RESUMO
The most toxic of the ochratoxins is ochratoxin A (OTA), which is primarily produced by species of Aspergillus and Penicillium that can be found in maize, wheat, coffee, red wine, and various grains. OTA induces immunotoxicity, nephrotoxicity, hepatotoxicity, teratogenicity, and carcinogenicity in both animals and humans. Thus, there is a need to identify mycotoxin detoxification agents that can effectively decontaminate OTA. Seeds of basil (Ocimum basilicum L.), chan (Hyptis suaveolens L.), and chia (Salvia hispanica L.) are functional foods capable of eliminating harmful substances. Despite this potential, the impact of these seeds on OTA detoxification remains unclear. This study reveals that milled basil, chan, and chia seeds adsorb significant levels of OTA, with chia demonstrating the highest adsorption capacity, followed by chan and basil seeds showing the least efficiency. Furthermore, milled basil, chan, and chia seeds effectively reduced OTA residues in artificial gastric and intestinal fluids, where they achieved up to 93% OTA adsorption in the former. In addition, these milled seeds were able to remove OTAs from canned, drip, and instant coffee. This study is the first to report the OTA elimination potential of basil, chan, and chia seeds.
Assuntos
Ocratoxinas , Ocimum basilicum , Humanos , Animais , Ocratoxinas/análise , Café/química , Sementes/químicaRESUMO
Dibenzoylmethane (DBM) belongs to the flavonoid family and is a minor constituent of the root extract of licorice and the ß-diketone analogue of curcumin. It exhibits antimutagenic, anticancer, and chemopreventive effects. Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway, plays an important role in growth, proliferation, and transformation. Our previous studies showed ODC overexpression prevented etoposide-, paclitaxel-, and cisplatin-induced apoptosis. Here, we investigated one mechanism of DBM-induced apoptosis and the antiapoptotic effects of ODC during DBM treatment. We found that DBM induced apoptosis, promoted reactive oxygen species (ROS) generation, and disrupted the mitochondrial membrane potential (Δψ(m). N-acetylcysteine, a ROS scavenger, reduced DBM-induced apoptosis, which led to the loss of Δψ(m) due to reduced ROS. Overexpression of ODC in parental cells had the same effects as the ROS scavenger. The results demonstrated that DBM-induced apoptosis was a ROS-dependent pathway and ODC overexpression blocked DBM-induced apoptosis by inhibiting intracellular ROS production.
Assuntos
Acetilcisteína/farmacologia , Chalconas/farmacologia , Sequestradores de Radicais Livres/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Ornitina Descarboxilase/metabolismo , Extratos Vegetais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chalconas/química , Curcumina/química , Expressão Gênica , Glycyrrhiza/química , Células HL-60 , Humanos , Camundongos , Mitocôndrias/metabolismo , Ornitina Descarboxilase/genética , Extratos Vegetais/química , Plasmídeos , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer K(d), suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (K(d) value = 1.3 microm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a K(d) value of approximately 0.1 microm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Multimerização Proteica , Sítios de Ligação/genética , Proteínas de Transporte/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ornitina Descarboxilase/química , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Ligação Proteica , UltracentrifugaçãoRESUMO
Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway, plays an important role in cell cycle, tumor promotion and anti-apoptosis. In our previous studies, overexpression of ODC prevented apoptosis induced by tumor necrosis factor-alpha and methotrexate. We further investigated the apoptotic mechanisms of the cancer chemotherapeutic drugs, including etoposide (VP-16), paclitaxel (TAX) and cisplatin (CDDP), and the influences of ODC on apoptosis and cell cycle. Our results showed that the investigated drugs induced caspase-dependent apoptosis, the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (Deltapsi m) in HL-60 cells, all of which were reversed by putrescine, glutathione or N-acetyl-l-cysteine. Overexpression of ODC prevented the cancer chemotherapeutic drugs-induced apoptosis, ROS generation and the disruption of Deltapsi m. After drug administrations, the decline of Bcl-2, cytochrome c release and caspases' activation were inhibited by ODC overexpression. In cell cycle, ODC overexpressed cells seemed to overcome the G1 arrest and G2/M arrest, caused by VP-16 and TAX, respectively, and kept on the cell cycle rolling. Overexpression of ODC increased the expression of Cyclin A, D, E and Cdk4 and the enzyme activity of Cdk1 and Cdk2 after the treatment of VP-16 and TAX, respectively. In conclusions, the cancer chemotherapeutic drugs-induced apoptosis is through ROS-related, mitochondria-mediated and caspase-dependent pathways. With higher ODC activity, cells are resistant to the cancer chemotherapeutic drugs-induced apoptosis and keep on the cell cycle rolling with the significant interference in G1/S arrest caused by VP-16 and G2/M arrest by TAX.
