer Transcription Factor-1/metabolism , Symporters , Bile Acids and Salts/metabolism , Cholesterol/metabolism , Humans , Liver/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Organic Anion Transporters, Sodium-Dependent/metabolism , Organic Cation Transporter 1/metabolism , Peptides/metabolism , Symporters/metabolism , Taurocholic Acid
13.
Mol Biol (Mosk) ; 56(4): 595-603, 2022.
Article in Russian | MEDLINE | ID: mdl-35964316

ABSTRACT

Increased expression levels of the Oct-1 transcription factor is considered to be one of the key markers of poor cancer prognosis. In addition to the ubiquitous Oct-1A isoform, which is found in all cells, there also exists a tissue-specific Oct-1L isoform, which is expressed in hematopoietic cells. Oct-1L increases cell resistance to different stresses and also regulates the expression of genes controlling differentiation of hematopoietic and immune system cells. The tissue-specific Oct-1L isoform levels are significantly increased in the B-cell lymphoblastoma Namalwa and Raji lines and the T-cell lymphoblastoma Jurkat line compared to normal B and T cells. Apparently, aberrant Oct-1L overexpression not only enhances stress resistance but also leads to the disruption of developmental pathways in the cells promoting their malignant transformation. We report here that targeted suppression of the tissue-specific Oct-1L isoform expression reduces the proliferation rate of Namalwa B-lymphoblastic Burkitt's lymphoma cells, significantly increases cell death rate under hypoxic conditions, and makes cells more sensitive to chemotherapeutic agents such as docetaxel and doxorubicin. These results indicate that targeted therapy aimed at the suppression of the Oct-1 isoforms with increased expression levels in tumor cells rather than the total Oct-1, thus avoiding the traumatic effects of total Oct-1 knockdown, may be promising. Selective suppression of Oct-1 isoforms is a promising strategy in the treatment of lymphoid tumors and may contribute to mitigating the disease course and increasing survival rates in cancer patients.


Subject(s)
Antineoplastic Agents , Burkitt Lymphoma , Octamer Transcription Factor-1/metabolism , Antineoplastic Agents/pharmacology , Burkitt Lymphoma/drug therapy , Burkitt Lymphoma/genetics , Burkitt Lymphoma/metabolism , Humans , Protein Isoforms/genetics , T-Lymphocytes/metabolism
14.
Yonsei Med J ; 63(6): 591-600, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35619584

ABSTRACT

PURPOSE: SOX12 is overexpressed in many cancers, and we aimed to explore the biological function and mechanism of SOX12 in thyroid cancer. MATERIALS AND METHODS: We first analyzed the expression of SOX12 in thyroid cancer using data in The Cancer Genome Atlas. Immunohistochemistry and qRT-PCR were performed to identify SOX12 expression in thyroid cancer tissue and cells. Thyroid cancer cells were transfected with small interfering RNA targeting SOX12, and cellular functional experiments, including CCK8, wound healing, and Transwell assays, were performed. Protein expression was examined by Western blot analysis. A xenograft model was developed to evaluate the effect of SOX12 on tumor growth in vivo. RESULTS: SOX12 expression was increased in thyroid cancer tissue and cells. SOX12 promoted cell proliferation, migration, and invasion and accelerated tumor growth in vivo. The expression of PCNA, Cyclin D1, E-cadherin, Snail, MMP-2, and MMP-9 was affected by SOX12 knockdown. Bioinformatic analysis showed that SOX12 could interact with the POU family. SOX12 knockdown inhibited the expression of POU2F1, POU2F2, POU3F1 and POU3F2, and SOX12 expression showed a positive correlation with POU2F1, POU3F1, and POU3F2 expression in clinical data. POU2F1 and POU3F1 were able to reverse the effect of SOX12 knockdown on thyroid cancer cells. CONCLUSION: SOX12 affects the progression of thyroid cancer by regulating epithelial-mesenchymal transition and interacting with POU2F1 and POU3F1, which may be novel targets for thyroid cancer molecular therapy.


