RESUMO
Proteinase inhibitors play an important role in plant resistance of insects and pathogens. In this study, we characterized the serine proteinase inhibitor SaPIN2b, which is constitutively expressed in Solanum americanum trichomes and contains two conserved motifs of the proteinase inhibitor II (PIN2) family. The recombinant SaPIN2b (rSaPIN2b), which was expressed in Escherichia coli, was demonstrated to be a potent proteinase inhibitor against a panel of serine proteinases, including subtilisin A, chymotrypsin and trypsin. Moreover, rSaPIN2b also effectively inhibited the proteinase activities of midgut trypsin-like proteinases that were extracted from the devastating pest Helicoverpa armigera. Furthermore, the overexpression of SaPIN2b in transgenic tobacco plants resulted in enhanced resistance against H. armigera. Taken together, our results demonstrated that SaPIN2b is a potent serine proteinase inhibitor that may act as a protective protein in plant defense against insect attacks.
Assuntos
Proteínas de Plantas/farmacologia , Inibidores de Serina Proteinase/farmacologia , Solanum/química , Tricomas/química , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Expressão Gênica , Dados de Sequência Molecular , Mariposas/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/isolamento & purificação , Nicotiana/genética , Nicotiana/parasitologiaRESUMO
PURPOSE: To investigate the antitumor efficacy of pingyangmycin (PYM) in combination with anti-PD-1 antibody and determine the capability of PYM to induce immunogenic cell death (ICD) in cancer cells. METHODS: The murine 4T1 breast cancer and B16 melanoma models were used for evaluation of therapeutic efficacy of the combination of PYM with anti-PD-1 antibody. The ELISA kits were used to quantify the ICD related ATP and HMGB1 levels. The Transwell assay was conducted to determine the chemotaxis ability of THP-1 cell in vitro. The flow cytometry was used to measure reactive oxygen species level and analyze the ratio of immune cell subsets. RESULTS: PYM induced ICD in murine 4T1 breast cancer and B16 melanoma cells and increased the release of nucleic acid fragments that may further promote the monocytic chemotaxis. In the 4T1 murine breast cancer model, PYM alone, anti-PD-1 antibody alone, and their combination suppressed tumor growth by 66.3%, 16.1% and 77.6%, respectively. PYM markedly enhanced the therapeutic efficacy of anti-PD-1 antibody against 4T1 breast cancer. The calculated CDI (coefficient of drug interaction) indicated synergistic effect. Evaluated by graphic analysis, the nucleated cells intensity in the femur bone marrow remained unchanged. Histopathological observations revealed no noticeable toxico-pathological changes in the lung and various organs, indicating that the PYM and anti-PD-1 antibody combination exerted enhanced efficacy at well-tolerated dosage level. By the combination treatment, a panel of immunological changes emerged. The ratio of CD3+ cells, NK cells and NKT cells increased and Tregs decreased in peripheral blood. The DCs increased in the spleen. Prominent changes occurred in tumor infiltrating lymphocytes. The ratio of CD8+ cells increased, while that of CD4+ cells decreased; however, the ratio of CD3+ cells remained unchanged, implying that certain immunological responses emerged in the tumor microenvironment. PYM alone could also increase CD8+ cells and reduce CD4+ cells in tumor infiltrating lymphocytes. CONCLUSIONS: The studies indicate that PYM, as an ICD inducer with mild myelosuppression effect, may enhance the therapeutic efficacy of anti-PD-1 antibody in association with tumor infiltrating CD8+ T cell augmentation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Mamárias Animais/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bleomicina/administração & dosagem , Bleomicina/análogos & derivados , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Mamárias Animais/patologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologia , Microambiente Tumoral/imunologiaRESUMO
SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.
Assuntos
Proteínas de Plantas/metabolismo , Inibidores de Proteases/metabolismo , Solanum/metabolismo , Sequência de Aminoácidos , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Cisteína Endopeptidases/metabolismo , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Floema/genética , Floema/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Caules de Planta/genética , Caules de Planta/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Análise de Sequência de Proteína , Serina Endopeptidases/metabolismo , Solanum/genética , Subtilisina/antagonistas & inibidores , Subtilisina/metabolismo , Temperatura , Tripsina/metabolismoRESUMO
Methamphetamine (METH) is a psychostimulant drug. Abuse of METH produces long-term behavioral changes including behavioral, sensitization, tolerance, and dependence. It induces neurotoxic effects in several areas of the brain via enhancing dopamine (DA) level abnormally, which may cause a secondary release of glutamate (GLU). However, repeated administration of METH still increases release of GLU even when dopamine content in tissue is significantly depleted. It implies that some other mechanisms are likely to involve in METH-induced GLU release. The goal of this study was to observe METH affected glutamatergic synaptic transmission in rat primary cultured hippocampal neurons and to explore the mechanism of METH modulated GLU release. Using whole-cell patch-clamp recordings, we found that METH (0.1-50.0µM) increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). However, METH decreased the frequency of sEPSCs and mEPSCs at high concentration of 100µM. The postsynaptic NMDA receptor currents and P/Q-type calcium channel were not affected by the use of METH (10,100µM). METH did not present visible effect on N-type Ca(2+) channel current at the concentration lower than 50.0µM, but it was inhibited by use of METH at a 100µM. The effect of METH on glutamatergic synaptic transmission was not revered by pretreated with DA receptor antagonist SCH23390. These results suggest that METH directly modulated presynaptic GLU release at a different concentration, while dopaminergic system was not involved in METH modulated release of GLU in rat primary cultured hippocampal neurons.