RESUMEN
The abuse or misuse of antibiotics has caused the emergence of extensively drug-resistant (XDR) bacteria, rendering most antibiotics ineffective and increasing the mortality rate of patients with bacteremia or sepsis. Antimicrobial peptides (AMPs) are proposed to overcome this problem; however, many AMPs have attenuated antimicrobial activities with hemolytic toxicity in blood. Recently, AMPR-11 and its optimized derivative, AMPR-22, were reported to be potential candidates for the treatment of sepsis with a broad spectrum of antimicrobial activity and low hemolytic toxicity. Here, we performed molecular dynamics (MD) simulations to clarify the mechanism of lower hemolytic toxicity and higher efficacy of AMPR-22 at an atomic level. We found four polar residues in AMPR-11 bound to a model mimicking the bacterial inner/outer membranes preferentially over eukaryotic plasma membrane. AMPR-22 whose polar residues were replaced by lysine showed a 2-fold enhanced binding affinity to the bacterial membrane by interacting with bacterial specific lipids (lipid A or cardiolipin) via hydrogen bonds. The MD simulations were confirmed experimentally in models that partially mimic bacteremia conditions in vitro and ex vivo. The present study demonstrates why AMPR-22 showed low hemolytic toxicity and this approach using an MD simulation would be helpful in the development of AMPs.
Asunto(s)
Bacteriemia , Proteínas de la Membrana , Proteínas Mitocondriales , Simulación de Dinámica Molecular , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Bacteriemia/metabolismo , Bacterias , Membrana Celular/metabolismo , Hemólisis , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/farmacología , Pruebas de Sensibilidad Microbiana , Proteínas Mitocondriales/química , Proteínas Mitocondriales/farmacologíaRESUMEN
The emergence of multidrug-resistant (MDR) bacteria through the abuse and long-term use of antibiotics is a serious health problem worldwide. Therefore, novel antimicrobial agents that can cure an infection from MDR bacteria, especially gram-negative bacteria, are urgently needed. Antimicrobial peptides, part of the innate immunity system, have been studied to find bactericidal agents potent against MDR bacteria. However, they have many problems, such as restrained systemic activity and cytotoxicity. In a previous study, we suggested that the K58-R78 domain of Romo1, a mitochondrial protein encoded by the nucleus, was a promising treatment candidate for sepsis caused by MDR bacteria. Here, we performed sequence optimization to enhance the antimicrobial activity of this peptide and named it as AMPR-22 (antimicrobial peptide derived from Romo1). It showed broad-spectrum antimicrobial activity against 17 sepsis-causing bacteria, including MDR strains, by inducing membrane permeabilization. Moreover, treatment with AMPR-22 enabled a remarkable survival rate in mice injected with MDR bacteria in a murine model of sepsis. Based on these results, we suggest that AMPR-22 could be prescribed as a first-line therapy (prior to bacterial identification) for patients diagnosed with sepsis.
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Proteínas de la Membrana/química , Proteínas Mitocondriales/química , Fragmentos de Péptidos/uso terapéutico , Proteínas Citotóxicas Formadoras de Poros/uso terapéutico , Sepsis/tratamiento farmacológico , Animales , Células Cultivadas , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/farmacología , Dominios Proteicos , Sepsis/microbiologíaRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to behave as an attractive anti-cancer agent in various cancers. Despite its promise TRAIL has limitations such as short half-life and rapid development of resistance. In this regard, approaches to sensitizers of TRAIL that can overcome the limitations of TRAIL are necessary. However, the molecular targets and mechanisms underlying sensitization to TRAIL-induced apoptosis are not fully understood. Here, we propose that reactive oxygen species modulator-1 (Romo1) as an attractive sensitizer of TRAIL. Romo1 is a mitochondrial inner membrane channel protein that controls reactive oxygen species (ROS) production, and its expression is highly upregulated in various cancers, including colorectal cancer. In the present study, we demonstrated that Romo1 inhibition significantly increased TRAIL-induced apoptosis of colorectal cancer cells, but not of normal colon cells. The combined effect of TRAIL and Romo1 inhibition was correlated with the activation of mitochondrial apoptosis pathways. Romo1 silencing elevated the protein levels of BCL-2-associated X protein (Bax) by downregulating the ubiquitin proteasome system (UPS). Romo1 inhibition downregulated the interaction between Bax and Parkin. Furthermore, Romo1 knockdown triggered the mitochondrial dysfunction and ROS generation. We validated the effect of combination in tumor xenograft model in vivo. In conclusion, our study demonstrates that Romo1 inhibition induces TRAIL-mediated apoptosis by identifying the novel mechanism associated with the Bax/Parkin interaction. We suggest that targeting of Romo1 is essential for the treatment of colorectal cancer and may be a new therapeutic approach in the future and contribute to the drug discovery.
