RESUMO
Vascular calcification (VC) and osteoporosis are age-related diseases and significant risk factors for the mortality of elderly. VC and osteoporosis may share common risk factors such as renin-angiotensin system (RAS)-related hypertension. In fact, inhibitors of RAS pathway, such as angiotensin type 1 receptor blockers (ARBs), improved both vascular calcification and hip fracture in elderly. However, a sex-dependent discrepancy in the responsiveness to ARB treatment in hip fracture was observed, possibly due to the estrogen deficiency in older women, suggesting that blocking the angiotensin signaling pathway may not be effective to suppress bone resorption, especially if an individual has underlying osteoclast activating conditions such as estrogen deficiency. Therefore, it has its own significance to find alternative modality for inhibiting both vascular calcification and osteoporosis by directly targeting osteoclast activation to circumvent the shortcoming of ARBs in preventing bone resorption in estrogen deficient individuals. In the present study, a natural compound library was screened to find chemical agents that are effective in preventing both calcium deposition in vascular smooth muscle cells (vSMCs) and activation of osteoclast using experimental methods such as Alizarin red staining and Tartrate-resistant acid phosphatase staining. According to our data, citreoviridin (CIT) has both an anti-VC effect and anti-osteoclastic effect in vSMCs and in Raw 264.7 cells, respectively, suggesting its potential as an effective therapeutic agent for both VC and osteoporosis.
Assuntos
Aurovertinas , Reabsorção Óssea , Osteoporose , Calcificação Vascular , Humanos , Antagonistas de Receptores de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Reabsorção Óssea/metabolismo , Cálcio/metabolismo , Estrogênios/farmacologia , Músculo Liso Vascular , Miócitos de Músculo Liso , Osteoporose/metabolismo , Calcificação Vascular/metabolismo , Animais , Camundongos , Células RAW 264.7 , Aurovertinas/farmacologiaRESUMO
Citreoviridin (CTV) is a mycotoxin produced by various fungi, including Penicillium citreonigrum. One of the toxicities reportedly associated with CTV is neurotoxicity. CTV is also suspected to be associated with acute cardiac beriberi (also known as "Shoshin-kakke") and Keshan disease, which can have adverse effects on the heart, so the in vivo and in vitro toxicity of CTV on the heart or cardiomyocytes in experimental animal models have been reported. However, the toxicity of CTV for the human heart, especially its electrophysiological effect, remains poorly understood. Therefore, to investigate the electrophysiological effect of CTV on the human cardiomyocytes, we conducted a multi-electrode array (MEA) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The MEA revealed that 30 µmol/L of CTV stopped the beating of hiPSC-CMs, and the field potential duration and first peak amplitude were shortened at 10 µmol/L. Before the hiPSC-CMs stopped beating, the length of the inter-spike interval varied two- to four-fold. These results demonstrated that CTV induced an electrophysiological disturbance on human cardiomyocytes. This is first paper to elucidate the electrophysiological effect of CTV on human heart directly and may aid in analyzing the risk associated with CTV to ensure food safety.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Aurovertinas/farmacologia , Células CultivadasRESUMO
Increasing evidences indicate the vital role of exosomes-mediated intercellular communication in the pathogenesis of liver fibrosis. However, the underlying mechanisms are still not clearly defined. In this study, we found that citreoviridin (CIT), a mycotoxin and ectopic ATP synthase (e-ATPS) inhibitor, induced liver fibrosis in mice. The exosomes derived from CIT-treated L-02 hepatocytes activated hepatic stellate cells (HSC) LX-2. With exosomal small RNA sequencing, we found 156 differentially expressed miRNAs in the exosomes from CIT-treated L-02 cells, and the predicted target genes of exosomal miRNAs were enriched in calcium signaling pathway. The exosomes from CIT-treated L-02 cells induced mitochondrial calcium accumulation in LX-2 cells. And pharmacological inhibition of mitochondrial calcium uptake relieved exosomes-activated fibrogenic response in LX-2 cells. The miR-181a-2-3p that was predicted to target-regulate mitochondrial calcium uptake 1 (MICU1) was significantly increased in the exosomes from CIT-treated L-02 cells. Exosomes-induced reduction of MICU1, mitochondrial calcium overload and activation of LX-2 cells were reversed by AntagomiR-181a-2-3p. In this study, we pointed out that exosomal miR-181a-2-3p from CIT-treated hepatocytes induced mitochondrial calcium accumulation and activated HSC subsequently through inhibiting the expression of MICU1, shedding new light on the mechanism underlying liver fibrosis and CIT hepatotoxicity.
