RESUMO
Bidirectional transcription of mammalian mitochondrial DNA generates overlapping transcripts that are capable of forming double-stranded RNA (dsRNA) structures. Release of mitochondrial dsRNA into the cytosol activates the dsRNA-sensing immune signaling, which is a defense mechanism against microbial and viral attack and possibly cancer, but could cause autoimmune diseases when unchecked. A better understanding of the process is vital in therapeutic application of this defense mechanism and treatment of cognate human diseases. In addition to exporting dsRNAs, mitochondria also export and import a variety of non-coding RNAs. However, little is known about how these RNAs are transported across mitochondrial membranes. Here we provide direct evidence showing that adenine nucleotide translocase-2 (ANT2) functions as a mammalian RNA translocon in the mitochondrial inner membrane, independent of its ADP/ATP translocase activity. We also show that mitochondrial dsRNA efflux through ANT2 triggers innate immunity. Inhibiting this process alleviates inflammation in vivo, providing a potential therapeutic approach for treating autoimmune diseases.
Assuntos
Translocador 2 do Nucleotídeo Adenina , Mitocôndrias , Membranas Mitocondriais , RNA de Cadeia Dupla , Animais , Translocador 2 do Nucleotídeo Adenina/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Humanos , RNA de Cadeia Dupla/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Camundongos , Imunidade Inata , Transporte de RNA , Células HEK293 , Camundongos Endogâmicos C57BLRESUMO
Successful completion of spermatogenesis is crucial for the perpetuation of the species. In Drosophila, spermatid individualization, a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm. Ant2, encoding a mitochondrial adenine nucleotide translocase, is highly expressed in male testes and plays a role in energy metabolism in the mitochondria. However, its molecular function remains unclear. Here, we identified an important role of Ant2 in spermatid individualization. In Ant2 knockdown testes, spermatid individualization complexes composed of F-actin cones exhibited a diffuse distribution, and mature sperms were absent in the seminal vesicle, thus leading to male sterility. The most striking effects in Ant2-knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function. Excessive apoptotic cells were also observed in Ant2-knockdown testes. To further investigate the phenotype of Ant2 knockdown in testes at the molecular level, complementary transcriptome and proteome analyses were performed. At the mRNA level, 868 differentially expressed genes were identified, of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown. iTRAQ-labeling proteome analysis revealed 350 differentially expressed proteins, of which 117 proteins were upregulated and 233 were downregulated. The expression of glutathione transferase (GstD5, GstE5, GstE8, and GstD3), proteins involved in reproduction were significantly regulated at both the mRNA and protein levels. These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Espermatogênese , Animais , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Testículo/metabolismo , Translocador 2 do Nucleotídeo Adenina/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Espermátides/metabolismoRESUMO
Adenine Nucleotide Translocator isoforms (ANTs) exchange ADP/ATP across the inner mitochondrial membrane, are also voltage-activated proton channels and regulate mitophagy and apoptosis. The ANT1 isoform predominates in heart and muscle while ANT2 is systemic. Here, we report the creation of Ant mutant mouse myoblast cell lines with normal Ant1 and Ant2 genes, deficient in either Ant1 or Ant2, and deficient in both the Ant1 and Ant2 genes. These cell lines are immortal under permissive conditions (IFN-γ + serum at 32 °C) permitting expansion but return to normal myoblasts that can be differentiated into myotubes at 37 °C. With this system we were able to complement our Ant1 mutant studies by demonstrating that ANT2 is important for myoblast to myotube differentiation and myotube mitochondrial respiration. ANT2 is also important in the regulation of mitochondrial biogenesis and antioxidant defenses. ANT2 is also associated with increased oxidative stress response and modulation for Ca++ sequestration and activation of the mitochondrial permeability transition (mtPTP) pore during cell differentiation.