RESUMEN
To overcome increasing bacterial resistance to conventional antibiotics, many antimicrobial peptides (AMPs) derived from host defense proteins have been developed. However, there are considerable obstacles to their application to systemic infections because of their low bioavailability. In the present study, we developed an AMP derived from Romo1 (AMPR-11) that exhibits a broad spectrum of antimicrobial activity. AMPR-11 showed remarkable efficacy against sepsis-causing bacteria, including multidrug-resistant strains, with low toxicity in a murine model of sepsis after intravenous administration. It seems that AMPR-11 disrupts bacterial membranes by interacting with cardiolipin and lipid A. From the results of this study, we suggest that AMPR-11 is a new class of agent for overcoming low efficacy in the intravenous application of AMPs and is a promising candidate to overcome multidrug resistance.IMPORTANCE Abuse of antibiotics often leads to increase of multidrug-resistant (MDR) bacteria, which threatens the life of human beings. To overcome threat of antibiotic resistance, scientists are developing a novel class of antibiotics, antimicrobial peptides, that can eradicate MDR bacteria. Unfortunately, these antibiotics have mainly been developed to cure bacterial skin infections rather than others, such as life-threatening sepsis. Major pharmaceutical companies have tried to develop antiseptic drugs; however, they have not been successful. Here, we report that AMPR-11, the antimicrobial peptide (AMP) derived from mitochondrial nonselective channel Romo1, has antimicrobial activity against Gram-positive and Gram-negative bacteria comprising many clinically isolated MDR strains. Moreover, AMPR-11 increased the survival rate in a murine model of sepsis caused by MDR bacteria. We propose that AMPR-11 could be a novel antiseptic drug candidate with a broad antimicrobial spectrum to overcome MDR bacterial infection.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/uso terapéutico , Bacterias/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple , Proteínas de la Membrana/uso terapéutico , Proteínas Mitocondriales/uso terapéutico , Sepsis/tratamiento farmacológico , Administración Intravenosa , Animales , Antiinfecciosos/uso terapéutico , Membrana Externa Bacteriana/efectos de los fármacos , Biopelículas/efectos de los fármacos , Modelos Animales de Enfermedad , Células HeLa , Humanos , Masculino , Proteínas de la Membrana/química , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Proteínas Mitocondriales/químicaRESUMEN
Hepatitis C virus (HCV) p7 is known to be a nonselective cation channel for HCV maturation. Because the interaction of HCV proteins with host lipids in the endoplasmic reticulum membrane is crucial for the budding process, the identification of p7-lipid interactions could be important for understanding the HCV life cycle. Here, we report that p7 interacts with phosphatidylserine (PS) to induce membrane permeabilization. The interaction of p7 with PS was not inhibited by Gd3+ ions, which have been known to interact with negatively charged lipids, but channel activity and p7-induced mitochondrial depolarization were inhibited by Gd3+ ions. From the present results, we suggest that the p7-PS interaction plays an essential role in regulating its ion channel function and could be a potential molecular target for anti-HCV therapy.