Assuntos
Cálcio , Células Estreladas do Fígado , Cirrose Hepática , MicroRNAs , Mitocôndrias Hepáticas , Animais , Aurovertinas/farmacologia , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Exossomos/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transdução de Sinais/genéticaRESUMO
Aurovertin B, a natural compound from Calcarisporium arbuscular, exhibits potent antiproliferative activity particularly against triple-negative breast cancer cells (TNBC), while having less cytotoxicity on normal breast cell MCF10A. However, very little is known about the in vivo antitumor activity of aurovertin B and the possible mechanism of the selective effect on triple-negative breast cancer cells. In this study, flow cytometry and DAPI staining analysis showed that aurovertin B treatment in human triple-negative breast cancer cell MDA-MB-231 could induce more apoptotic cells than taxol treatment group. Furthermore, the present study also revealed that aurovertin B induced apoptosis was due to regulation of ATP synthase activity rather than changes in gene expression. Interestingly, the cancer genome atlas (TCGA) data analysis implied that the expression level of DUSP1, a member of the dual-specificity phosphatases, was highly downregulated in breast tissue of TNBC patients compared with their adjacent normal tissues. Real-time PCR and western blot analyses further demonstrated that aurovertin B could dramatically increase mRNA and protein expression levels of DUSP1 in MDA-MB-231 cells but not in MCF10A cells. The potent anti-tumor activity of aurovertin B was further verified in a human MDA-MB-231 xenograft mouse model.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Aurovertinas/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fosfatase 1 de Especificidade Dupla/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Citreoviridin (CIT), a mycotoxin and ATP synthase inhibitor, is regarded as one of aetiology factors of cardiac beriberi and Keshan disease. Thiamine (VB1) and selenium (Se) improve the recovery of these two diseases respectively. The underlying mechanisms of cardiotoxic effect of CIT and cardioprotective effect of VB1 and Se have not been fully elucidated. In this study, we found that ectopic ATP synthase was more sensitive to CIT treatment than mitochondrial ATP synthase in H9c2 cardiomyocytes. CIT inhibited the transcriptional activity of peroxisome proliferator activated receptor gamma (PPAR-γ) in mice hearts and H9c2 cells. PPAR-γ agonist attenuated the inhibitory effect of CIT on mechanistic target of rapamycin complex 2 (mTORC2) and stimulatory effect of CIT on autophagy in cardiomyocytes. CIT induced apoptosis through lysosomal-mitochondrial axis in cardiomyocytes. PPAR-γ agonist and autophagy inhibitor alleviated CIT-induced apoptosis and accelerated cardiac biomarker. VB1 and Se accelerated the basal transcriptional activity of PPAR-γ in mice hearts and H9c2 cells. Furthermore, VB1 and Se reversed the effect of CIT on PPAR-γ, autophagy and apoptosis. Our findings defined PPAR-γ-mTORC2-autophagy pathway as the key link between CIT cardiotoxicity and cardioprotective effect of VB1 and Se. The present study would shed new light on the pathogenesis of cardiomyopathy and the cardioprotective mechanism of micronutrients.
