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1.
Pharmacol Rev ; 76(2): 300-320, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38351074

RESUMO

In humans, there are two arylamine N-acetyltransferase genes that encode functional enzymes (NAT1 and NAT2) as well as one pseudogene, all of which are located together on chromosome 8. Although they were first identified by their role in the acetylation of drugs and other xenobiotics, recent studies have shown strong associations for both enzymes in a variety of diseases, including cancer, cardiovascular disease, and diabetes. There is growing evidence that this association may be causal. Consistently, NAT1 and NAT2 are shown to be required for healthy mitochondria. This review discusses the current literature on the role of both NAT1 and NAT2 in mitochondrial bioenergetics. It will attempt to relate our understanding of the evolution of the two genes with biologic function and then present evidence that several major metabolic diseases are influenced by NAT1 and NAT2. Finally, it will discuss current and future approaches to inhibit or enhance NAT1 and NAT2 activity/expression using small-molecule drugs. SIGNIFICANCE STATEMENT: The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 share common features in their associations with mitochondrial bioenergetics. This review discusses mitochondrial function as it relates to health and disease, and the importance of NAT in mitochondrial function and dysfunction. It also compares NAT1 and NAT2 to highlight their functional similarities and differences. Both NAT1 and NAT2 are potential drug targets for diseases where mitochondrial dysfunction is a hallmark of onset and progression.


Assuntos
Arilamina N-Acetiltransferase , Doenças Metabólicas , Doenças Mitocondriais , Humanos , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Especificidade por Substrato , Doenças Metabólicas/tratamento farmacológico , Doenças Mitocondriais/tratamento farmacológico
2.
Nanomedicine ; 56: 102733, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38199450

RESUMO

Anti-cancer monoclonal antibodies often fail to provide therapeutic benefit in receptor-positive patients due to rapid endocytosis of antibody-bound cell surface receptors. High dose co-administration of prochlorperazine (PCZ) inhibits endocytosis and sensitises tumours to mAbs by inhibiting dynamin II but can also introduce neurological side effects. We examined the potential to use PEGylated liposomal formulations of PCZ (LPCZ) to retain the anti-cancer effects of PCZ, but limit brain uptake. Uncharged liposomes showed complete drug encapsulation and pH-dependent drug release, but cationic liposomes showed limited drug encapsulation and lacked pH-dependent drug release. Uncharged LPCZ showed comparable inhibition of EGFR internalisation to free PCZ in KJD cells. After IV administration to rats, LPCZ reduced the plasma clearance and brain uptake of PCZ compared to IV PCZ. The results suggest that LPCZ may offer some benefit over PCZ as an adjunct therapy in cancer patients receiving mAb treatment.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Ratos , Animais , Proclorperazina/efeitos adversos , Dinamina II/metabolismo , Lipossomos/uso terapêutico , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/metabolismo , Encéfalo/metabolismo , Polietilenoglicóis/uso terapêutico
3.
Biochemistry ; 62(14): 2093-2097, 2023 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-37318062

RESUMO

There are two human arylamine N-acetyltransferases (NAT1 and NAT2) that have evolved separately and differ in their substrate specificity and tissue localization. In addition to its acetyltransferase activity, NAT1 can hydrolyze acetyl coenzyme A to coenzyme A in the presence of folate. Here, we show that NAT1 is rapidly inactivated at temperatures above 39 °C whereas NAT2 is more stable. NAT1 acetyltransferase activity is also rapidly lost in whole cells at a rate similar to that of recombinant protein, suggesting it is not protected by intracellular chaperones. By contrast, the hydrolase activity of NAT1 is resistant to heat-induced inactivation, in part because folate stabilizes the protein. Heat generated by mitochondria following the dissipation of the inner membrane potential was sufficient to inactivate NAT1 in whole cells. Within the physiological range of core body temperatures (36.5-37.5 °C), NAT1 acetyltransferase activity decreased by 30% while hydrolase activity increased by >50%. This study demonstrates the thermal regulation of NAT1, but not NAT2, and suggests that NAT1 may switch between an acetyltransferase and a hydrolase within a narrow temperature range in the presence of folate.


