RESUMEN

Ferroptosis holds promise for cancer therapy. A recent study by Yang et al. in Cell Metabolism reveals that VKORC1L1-mediated reduction of vitamin K inhibits ferroptosis and establishes a direct p53-VKORC1L1 link in its regulation. As warfarin can inhibit VKORC1L1, the study further underscores this drug's potential as a cancer therapy.

Asunto(s)

Ferroptosis , Neoplasias , Humanos , Warfarina/uso terapéutico , Warfarina/farmacología , Vitamina K Epóxido Reductasas/metabolismo , Neoplasias/tratamiento farmacológico , Vitamina K/metabolismo , Proteína p53 Supresora de Tumor

RESUMEN

Here, we identified vitamin K epoxide reductase complex subunit 1 like 1 (VKORC1L1) as a potent ferroptosis repressor. VKORC1L1 protects cells from ferroptosis by generating the reduced form of vitamin K, a potent radical-trapping antioxidant, to counteract phospholipid peroxides independent of the canonical GSH/GPX4 mechanism. Notably, we found that VKORC1L1 is also a direct transcriptional target of p53. Activation of p53 induces downregulation of VKORC1L1 expression, thus sensitizing cells to ferroptosis for tumor suppression. Interestingly, a small molecular inhibitor of VKORC1L1, warfarin, is widely prescribed as an FDA-approved anticoagulant drug. Moreover, warfarin represses tumor growth by promoting ferroptosis in both immunodeficient and immunocompetent mouse models. Thus, by downregulating VKORC1L1, p53 executes the tumor suppression function by activating an important ferroptosis pathway involved in vitamin K metabolism. Our study also reveals that warfarin is a potential repurposing drug in cancer therapy, particularly for tumors with high levels of VKORC1L1 expression.

Asunto(s)

Proteína p53 Supresora de Tumor , Warfarina , Animales , Ratones , Anticoagulantes/farmacología , Anticoagulantes/uso terapéutico , Vitamina K/metabolismo , Vitamina K Epóxido Reductasas/genética , Vitamina K Epóxido Reductasas/metabolismo , Warfarina/farmacología , Warfarina/uso terapéutico

RESUMEN

Sodium dehydroacetate (Na-DHA) is widely used as an antibacterial and preservative additive in food and cosmetics. Previously, we reported that repeated oral administration of Na-DHA induces coagulation disorders, and inhibited liver vitamin K epoxide reductase complex subunit 1 (VKORC1) and VKORC1-like protein 1 (VKORC1L1) in rats. However, the effects of Na-DHA on coagulation factors in rat hepatocytes and the mechanism of VKORC1 and VKORC1L1 signaling in that process are unclear. Here, we constructed stable Vkorc1 and Vkorc1l1 overexpressing cell lines using lentiviruses and transfected small interfering RNAs into buffalo rat liver BRL3A cells for Vkorc1 and Vkorc1l1 overexpression and silencing, respectively. After treatment with 5 mmol/L Na-DHA for 24 h, VKORC1 and VKORC1L1 expression levels were detected by real-time PCR and western blotting. Vitamin K (VK) and factor IX (FIX) contents were detected using enzyme linked immunosorbent assays. We observed that Na-DHA inhibited VKORC1 and VKORC1L1 expression levels and reduced VK and FIX levels in rat hepatocytes. Overexpression or silencing of Vkorc1 and Vkorc1l1 increased or decreased, respectively, the production and secretion of VK and FIX in rat hepatocytes, and alleviated or aggravated the inhibitory effects of Na-DHA on VKORC1 and VKORC1L1 expression levels. Taken together, the results indicated that both VKORC1 and VKORC1L1 signaling play regulatory roles in the effects of Na-DHA on coagulation factors in rat hepatocytes.

Asunto(s)

Hepatocitos , Vitamina K , Ratas , Animales , Vitamina K Epóxido Reductasas/genética , Vitamina K Epóxido Reductasas/metabolismo , Vitamina K/metabolismo , Hepatocitos/metabolismo , Factores de Coagulación Sanguínea