Assuntos
Apoptose/efeitos dos fármacos , Aurovertinas/farmacologia , Autofagia/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Selênio/farmacologia , Tiamina/farmacologia , Animais , Aquaporinas/genética , Aquaporinas/metabolismo , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Miocárdio/metabolismo , Miocárdio/patologia , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Proteína X Associada a bcl-2/metabolismoRESUMO
The natural killer group 2D (NKG2D) receptor on natural killer (NK) cells play an important role in immunosurveillance to cancer cells, which could mediate the eradication of tumor cells through specific interactions with NKG2D ligands on tumor cells. Here we report one natural compound aurovertin B from basidiomycete Albatrellus confluens significantly stimulates the expression of NKG2D ligands on tumor cells, which greatly sensitizes its recognition and lysis by NK cell. It is completely a novel role for aurovertin B to target tumor cells to death mediated by NK cells and our findings indicate aurovertin B may deserve further development as sensitizing agent in NK cell mediated cancer immunotherapy.
Assuntos
Antineoplásicos/farmacologia , Aurovertinas/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Citotoxicidade Imunológica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Antineoplásicos/química , Aurovertinas/química , Basidiomycota/química , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células Matadoras Naturais/imunologia , Regulação para Cima/genéticaRESUMO
Porphyromonas gingivalis is a well-known Gram-negative bacterium that causes periodontal disease. The bacterium metabolizes amino acids and peptides to obtain energy. An ion gradient across its plasma membrane is thought to be essential for nutrient import. However, it is unclear whether an ion-pumping ATPase responsible for the gradient is required for bacterial growth. Here, we report the inhibitory effect of protonophores and inhibitors of a proton-pumping ATPase on the growth of P. gingivalis. Among the compounds examined, curcumin and citreoviridin appreciably reduced the bacterial growth. Furthermore, these compounds inhibited the ATPase activity in the bacterial membrane, where the A-type proton-pumping ATPase (A-ATPase) is located. This study suggests that curcumin and citreoviridin inhibit the bacterial growth by inhibiting the A-ATPase in the P. gingivalis membrane.
Assuntos
Porphyromonas gingivalis/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , Aurovertinas/farmacologia , Proteínas de Bactérias , Membrana Celular/enzimologia , Curcumina/farmacologia , Doenças Periodontais/prevenção & controle , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/crescimento & desenvolvimento , Inibidores da Bomba de Prótons/farmacologia , Bombas de Próton/químicaRESUMO
Citreoviridin (CIT) is a mycotoxin produced by Penicillum citreonigrum, Aspergillus terreus and Eupenicillium ochrosalmoneum. CIT occurs naturally in moldy rice and corn. CIT is associated with the development of atherosclerosis in the general population. Alteration in hepatic lipid metabolism is a pathogenic factor in atherosclerosis. However the effect and the underlying mechanism of CIT on hepatic lipid metabolism are largely unknown. In this study, we reported that CIT induced triglyceride accumulation in mice liver and human liver HepG2 cells as shown in oil red O staining. CIT (0.1 mg/kg-0.3 mg/kg) for 6 weeks elevated liver triglyceride contents in mice. CIT inhibited the transactivation activity of peroxisome proliferator-activated receptor-α (PPAR-α) in hepatocyte in vivo and in vitro, as shown by the reduced mRNA levels of PPAR-α target genes which play key roles in lipid metabolism in various aspects. PPAR-α agonist fenofibrate attenuated CIT-induced triglyceride accumulation in HepG2 cells. Furthermore, CIT increased serum total cholesterol/high-density lipoprotein cholesterol ratio, a strong risk factor for cardiovascular disease. In summary, we reported that CIT induced PPAR-α-dependent hepatic triglyceride accumulation and dyslipidemia. Our data will provide new mechanistic insights into CIT-induced lipid alterations.