Assuntos
Arilamina N-Acetiltransferase , Humanos , Arilamina N-Acetiltransferase/metabolismo , Temperatura , Acetil-CoA Hidrolase , Acetiltransferases/metabolismo , Ácido Fólico
4.
Mol Pharm ; 20(7): 3494-3504, 2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37256791

RESUMO

PEGylated lipid nanoparticle-based Covid-19 vaccines, including Pfizer's BNT162b2 and Moderna's mRNA-1273, have been shown to stimulate variable anti-PEG antibody production in humans. Anti-PEG antibodies have the potential to accelerate the plasma clearance of PEGylated therapeutics, such as PEGylated liposomes and proteins, and compromise their therapeutic efficacy. However, it is not yet clear whether antibody titers produced by PEGylated Covid-19 vaccines significantly affect the clearance of PEGylated therapeutics. This study examined how anti-PEG IgM levels affect the pharmacokinetics of PEGylated liposomal doxorubicin (PLD) and compared the immunogenicity of a lipid nanoparticle formulation of linear DNA (DNA-LNP) to standard PEG-HSPC liposomes. DNA-LNP was prepared using the same composition and approach as Pfizer's BNT162b2, except linear double-stranded DNA was used as the genetic material. PEGylated HSPC-based liposomes were formulated using the lipid rehydration and extrusion method. Nanoparticles were dosed IM to rats at 0.005-0.5 mg lipid/kg body weight 1 week before evaluating the plasma pharmacokinetics of clinically relevant doses of PLD (1 mg/kg, IV) or PEGylated interferon α2a (Pegasys, 5 µg/kg, SC). Plasma PEG IgM was compared between pre- and 1-week post-dose blood samples. The results showed that anti-PEG IgM production increased with increasing PEG-HSPC liposome dose and that IgM significantly correlated with the plasma half-life, clearance, volume of distribution, and area under the curve of a subsequent dose of PLD. The plasma exposure of Pegasys was also significantly reduced after initial delivery of 0.005 mg/ml PEG-HSPC liposome. However, a single 0.05 mg/kg IM dose of DNA-LNP did not significantly elevate PEG IgM and did not alter the IV pharmacokinetics of PLD. These data showed that PEGylated Covid-19 vaccines are less immunogenic compared to standard PEGylated HSPC liposomes and that there is an antibody threshold for accelerating the clearance of PEGylated therapeutics.


Assuntos
COVID-19 , Nanopartículas , Ratos , Humanos , Animais , Lipossomos , Vacina BNT162 , Vacinas contra COVID-19 , Imunoglobulina M , Polietilenoglicóis/farmacocinética , DNA , Fosfatidilcolinas
5.
Mol Pharm ; 20(9): 4468-4477, 2023 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-37548597

RESUMO

Dynamin II (dynII) plays a significant role in the internalization pathways of endocytic cells, by allowing membrane invaginations to "bud off". An important class of dynII inhibitors that are used clinically are phenothiazines, such as prochlorperazine (PCZ). PCZ is an antipsychotic drug but is also currently in clinical trials at higher concentrations as an adjuvant in cancer patients that increases the efficacy of monoclonal antibodies at high intravenous doses. It is unknown, however, whether high-dose dynII inhibitors have the potential to alter the pharmacokinetics of co-administered chemotherapeutic nanomedicines that are largely cleared via the mononuclear phagocyte system. This work therefore sought to investigate the impact of clinically relevant concentrations of phenothiazines, PCZ and thioridazine, on in vitro liposome endocytosis and in vivo liposome pharmacokinetics after PCZ infusion in rats. The uptake of fluorescently labeled PEGylated liposomes into differentiated and undifferentiated THP-1 and RAW246.7 cells, and primary human peripheral white blood cells, was investigated via flow cytometry after co-incubation with dynII inhibitors. The IV pharmacokinetics of PEGylated liposomes were also investigated in rats after a 20 min infusion with PCZ. Phenothiazines and dyngo4a reduced the uptake of PEGylated liposomes by THP-1 and RAW264.7 cells in a concentration-dependent manner in vitro. However, dynII inhibitors did not alter the mean uptake of liposomes by human peripheral white blood cells, but endocytic white cells from some donors exhibited sensitivity to phenothiazine exposure. When a clinically relevant dose of PCZ was co-administered with PEGylated liposomal doxorubicin (Caelyx/Doxil) in rats, the pharmacokinetics and biodistribution of liposomes were unaltered. These data suggest that while clinically relevant doses of dynII inhibitors can inhibit the uptake of liposomes by endocytic cells in vitro, they are unlikely to significantly affect the pharmacokinetics of long-circulating, co-administered liposomes.