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Hepacivirus/fisiología , Hepatitis C/virología , Canales Iónicos/antagonistas & inhibidores , Fosfatidilserinas/metabolismo , Proteínas Virales/metabolismo , Permeabilidad de la Membrana Celular , Retículo Endoplásmico/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virología , Mitocondrias/metabolismoRESUMEN
Hepatitis B virus X protein (HBx) functions in a variety of cellular events during the HBV life cycle. In a previous study, we reported that the HBx protein is sufficient to induce mitochondrial membrane permeabilization; however, the exact mechanism of HBx-induced mitochondrial membrane permeabilization has been not proposed. In this study, we report that HBx specifically targets cardiolipin (CL) and induces membrane permeabilization depending on CL concentration in mitochondrial outer membrane-mimic artificial liposomes. Interestingly, HBx-induced membrane permeabilization was enhanced by liposomes containing phosphatidylethanolamine, which plays a crucial role in forming a negative curvature on the membrane. We also show that the 68-117 region of HBx, which interacts with mitochondria, is necessary for membrane permeabilization. We examined the size of the pores formed by HBx and found that HBx permeates fluorescent dyes depending on the hydrodynamic diameter with a pore size of approximately 10â¯nm. The results of this study suggest that CL is necessary for HBx-induced membrane permeabilization and provide important information that suggests a new strategy for anti-HBV therapy.
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Cardiolipinas/química , Virus de la Hepatitis B/química , Mitocondrias Hepáticas/química , Membranas Mitocondriales/química , Transactivadores/química , Animales , Cardiolipinas/metabolismo , Virus de la Hepatitis B/metabolismo , Liposomas/química , Ratones , Mitocondrias Hepáticas/metabolismo , Membranas Mitocondriales/metabolismo , Permeabilidad , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias ViralesRESUMEN
We previously reported that podophyllotoxin acetate (PA) inhibits the growth and proliferation of non-small cell lung cancer (NSCLC) cells and also makes them more sensitive to radiation and chemotherapeutic agents. In an attempt to enhance PA activity, we synthesized 34 derivatives based on podophyllotoxin (PPT). Screening of the derivative compounds for anti-cancer activity against NSCLC led to the identification of ß-apopicropodophyllin (APP) as a strong anti-cancer agent. In addition to its role as an immunosuppressive regulator of the T-cell mediated immune response, the compound additionally showed anti-cancer activity against A549, NCI-H1299 and NCI-460 cell lines with IC50 values of 16.9, 13.1 and 17.1â¯nM, respectively. The intracellular mechanisms underlying the effects of APP were additionally examined. APP treatment caused disruption of microtubule polymerization and DNA damage, which led to cell cycle arrest, as evident from accumulation of phospho-CHK2, p21, and phospho-Cdc2. Moreover, APP stimulated the pro-apoptotic ER stress signaling pathway, indicated by elevated levels of BiP, phospho-PERK, phospho-eIF2α, CHOP and ATF4. We further observed activation of caspase-3, -8 and -9, providing evidence that both intrinsic and extrinsic apoptotic pathways were triggered. In vivo, APP inhibited tumor growth of NSCLC xenografts in nude mice by promoting apoptosis. Our results collectively support a novel role of APP as an anticancer agent that evokes apoptosis by inducing microtubule disruption, DNA damage, cell cycle arrest and ER stress.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Podofilino/farmacología , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Estructura Molecular , Podofilino/síntesis química , Podofilino/químicaRESUMEN
INTRODUCTION: Reactive oxygen species modulator-1 (Romo1) is a protein that modulates levels of reactive oxygen species (ROS) and has been reported to affect cancer cell invasion and proliferation via persistent inflammation. Several studies have demonstrated the clinical application of Romo1 as a prognostic marker in non-small cell lung cancer (NSCLC); however, there have been no studies investigating the mechanism by which Romo1 adversely affects the prognosis of these patients. METHODS: We examined Romo1, ROS, and vascular endothelial growth factor (VEGF) in tumor tissues immunohistochemically. We conducted survival analyses of patients who had curative resection (n=30) in accordance with clinical parameters including levels of Romo1 expression. RESULTS: Romo1 levels were associated with serologic inflammatory markers and high lymphatic metastatic tendencies. Significantly longer disease-free survival (68.7 vs 24.2 months, P=0.031) and overall survival (92.7 vs 51.6 months) were observed in the group with low Romo1 compared with high Romo1. Survival outcomes were also significantly associated with serologic inflammatory markers. Spearman's correlation analyses demonstrated significant positive correlations of Romo1 expression with VEGF-C (P=0.008, R=0.478) and ROS (P=0.016, R=0.436) in tumor samples. CONCLUSION: The current study demonstrates that Romo1 induces lymphatic metastasis of NSCLC by modulating persistent inflammation and oxidative stress (ROS)/VEGF signaling. Lymphatic metastasis associated with elevated Romo1 was shown to be a key reason for unfavorable survival rates.