Assuntos
Translocador 2 do Nucleotídeo Adenina , Nucleotídeos de Adenina , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Camundongos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Desenvolvimento Muscular/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a lack of dystrophin, a protein essential for myocyte integrity. Mitochondrial dysfunction is reportedly responsible for DMD. This study examines the effect of glucocorticoid deflazacort on the functioning of the skeletal-muscle mitochondria of dystrophin-deficient mdx mice and WT animals. Deflazacort administration was found to improve mitochondrial respiration of mdx mice due to an increase in the level of ETC complexes (complexes III and IV and ATP synthase), which may contribute to the normalization of ATP levels in the skeletal muscle of mdx animals. Deflazacort treatment improved the rate of Ca2+ uniport in the skeletal muscle mitochondria of mdx mice, presumably by affecting the subunit composition of the calcium uniporter of organelles. At the same time, deflazacort was found to reduce the resistance of skeletal mitochondria to MPT pore opening, which may be associated with a change in the level of ANT2 and CypD. In this case, deflazacort also affected the mitochondria of WT mice. The paper discusses the mechanisms underlying the effect of deflazacort on the functioning of mitochondria and contributing to the improvement of the muscular function of mdx mice.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/tratamento farmacológico , Pregnenodionas/farmacologia , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Peptidil-Prolil Isomerase F/genética , Peptidil-Prolil Isomerase F/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologiaRESUMO
BACKGROUND: The aberrant expression of genes involved in androgen metabolism and genetic contribution are unclear in hypospadias. METHODS: We compared gene expression profiles by RNA sequencing from five non-hypospadiac foreskins, five mild hypospadiac foreskins, and five severe hypospadiac foreskins. In addition, to identify rare coding variants with large effects on hypospadias risk, we carried out whole exome sequencing in three patients in a hypospadias family. RESULTS: The average expression of androgen receptor (AR) and CYP19A1 were significantly decreased in severe hypospadias (p < .01) and mild hypospadias (p < .05), whereas expression of several other androgen metabolism enzymes, including CYP3A4, HSD17B14, HSD3B7, HSD17B7, CYP11A1 were exclusively significantly expressed in severe hypospadias (p < .05). Compound rare damaging mutants of AR gene with HSD3B1 and SLC25A5 genes were identified in the different severe hypospadias. CONCLUSIONS: In conclusion, our findings demonstrated that dysregulation of AR and CYP19A1 could play a crucial role in the development of hypospadias. Inconsistent AR expression may be caused by the feedback loop of ESR1 signaling or combined genetic effects with other risk genes. This findings complement the possible role of AR triggered mechanism in the development of hypospadias.
Assuntos
Androgênios/genética , Hipospadia/genética , Mutação , Transcriptoma , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Androgênios/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Criança , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Exoma , Humanos , Hipospadia/patologia , Masculino , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Progesterona Redutase/genética , Progesterona Redutase/metabolismo , Proteína A6 Ligante de Cálcio S100/genética , Proteína A6 Ligante de Cálcio S100/metabolismo , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca2+ homeostasis. This work examines the kinetic parameters of Ca2+ transport and the opening of the Ca2+-dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca2+ uniport, with the kinetics of Na+-dependent Ca2+ efflux not changing. The data obtained indicate that the decreased rate of Ca2+ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca2+ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca2+ homeostasis of skeletal-muscle mitochondria.