Assuntos
Antineoplásicos/antagonistas & inibidores , Apoptose , Leucemia/tratamento farmacológico , Ornitina Descarboxilase/metabolismo , Acetilcisteína/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Ciclo Celular , Cisplatino/antagonistas & inibidores , Etoposídeo/antagonistas & inibidores , Glutationa/farmacologia , Células HL-60 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Paclitaxel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Putrescina/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ornithine decarboxylase (ODC), a tumor promoter, provokes cell proliferation, and inhibits cell death; but the mechanism involved in cell differentiation remains unknown. Herein, we examine whether it functions during macrophage-like differentiation. Previous studies reveal that ODC, a rate-limiting enzyme of polyamine biosynthesis, and polyamines are involved in restraining immune response in activated macrophage. By using 12-O-tetradecanoylphorbol-13-acetate (TPA)-differentiated human promyelocytic HL-60 and promonocytic U-937 cells, we discover that polyamines block the expression, secretion and activation of MMP-9. Meanwhile conventional expression of ODC represses tumor necrosis factor-alpha (TNF-alpha) expression and nuclear factor-kappaB (NF-kappaB) activation as well as MMP-9 enzyme activity. Following stimulation by TNF-alpha, the secretion of MMP-9 is restored in ODC-overexpressed cells. In addition, the NF-kappaB inhibitors (pyrrolidinedithiocarbamate, BAY-11-7082 and lactacystin) suppress the TPA-induced MMP-9 enzyme activity. Concurrently, both the irreversible inhibitor of ODC, alpha-difluoromethylornithine, and TNF-alpha could not recover MMP-9 activation following NF-kappaB inhibitor treatment in parental cells. Furthermore, ODC could directly inhibit and attenuate NF-kappaB DNA binding and transcriptional activation. Therefore, we suggest that ODC inhibits the TNF-alpha-elevated MMP-9 activation via NF-kappaB as TPA-induced macrophage-like differentiation and this interrupting mechanism may provide a new conceivable resolution why leukemia is poorly differentiated besides atypical growth.
Assuntos
Diferenciação Celular/fisiologia , Macrófagos/citologia , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/fisiologia , Ornitina Descarboxilase/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Sequência de Bases , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Células HL-60 , Humanos , Ornitina Descarboxilase/fisiologiaRESUMO
Curcumin, a well-known dietary pigment derived from the food flavoring turmeric (Curcuma longa) exhibits anti-proliferative, anti-inflammatory, and anti-oxidative activities. Recently, studies have shown that a chemopreventive effect of curcumin could be due to the hyperproduction of reactive oxygen species (ROS) inducing apoptosis in tumor cells. In our previous studies, ornithine decarboxylase (ODC) overexpression prevented tumor necrosis factor alpha (TNF-alpha)- and methotrexate-induced apoptosis via reduction of ROS. Furthermore, ODC is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. In this study, we found that enzyme activity and protein expression of ODC were reduced during curcumin treatment. Overexpression of ODC in human promyelocytic leukemia HL-60 parental cells could reduce curcumin-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsi(m)), through reducing intracellular ROS. Moreover, ODC overexpression prevented cytochrome c release and the activation of caspase-9 and caspase-3 following curcumin treatment. These results demonstrate that curcumin-induced apoptosis occurs through a mechanism of down-regulating ODC and along a ROS-dependent mitochondria-mediated pathway.