Assuntos
Dinamina II , Lipossomos , Ratos , Humanos , Animais , Distribuição Tecidual , Doxorrubicina , Polietilenoglicóis , Fenotiazinas , Proclorperazina
6.
Breast Cancer Res Treat ; 195(3): 223-236, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35918499

RESUMO

PURPOSE: Arylamine N-acetyltransferase 1 (NAT1) deficiency has been associated with drug resistance and poor outcomes in breast cancer patients. The current study aimed to investigate drug resistance in vitro using normal breast cancer cell lines and NAT1-deficient cell lines to understand the changes induced by the lack of NAT1 that resulted in poor drug response. METHODS: The response to seven chemotherapeutic agents was quantified following NAT1 deletion using CRISPR-Cas 9 in MDA-MB-231 and T-47D cells. Apoptosis was monitored by annexin V staining and caspase 3/7 activity. Cytochrome C release and caspase 8 and 9 activities were measured by Western blots. Caspase 8 was inhibited using Z-IETD-FMK and necroptosis was inhibited using necrostatin and necrosulfonamide. RESULTS: Compared to parental cells, NAT1 depleted cells were resistant to drug treatment. This could be reversed following NAT1 rescue of the NAT1 deleted cells. Release of cytochrome C in response to treatment was decreased in the NAT1 depleted cells, suggesting suppression of the intrinsic apoptotic pathway. In addition, NAT1 knockout resulted in a decrease in caspase 8 activation. Treatment with necrosulfonamide showed that NAT1 deficient cells switched from intrinsic apoptosis to necroptosis when treated with the anti-cancer drug cisplatin. CONCLUSIONS: NAT1 deficiency can switch cell death from apoptosis to necroptosis resulting in decreased response to cytotoxic drugs. The absence of NAT1 in patient tumours may be a useful biomarker for selecting alternative treatments in a subset of breast cancer patients.


Assuntos
Antineoplásicos , Arilamina N-Acetiltransferase , Neoplasias da Mama , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Arilamina N-Acetiltransferase/deficiência , Arilamina N-Acetiltransferase/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Caspase 8/uso terapêutico , Morte Celular , Citocromos c/metabolismo , Citocromos c/uso terapêutico , Feminino , Humanos , Isoenzimas/deficiência , Isoenzimas/genética , Necroptose
7.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Nitrobenzenos , Conformação Proteica , Pirazóis/farmacologia , Pirazolonas , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
8.
Drug Metab Dispos ; 48(5): 337-344, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32152050

RESUMO

Sulfotransferase (SULT) 4A1 is a brain-selective sulfotransferase-like protein that has recently been shown to be essential for normal neuronal development in mice. In the present study, SULT4A1 was found to colocalize with SULT1A1/3 in human brain neurons. Using immunoprecipitation, SULT4A1 was shown to interact with both SULT1A1 and SULT1A3 when expressed in human cells. Mutation of the conserved dimerization motif located in the C terminus of the sulfotransferases prevented this interaction. Both ectopically expressed and endogenous SULT4A1 decreased SULT1A1/3 protein levels in neuronal cells, and this was also prevented by mutation of the dimerization motif. During differentiation of neuronal SH-SY5Y cells, there was a loss in SULT1A1/3 protein but an increase in SULT4A1 protein. This resulted in an increase in the toxicity of dopamine, a substrate for SULT1A3. Inhibition of SULT4A1 using small interference RNA abrogated the loss in SULT1A1/3 and reversed dopamine toxicity. These results show a reciprocal relationship between SULT4A1 and the other sulfotransferases, suggesting that it may act as a chaperone to control the expression of SULT1A1/3 in neuronal cells. SIGNIFICANCE STATEMENT: The catalytically inactive sulfotransferase (SULT) 4A1 may regulate the function of other SULTs by interacting with them via a conserved dimerization motif. In neuron-like cells, SULT4A1 is able to modulate dopamine toxicity by interacting with SULT1A3, potentially decreasing the metabolism of dopamine.