RESUMEN
Reactive oxygen species (ROS) modulator 1 (Romo1) is a nuclear-encoded mitochondrial inner membrane protein known to regulate mitochondrial ROS production and to act as an essential redox sensor in mitochondrial dynamics. Although its physiological roles have been studied for a decade, the biophysical mechanisms that explain these activities of Romo1 are unclear. In this study, we report that Romo1 is a unique mitochondrial ion channel that differs from currently identified eukaryotic ion channels. Romo1 is a highly conserved protein with structural features of class II viroporins, which are virus-encoded nonselective cation channels. Indeed, Romo1 forms a nonselective cation channel with its amphipathic helical transmembrane domain necessary for pore-forming activity. Notably, channel activity was specifically inhibited by Fe2+ ions, an essential transition metal ion in ROS metabolism. Using structural bioinformatics, we designed an experimental data-guided structural model of Romo1 with a rational hexameric structure. We propose that Romo1 establishes a new category of viroporin-like nonselective cation channel in eukaryotes.
Asunto(s)
Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencia de Aminoácidos , Cationes , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Secuencia Conservada , Células HEK293 , Humanos , Canales Iónicos/química , Hierro/farmacología , Proteínas de la Membrana/química , Proteínas Mitocondriales/química , Modelos Moleculares , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
Hepatitis C virus (HCV)encoded protein p7 is a viroporin that acts as an ion channel and is indispensable for HCV particle production. Although the main target of HCV p7 is the endoplasmic reticulum, it also targets mitochondria. HCVinfected cells show mitochondrial depolarization and ATP depletion; however, the function of HCV p7 in mitochondria is not fully understood. The present study demonstrated that treatment of isolated mouse liver mitochondria with the synthesized HCV p7 protein induced mitochondrial dysfunction. It also demonstrated that HCV p7 targeted isolated mouse liver mitochondria and induced mitochondrial depolarization. In addition, HCV p7 triggered matrix acidification and, ultimately, a decrease in ATP synthesis in isolated mitochondria. These findings indicate that targeting of mitochondria by HCV p7 in infected cells causes mitochondrial dysfunction to support HCV particle production. The present study provided evidence for the role of HCV p7 in mitochondria, and may lead to the development of novel strategies for HCV therapy.
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Potencial de la Membrana Mitocondrial , Mitocondrias Hepáticas/metabolismo , Proteínas Virales/metabolismo , Ácidos/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Masculino , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Reactive oxygen species modulator-1 (Romo1) is a novel protein that has been reported to be crucial for cancer cell proliferation and invasion. However, its clinical implications in colorectal cancer patients are not well-known. For the first time, we investigated the association between Romo1 and the clinical outcomes of colorectal cancer patients. STUDY: We examined Romo1 expression in resected tumor tissues immunohistochemically and assessed it with histological scores. We conducted survival analyses for patients who had curative resection (n = 190) in accordance with clinical parameters including level of Romo1 expression, and we examined the association between Romo1 expression and cell invasion using Matrigel invasion assay in colorectal cancer cells. RESULTS: We observed significantly longer mean disease-free survival in the low Romo1 group compared with the high Romo1 group (161 vs 127.6 months, p = 0.035), and the median overall survival of the low Romo1 group was significantly longer than that of the high Romo1 group (196.9 vs 171.3 months, p = 0.036). Cell invasiveness decreased in the Romo1 knockdown colorectal cancer cells in contrast to the controlled cells. Romo1 overexpression in tumor tissue was associated with a high lymph node ratio between the metastatic and examined lymph nodes (p = 0.025). CONCLUSIONS: Romo1 overexpression in tumor tissue was significantly associated with survival in curatively resected colorectal cancer patients, suggesting Romo1 expression as a potential adverse prognostic marker. Increased Romo1 expression was found to be associated with high lymph node ratio. Cancer invasiveness appeared to be a key reason for the poor survival related to highly expressed Romo1.