Assuntos
Anticarcinógenos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Ornitina Descarboxilase/metabolismo , Caspase 3/biossíntese , Caspase 9/biossíntese , Citocromos c/metabolismo , Citosol/efeitos dos fármacos , Citosol/enzimologia , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Células HL-60 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Inibidores da Ornitina Descarboxilase , Espécies Reativas de Oxigênio/metabolismoRESUMO
Aloe-emodin (AE) is derived from Aloe vera and rhubarb (Rheum palmatum) and exhibits anticancer activities via multiple regulatory mechanisms in various cancers. AE can also enhance the anticancer efficacy of cisplatin, doxorubicin, docetaxel, and 5-fluorouracil; however, its effects remain poorly characterized. MCF-7, MDA-MB-231, MDA-MB-468, BT-474, and HCC-1954 breast cancer cell lines were treated with the indicated conditions of AE, and cell viability assays were performed. The expression levels of signaling proteins were determined by western blot analysis, intracellular reactive oxygen species (ROS), cell cycle distributions, and rates of apoptosis as estimated by flow cytometry. In comparison with other cells, MCF-7 cells were more sensitive to AE treatment; AE enhanced the cytotoxicity of 9[Formula: see text][Formula: see text]g/ml tamoxifen by reducing EGFR, ER[Formula: see text], Ras, ERK, c-Myc, and mTOR protein expression and blocking PI3K and mTOR activation. Finally, although co-treatment of AE with tamoxifen increased intracellular ROS, there were no effects on cell cycle progression. Besides facilitating tamoxifen-induced cell death, AE also enhanced the antiproliferative activity of tamoxifen by blocking Ras/ERK and PI3K/mTOR pathways in breast cancer cells, thus demonstrating the chemosensitizing potential of AE.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos Fitogênicos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tamoxifeno/farmacologia , Proteínas ras/metabolismo , Aloe/química , Antraquinonas/isolamento & purificação , Sinergismo Farmacológico , Rheum/química , Células Tumorais CultivadasRESUMO
Rottlerin is a widely selective protein kinase C delta (PKCdelta) inhibitor isolated from Mallotus philippinensis. It shown to be effective against several human tumor cell lines and in potentiating chemotherapy-induced cytotoxcicity. Using the trypan blue exclusion assay, we demonstrated that rottlerin reduced the viability in a dose- and time-dependent manner of human leukemia HL60 cells, human acute T cell leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. Rottlerin caused apoptosis and the apaptotic processing was inhibited by a caspase inhibitor, z-VAD-fmk, in these haematopoietic cells. The apoptosis-inducing activities were determined by nuclear condensation, sub-G1 appearance, DNA fragmentation, loss of mitochondrial membrane potential (Deltapsim), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Expression of PKCdelta and Bcl-2 protein inhibited Deltapsim change and repressed cell death. These studies suggest that the cytotoxic effects of rottlerin through inhibition of PKCdelta cause mitochondrial dysfunction, cytochrome c release from mitochondria into cytoplasm and the activation of caspases' cascade.
Assuntos
Acetofenonas/farmacologia , Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Caspases/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Animais , Apoptose/fisiologia , Caspases/fisiologia , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Inibidores Enzimáticos/farmacologia , Células-Tronco Hematopoéticas/enzimologia , Humanos , Membranas Intracelulares/enzimologia , Potenciais da Membrana , Camundongos , Mitocôndrias/enzimologia , Proteína Quinase C/fisiologia , Proteína Quinase C-delta , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Dibenzoylmethane (DB), a minor constituent of the root extract of licorice, belongs to the flavonoid family. Hydroxydibenzoylmethane (HDB) and hydroxymethyldibenzoylmethane (HMDB) have an identical structure to DB, but also possess a hydroxyl group and a hydroxyl and methyl group bonded to aromatic rings, respectively. They inhibit cellular proliferation and induce apoptosis in a variety of types of cancer cell, however, the antimetastatic effects of DB, HDB and HMDB on human breast carcinoma cells remain to be elucidated. The present study aimed to clarify the molecular mechanisms underlying the effects of DB and its analogues on phorbol12myristate 13acetate (PMA)induced MCF7 cell metastasis. The results revealed that DB, HDB and HMDB inhibited cell migration and invasion. In addition, PMAmediated MCF7 cell invasion was inhibited by DB, HDB and HMDB by inhibiting the expression of matrix metalloproteinase (MMP)9. Rottlerin, a protein kinase C (PKC)δ inhibitor and LY294002, a phosphatidylinositide 3kinase (PI3K) inhibitor, reduced the PMAmediated expression of MMP9 and cell invasion. Furthermore, DB, HDB and HMDB prevented the activation of PKCδ and PI3K by inhibiting their phosphorylation. The present study was the first, to the best of our knowledge, to demonstrate the antimetastatic potential of DB, HDB and HDMB, which decreased cancer cell invasion through the PI3K/PKCδmediated MMP9 pathway.