Subject(s)
SOXC Transcription Factors , Thyroid Neoplasms , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Humans , Octamer Transcription Factor-1/genetics , Octamer Transcription Factor-1/metabolism , SOXC Transcription Factors/genetics , Thyroid Neoplasms/genetics
15.
Dokl Biochem Biophys ; 503(1): 76-79, 2022 Apr.
Article in English | MEDLINE | ID: mdl-35538282

ABSTRACT

Overexpression of the transcription factor POU2F1 (Oct-1) increases the malignant potential of the tumor and determines the unfavorable prognosis for both solid and hematological cases of the disease in human carcinogenesis. The Oct-1 level determines the rate of development of the disease in acute myelodysplastic leukemia (AML), and a decrease in its expression significantly delays the development of leukemia in mice; however, a complete knockout of Oct-1 leads to the death of the animals. POU2F1 (Oct-1) is expressed as several isoforms transcribed from alternative promoters. They include both ubiquitous and tissue-specific isoforms. It was shown that in Burkitt's lymphoma Namalwa cells 5-azacytidine specifically suppresses the expression of the tissue-specific isoform Oct-1L mRNA (level of Oct-1L is abnormally increased in these cells), while not causing changes in the amount of the ubiquitous isoform Oct-1A mRNA. These results show that it is possible to selectively reduce the transcription level of the Oct-1L isoform aberrantly expressed in human tumor cells.


Subject(s)
Azacitidine , Burkitt Lymphoma , Leukemia , Octamer Transcription Factor-1 , Animals , Azacitidine/pharmacology , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , Cell Culture Techniques , Mice , Octamer Transcription Factor-1/antagonists & inhibitors , Octamer Transcription Factor-1/genetics , Octamer Transcription Factor-1/metabolism , Protein Isoforms , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Cells, Cultured
16.
J Transl Med ; 20(1): 220, 2022 05 13.
Article in English | MEDLINE | ID: mdl-35562740

ABSTRACT

BACKGROUND: An increasing number of studies have demonstrated that long non-coding RNAs (lncRNAs) serve as key regulators in tumor development and progression. However, only a few lncRNAs have been functionally characterized in gastric cancer (GC). METHODS: Bioinformatics analysis was conducted to find lncRNAs that are associated with GC metastasis. RNA FISH, RIP, and RNA pull down assays were used to study the complementary binding of LINC01564 complementary to the 3'UTR of transcription factor POU2F1. The transcription activation of LINC01564 by POU2F1 as a transcription factor was examined by ChIP assay. In vitro assays such as MTT, cell invasion assay, and clonogenic assay were conducted to examined the impacts of LINC01564 and POU2F1 on GC cell proliferation and invasion. Experiments in vivo were performed to access the impacts of LINC01564 and POU2F1 on GC metastasis. RESULTS: The results showed that LINC01564 complementary bound to the 3'UTR of POU2F1 to form an RNA duplex, whereby stabilizing POU2F1 mRNA and increasing the enrichment in cells. The level of LINC01564 was also increased by POU2F1 through transcription activation. In vitro assays showed that LINC01564 promoted the proliferation, invasion and migration of GC cells through increasing POU2F1. In vivo experiments indicate the promotion of GC proliferation and metastasis by the interaction between LINC01564 and POU2F1. CONCLUSION: Taken together, our results indicate that the interaction between LINC01564 and POU2F1 promotes the proliferation, migration and invasion of GC cells.