Assuntos
Aurovertinas/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , PPAR alfa/antagonistas & inibidores , Triglicerídeos/metabolismo , Animais , Aurovertinas/administração & dosagem , Relação Dose-Resposta a Droga , Células Hep G2 , Hepatócitos/patologia , Humanos , Injeções Intraperitoneais , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , PPAR alfa/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The F1 sector of ATP synthase (FOF1) synthesizes or hydrolyses ATP via a rotational catalysis mechanism that couples chemical reaction with subunit rotation. Phytopolyphenols such as curcumin can inhibit bulk phase F1 ATPase activity by extending the catalytic dwell time during subunit rotation (Sekiya, M., Hisasaka, R., Iwamoto-Kihara, A., Futai, M., Nakanishi-Matsui, M., Biochem. Biophys. Res. Commun. 452 (2014) 940-944). Citreoviridin, a polyene α-pyrone mycotoxin isolated from Penicillium sp, also inhibits ATPase activity. Molecular docking and mutational analysis indicated that these compounds interact with a region near the ß-subunit Arg398 residue that lies at the interface with the α-subunit. Binding of these inhibitors lowered the rotation rate and increased the duration of the catalytic dwell synergistically with substitution of ß-subunit Ser174 to Phe (ßS174F), which rendered the enzyme defective for conformational transmission between ß-subunits of different catalytic stages. Furthermore, substitution of α-subunit Glu402 to Ala (αE402A) in the α/ß-interface also decreased the rotation rate by increasing the duration of the catalytic dwell. Interestingly, this mutation restored the catalytic dwell of the ßS174F variant to that of the wild-type enzyme. These results suggest that the α/ß-interface is involved in conformational changes of the ß-subunit during rotational catalysis.
Assuntos
Biocatálise , Inibidores Enzimáticos/farmacologia , Mutação , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Animais , Aurovertinas/metabolismo , Aurovertinas/farmacologia , Sítios de Ligação , Bovinos , Curcumina/metabolismo , Curcumina/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/metabolismo , Escherichia coli/enzimologia , Hidrólise , Simulação de Acoplamento Molecular , Mutação/efeitos dos fármacos , Conformação Proteica , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , RotaçãoRESUMO
Citreoviridin (1) belongs to a class of F1-ATPase ß-subunit inhibitors that are synthesized by highly reducing polyketide synthases. These potent mycotoxins share an α-pyrone polyene structure, and they include aurovertin, verrucosidin, and asteltoxin. The identification of the citreoviridin biosynthetic gene cluster in Aspergillus terreus var. aureus and its reconstitution using heterologous expression in Aspergillus nidulans are reported. Two intermediates were isolated that allowed the proposal of the biosynthetic pathway of citreoviridin.
Assuntos
Aspergillus nidulans/química , Aurovertinas/química , Micotoxinas/química , Policetídeo Sintases/metabolismo , Pironas/química , Aspergillus nidulans/genética , Aurovertinas/isolamento & purificação , Aurovertinas/farmacologia , Vias Biossintéticas , Estrutura Molecular , Família Multigênica , Micotoxinas/isolamento & purificação , Micotoxinas/farmacologia , Policetídeos/metabolismo , Pironas/isolamento & purificação , Pironas/farmacologiaRESUMO
A comparative metabolomic study of a marine derived fungus (Aspergillus terreus) grown under various culture conditions is presented. The fungus was grown in eleven different culture conditions using solid agar, broth cultures, or grain based media (OSMAC). Multivariate analysis of LC/MS data from the organic extracts revealed drastic differences in the metabolic profiles and guided our subsequent isolation efforts. The compound 7-desmethylcitreoviridin was isolated and identified, and is fully described for the first time. In addition, 16 known fungal metabolites were also isolated and identified. All compounds were elucidated by detailed spectroscopic analysis and tested for antibacterial activities against five human pathogens and tested for cytotoxicity. This study demonstrates that LC/MS based multivariate analysis provides a simple yet powerful tool to analyze the metabolome of a single fungal strain grown under various conditions. This approach allows environmentally-induced changes in metabolite expression to be rapidly visualized, and uses these differences to guide the discovery of new bioactive molecules.