RESUMEN

Sodium dehydroacetate (Na-DHA), a fungicide used in food, feed, cosmetics, and medicine, has been found to cause coagulation aberration accompanied by the inhibition of vitamin K epoxide reductase (VKOR) in the liver in rats. VKOR complex 1 (VKORC1) and VKORC1 like-1 (VKORC1L1) are two homologous VKOR proteins. Little information is available on the effect of Na-DHA on VKORC1L1 in the liver or VKORC1/VKORC1L1 in extrahepatic tissue and sex differences in Na-DHA metabolism. In the present study, after administration of 200 mg/kg Na-DHA by gavage, significant inhibition of VKORC1 or VKORC1L1 expression in tissues, as well as prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT), were observed. The PT/APTT in the Na-DHA-exposed males were 1.27- to 1.48-fold/1.17- to 1.37-fold, while the corresponding values in the Na-DHA-exposed females were 1.36- to 2.02-fold/1.20- to 1.70-fold. Serum or tissue Na-DHA concentrations were significantly higher in females than in males. The pharmacokinetic parameters (t1/2, Cmax, AUC0∼24 h, and MRT0∼24 h) of Na-DHA in female rats were significantly higher than those in male rats. Furthermore, cytochrome P450 (CYP) activity was investigated using the cocktail probe method. The results revealed that Na-DHA exhibited an inductive effect on CYP1A2, 2D1/2, and 3A1/2 activities by changing the main pharmacokinetic parameters of probe drugs in male rats. However, no significant change in CYP2E1 activity was found. There were sex differences in the metabolism and coagulation in rats exposed to Na-DHA. The lower metabolism and higher blood Na-DHA concentration in females may be the reasons for higher coagulation sensitivity in female rats.

RESUMEN

Worldwide use of anticoagulant rodenticides (ARs) for rodents control has frequently led to secondary poisoning of non-target animals, especially raptors. In order to suggest some factors that may help considering the mechanism of the incidents, this study focused on the avian vitamin K 2, 3-epoxide reductase (VKOR) that is the target protein of ARs. We addressed the interspecific differences in VKOR activity and inhibition related to amino acid sequence and mRNA expression of VKORC1 and VKORC1-like1 (VKORC1L1). Poultry have been considered to be more tolerant to ARs than mammals. However, VKOR activity of owls, hawks, falcon and surprisingly, canaries, was lower and inhibited by warfarin more easily than that of chickens and turkeys. The amino acid sequence of VKORC1 and VKORC1L1 implied that the value of Ki for VKOR activity to ARs could depend on the amino acid at position 140 in the TYX warfarin-binding motif in VKORC1, and other amino acid mutations in VKORC1L1. The mRNA expression ratio of VKORC1:VKORC1L1 differed between turkey (8:1) and chicken (2:3) liver. VKORC1L1 has been reported to be resistant to warfarin compared to VKORC1. Hence, both the Ki of specific VKORC1 and VKORC1L1, and the mRNA expression ratio would cause avian interspecific difference of the VKOR inhibition. Our study also suggested the high inhibition of VKOR activities in raptors and surprisingly that in canaries as well. These factors are the most likely to contribute to the high sensitivity to ARs found in raptors.

Asunto(s)

Anticoagulantes/envenenamiento , Canarios/genética , Resistencia a Medicamentos/genética , Rapaces/genética , Rodenticidas/envenenamiento , Vitamina K Epóxido Reductasas/antagonistas & inhibidores , Warfarina/envenenamiento , Secuencia de Aminoácidos/genética , Animales , Mutación , ARN Mensajero/biosíntesis , Especificidad de la Especie , Vitamina K Epóxido Reductasas/química , Vitamina K Epóxido Reductasas/genética

RESUMEN

ALK-rearranged lung cancer defines a distinctive molecular cohort of patients whose outcomes are significantly improved by the availability of ALK inhibitors. Thus, it is imperative for clinicians to screen appropriate patients for this driver mutation with a molecular testing platform capable of capturing all ALK fusions. Here, we report a novel VKORC1L1-ALK fusion and an ALK T1151K resistance mutation detected in a lung cancer patient who had been on crizotinib for over 8 years. Alectinib induced a dramatic response in this patient demonstrating its clinical activity against T1151K. This case illustrates the importance of performing repeat biopsy to explore mechanism(s) of resistance when patients experience disease progression on an ALK inhibitor. The approach has a direct therapeutic impact particularly when an ALK resistance mutation is identified.