Assuntos
Cálcio/metabolismo , Mitocôndrias Musculares/patologia , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria/genética , Distrofia Muscular de Duchenne/patologia , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Animais , Cátions Bivalentes/metabolismo , Modelos Animais de Doenças , Distrofina/genética , Distrofina/metabolismo , Humanos , Transporte de Íons/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos mdx , Microscopia Eletrônica , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Distrofia Muscular de Duchenne/genética , Fosforilação OxidativaRESUMO
ADP/ATP carriers (AACs) are mitochondrial transport proteins playing a strategic role in maintaining the respiratory chain activity, fueling the cell with ATP, and also regulating mitochondrial apoptosis. To understand if AACs might represent a new molecular target for cancer treatment, we evaluated AAC expression levels in cancer/normal tissue pairs available on the Tissue Cancer Genome Atlas database (TCGA), observing that AACs are dysregulated in most of the available samples. It was observed that at least two AACs showed a significant differential expression in all the available kidney cancer/normal tissue pairs. Thus, we investigated AAC expression in the corresponding kidney non-cancer (HK2)/cancer (RCC-Shaw and CaKi-1) cell lines, grown in complete medium or serum starvation, for investigating how metabolic alteration induced by different growth conditions might influence AAC expression and resistance to mitochondrial apoptosis initiators, such as "staurosporine" or the AAC highly selective inhibitor "carboxyatractyloside". Our analyses showed that AAC2 and AAC3 transcripts are more expressed than AAC1 in all the investigated kidney cell lines grown in complete medium, whereas serum starvation causes an increase of at least two AAC transcripts in kidney cancer cell lines compared to non-cancer cells. However, the total AAC protein content is decreased in the investigated cancer cell lines, above all in the serum-free medium. The observed decrease in AAC protein content might be responsible for the decrease of OXPHOS activity and for the observed lowered sensitivity to mitochondrial apoptosis induced by staurosporine or carboxyatractyloside. Notably, the cumulative probability of the survival of kidney cancer patients seriously decreases with the decrease of AAC1 expression in KIRC and KIRP tissues making AAC1 a possible new biomarker of metabolic remodeling and survival in kidney cancers.
Assuntos
Translocador 2 do Nucleotídeo Adenina/genética , Translocador 3 do Nucleotídeo Adenina/genética , Arilamina N-Acetiltransferase/genética , Isoenzimas/genética , Neoplasias Renais/genética , Translocases Mitocondriais de ADP e ATP/genética , Sequência de Aminoácidos/genética , Apoptose/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Rim/metabolismo , Rim/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Translocases Mitocondriais de ADP e ATP/metabolismo , Fosforilação OxidativaRESUMO
In addition to skeletal muscle dysfunction, cancer cachexia is a systemic disease involving remodeling of nonmuscle organs such as adipose and liver. Impairment of mitochondrial function is associated with multiple chronic diseases. The tissue-specific control of mitochondrial function in cancer cachexia is not well defined. This study determined mitochondrial respiratory capacity and coupling control of skeletal muscle, white adipose tissue (WAT), and liver in colon-26 (C26) tumor-induced cachexia. Tissues were collected from PBS-injected weight-stable mice, C26 weight-stable mice and C26 mice with moderate (10% weight loss) and severe cachexia (20% weight loss). The respiratory control ratio [(RCR) an index of oxidative phosphorylation (OXPHOS) coupling efficiency] was low in WAT during the induction of cachexia because of high nonphosphorylating LEAK respiration. Liver RCR was low in C26 weight-stable and moderately cachexic mice because of reduced OXPHOS. Liver RCR was further reduced with severe cachexia, where Ant2 but not Ucp2 expression was increased. Ant2 was inversely correlated with RCR in the liver (r = -0.547, P < 0.01). Liver cardiolipin increased in moderate and severe cachexia, suggesting this early event may also contribute to mitochondrial uncoupling. Impaired skeletal muscle mitochondrial respiration occurred predominantly in severe cachexia, at complex I. These findings suggest that mitochondrial function is subject to tissue-specific control during cancer cachexia, whereby remodeling in WAT and liver arise early and may contribute to altered energy balance, followed by impaired skeletal muscle respiration. We highlight an under-recognized role of liver and WAT mitochondrial function in cancer cachexia and suggest mitochondrial function of multiple tissues to be therapeutic targets.