Subject(s)
MicroRNAs , RNA, Long Noncoding , Stomach Neoplasms , 3' Untranslated Regions , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , Octamer Transcription Factor-1/genetics , Octamer Transcription Factor-1/metabolism , RNA, Long Noncoding/genetics , Stomach Neoplasms/pathology , Transcription Factors/metabolism
17.
Sci Rep ; 12(1): 6094, 2022 04 12.
Article in English | MEDLINE | ID: mdl-35413990

ABSTRACT

Androgen and androgen receptor (AR) targeted therapies are the main treatment for most prostate cancer (PC) patients. Although AR signaling inhibitors are effective, tumors can evade this treatment by transforming to an AR-negative PC via lineage plasticity. OCT1 is a transcription factor interacting with the AR to enhance signaling pathways involved in PC progression, but its role in the emergence of the AR-negative PC is unknown. We performed chromatin immunoprecipitation sequencing (ChIP-seq) in patient-derived castration-resistant AR-negative PC cells to identify genes that are regulated by OCT1. Interestingly, a group of genes associated with neural precursor cell proliferation was significantly enriched. Then, we focused on neural genes STNB1 and PFN2 as OCT1-targets among them. Immunohistochemistry revealed that both STNB1 and PFN2 are highly expressed in human AR-negative PC tissues. Knockdown of SNTB1 and PFN2 by siRNAs significantly inhibited migration of AR-negative PC cells. Notably, knockdown of PFN2 showed a marked inhibitory effect on tumor growth in vivo. Thus, we identified OCT1-target genes in AR-negative PC using a patient-derived model, clinicopathologial analysis and an animal model.


Subject(s)
Prostatic Neoplasms , Receptors, Androgen , Androgens/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Humans , Male , Octamer Transcription Factor-1 , Profilins/genetics , Prostatic Neoplasms/pathology , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Signal Transduction
18.
J Virol ; 96(9): e0034922, 2022 05 11.
Article in English | MEDLINE | ID: mdl-35404085

ABSTRACT

Herpes simplex virus 2 (HSV-2) establishes latent infection in dorsal root ganglion (DRG) neurons after productive (lytic) infection in peripheral tissues. A neuron-specific microRNA, miR-138, favors HSV-1 latency by repressing viral ICP0 and host Oct-1 and Foxc1 genes, yet the role of miR-138 in HSV-2 infection was unknown. The ICP0 mRNAs of HSV-1, HSV-2, and chimpanzee herpesvirus each have one to two canonical miR-138 binding sites. The sites are 100% conserved in 308 HSV-1 and 300 HSV-2 published sequences of clinical isolates. In cotransfection assays, miR-138 repressed HSV-2 ICP0 expression through the seed region and surrounding interactions that are different from HSV-1. An HSV-2 mutant with disrupted miR-138 binding sites on ICP0 showed increased ICP0 expression in Neuro-2a cells. Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation confirmed miR-138 binding to HSV-2 ICP0 and identified UL19 and UL20 as additional targets whose expression was repressed by miR-138 during cotransfection. In Neuro-2a cells, transfected miR-138 and its antagomir decreased and increased HSV-2 replication, respectively, and a knockout experiment showed that miR-138's host targets OCT-1 and FOXC1 were important for HSV-2 replication. In primary mouse DRG neurons, both ICP0 and FOXC1 positively regulated HSV-2 replication, but both overexpressed and endogenous miR-138 suppressed HSV-2 replication primarily by repressing ICP0 expression. Thus, miR-138 can suppress HSV-2 neuronal replication through multiple viral and host pathways. These results reveal functional similarities and mechanistic differences in how miR-138 regulates HSV-1 and HSV-2 infection and indicate an evolutionary advantage of using miR-138 to repress lytic infection in neurons. IMPORTANCE HSV-1 and HSV-2 are closely related viruses with major differences. Both viruses establish latency in neurons from which they reactivate to cause disease. A key aspect of HSV latency is repression of productive infection in neurons. Based on previous work with HSV-1, we investigated the role of a neuron-specific microRNA, miR-138, in HSV-2 infection and established it as a repressor of HSV-2 productive infection in neuronal cells. This repression is mediated mainly by targeting viral ICP0 and host Foxc1 mRNAs, but other pathways also contribute. Despite functional conservation of the role of miR-138 between HSV-1 and HSV-2, many molecular mechanisms differ, including how miR-138 represses ICP0 expression and miR-138 targeting of HSV-2 but not HSV-1 UL19 and UL20. To our knowledge, this study provides the first example of host microRNA regulation of HSV-2 infection.