RESUMEN

Vitamin K is an essential nutrient involved in the regulation of blood clotting and tissue mineralization. Vitamin K oxidoreductase (VKORC1) converts vitamin K epoxide into reduced vitamin K, which acts as the co-factor for the γ-carboxylation of several proteins, including coagulation factors produced by the liver. VKORC1 is also the pharmacological target of warfarin, a widely used anticoagulant. Vertebrates possess a VKORC1 paralog, VKORC1-like 1 (VKORC1L1), but until very recently, the importance of VKORC1L1 for protein γ-carboxylation and hemostasis in vivo was not clear. Here, we first review the current knowledge on the structure, function and expression pattern of VKORC1L1, including recent data establishing that, in the absence of VKORC1, VKORC1L1 can support vitamin K-dependent carboxylation in the liver during the pre- and perinatal periods in vivo. We then provide original data showing that the partial redundancy between VKORC1 and VKORC1L1 also exists in bone around birth. Recent studies indicate that, in vitro and in cell culture models, VKORC1L1 is less sensitive to warfarin than VKORC1. Genetic evidence is presented here, which supports the notion that VKORC1L1 is not the warfarin-resistant vitamin K quinone reductase present in the liver. In summary, although the exact physiological function of VKORC1L1 remains elusive, the latest findings clearly established that this enzyme is a vitamin K oxidoreductase, which can support γ-carboxylation in vivo.

Asunto(s)

Coagulación Sanguínea , Ácidos Carboxílicos/metabolismo , Hígado/enzimología , Vitamina K 1/análogos & derivados , Vitamina K Epóxido Reductasas/metabolismo , Animales , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Oxidación-Reducción , Conformación Proteica , Procesamiento Proteico-Postraduccional , Relación Estructura-Actividad , Vitamina K 1/metabolismo , Vitamina K Epóxido Reductasas/antagonistas & inhibidores , Vitamina K Epóxido Reductasas/química , Vitamina K Epóxido Reductasas/genética , Warfarina/farmacología

RESUMEN

The aim of this study was to investigate the possible effects of EPHX1 and VKORC1L1 polymorphisms on variability of responses to warfarin. Sixteen single nucleotide polymorphisms (SNPs) in 201 patients with stable warfarin doses were analyzed including genes of VKORC1, CYP2C9, CYP4F2, GGCX, EPHX1 and VKORC1L1. Univariate analysis was conducted for the association of genotypes with stable warfarin doses. Multiple linear regression analysis was used to investigate factors that independently affected the inter-individual variability of warfarin dose requirements. The rs4072879 of VKORC1L1 (A>G) was significantly associated with stable warfarin doses; wild homozygote carriers (AA) required significantly lower stable warfarin doses than those with the variant G allele (5.02±1.56 vs. 5.96±2.01mg; p=0.001). Multivariate analysis showed that EPHX1 rs1877724 and VKORC1L1 rs4072879 accounted for 1.5% and 1.3% of the warfarin dose variability. Adding EPHX1 and VKORC1L1 SNPs to the base model including non-genetic variables (operation age, body weight and the therapy of ACEI or ARB) and genetic variables (VKORC1 rs9934438, CYP2C9 rs1057910, and CYP4F2 rs2108622) gave a number needed to genotype of 34. This study showed that polymorphisms of EPHX1 and VKORC1L1 could be determinants of stable warfarin doses.

Asunto(s)

Anticoagulantes/administración & dosificación , Anticoagulantes/uso terapéutico , Epóxido Hidrolasas/genética , Vitamina K Epóxido Reductasas/genética , Warfarina/administración & dosificación , Warfarina/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Frecuencia de los Genes , Implantación de Prótesis de Válvulas Cardíacas , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Vitamina K/metabolismo

RESUMEN

BACKGROUND: There has been limited characterization of biological variables that impact vitamin K metabolism. This gap in knowledge can limit the translation of data obtained from preclinical animal studies to future human studies. OBJECTIVE: The purpose of this study was to determine the effects of diet, sex, and housing on serum, tissue, and fecal vitamin K concentrations and gene expression in C57BL6 mice during dietary vitamin K manipulation. METHODS: C57BL6 4-mo-old male and female mice were randomly assigned to conventional or suspended-wire cages and fed control [1400 ± 80 µg phylloquinone (PK)/kg] or deficient (31 ± 0.45 µg PK/kg) diets for 28 d in a factorial design. PK and menaquinone (MK) 4 plasma and tissue concentrations were measured by HPLC. Long-chain MKs were measured in all matrices by LC-atmospheric pressure chemical ionization-mass spectrometry. Gene expression was quantified by reverse transcriptase-polymerase chain reaction in the liver, brain, kidney, pancreas, and adipose tissue. RESULTS: Male and female mice responded differently to dietary manipulation in a tissue-dependent manner. In mice fed the control diet, females had ∼3-fold more MK4 in the brain and mesenteric adipose tissue than did males and 100% greater PK concentrations in the liver, kidney, and mesenteric adipose tissue than did males. In mice fed the deficient diet, kidney MK4 concentrations were ∼4-fold greater in females than in males, and there were no differences in other tissues. Males and females differed in the expression of vitamin K expoxide reductase complex 1 (Vkorc1) in mesenteric adipose tissue and the pancreas and ubiA domain-containing protein 1 (Ubiad1) in the kidney and brain. There was no effect of housing on serum, tissue, or fecal concentrations of any vitamin K form. CONCLUSIONS: Vitamin K concentrations and expression of key metabolic enzymes differ between male and female mice and in response to the dietary PK concentration. Identifying factors that may impact study design and outcomes of interest is critical to optimize study parameters examining vitamin K metabolism in animal models.