Assuntos
Caquexia/metabolismo , Mitocôndrias Musculares/metabolismo , Neoplasias Experimentais/metabolismo , Consumo de Oxigênio/fisiologia , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Animais , Cardiolipinas/metabolismo , Neoplasias do Colo , Fígado/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Acoplamento Oxidativo , Distribuição Aleatória , Espécies Reativas de Oxigênio , Redução de PesoRESUMO
Cellular senescence is a form of cell cycle arrest that limits the proliferative potential of cells, including tumour cells. However, inability of immune cells to subsequently eliminate senescent cells from the organism may lead to tissue damage, inflammation, enhanced carcinogenesis and development of age-related diseases. We found that the anticancer agent mitochondria-targeted tamoxifen (MitoTam), unlike conventional anticancer agents, kills cancer cells without inducing senescence in vitro and in vivo. Surprisingly, it also selectively eliminates both malignant and non-cancerous senescent cells. In naturally aged mice treated with MitoTam for 4 weeks, we observed a significant decrease of senescence markers in all tested organs compared to non-treated animals. Mechanistically, we found that the susceptibility of senescent cells to MitoTam is linked to a very low expression level of adenine nucleotide translocase-2 (ANT2), inherent to the senescent phenotype. Restoration of ANT2 in senescent cells resulted in resistance to MitoTam, while its downregulation in non-senescent cells promoted their MitoTam-triggered elimination. Our study documents a novel, translationally intriguing role for an anticancer agent targeting mitochondria, that may result in a new strategy for the treatment of age-related diseases and senescence-associated pathologies.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Antineoplásicos Hormonais/farmacologia , Senescência Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Tamoxifeno/farmacologia , Translocador 2 do Nucleotídeo Adenina/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Mitocôndrias/metabolismo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. More than 80% of patients with HCC are not good candidates for curative surgical resection due to advanced liver cirrhosis caused by underlying chronic hepatitis virus (B or C) infection. Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced HCC. Although, sorafenib currently sets the new standard for advanced HCC treatment, tumor response rates are usually quite low. An understanding of the underlying mechanisms for sorafenib resistance is critical. In the present study, we found that adenine nucleotide translocator 2 (ANT2) was upregulated in sorafenibresistant HCC Huh7 cells (Huh7-R) and its overexpression promoted sorafenib resistance. ANT2 induced the formation of cancer-initiating cell (CIC) phenotypes and promoted metastasis-associated traits in the Huh7 cells. Silencing of miR-137 upregulated ANT2 protein expression in the Huh7 cells. miR-137 was downregulated in the Huh7-R cells, compared with that in the Huh7 cells and its restoration reversed sorafenib resistance in the Huh7-R cells. Restoration of miR-137 inhibited formation of CIC traits and attenuated the abilities of migration and invasion in the Huh7-R cells. Moreover, we demonstrated that high-intensity focused ultrasound (HIFU) in unresectable HCC upregulated serum miR-137. Combining HIFU and sorafenib may be a wise option for advanced and unresectable HCC.