Assuntos
Antibacterianos/química , Aspergillus/química , Produtos Biológicos/química , Bioprospecção , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Aspergillus/crescimento & desenvolvimento , Aurovertinas/química , Aurovertinas/isolamento & purificação , Aurovertinas/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Cromatografia Líquida , Humanos , Espectrometria de Massas , Micotoxinas/química , Micotoxinas/isolamento & purificação , Micotoxinas/farmacologiaRESUMO
Citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. However, the mechanism of citreoviridin triggering dynamic molecular responses in cancer cells remains unclear. Here, we performed temporal phosphoproteomics to elucidate the dynamic changes after citreoviridin treatment in cells and xenograft model. We identified a total of 829 phosphoproteins and demonstrated that citreoviridin treatment affects protein folding, cell cycle, and cytoskeleton function. Furthermore, response network constructed by mathematical modeling shows the relationship between the phosphorylated heat shock protein 90 ß and mitogen-activated protein kinase signaling pathway. This work describes that citreoviridin suppresses cancer cell growth and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 and provides perspectives in cancer therapeutic strategies.
Assuntos
Aurovertinas/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Animais , Aurovertinas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Modelos Teóricos , Fosforilação , Dobramento de Proteína/efeitos dos fármacos , Proteoma/química , Proteoma/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Citreoviridin (CIT) is a mycotoxin derived from fungal species in moldy cereals. In our previous study, we reported that CIT stimulated autophagosome formation in human liver HepG2 cells. Here, we aimed to explore the relationship of autophagy with lysosomal membrane permeabilization and apoptosis in CIT-treated cells. Our data showed that CIT increased the expression of LC3-II, an autophagosome biomarker, from the early stage of treatment (6 h). After treatment with CIT for 12 h, lysosomal membrane permeabilization occurred, followed by the release of cathepsin D in HepG2 cells. Inhibition of autophagosome formation with siRNA against Atg5 attenuated CIT-induced lysosomal membrane permeabilization. In addition, CIT induced collapse of mitochondrial transmembrane potential as assessed by JC-1 staining. Furthermore, caspase-3 activity assay showed that CIT induced apoptosis in HepG2 cells. Inhibition of autophagosome formation attenuated CIT-induced apoptosis, indicating that CIT-induced apoptosis was autophagy-dependent. Cathepsin D inhibitor, pepstatin A, relieved CIT-induced apoptosis as well, suggesting the involvement of the lysosomal-mitochondrial axis in CIT-induced apoptosis. Taken together, our data demonstrated that CIT induced autophagy-dependent apoptosis through the lysosomal-mitochondrial axis in HepG2 cells. The study thus provides essential mechanistic insight, and suggests clues for the effective management and treatment of CIT-related diseases.
Assuntos
Apoptose/efeitos dos fármacos , Aurovertinas/farmacologia , Autofagia/efeitos dos fármacos , Micotoxinas/farmacologia , Caspase 3/metabolismo , Células Hep G2 , Humanos , Membranas Intracelulares/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Permeabilidade/efeitos dos fármacosRESUMO
Nematophagous fungi are globally distributed soil fungi and well-known natural predators of soil-dwelling nematodes. Pochonia chlamydosporia can be found in diverse nematode-suppressive soils as a parasite of nematode eggs and is one of the most studied potential biological control agents of nematodes. However, little is known about the functions of small molecules in the process of infection of nematodes by this parasitic fungus or about small-molecule-mediated interactions between the pathogenic fungus and its host. Our recent study demonstrated that a P. chlamydosporia strain isolated from root knots of tobacco infected by the root-knot nematode Meloidogyne incognita produced a class of yellow pigment metabolite aurovertins, which induced the death of the free-living nematode Panagrellus redivevus. Here we report that nematicidal P. chlamydosporia strains obtained from the nematode worms tended to yield a total yellow pigment aurovertin production exceeding the inhibitory concentration shown in nematicidal bioassays. Aurovertin D was abundant in the pigment metabolites of P. chlamydosporia strains. Aurovertin D showed strong toxicity toward the root-knot nematode M. incognita and exerted profound and detrimental effects on the viability of Caenorhabditis elegans even at a subinhibitory concentration. Evaluation of the nematode mutation in the ß subunit of F1-ATPase, together with the application of RNA interference in screening each subunit of F1FO-ATPase in the nematode worms, demonstrated that the ß subunit of F1-ATPase might not be the specific target for aurovertins in nematodes. The resistance of C. elegans daf-2(e1370) and the hypersensitivity of C. elegans daf-16(mu86) to aurovertin D indicated that DAF-16/FOXO transcription factor in nematodes was triggered in response to the aurovertin attack. These findings advance our understanding of the roles of aurovertin production in the interactions between nematodes and the pathogen fungus P. chlamydosporia.