Asunto(s)

Tejido Adiposo/metabolismo , Encéfalo/metabolismo , Dieta , Riñón/metabolismo , Hígado/metabolismo , Páncreas/metabolismo , Vitamina K/metabolismo , Tejido Adiposo/enzimología , Animales , Dimetilaliltranstransferasa/metabolismo , Femenino , Vivienda , Vivienda para Animales , Masculino , Proteínas de la Membrana/metabolismo , Mesenterio/enzimología , Mesenterio/metabolismo , Ratones Endogámicos C57BL , Páncreas/enzimología , Factores Sexuales , Distribución Tisular , Vitamina K/administración & dosificación , Vitamina K 1/administración & dosificación , Vitamina K 1/metabolismo , Vitamina K 2/metabolismo , Deficiencia de Vitamina K/enzimología , Deficiencia de Vitamina K/metabolismo , Vitamina K Epóxido Reductasas/metabolismo

RESUMEN

Among all cellular life on earth, with the exception of yeasts, fungi, and some prokaryotes, VKOR family homologs are ubiquitously encoded in nuclear genomes, suggesting ancient and important biological roles for these enzymes. Despite single gene and whole genome duplications on the largest evolutionary timescales, and the fact that most gene duplications eventually result in loss of one copy, it is surprising that all jawed vertebrates (gnathostomes) have retained two paralogous VKOR genes. Both VKOR paralogs function as entry points for nutritionally acquired and recycled K vitamers in the vitamin K cycle. Here we present phylogenetic evidence that the human paralogs likely arose earlier than gnathostomes, possibly in the ancestor of crown chordates. We ask why gnathostomes have maintained these paralogs throughout evolution and present a current summary of what we know. In particular, we look to published studies about tissue- and developmental stage-specific expression, enzymatic function, phylogeny, biological roles and associated pathways that together suggest subfunctionalization as a major influence in evolutionary fixation of both paralogs. Additionally, we investigate on what evolutionary timescale the paralogs arose and under what circumstances in order to gain insight into the biological raison d'être for both VKOR paralogs in gnathostomes.

Asunto(s)

Evolución Biológica , Filogenia , Vertebrados/genética , Vitamina K Epóxido Reductasas/genética , Vitamina K/metabolismo , Animales , Humanos , Homología de Secuencia de Aminoácido , Vitamina K Epóxido Reductasas/metabolismo

RESUMEN

In humans and other vertebrate animals, vitamin K 2,3-epoxide reductase (VKOR) family enzymes are the gatekeepers between nutritionally acquired K vitamins and the vitamin K cycle responsible for posttranslational modifications that confer biological activity upon vitamin K-dependent proteins with crucial roles in hemostasis, bone development and homeostasis, hormonal carbohydrate regulation and fertility. We report a phylogenetic analysis of the VKOR family that identifies five major clades. Combined phylogenetic and site-specific conservation analyses point to clade-specific similarities and differences in structure and function. We discovered a single-site determinant uniquely identifying VKOR homologs belonging to human pathogenic, obligate intracellular prokaryotes and protists. Building on previous work by Sevier et al. (Protein Science 14:1630), we analyzed structural data from both VKOR and prokaryotic disulfide bond formation protein B (DsbB) families and hypothesize an ancient evolutionary relationship between the two families where one family arose from the other through a gene duplication/deletion event. This has resulted in circular permutation of primary sequence threading through the four-helical bundle protein folds of both families. This is the first report of circular permutation relating distant a-helical membrane protein sequences and folds. In conclusion, we suggest a chronology for the evolution of the five extant VKOR clades.