Assuntos
Translocador 2 do Nucleotídeo Adenina/genética , Carcinoma Hepatocelular/genética , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/genética , MicroRNAs/genética , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Regulação para Cima , Regiões 3' não Traduzidas , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Ablação por Ultrassom Focalizado de Alta Intensidade , Humanos , Masculino , Células-Tronco Neoplásicas/efeitos dos fármacos , Niacinamida/farmacologia , SorafenibeRESUMO
Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Fígado Gorduroso/metabolismo , Resistência à Insulina , Mitocôndrias Hepáticas/metabolismo , Substâncias Protetoras/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Animais , Atractilosídeo/análogos & derivados , Dieta Hiperlipídica , Modelos Animais de Doenças , Fígado Gorduroso/terapia , Feminino , Técnica Clamp de Glucose , Hiperinsulinismo , Metabolismo dos Lipídeos , Lipogênese , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/terapia , Obesidade/metabolismo , Obesidade/terapia , Ácido Pirúvico/metabolismoRESUMO
The mouse fetal and adult hearts express two adenine nucleotide translocator (ANT) isoform genes. The predominant isoform is the heart-muscle-brain ANT-isoform gene 1 (Ant1) while the other is the systemic Ant2 gene. Genetic inactivation of the Ant1 gene does not impair fetal development but results in hypertrophic cardiomyopathy in postnatal mice. Using a knockin X-linked Ant2 allele in which exons 3 and 4 are flanked by loxP sites combined in males with a protamine 1 promoter driven Cre recombinase we created females heterozygous for a null Ant2 allele. Crossing the heterozygous females with the Ant2(fl), PrmCre(+) males resulted in male and female ANT2-null embryos. These fetuses proved to be embryonic lethal by day E14.5 in association with cardiac developmental failure, immature cardiomyocytes having swollen mitochondria, cardiomyocyte hyperproliferation, and cardiac failure due to hypertrabeculation/noncompaction. ANTs have two main functions, mitochondrial-cytosol ATP/ADP exchange and modulation of the mitochondrial permeability transition pore (mtPTP). Previous studies imply that ANT2 biases the mtPTP toward closed while ANT1 biases the mtPTP toward open. It has been reported that immature cardiomyocytes have a constitutively opened mtPTP, the closure of which signals the maturation of cardiomyocytes. Therefore, we hypothesize that the developmental toxicity of the Ant2 null mutation may be the result of biasing the cardiomyocyte mtPTP to remain open thus impairing cardiomyocyte maturation and resulting in cardiomyocyte hyperproliferation and failure of trabecular maturation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Assuntos
Translocador 2 do Nucleotídeo Adenina/deficiência , Cardiopatias Congênitas/genética , Insuficiência Cardíaca/genética , Ventrículos do Coração/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Adenina/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Animais , Transporte Biológico , Proliferação de Células , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Insuficiência Cardíaca/embriologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/anormalidades , Ventrículos do Coração/embriologia , Integrases , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/patologia , Dilatação Mitocondrial/genética , Miócitos Cardíacos/patologia , Organogênese , FenótipoRESUMO
MicroRNAs (miRNAs) are negative regulators of gene expression, and miRNA deregulation is found in various tumors. We previously reported that suppression of adenine nucleotide translocase 2 (ANT2) by short hairpin RNA (shRNA) inhibits hepatocellular carcinoma (HCC) development by rescuing miR-636 expression. However, the tumor-suppressive mechanisms of ANT2 shRNA are still poorly understood in HCC. Here, we hypothesized that miRNAs that are specifically downregulated by ANT2 shRNA might function as oncomiRs, and we investigated the roles of ANT2 shRNA-regulated miRNAs in the pathogenesis of HCC. Our data show that miR-19a and miR-96, whose expression is regulated by ANT2 suppression, were markedly upregulated in HCC cell lines and clinical samples. Ectopic expression of miR-19a and miR-96 dramatically induced the proliferation and colony formation of hepatoma cells in vitro, whereas inhibition of miR-19a and miR-96 reduced these effects. To investigate the in vivo function, we implanted miR-96-overexpressing HepG2 cells in a xenograft model and demonstrated that the increase in miR-96 promoted tumor growth. We also found that miR-19a and miR-96 inhibited expression of tissue inhibitor of metalloproteinase-2. Taken together, our results suggest that ANT2-regulated miR-19a and miR-96 play an important role in promoting the proliferation of human HCC cells, and the knockdown of ANT2 directly downregulates miR-19a and miR-96, ultimately resulting in the suppression of tumor growth.