Assuntos
Arilsulfotransferase/genética , Encéfalo/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Sulfotransferases/metabolismo , Arilsulfotransferase/metabolismo , Encéfalo/citologia , Diferenciação Celular , Linhagem Celular Tumoral , Dopamina/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Mutação , Neurônios/enzimologia , Multimerização Proteica/genética , Sulfotransferases/genética
9.
Pharm Res ; 37(11): 224, 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33078255

RESUMO

PURPOSE: The aim of this work was to identify whether biochemical and physiological sources of mAb pharmacokinetic sex-effects could be identified in the rat model where target-mediated disposition is avoided. METHODS: Plasma and lymphatic pharmacokinetics of the humanised anti-EGFR antibody cetuximab, along with potential physiological and biochemical drivers of pharmacokinetic sex differences, were examined in male and female rats. Cetuximab was used as a model mAb since plasma clearance is slower in female patients. RESULTS: When plasma concentrations were normalised to dose, female rats displayed slower plasma clearance than males, but no significant differences were observed in liver and spleen biodistribution. Sex differences in apparent plasma clearance, however, were abolished after normalisation to body weight, surface area or fat-free mass. Significant sex differences were observed in plasma testosterone, endogenous IgG and fat free mass, but did not correlate with apparent clearance. Females did, however, show two-fold higher lymphatic exposure compared to males. CONCLUSIONS: These data suggested that mAbs more efficiently access lymph in females, but this does not affect plasma pharmacokinetics or biodistribution. Further, the data suggest that sex differences observed in humans could be a function of antigen density.


Assuntos
Antineoplásicos Imunológicos/farmacocinética , Cetuximab/farmacocinética , Sistema Linfático/metabolismo , Administração Intravenosa , Animais , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/sangue , Cetuximab/administração & dosagem , Cetuximab/sangue , Feminino , Masculino , Taxa de Depuração Metabólica , Ratos Sprague-Dawley , Fatores Sexuais , Distribuição Tecidual
10.
Mol Pharmacol ; 96(5): 573-579, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31444237

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a drug-metabolizing enzyme that influences cancer cell proliferation and survival. However, the mechanism for these effects is unknown. Because of previous observations that NAT1 inhibition decreases invasiveness, we investigated the expression of the metalloproteinase matrix metalloproteinase 9 (MMP9) in human breast cancer samples and in cancer cells. We found a negative correlation between the expression of NAT1 and MMP9 in 1904 breast cancer samples. Moreover, when NAT1 was deleted in highly invasive breast cancer cells, MMP9 mRNA and protein significantly increased, both of which were reversed by reintroducing NAT1 into the knockout cells. After NAT1 deletion, there was an increased association of acetylated histone H3 with the SET and MYND-domain containing 3 (SMYD3) element in the MMP9 promoter, consistent with an increase in MMP9 transcription. NAT1 deletion also up-regulated hypoxia-inducible factor 1-α (HIF1-α). Treatment of the NAT1 knockout cells with small interfering RNA directed toward HIF1-α mRNA inhibited the increased expression of MMP9. Taken together, these results show a direct inverse relationship between NAT1 and MMP9 and suggest that HIF1-α may be essential for the regulation of MMP9 expression by NAT1. SIGNIFICANCE STATEMENT: The expression of the enzyme NAT1 was found to be negatively correlated with MMP9 expression in tumor tissue from breast cancer patients. In cells, NAT1 regulated MMP9 expression at a transcriptional level via HIF1-α. This finding is important as it may explain some of the pathological features associated with changes in NAT1 expression in cancer.