Assuntos
Chalconas/farmacologia , Cetonas/farmacologia , Propano/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/toxicidade , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Cromonas/farmacologia , Feminino , Humanos , Células MCF-7 , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Propano/farmacologia , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/metabolismoRESUMO
Peptidylarginine deiminase type 2 (PADI2) deiminates (or citrullinates) arginine residues in protein to citrulline residues in a Ca2+-dependent manner, and is found in lymphocytes and macrophages. Vimentin is an intermediate filament protein and a well-known substrate of PADI2. Citrullinated vimentin is found in ionomycin-induced macrophage apoptosis. Citrullinated vimentin is the target of anti-Sa antibodies, which are specific to rheumatoid arthritis, and play a critical role in the pathogenesis of the disease. To investigate the role of PADI2 in apoptosis, we generated a Jurkat cell line that overexpressed the PADI2 transgene from a tetracycline-inducible promoter, and used a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin to activate Jurkat cells. We found that PADI2 overexpression reduced the cell viability of activated Jurkat cells in1a dose- and time-dependent manner. The PADI2-overexpressed and -activated Jurkat cells presented typical manifestations of apoptosis, and exhibited greater levels of citrullinated proteins, including citrullinated vimentin. Vimentin overexpression rescued a portion of the cells from apoptosis. In conclusion, PADI2 overexpression induces apoptosis in activated Jurkat cells. Vimentin is involved in PADI2-induced apoptosis. Moreover, PADI2-overexpressed Jurkat cells secreted greater levels of vimentin after activation, and expressed more vimentin on their cell surfaces when undergoing apoptosis. Through artificially highlighting PADI2 and vimentin, we demonstrated that PADI2 and vimentin participate in the apoptotic mechanisms of activated T lymphocytes. The secretion and surface expression of vimentin are possible ways of autoantigen presentation to the immune system.
Assuntos
Apoptose , Hidrolases/metabolismo , Vimentina/fisiologia , Membrana Celular/metabolismo , Citrulina/metabolismo , Expressão Gênica , Humanos , Células Jurkat , Ativação Linfocitária , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em ProteínasRESUMO
This study investigated the functional roles of the N-terminal Ca(2+) ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca(2+)-binding site of PAD4 were mutated to disrupt the binding of Ca(2+) ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k(cat)/K(m,BAEE) values were 0.02, 0.63 and 0.01 s(-1)mM(-1) (20.8 s(-1)mM(-1) for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k(cat) value of 0.3 s(-1) (13.3 s(-1) for wild-type), whereas D176A retained some catalytic power with a k(cat) of 9.7 s(-1). Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k(cat)/K(m,BAEE) values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca(2+) indicated that the conformational stability of the enzyme is highly dependent on Ca(2+) ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca(2+) ions in the N-terminal Ca(2+)-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca(2+) ions play critical roles in the full activation of the PAD4 enzyme.
Assuntos
Aminoácidos/química , Proteínas de Ligação ao Cálcio/química , Cálcio/metabolismo , Hidrolases/química , Aminoácidos/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Humanos , Hidrolases/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em ProteínasRESUMO
Anisomeles indica popularly known in Taiwan as 'yu-chen-tsao' has been traditionally used as an anticancer and anti-inflammatory agent; however, little is known about its anti-metastatic potential. Therefore, we attempted in this study to examine the anti-metastatic potential of A. indica aqueous extract (AI), its isolated compounds apigenin, ovatodiolide, ß-sitosterol and acteoside in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced human breast adenocarcinoma MCF-7 cells. Among the test agents, crude extract AI and pure compound apigenin potently suppressed the TPA-induced MCF-7 cells migration and invasion. In addition, AI and apigenin time- and dose-dependently down regulated the matrix metalloproteinase (MMP)-9 enzymatic activities and its mRNA expression. Furthermore, AI and apigenin also down regulated the nuclear factor (NF)-κB subunit p65, and activator protein (AP)-1 subunit c-Fos proteins expression in nucleus and, transcriptional activity of NF-κB and AP-1. This is the first report on the anti-metastatic potential of A. indica that suppressed the cancer cell invasion through the inhibition of MMP-9 enzyme via NF-κB/AP-1 signaling. Taken together, our data indicate that A. indica can be considered as a source of new anti-metastatic agent for food and pharmaceutical industries.
Assuntos
Neoplasias da Mama/patologia , Lamiaceae/química , Inibidores de Metaloproteinases de Matriz , NF-kappa B/antagonistas & inibidores , Metástase Neoplásica/prevenção & controle , Extratos Vegetais/farmacologia , Fator de Transcrição AP-1/antagonistas & inibidores , Sequência de Bases , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Primers do DNA , Feminino , Humanos , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Invasividade Neoplásica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/metabolismo , Água/químicaRESUMO
Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Δψ(m)), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.