Assuntos
Translocador 2 do Nucleotídeo Adenina/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , MicroRNAs/genética , RNA Interferente Pequeno/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de SinaisRESUMO
EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy has achieved favorable clinical outcomes in non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, patients eventually develop resistance to EGFR-TKIs by several mechanisms. Adenine nucleotide translocase-2 (ANT2) is an oncogenic mitochondrial membrane-associated protein. We investigated the therapeutic potential of ANT2 inhibition to EGFR-TKI resistance in NSCLC using gefitinib-sensitive (PC9 and HCC827) and gefitinib-resistant (H1975 and HCC827/GR) NSCLC cell lines. ANT2 was inhibited by transfecting cells with an ANT2-specific shRNA. ANT2 expression was elevated in the H1975 and HCC827/GR cells compared with the PC9 and HCC827 cells. ANT2 upregulation in gefitinib-resistant cells was associated with increased SP1 binding to the ANT2 promoter. ANT2-specific shRNA decreased NSCLC cell viability. Moreover, ANT2-specific shRNA sensitized the H1975 and HCC827/GR cells to gefitinib, accompanied by HSP90 and EGFR downregulation. ANT2-specific shRNA also inactivated the PI3K/Akt signaling pathway in the H1975 and HCC827/GR cells, which was mediated by the suppression of miR-221/222 levels and by the subsequent restoration of PTEN. In EGFR-TKI-treated NSCLC patients, ANT2 expression was higher in patients exhibiting poor responses compared with patients showing excellent responses. Furthermore, ANT2 expression increased in tumor tissues biopsied after acquiring gefitinib resistance compared with tissues before gefitinib treatment. These findings suggest that ANT2 overexpression contributes to EGFR-TKI resistance in NSCLC and that ANT2 targeting may be considered a novel strategy for overcoming this resistance. Mol Cancer Ther; 15(6); 1387-96. ©2016 AACR.
Assuntos
Translocador 2 do Nucleotídeo Adenina/genética , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Quinazolinas/farmacologia , RNA Interferente Pequeno/farmacologia , Translocador 2 do Nucleotídeo Adenina/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Gefitinibe , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Terapia de Alvo Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Adenine nucleotide translocases (ANTs) transport ADP and ATP through mitochondrial inner membrane, thus playing an essential role for energy metabolism of eukaryotic cells. Mice have three ANT paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. While knockout mice have been characterized with Ant1 and Ant4 genes, which resulted in exercise intolerance and male infertility, respectively, the role of the ubiquitously expressed Ant2 gene in animal development has not been fully demonstrated. Here, we generated Ant2 hypomorphic mice by targeted disruption of the gene, in which Ant2 expression is largely depleted. The mice showed apparently normal embryonic development except pale phenotype along with a reduced birth rate. However, postnatal growth was severely retarded with macrocytic anemia, B lymphocytopenia, lactic acidosis and bloated stomach, and died within 4 weeks. Ant2 depletion caused anemia in a cell-autonomous manner by maturation arrest of erythroid precursors with increased reactive oxygen species and premature deaths. B-lymphocyte development was similarly affected by Ant2 depletion, and splenocytes showed a reduction in maximal respiration capacity and cellular ATP levels as well as an increase in cell death accompanying mitochondrial permeability transition pore opening. In contrast, myeloid, megakaryocyte and T-lymphocyte lineages remained apparently intact. Erythroid and B-cell development may be particularly vulnerable to Ant2 depletion-mediated mitochondrial dysfunction and oxidative stress.