Assuntos
Ascomicetos/fisiologia , Aurovertinas/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Nematoides/fisiologia , Animais , Antinematódeos , Aurovertinas/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Raízes de Plantas/parasitologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , Interferência de RNA , Nicotiana/parasitologia , Tylenchoidea/efeitos dos fármacosRESUMO
A set of isolates very similar to or potentially conspecific with an unidentified Penicillium isolate NRRL 735, was assembled using a BLAST search of ITS similarity among described (GenBank) and undescribed Penicillium isolates in our laboratories. DNA was amplified from six loci of the assembled isolates and sequenced. Two species in section Cinnamopurpurea are self-compatible sexual species, but the asexual species had polymorphic loci suggestive of sexual reproduction and variation in conidium size suggestive of ploidy level differences typical of heterothallism. Accordingly we use genealogical concordance analysis, a technique valid only in heterothallic organisms, for putatively asexual species. Seven new species were revealed in the analysis and are described here. Extrolite analysis showed that two of the new species, P. colei and P. monsserratidens produce the mycotoxin citreoviridin that has demonstrated pharmacological activity against human lung tumors. These isolates could provide leads in pharmaceutical research.
Assuntos
Aurovertinas/farmacologia , DNA Fúngico/isolamento & purificação , Micotoxinas/farmacologia , Penicillium/genética , Aurovertinas/isolamento & purificação , Linhagem Celular Tumoral , DNA Fúngico/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Micotoxinas/isolamento & purificação , Penicillium/química , Penicillium/isolamento & purificação , Filogenia , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
F1Fo ATP synthase is present in all organisms and is predominantly located on the inner membrane of mitochondria in eukaryotic cells. The present study demonstrated that ATP synthase and electron transport chain complexes were ectopically expressed on the surface of breast cancer cells and could serve as a potent anticancer target. We investigated the anticancer effects of the ATP synthase inhibitor citreoviridin on breast cancer cells through proteomic approaches and revealed that differentially expressed proteins in cell cycle regulation and in the unfolded protein response were functionally enriched. We showed that citreoviridin triggered PERK-mediated eIF2α phosphorylation, which in turn attenuated general protein synthesis and led to cell cycle arrest in the G0/G1 phase. We further showed that the combination of citreoviridin and the 26S proteasome inhibitor bortezomib could improve the anticancer activity by enhancing ER stress, by ameliorating citreoviridin-caused cyclin D3 compensation, and by contributing to CDK1 deactivation and PCNA downregulation. More interestingly, the combined treatment triggered lethality through unusual non-apoptotic caspase- and autophagy-independent cell death with a cytoplasmic vacuolization phenotype. The results imply that by boosting ER stress, the combination of ATP synthase inhibitor citreoviridin and 26S proteasome inhibitor bortezomib could potentially be an effective therapeutic strategy against breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Estresse do Retículo Endoplasmático , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Terapia de Alvo Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Aurovertinas/farmacologia , Aurovertinas/uso terapêutico , Autofagia/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Bortezomib , Neoplasias da Mama/patologia , Cálcio/metabolismo , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ciclina D3/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Humanos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Receptores Purinérgicos/metabolismo , Ensaio Tumoral de Célula-Tronco , Ubiquitina/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , eIF-2 Quinase/metabolismoRESUMO