Assuntos
Apoptose/efeitos dos fármacos , Chalconas/farmacologia , Leucemia Mieloide/enzimologia , Ornitina Descarboxilase/metabolismo , Caspase 3/metabolismo , Chalconas/metabolismo , Quimioprevenção , Citocromos c/biossíntese , Citocromos c/metabolismo , Regulação para Baixo , Expressão Gênica , Células HL-60 , Humanos , Immunoblotting , Leucemia Mieloide/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/enzimologia , Ornitina Descarboxilase/genética , Inibidores da Ornitina Descarboxilase , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, has paradoxical roles in apoptosis. Our published papers show overexpression of ODC prevents the apoptosis induced by many cytotoxic drugs. Thapsigargin (TG) is an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) ATPase (SERCA) pumps and causes ER stress-induced apoptosis. We used ODC overexpressing cell lines to examine whether overexpression of ODC inhibits TG-induced apoptosis. Our results indicated overexpression of ODC attenuated TG-induced apoptosis. Overexpression of ODC blocked procaspase-4 cleavage and phosphorylation of protein kinase-like ER-resident kinase (PERK), triggered by TG. It also attenuated the increase in CAAT/enhancer binding protein homologous protein (CHOP). Cells with overexpressed ODC had greater Bcl-2 expression. Overexpression of ODC preserved the expression of Bcl-2, inhibited the increase in Bak and stabilized mitochondrial membrane potential without the influences of TG. Cytochrome c release and down-stream caspase activation were blocked. That is, overexpression of ODC inhibits the mitochondria-mediated apoptotic pathway, induced by TG. Finally, overexpression of ODC maintains the protein and mRNA expression of SERCA. In conclusion, overexpression of ODC suppresses TG-induced apoptosis by blocking caspase-4 activation and PERK phosphorylation, attenuating CHOP expression and inhibiting the mitochondria-mediated apoptotic pathway.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Ornitina Descarboxilase , Tapsigargina/farmacologia , Animais , Inibidores de Caspase , Caspases/análise , Caspases/metabolismo , Citocromos c/análise , Citocromos c/metabolismo , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Células HL-60 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Poliaminas/análise , Poliaminas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Peptidylarginine deiminase IV (PADI4) posttranslationally converts peptidylarginine to citrulline. It plays an essential role in immune cell differentiation and apoptosis. A haplotype of single-nucleotide polymorphisms (SNPs) in PADI4 is functionally relevant as a rheumatoid arthritis (RA) gene. It could increase enzyme activity leading to raised levels of citrullinated protein and stimulating autoantibody. Previously, our study showed that inducible PADI4 causes haematopoietic cell death. Herein, we further investigate whether RA risk PADI4 haplotype (SNP PADI4; S55G, A82V and A112G) and the increase of its enzymatic activity induce apoptosis. In the tetracycline (Tet)-On Jurkat T cells, ionomycin (Ion) only treatment didn't induce apoptosis however it promoted inducible PADI4-decreased cell viability and -enhanced apoptosis. Through in vitro and in vivo PADI enzyme activity assay, we demonstrated that PADI4 enzyme activity of SNP PADI4 was higher than RA non-risk PADI4 haplotype (WT PADI4). The effect of SNP PADI4-induced apoptosis was superior to WT PADI4. In addition, both Ion and SNP PADI4 synergistically provoked apoptosis were compared with both Ion and WT PADI4. Concurrently, in the conditionally inducible SNP PADI4 cells of Ion treatment-induced apoptosis, not only the expression of Bcl-xL was down-regulated and Bax up-regulated, but also cytochrome c was released from mitochondria to cytoplasm in significant amounts. Western blotting data showed the increase in apoptosomal caspase activation during programmed cell death in the inducible SNP PADI4 cells subsequent to Ion treatment. These data demonstrated that both SNP PADI4 increasing their enzyme activity could enhance apoptosis through the mitochondrial pathway and further provide a conceivable explanation in the pathogenesis of RA following the upregulation of PADI4 activity in its SNPs.
Assuntos
Apoptose/fisiologia , Artrite Reumatoide , Haplótipos , Hidrolases/metabolismo , Artrite Reumatoide/enzimologia , Artrite Reumatoide/genética , Caspases/metabolismo , Sobrevivência Celular , Ativação Enzimática , Predisposição Genética para Doença , Humanos , Hidrolases/genética , Células Jurkat , Mutagênese Sítio-Dirigida , Polimorfismo de Nucleotídeo Único , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em ProteínasRESUMO
A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.