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Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/patología , Proteínas de la Membrana/fisiología , Proteínas Mitocondriales/fisiología , Adulto , Anciano , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Análisis de SupervivenciaRESUMEN
Alterations in mitochondrial respiration contribute to the development and progression of cancer via abnormal biogenesis, including generation of reactive oxygen species. Ubiquinol-cytochrome c reductase hinge protein (UQCRH) consists of the cytochrome bc1 complex serving respiration in mitochondria. In the present study, we analyzed UQCRH abnormalities in hepatocellular carcinoma (HCC) and its association with clinical outcomes of patients. UQCRH expression in HCC was determined via semiquantitative and quantitative real-time reverse transcriptase polymerase chain reaction of 96 surgically resected HCC tissues positive for hepatitis B virus surface antigen. UQCRH was frequently overexpressed in HCC tissues (46.8%, based on 2.1-fold cutoff). UQCRH overexpression was observed in HCCs with larger tumor size, poorer differentiation, or vascular invasion. Kaplan-Meier analysis revealed significantly shorter overall (P = 0.005) and recurrence-free survival (P = 0.027) in patients with tumors overexpressing UQCRH. The prognostic impact of UQCRH was significant in subgroups of patients divided according to the α-fetoprotein (AFP) level. The patient subgroup with higher AFP levels (≥20 ng/mL) exhibited significant differences in 5-year overall (18.5% vs. 67.9%) and recurrence-free survival rates (11.1% vs. 46.4%) between groups with and without UQCRH overexpression. In contrast, no marked survival differences were observed between subgroups with lower AFP levels (<20 ng/mL). Multivariate analysis defined UQCRH as an independent poor prognostic factor. Conclusively, our results indicate that UQCRH overexpression is correlated with poor outcomes of HCC patients. Furthermore, in patients grouped as high risk based on elevated AFP, lack of UQCRH overexpression could be a useful indicator for clinical treatment.
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Carcinoma Hepatocelular/patología , Complejo III de Transporte de Electrones/genética , Hepatitis B/inmunología , Neoplasias Hepáticas/patología , Regulación hacia Arriba , Adulto , Anciano , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/virología , Femenino , Regulación Neoplásica de la Expresión Génica , Antígenos de Superficie de la Hepatitis B/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Pronóstico , Carga TumoralRESUMEN
Viroporin p7 of the hepatitis C virus (HCV) acts as an ion channel for pH equilibration to stabilize HCV particles; most studies of p7 have focused on this role. However, pH equilibration by p7 via its ion channel activity does not fully explain the importance of p7 in HCV particle production. Indeed, several researchers have suggested p7 to have an unidentified ion channel-independent function. Here, we show that p7 has a novel role as a lipid raft adhesion factor, which is independent of its ion channel activity. We found that p7 targets not only the liquid-disordered (Ld) phase, but also the negatively-charged liquid-ordered (Lo) phase that can be represented as a lipid raft. p7 clusters at the phase boundary of the neutral Ld phase and the negatively-charged Lo phase. Interestingly, p7 targeting the Lo phase facilitates membrane-to-membrane adhesion, and this activity is not inhibited by p7 ion channel inhibitors. Our results demonstrated that HCV p7 has dual roles as a viroporin and as a lipid raft adhesion factor. This ion channel-independent function of p7 might be an attractive target for development of anti-HCV compounds.