Assuntos
Arilamina N-Acetiltransferase/deficiência , Neoplasias da Mama/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Isoenzimas/deficiência , Metaloproteinase 9 da Matriz/biossíntese , Arilamina N-Acetiltransferase/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Células HT29 , Células HeLa , Humanos , Isoenzimas/genética , Metaloproteinase 9 da Matriz/deficiência
11.
Drug Metab Dispos ; 47(3): 314-319, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30606728

RESUMO

Sulfotransferase 4A1 (SULT4A1) is a sulfotransferase-like protein that is highly conserved between species. In human tissues, there are two transcripts, one that produces a full-length protein and one that produces an unstable truncated protein. The second transcript, which includes a pseudo-exon between exons 6 and 7 (6p), is widely expressed, whereas the first is more restricted. Differentiation of neuronal cells results in the removal of the pseudo-exon and subsequent SULT4A1 protein expression. Recent studies with SULT4A1 knockout mice showed that the protein is essential for normal development and that its absence leads to a severe neurologic phenotype. Here, the regulation of SULT4A1 6p splicing was investigated during neuronal differentiation using SH-SY5Y cells, human induced pluripotent stem cells, and mouse embryonic tissue. In all three models, pseudo-exon 6p was removed during differentiation, resulting in stable SULT4A1 protein expression. Using a minigene splicing assay, a region upstream of pseudo-exon 6p was identified that is essential for correct splicing of SULT4A1 mRNA. Within this region, there were binding motifs for four RNA processing factors (MBNL-1, MBNL-2, CELF-1, and CELF-2). Time-dependent changes in SULT4A1 protein and MBNL/CELF protein during differentiation supported their role in correctly splicing the SULT4A1 mRNA. Furthermore, ectopic expression of each factor produced efficient splicing in the minigene assay as well as correct splicing of the endogenous SULT4A1 mRNA. These results show that SULT4A1 mRNA is a target for MBNL/CELF-dependent splicing, which may be essential in producing stable, functional SULT4A1.


Assuntos
Processamento Alternativo/fisiologia , Neurônios/fisiologia , Fatores de Processamento de RNA/metabolismo , Sulfotransferases/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Embrião de Mamíferos , Éxons/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
BMC Genomics ; 19(1): 513, 2018 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-29969986

RESUMO

BACKGROUND: Arylamine N-acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that has been associated with cancer cell proliferation in vitro and with survival in vivo. NAT1 expression has been associated with the estrogen receptor and it has been proposed as a prognostic marker for estrogen receptor positive cancers. However, little is known about the distribution of NAT1 mRNA across an entire patient population or its effects on outcomes. To address this, gene expression data from breast cancer patient cohorts were investigated to identify sub-populations based on the level of NAT1 expression. Patient survival and drug response was examined to determine whether NAT1 mRNA levels influenced any of these parameters. RESULTS: NAT1 expression showed a trimodal distribution in breast cancer samples (n = 1980) but not in tumor tissue from ovarian, prostate, cervical or colorectal cancers. In breast cancer, NAT1 mRNA in each sub-population correlated with a separate set of genes suggesting different mechanisms of NAT1 gene regulation. Kaplan-Meier plots showed significantly better survival in patients with highest NAT1 mRNA compared to those with intermediate or low expression. While NAT1 expression was elevated in estrogen receptor-positive patients, it did not appear to be dependent on estrogen receptor expression. Overall survival was analyzed in patients receiving no treatment, hormone therapy or chemotherapy. NAT1 expression correlated strongly with survival in the first 5 years in those patients receiving chemotherapy but did not influence survival in the other two groups. This suggests that low NAT1 expression is associated with chemo-resistance. The sensitivity of NAT1 mRNA levels as a single parameter to identify non-responders to chemotherapy was 0.58 at a log(2) < 6.5. CONCLUSIONS: NAT1 mRNA can be used to segregate breast cancer patients into sub-populations that demonstrate different overall survival. Moreover, low NAT1 expression shows a distinct poor response to chemotherapy. Analysis of NAT1 expression may be useful for identifying specific individuals who would benefit from alternative therapy or drug combinations. However, additional information is required to increase the sensitivity of identifying non-responders.