Subject(s)
Herpes Simplex , Herpesvirus 2, Human , MicroRNAs , Neurons , Animals , Forkhead Transcription Factors , Gene Expression Regulation, Viral , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/physiology , Herpesvirus 2, Human/genetics , Herpesvirus 2, Human/physiology , Immediate-Early Proteins/metabolism , Mice , MicroRNAs/genetics , Neurons/virology , Octamer Transcription Factor-1 , Ubiquitin-Protein Ligases/metabolism , Virus Latency/genetics , Virus Replication
19.
Genomics ; 114(2): 110314, 2022 03.
Article in English | MEDLINE | ID: mdl-35167937

ABSTRACT

Farnesyltransferase (FTase) enables about 100 proteins to interact with cellular membranes by catalyzing the posttranslational addition of a farnesyl group. Farnesylated proteins provide important functions and inhibitors against the ß-subunit of the heterodimer of FTase are intensively studied in clinical and preclinical trials. However, very little is known about the transcriptional regulation of the ß-subunit. The examined promoter region of the human FTase ß-subunit gene (FNTB) showed significant basal promoter activity in HEK-293 and in HeLa cells. We were able to locate the core promoter at -165 to -74. Ten potential binding sites of the transcription factor OCT-1 were detected. Three could be confirmed using EMSA super shift experiments. OCT-1 overexpression and knockdown confirmed it as an important regulator of FNTB expression. Our results provide a basis for further research on FNTB/OCT-1 regulation, its inhibitors and diseases influenced by both such as colon carcinoma or diabetes mellitus.


Subject(s)
Alkyl and Aryl Transferases , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Farnesyltranstransferase/genetics , Farnesyltranstransferase/metabolism , HEK293 Cells , HeLa Cells , Humans , Octamer Transcription Factor-1/genetics , Octamer Transcription Factor-1/metabolism , Promoter Regions, Genetic
20.
Int J Mol Sci ; 23(4)2022 Feb 11.
Article in English | MEDLINE | ID: mdl-35216120

ABSTRACT

The organic cation transporter 1 (OCT1, SLC22A1) transports a large number of structurally diverse endogenous and exogenous substrates. There are numerous known competitive and non-competitive inhibitors of OCT1, but there are no studies systematically analyzing the relationship between transport, stimulation, and inhibition. Here, we tested in vitro OCT1 inhibition by OCT1 substrates and transport of OCT1 inhibitors under uniform analytical conditions. Beyond inhibition testing with two model substrates, we tested nine additional OCT1 substrates for their mutual inhibition. Inhibition of ASP+ uptake by most OCT1 substrates was weak. The model substrate sumatriptan, with its moderately stronger inhibitability, was used to confirm this. Interestingly, OCT1 substrates exhibiting stronger OCT1 inhibition were mainly biaromatic ß-agonistic drugs, such as dobutamine, fenoterol, ractopamine and ritodrine. Biaromatic organic cations were both, strong inhibitors and good substrates, but many OCT1 substrates showed little pairwise inhibition. Surprisingly, sumatriptan did significantly enhance dobutamine uptake. This effect was concentration dependent and additional experiments indicated that efflux inhibition may be one of the underlying mechanisms. Our data suggests, that OCT1 substrates are mainly weak OCT1 inhibitors and among those inhibiting well, noncompetitive inhibition could be responsible. Weak competitive inhibition confirms that OCT1 inhibition screenings poorly predict OCT1 substrates. Additionally, we showed that the OCT1 substrate sumatriptan can enhance uptake of some other OCT1 substrates. OCT1 transport stimulation was already observed earlier but is still poorly understood. Low OCT1 uptake inhibition and strong OCT1 efflux inhibition could be mechanisms exploitable for enhancing transport.


Subject(s)
Biological Transport/physiology , Octamer Transcription Factor-1/metabolism , Cell Line , HEK293 Cells , Humans
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