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Hepacivirus/genética , Hepatitis C/genética , Proteínas Virales/genética , Secuencia de Aminoácidos/genética , Adhesión Celular/genética , Línea Celular , Regulación Viral de la Expresión Génica , Hepacivirus/metabolismo , Hepacivirus/patogenicidad , Hepatitis C/patología , Hepatitis C/virología , Humanos , Concentración de Iones de Hidrógeno , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
In this paper, the simple and biocompatible micropatterning of cell-repellent poly(N-isopropylacrylamide) (PNIPAAm) on a cell-adhesive substrate by ion beam micropatterning to control cell adhesion is described. Cell-repellent PNIPAAm films spin-coated on cell-adhesive tissue culture polystyrene (TCPS) substrates were selectively irradiated by energetic proton ions at various fluences through a pattern mask, and subsequently developed to create the micropatterns of PNIPAAm. Well-defined negative-type PNIPAAm micropatterns were successfully created on the TCPS substrates at fluences higher than 5 x 10¹4 ions/cm², and their chemical properties were dependent on the fluence. Moreover, based on the results of the protein adsorption and in-vitro cell culture tests, 200 µm well-defined micropatterns of mammalian cells were clearly formed on the PNIPAAm-micropatterned TCPS substrates though the preferential adsorption and growth of cells on the TCPS regions due to the strong cell-repellency of PNIPAAm.
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Resinas Acrílicas/farmacología , Materiales Biocompatibles/farmacología , Microtecnología/métodos , Polímeros/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células HeLa , Humanos , Iones , Ratones , Microscopía Fluorescente , Espectroscopía Infrarroja por Transformada de Fourier , Agua/químicaRESUMEN
Reactive oxygen species (ROS) are important contributors to tumor cell invasion. ROS enhanced by reactive oxygen species modulator 1 (Romo1) expression has been reported to increase invasive potential and constitutive activation of nuclear factor-κB (NF-κB) in hepatocellular carcinoma (HCC). Therefore, we investigated whether constitutive NF-κB activation due to Romo1 expression is associated with breast cancer tumor cell invasion. In this study, we show that oxidative stress-induced invasion is mediated by Romo1 expression. The Romo1-induced increase of invasive activity was blocked by an inhibitor of κB kinase (IKK). These results demonstrate that tumor cell invasion in response to oxidative stress is associated with Romo1 expression and the NF-κB signaling pathway. Romo1 is therefore a promising therapeutic target for diseases characterized by NF-κB deregulation.
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Neoplasias de la Mama/patología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo , Western Blotting , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Invasividad NeoplásicaRESUMEN
OBJECTIVES: Reactive oxygen species modulator 1 (Romo1) is a novel protein that plays an important role in intracellular reactive oxygen species generation. Romo1 is overexpressed in most cancer cell lines and related to invasiveness and chemoresistance in vitro. However, little information is available on its clinical implications. We investigated the association between Romo1 expression and the clinical outcomes of non-small cell lung cancer (NSCLC) patients who underwent surgical resection. MATERIALS AND METHODS: Romo1 protein expressions were evaluated immunohistochemically in resected tumor specimens. Survival analyses for overall population (n=110) and early-stage patients (n=97) were performed according to clinical parameters including level of Romo1 expression. RESULTS: Multivariate analyses showed that high Romo1 expression in tumor tissues was significantly associated with short disease-free survival (hazard ratio [HR]=3.16, 95% confidence interval [CI]: 1.21-8.22), and with short overall survival (HR=3.22, 95% CI: 1.02-10.21). Stronger associations were observed between Romo1 expression and disease-free survival (HR=3.69, 95% CI: 1.39-9.97) and overall survival (HR=4.21, 95% CI: 1.12-14.67) in stage I and II patients than in the overall population. Romo1 expression was not associated with any clinical parameter including age, gender, smoking status, stage, differentiation, or tumor histology. CONCLUSIONS: Increased Romo1 expression in surgically resected NSCLC was found to be significantly associated with early recurrence and poor survival. Romo1 overexpression could be a potential adverse prognostic marker in this setting.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Terapia Combinada , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/cirugía , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Resultado del TratamientoRESUMEN
Deregulation of nuclear factor-κB (NF-κB) and related pathways contribute to tumor cell proliferation and invasion. Mechanisms for constitutive NF-κB activation are not fully explained; however, the underlying defects appear to generate and maintain pro-oxidative conditions. In hepatocellular carcinoma (HCC) tissues, up-regulation of reactive oxygen species modulator 1 (Romo1) correlates positively with tumor size. In the present study, we showed that Romo1 expression is required to maintain constitutive nuclear DNA-binding activity of NF-κB and transcriptional activity through constitutive IκBα phosphorylation. Overexpression of Romo1 promoted p65 nuclear translocation and DNA-binding activity. We also show that Romo1 depletion suppressed anchorage-independent colony formation by HCC cells and suppressed tumor growth in vivo. Based on these findings, Romo1 may be a principal regulatory factor in the maintenance of constitutive NF-κB activation in tumor cells. In the interest of anti-proliferative treatments for cancer, Romo1 may also present a productive target for drug development.