Assuntos
Translocador 2 do Nucleotídeo Adenina/deficiência , Linfócitos B/citologia , Eritrócitos/citologia , Eritropoese/fisiologia , Linfopoese/fisiologia , Translocases Mitocondriais de ADP e ATP/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Anemia/genética , Anemia/metabolismo , Animais , Linfócitos B/metabolismo , Eritrócitos/metabolismo , Eritropoese/genética , Feminino , Técnicas de Inativação de Genes , Transtornos do Crescimento/genética , Transtornos do Crescimento/metabolismo , Humanos , Linfopoese/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismoRESUMO
Oxidative stress and persistent activation of DNA damage response (DDR) are causally involved in the development of cellular senescence, a phenomenon implicated in fundamental (patho)physiological processes such as aging, fetal development and tumorigenesis. Here, we report that adenine nucleotide translocase-2 (ANT2) is consistently down-regulated in all three major forms of cellular senescence: replicative, oncogene-induced and drug-induced, in both normal and cancerous human cells. We previously reported formation of novel NF1/Smad transcription repressor complexes in growth-arrested fibroblasts. Here we show that such complexes form in senescent cells. Mechanistically, binding of the NF1/Smad complexes to the NF1-dependent repressor elements in the ANT2 gene promoter repressed ANT2 expression. Etoposide-induced formation of these complexes and repression of ANT2 were relatively late events co-incident with production and secretion of, and dependent on, TGF-ß. siRNA-mediated knock-down of ANT2 in proliferating cells resulted in increased levels of reactive oxygen species (ROS) and activation of the DDR. Knock-down of ANT2, together with etoposide treatment, further intensified ROS production and DNA damage signaling, leading to enhanced apoptosis. Together, our data show that TGF-ß-mediated suppression of ANT2 through NF1/Smad4 complexes contributes to oxidative stress and DNA damage during induction of cellular senescence.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Senescência Celular , Fatores de Transcrição NFI/metabolismo , Estresse Oxidativo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Senescência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Dano ao DNA , Regulação para Baixo/efeitos dos fármacos , Etoposídeo/farmacologia , Humanos , Mutação , Estresse Oxidativo/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismoRESUMO
PURPOSE: The purpose of this study was to investigate the anticancer effects of combined RNA interference (RNAi) of the adenine nucleotide translocase-2 (ANT2) gene and ganciclovir (GCV) therapy for treatment of hepatocellular carcinoma cells (Huh 7) in an animal model. METHODS: The Huh 7/NTG stable cell line was established by transfection of a vector with the human sodium iodide symporter (hNIS), HSV1-sr39 thymidine kinase (tk), and enhanced green florescent protein (EGFP) fusion gene into Huh 7 cells. mRNA expressions of these genes were evaluated by RT-PCR analysis. The functions of hNIS and HSV1-sr39tk were verified with (125)I uptake and (3)H-penciclovir (PCV) uptake tests. EGFP and hNIS expression was confirmed with confocal microscopy after immunocytochemical staining. We treated the tumor cells with ANT2 shRNA or GCV or both ANT2 shRNA and GCV and treated the in vivo mouse model with a Huh 7/NTG tumor xenograft. The therapeutic effects of the in vivo study were assessed with caliper measurements and gamma camera imaging using (99m)Tc-pertechnetate. RESULTS: Huh 7/NTG cells showed a cell number-dependent increase in (125)I uptake and a 24-fold higher (3)H-PCV uptake compared to parent Huh 7 cells. Huh 7/NTG cells transfected with ANT2 shRNA had lower ANT2 mRNA expression and more impaired proliferation activity than cells transfected with scramble shRNA. Proliferation of Huh 7/NTG cells was also inhibited by GCV treatment. Combined GCV and ANT2 shRNA therapy further inhibited cell proliferation in the in vitro study. The combined therapy with GCV and ANT2 shRNA showed a further decrease in tumor growth in the mouse model. CONCLUSIONS: Our results suggest that the combined RNA interference with ANT2 and GCV therapy inhibited hepatocellular carcinoma cell proliferation more than single GCV therapy or ANT2 shRNA therapy in vitro and in vivo. Therefore it could be applied treating incurable hepatocellular carcinoma.