Assuntos
Arilamina N-Acetiltransferase/genética , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Isoenzimas/genética , Antineoplásicos/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Modelos de Riscos Proporcionais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo
13.
Cytometry A ; 87(6): 549-57, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25492545

RESUMO

The antinuclear antibody (ANA) test via indirect immunofluorescence applied on Human Epithelial type 2 (HEp-2) cells is a pathology test commonly used to identify connective tissue diseases (CTDs). Despite its effectiveness, the test is still considered labor intensive and time consuming. Applying image-based computer aided diagnosis (CAD) systems is one of the possible ways to address these issues. Ideally, a CAD system should be able to classify ANA HEp-2 images taken by a camera fitted to a fluorescence microscope. Unfortunately, most prior works have primarily focused on the HEp-2 cell image classification problem which is one of the early essential steps in the system pipeline. In this work we directly tackle the specimen image classification problem. We aim to develop a system that can be easily scaled and has competitive accuracy. ANA HEp-2 images or ANA images are generally comprised of a number of cells. Patterns exhibiting in the cells are then used to make inference on the ANA image pattern. To that end, we adapted a popular approach for general image classification problems, namely a bag of visual words approach. Each specimen is considered as a visual document containing visual vocabularies represented by its cells. A specimen image is then represented by a histogram of visual vocabulary occurrences. We name this approach as the Bag of Cells approach. We studied the performance of the proposed approach on a set of images taken from 262 ANA positive patient sera. The results show the proposed approach has competitive performance compared to the recent state-of-the-art approaches. Our proposal can also be expanded to other tests involving examining patterns of human cells to make inferences.


Assuntos
Anticorpos Antinucleares/imunologia , Núcleo Celular/imunologia , Doenças do Tecido Conjuntivo/diagnóstico , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Ciclo Celular , Linhagem Celular , Biologia Computacional/métodos , Células Epiteliais/imunologia , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos
14.
Pharmacol Rev ; 64(1): 147-65, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22090474

RESUMO

The human arylamine N-acetyltransferases first attracted attention because of their role in drug metabolism. However, much of the current literature has focused on their role in the activation and detoxification of environmental carcinogens and how genetic polymorphisms in the genes create predispositions to increased or decreased cancer risk. There are two closely related genes on chromosome 8 that encode the two human arylamine N-acetyltransferases--NAT1 and NAT2. Although NAT2 has restricted tissue expression, NAT1 is found in almost all tissues of the body. There are several single-nucleotide polymorphisms in the protein coding and 3'-untranslated regions of the gene that affect enzyme activity. However, NAT1 is also regulated by post-translational and environmental factors, which may be of greater importance than genotype in determining tissue NAT1 activities. Recent studies have suggested a novel role for this enzyme in cancer cell growth. NAT1 is up-regulated in several cancer types, and overexpression can lead to increased survival and resistance to chemotherapy. Although a link to folate homeostasis has been suggested, many of the effects attributed to NAT1 and cancer cell growth remain to be explained. Nevertheless, the enzyme has emerged as a viable candidate for drug development, which should lead to small molecule inhibitors for preclinical and clinical evaluation.


Assuntos
Arilamina N-Acetiltransferase , Descoberta de Drogas , Isoenzimas , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Arilamina N-Acetiltransferase/antagonistas & inibidores , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/fisiologia , Regulação para Baixo , Epigênese Genética , Ácido Fólico/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/fisiologia , Estrutura Molecular , Neoplasias/enzimologia , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Especificidade por Substrato
15.
J Biol Chem ; 288(48): 34364-74, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-24136195

RESUMO

Dopamine neurotoxicity is associated with several neurodegenerative diseases, and neurons utilize several mechanisms, including uptake and metabolism, to protect them from injury. Metabolism of dopamine involves three enzymes: monoamine oxidase, catechol O-methyltransferase, and sulfotransferase. In primates but not lower order animals, a sulfotransferase (SULT1A3) is present that can rapidly metabolize dopamine to dopamine sulfate. Here, we show that SULT1A3 and a closely related protein SULT1A1 are highly inducible by dopamine. This involves activation of the D1 and NMDA receptors. Both ERK1/2 phosphorylation and calcineurin activation are required for induction. Pharmacological agents that inhibited induction or siRNA targeting SULT1A3 significantly increased the susceptibility of cells to dopamine toxicity. Taken together, these results show that dopamine can induce its own metabolism and protect neuron-like cells from damage, suggesting that SULT1A3 activity may be a risk factor for dopamine-dependent neurodegenerative diseases.