In some areas of China, citreoviridin (CIT) is considered one of the risk factors for development of cardiovascular disease (CVD). Apoptosis of endothelial cell may induce vascular endothelium injury and atherosclerosis, which result in CVD probably. In this study, we investigated the effect of CIT on apoptosis and proliferation of human umbilical vein endothelial cells (HUVECs). The MTT assay was used to determinate HUVECs proliferation. Distribution of the cell cycle was analyzed by flow cytometry. The Annexin-V/PI staining was used to investigate cell apoptosis. Western blotting analysis was used to indicate changes in the expression level of apoptosis-related proteins. The results indicated that CIT inhibited HUVECs proliferation and the cells were arrested at G0/G1 phase, which is associated with decreased levels of cyclinD1 and increased expression of p53 and p21. The apoptosis rate of HUVECs was improved by CIT. The expression of Bcl-2 were down-regulated after CIT treatment, whereas the levels of Bax was significantly up-regulated. Furthermore, CIT-induced apoptosis was accompanied by the activation of caspase-3, -9. These findings demonstrate that CIT inhibits cell proliferation via DNA synthesis reduction and induces caspase-dependent apoptosis in HUVECs. CIT plays a pivotal role in the process of endothelial cell apoptosis, may thereby play an important role in the improvement of CVD in areas of China that have a high prevalence of CIT contamination.
Assuntos
Aurovertinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Micotoxinas/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.
Assuntos
Aurovertinas/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Algoritmos , Animais , Aurovertinas/química , Cátions , Membrana Celular/metabolismo , Proliferação de Células , Cromatografia Líquida , Biologia Computacional , Inibidores Enzimáticos/química , Feminino , Gluconeogênese , Glucose/metabolismo , Glicólise , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Nus , Proteoma , Espectrometria de Massas em Tandem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To investigate the effects of citreoviridin (CIT) on the expression of MCP-1, IL-1beta, IL-6 and IL-8 induced by TNF-alpha in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs isolated from the umbilical cord of neonates within 1 hour after birth (informed with consent form) were cultured in DMEM/ F12 media. After 80% of HUVECs were confluent, the cells were divided into four groups and treated with CIT (2 micromol/L) and/or TNF-alpha (10 microg/L). The levels of MCP-1, IL-1beta, IL-6 and IL-8 in the supernatant of cell culture media were measured by ELISA, the activation of NF-kappaB in HUVECs was detected by immunofluorescence staining, and the expression of MCP-1 mRNA was determinated by RT-PCR assay. RESULT: The levels of MCP-1, IL-1beta, IL-6 and IL-8 in the supernatant and the expression of NF-kappaB P65 and MCP-1 mRNA of HUVECs were higher in TNF-alpha group and TNF-alpha + CIT group than those in the control group (P < 0.05), and those of TNF-alpha + CIT group was higher than TNF-alpha group (P < 0.05). CONCLUSION: The expression of NF-kappaB, MCP-1, IL-1beta, IL-6 and IL-8 in HUVECs up-regulated by TNF-alpha was promoted by CIT.
Assuntos
Aurovertinas/farmacologia , Quimiocina CCL2/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Interleucinas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Quimiocina CCL2/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Interleucinas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
The mitochondrial F(1)F(o)-ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F(1)F(o)-ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the beta subunits in the F(1) domain and inhibits F(1)F(o)-ATPase-catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F(1)F(o)-ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K(i) values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F(1)F(o)-ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single-molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F(1)F(o)-ATPase catalyzes ATP synthesis and hydrolysis.