Assuntos
Translocador 2 do Nucleotídeo Adenina/deficiência , Translocador 2 do Nucleotídeo Adenina/genética , Carcinoma Hepatocelular/terapia , Ganciclovir/farmacologia , Neoplasias Hepáticas/terapia , Interferência de RNA , Animais , Transporte Biológico , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Transformação Celular Neoplásica , Ganciclovir/metabolismo , Ganciclovir/uso terapêutico , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Camundongos , Pró-Fármacos/farmacologia , Cintilografia , Pertecnetato Tc 99m de Sódio/metabolismo , Timidina Quinase/genéticaRESUMO
Loss-of-function mutations in several different neuronal pathways have been related to intellectual disability (ID). Such mutations often are found on the X chromosome in males since they result in functional null alleles. So far, microdeletions at Xq24 reported in males always have been associated with a syndromic form of ID due to the loss of UBE2A. Here, we report on overlapping microdeletions at Xq24 that do not include UBE2A or affect its expression, in patients with non-syndromic ID plus some additional features from three unrelated families. The smallest region of overlap, confirmed by junction sequencing, harbors two members of the mitochondrial solute carrier family 25, SLC25A5 and SLC25A43. However, identification of an intragenic microdeletion including SLC25A43 but not SLC25A5 in a healthy boy excluded a role for SLC25A43 in cognition. Therefore, our findings point to SLC25A5 as a novel gene for non-syndromic ID. This highly conserved gene is expressed ubiquitously with high levels in cortex and hippocampus, and a presumed role in mitochondrial exchange of ADP/ATP. Our data indicate that SLC25A5 is involved in memory formation or establishment, which could add mitochondrial processes to the wide array of pathways that regulate normal cognitive functions.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Deleção Cromossômica , Cromossomos Humanos X/genética , Deficiência Intelectual/genética , Mitocôndrias/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Elementos Alu , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/patologia , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Heterozigoto , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Mitocôndrias/genética , Dados de Sequência Molecular , Linhagem , Inativação do Cromossomo XRESUMO
BACKGROUND: It is important to simultaneously induce strong cell death and antitumor immunity in cancer patients for successful cancer treatment. Here, we investigated the cytotoxic and phenotypic modulation effects of the combination of ANT2 shRNA and human sodium iodide symporter (hNIS) radioiodine gene therapy in vitro and in vivo and visualized the antitumor effects in an immunocompromised mouse colon cancer model. METHODS: A mouse colon cancer cell line co-expressing hNIS and the luciferase gene (CT26/hNIS-Fluc, named CT26/NF) was established. CT26/NF cells and tumor-bearing mice were treated with HBSS, scramble, ANT2 shRNA, I-131, and ANT2 shRNA + I-131. The apoptotic rates (%) and MHC class I and Fas gene expression levels were determined in treated CT26/NF cells using flow cytometry. Concurrently, the level of caspase-3 activation was determined in treated cells in vitro. For in vivo therapy, tumor-bearing mice were treated with scramble, ANT2 shRNA, I-131, and the combination therapy, and the anti-tumor effects were monitored using bioluminescence. The killing activity of cytotoxic T cells (CTLs) was measured with a lactate dehydrogenase (LDH) assay. RESULTS: For the in vitro experiments, the combination of ANT2 shRNA and I-131 resulted in a higher apoptotic cell death rate compared with ANT2 shRNA or I-131 alone, and the levels of MHC class I and Fas-expressing cancer cells were highest in the cells receiving combination treatment, while single treatment modestly increased the level of MHC class I and Fas gene expression. The combination of ANT2 shRNA and I-131 resulted in a higher caspase-3 activation than single treatments. Interestingly, in vivo combination treatment led to increased gene expression of MHC class I and Fas than the respective mono-therapies; furthermore, bioluminescence showed increased antitumor effects after combination treatment than monotherapies. The LDH assay revealed that the CTL killing activity against CT26/NF cells was most effective after combination therapy. CONCLUSIONS: Increased cell death and phenotypic modulation of cancer cells in vitro and in vivo were achieved simultaneously after combination therapy with ANT2 shRNA and I-131, and this combination therapy induced remarkable antitumor outcomes through improvements in CTL immunity against CT26/NF. Our results suggest that combination therapy can be used as a new therapeutic strategy for cancer patients who show resistance to single therapy such as radiation or immunotherapy.