Assuntos
Arilsulfotransferase/metabolismo , Dopamina/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Arilsulfotransferase/antagonistas & inibidores , Arilsulfotransferase/genética , Benzazepinas/farmacologia , Calcineurina/metabolismo , Linhagem Celular , Dopamina/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/genética , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Fosforilação , RNA Interferente Pequeno/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Fatores de Risco , Transdução de Sinais
16.
Drug Metab Dispos ; 42(3): 377-83, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24346836

RESUMO

Humans are exposed to nanoparticles in the environment as well as those in nanomaterials developed for biomedical applications. However, the safety and biologic effects of many nanoparticles remain to be elucidated. Over the past decade, our understanding of the interaction of proteins with various nanomaterials has grown. The protein corona can determine not only how nanoparticles interact with cells but also their biologic effects and toxicity. In this study, we describe the effects that several different classes of nanoparticles exert on the enzymatic activity of the cytosolic protein human arylamine N-acetyltransferase 1 (NAT1), a drug-metabolizing enzyme widely distributed in the body that is also responsible for the activation and detoxification of known carcinogens. We investigated three metal oxides (zinc oxide, titanium dioxide, and silicon dioxide), two synthetic clay nanoparticles (layered double hydroxide and layered silicate nanoparticles), and a self-assembling thermo-responsive polymeric nanoparticle that differ in size and surface characteristics. We found that the different nanoparticles induced very different responses, ranging from inhibition to marked enhancement of enzyme activity. The layered silicates did not directly inactivate NAT1, but was found to enhance substrate-dependent inhibition. These differing effects demonstrate the multiplicity of nanoparticle-protein interactions and suggest that enzyme activity may be compromised in organs exposed to nanoparticles, such as the lungs or reticulo-endothelial system.


Assuntos
Arilamina N-Acetiltransferase/metabolismo , Isoenzimas/metabolismo , Nanopartículas/toxicidade , Polímeros/toxicidade , Dióxido de Silício/toxicidade , Titânio/toxicidade , Óxido de Zinco/toxicidade , Arilamina N-Acetiltransferase/antagonistas & inibidores , Sítios de Ligação , Temperatura Alta , Humanos , Isoenzimas/antagonistas & inibidores , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Nanopartículas/química , Tamanho da Partícula , Polímeros/química , Desnaturação Proteica , Dióxido de Silício/química , Propriedades de Superfície , Titânio/química , Óxido de Zinco/química
17.
Biochim Biophys Acta ; 1813(1): 231-7, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20920535

RESUMO

SULT4A1 is a cytosolic sulfotransferase that shares little homology with other human sulfotransferases but is highly conserved between species. It is found in neurons located in several regions of the brain. Recently, the stability of SULT4A1 was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. Since Pin1 binds preferentially to phosphoproteins, these findings suggested that SULT4A1 is post-translationally modified. In this study, we show that the Thr(11) residue of SULT4A1, which is involved in Pin1 binding is phosphorylated. MEK inhibition was shown to abolish Pin1 mediated degradation of SULT4A1 while in vitro phosphorylation assays using alanine substitution mutants of SULT4A1 demonstrated phosphorylation of Thr(11) by ERK1. We also show that dephosphorylation was catalyzed by the protein phosphatase 2A. The PP2A regulatory subunit, Bß was identified from a yeast-2-hybrid screen of human brain cDNA as a SULT4A1 interacting protein. This was further confirmed by GST pull-downs and immunoprecipitation. Other members of the B subunit (αδγ) did not interact with SULT4A1. Taken together, these studies indicate that SULT4A1 stability is regulated by post-translational modification that involves the ERK pathway and PP2A. The phosphorylation of SULT4A1 allows interaction with Pin1, which then promotes degradation of the sulfotransferase.