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División Celular/fisiología , Proteínas de la Membrana/fisiología , Proteínas Mitocondriales/fisiología , FN-kappa B/metabolismo , Neoplasias/patología , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias/metabolismoRESUMEN
Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H2O2) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H2O2 treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.
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Células Epiteliales/patología , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Muerte Celular , Línea Celular , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/citología , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genéticaRESUMEN
BACKGROUND & AIMS: Chronic oxidative stress from reactive oxygen species (ROS) produced by the mitochondria promotes hepatocarcinogenesis and tumor progression. However, the exact mechanism by which mitochondrial ROS contributes to tumor cell invasion is not known. We investigated the role of ROS modulator 1 (Romo1) in hepatocellular carcinoma (HCC) development and tumor cell invasiveness. METHODS: We performed real-time, semi-quantitative, reverse transcriptase polymerase chain reaction; invasion and luciferase assays; and immunofluorescence and immunohistochemical analyses. The formation of pulmonary metastatic nodules after tumor cell injection was tested in severe combined immunodeficient mice. We analyzed Romo1 expression in HCC cell lines and tissues (n = 95). RESULTS: Expression of Romo1 was increased in HCC cells, compared with normal human lung fibroblast cells. Exogenous expression of Romo1 in HCC cells increased their invasive activity, compared with control cells. Knockdown of Romo1 in Hep3B and Huh-7 HCC cells reduced their invasive activity in response to stimulation with 12-O-tetradecanoylphorbol-13-acetate. Levels of Romo1 were increased compared with normal liver tissues in 63 of 95 HCC samples from patients. In HCC samples from patients, there was an inverse correlation between Romo1 overexpression and patient survival times. Increased levels of Romo1 also correlated with vascular invasion by the tumors, reduced differentiation, and larger tumor size. CONCLUSIONS: Romo1 is a biomarker of HCC progression that might be used in diagnosis. Reagents that inhibit activity of Romo1 and suppress mitochondrial ROS production, rather than eliminate up-regulated intracellular ROS, might be developed as cancer therapies.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular Tumoral , Distribución de Chi-Cuadrado , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Persona de Mediana Edad , Invasividad Neoplásica , Modelos de Riesgos Proporcionales , Ratas , Factores de RiesgoRESUMEN
BACKGROUND: The tissue environment in the region of hepatocellular carcinoma (HCC) influences both vascular invasion and recurrence. Thus, HCC patient prognosis depends on the characteristics not only of the tumor but also those of adjacent surrounding liver tissue. MATERIALS AND METHODS: Expression profiles of both tumor and adjacent liver tissue following curative resection were measured to discriminate 56 hepatitis B virus-positive HCC patients into subgroups based on survival risk. This approach was further tested in 40 patients. RESULTS: Expression profiles of both tumor and adjacent liver tissue successfully discriminated 56 training samples into 2 subgroups, those at low- or high-risk for survival and recurrence. However, the prognostic gene set selected for tumor tissue was quite different from that for adjacent tissues. This variation in prognostic genes resulted in a change in allocation of patients within each low- or high-risk group. Combination of survival subgroups from tumor and adjacent liver tissue significantly improved the prediction of prognostic outcome. This integrative approach was confirmed to be effective in a further 40 test patients. A clinicopathological study showed that survival subgroups divided by tumor and adjacent liver tissue gene expression were also statistically associated with UICC stage and extent of cell differentiation, respectively. CONCLUSIONS: Variation in gene expression during the nontumor stage as well as the tumor stage may affect the prognosis of HCC patients, and integration of the gene expression profiles of HCC and adjacent liver tissue increases discriminatory effectiveness between patient groups, predicting clinical outcomes with enhanced statistical reliability.