Asunto(s)

Evolución Biológica , Disulfuros , Filogenia , Receptores Fc/genética , Vitamina K Epóxido Reductasas/genética , Vitamina K/metabolismo , Secuencia de Aminoácidos , Animales , Archaea , Bacterias , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Vitamina K Epóxido Reductasas/química

RESUMEN

VKORC1 and VKORC1L1 are enzymes that both catalyze the reduction of vitamin K2,3-epoxide via vitamin K quinone to vitamin K hydroquinone. VKORC1 is the key enzyme of the classical vitamin K cycle by which vitamin K-dependent (VKD) proteins are γ-carboxylated by the hepatic γ-glutamyl carboxylase (GGCX). In contrast, the VKORC1 paralog enzyme, VKORC1L1, is chiefly responsible for antioxidative function by reduction of vitamin K to prevent damage by intracellular reactive oxygen species. To investigate tissue-specific vitamin K 2,3-epoxide reductase (VKOR) function of both enzymes, we quantified mRNA levels for VKORC1, VKORC1L1, GGCX, and NQO1 and measured VKOR enzymatic activities in 29 different mouse tissues. VKORC1 and GGCX are highly expressed in liver, lung and exocrine tissues including mammary gland, salivary gland and prostate suggesting important extrahepatic roles for the vitamin K cycle. Interestingly, VKORC1L1 showed highest transcription levels in brain. Due to the absence of detectable NQO1 transcription in liver, we assume this enzyme has no bypass function with respect to activation of VKD coagulation proteins. Our data strongly suggest diverse functions for the vitamin K cycle in extrahepatic biological pathways.

Asunto(s)

Proteínas de la Membrana/metabolismo , Vitamina K Epóxido Reductasas/metabolismo , Animales , Encéfalo/metabolismo , Ligasas de Carbono-Carbono/genética , Ligasas de Carbono-Carbono/metabolismo , Glándulas Exocrinas/metabolismo , Femenino , Hígado/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Microsomas/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Tisular , Vitamina K/metabolismo , Vitamina K Epóxido Reductasas/genética

RESUMEN

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.

Asunto(s)

Anticoagulantes/farmacología , Hígado/enzimología , Oxigenasas de Función Mixta/metabolismo , Vitamina K Epóxido Reductasas/metabolismo , Animales , Biocatálisis/efectos de los fármacos , Línea Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Oxigenasas de Función Mixta/genética , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Vitamina K/antagonistas & inhibidores , Vitamina K/metabolismo , Vitamina K Epóxido Reductasas/deficiencia , Vitamina K Epóxido Reductasas/genética , Warfarina/farmacología

RESUMEN

BACKGROUND: Single nucleotide polymorphisms in the vitamin K epoxide reductase (VKOR) gene have been successfully used for warfarin dosage prediction. However, warfarin resistance studies of naturally occurring VKOR mutants do not correlate with their clinical phenotype. This discrepancy presumably arises because the in vitro VKOR activity assay is performed under artificial conditions using the non-physiological reductant dithiothreitol. OBJECTIVES: The aim of this study is to establish an in vivo VKOR activity assay in mammalian cells (HEK293) where VKOR functions in its native milieu without interference from endogenous enzymes. METHODS: Endogenous VKOR activity in HEK293 cells was knocked out by transcription activator-like effector nucleases (TALENs)-mediated genome editing. RESULTS AND CONCLUSIONS: Knockout of VKOR in HEK293 cells significantly decreased vitamin K-dependent carboxylation with vitamin K epoxide (KO) as substrate. However, the paralog of VKOR, VKORC1L1, also exhibits substantial ability to convert KO to vitamin K for carboxylation. Using both VKOR and VKORC1L1 knockout cells, we examined the enzymatic activity and warfarin resistance of 10 naturally occurring VKOR mutants that were reported previously to have no activity in an in vitro assay. All 10 mutants are fully active; five have increased warfarin resistance, with the order being W59R>L128R≈W59L>N77S≈S52L. Except for the L128R mutant, this order is consistent with the clinical anticoagulant dosages. The other five VKOR mutants do not change VKOR's warfarin sensitivity, suggesting that factors other than VKOR play important roles. In addition, we confirmed that the conserved loop cysteines in VKOR are not required for active site regeneration after each cycle of oxidation.

Asunto(s)

Resistencia a Medicamentos , Vitamina K Epóxido Reductasas/metabolismo , Warfarina/química , Anticoagulantes/química , Ditiotreitol/química , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Mutación , Oxígeno/química , Fenotipo , Polimorfismo de Nucleótido Simple , Transcripción Genética , Vitamina K/metabolismo , Vitamina K Epóxido Reductasas/genética