Assuntos
Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional , Sulfotransferases/metabolismo , Animais , Western Blotting , Células HeLa , Humanos , Imunoprecipitação , Peptidilprolil Isomerase de Interação com NIMA , Células PC12 , Fosforilação , Ligação Proteica , Ratos , Treonina , Técnicas do Sistema de Duplo-Híbrido
18.
Biochem Pharmacol ; 200: 115020, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35358480

RESUMO

Human arylamine N-acetyltransferase 1 (NAT1) encodes a drug-metabolising enzyme that plays a role in chemical-associated cancer risk, cancer cell survival and mitochondrial function. Its expression and protein activity are regulated by transcriptional, translational, and post-translational processes, including microRNAs such as miR-1290. Several studies have shown the presence of multiple polyadenylation sites in the NAT1 gene. However, their role in NAT1 expression is poorly understood. Here, we have investigated the genetic sequence of the NAT1 gene in human cell lines, peripheral blood mononuclear cells and breast tumour tissue. We identified five potential polyadenylation signals, two of which carry known single nucleotide polymorphism that affect site usage. Cells that are homozygous for adenine at base 1642, the most distal polyadenylation site, use this site whereas those homozygous for cytosine at base 1642 could not. We also found that the presence of adenine at base 1642 is associated with the NAT1*10 haplotype. Because the putative binding site for miR-1290 is located between the last two polyadenylation sites, we hypothesised that cells that do not use the most distal site will be unaffected by miR-1290. However, this was not the case. NAT1 activity was positively correlated with miR-1290, and induction of miR-1290 in SH-SY5Y cells was associated with induction, not inhibition, of NAT1 activity. The use of PolyA1264 or PolyA1642 did not alter NAT1 activity following ectopic expression of a miR-1290 mimic. These results suggest that the role of miR-1290 in the regulation of NAT1 activity is more complex than previously reported.


Assuntos
Arilamina N-Acetiltransferase , MicroRNAs , Adenina , Arilamina N-Acetiltransferase/genética , Humanos , Isoenzimas/genética , Leucócitos Mononucleares/metabolismo , MicroRNAs/genética , Poliadenilação , Regiões não Traduzidas
19.
Drug Metab Dispos ; 39(1): 77-82, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20870783

RESUMO

Arylamine N-acetyltransferase-1 (NAT1) has been associated with disorders involving folate metabolism, such as spina bifida, as well as numerous human cancers. As a result, the transcriptional and post-transcriptional regulation of NAT1 activity has been extensively studied. However, little work has been reported on the epigenetic control of NAT1 expression. Here, we demonstrate that the histone deacetylase inhibitor trichostatin A (TSA) increases NAT1 activity in human cancer cells by increasing transcription from the proximal promoter NATb. A specific Sp1 binding site was identified as essential for optimal induction of NAT1 by TSA. However, TSA did not increase the expression of Sp1 in HeLa cells. Instead, TSA increased the acetylation of histones associated with the NATb promoter. This allowed recruitment of Sp1 to the promoter along with acetylated histones. We propose that NAT1 transcription is partially repressed by the local chromatin condensation in the vicinity of NATb and that histone deacetylase inhibition leads to up-regulation of NAT1 expression via a direct change in chromatin conformation.


Assuntos
Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Cromatina/metabolismo , Regulação Enzimológica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Acetilação , Células HeLa , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Regulação para Cima
20.
J Pharm Sci ; 110(8): 3099-3101, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34019905

RESUMO

Pegylated liposomal doxorubicin (PLD) is widely utilised in cancer chemotherapy, but exhibits large inter-individual pharmacokinetic variability and sex differences in plasma clearance. Population pharmacokinetic modelling has suggested PLD plasma clearance correlates with peripheral monocyte counts, while sex hormones are known to affect endocytosis and phagocytosis in mononuclear cells. This study investigated whether sex hormones affect the uptake of PLD by human monocytes and macrophages in vitro. 17ß-estradiol, but not testosterone, inhibited the uptake of PLD in a concentration dependent manner in undifferentiated (but not differentiated) THP-1 cells, and primary monocytes obtained from women, but not men. Effects of estradiol were only evident at very high concentrations seen during pregnancy. No differences were observed in monocyte count or monocyte subtypes between males and females. These data show that monocytes do not contribute to sex differences seen in PLD clearance in humans of reproductive age.


Assuntos
Monócitos , Caracteres Sexuais , Antibióticos Antineoplásicos , Doxorrubicina/análogos & derivados , Feminino , Humanos , Masculino , Polietilenoglicóis
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