RESUMO
Evidence shows that tropomodulin 1 (TMOD1) is a powerful diagnostic marker in the progression of several cancer types. However, the regulatory mechanism of TMOD1 in tumor progression is still unclear. Here, we showed that TMOD1 was highly expressed in acute myeloid leukemia (AML) specimens, and TMOD1-silencing inhibited cell proliferation by inducing autophagy in AML THP-1 and MOLM-13 cells. Mechanistically, the C-terminal region of TMOD1 directly bound to KPNA2, and TMOD1-overexpression promoted KPNA2 ubiquitylation and reduced KPNA2 levels. In contrast, TMOD1-silencing increased KPNA2 levels and facilitated the nuclear transfer of KPNA2, then subsequently induced autophagy and inhibited cell proliferation by increasing the nucleocytoplasmic transport of p53 and AMPK activation. KPNA2/p53 inhibitors attenuated autophagy induced by silencing TMOD1 in AML cells. Silencing TMOD1 also inhibited tumor growth by elevating KPNA2-mediated autophagy in nude mice bearing MOLM-13 xenografts. Collectively, our data demonstrated that TMOD1 could be a novel therapeutic target for AML treatment.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies lacking effective therapies. KRAS mutations that occur in over 90% of PDAC are major oncogenic drivers of PDAC. The MAPK signaling pathway plays a central role in KRAS-driven oncogenic signaling. However, pharmacological inhibitors of the MAPK pathway are poorly responded in KRAS-mutant PDAC, raising a compelling need to understand the mechanism behind and to seek new therapeutic solutions. Herein, we perform a screen utilizing a library composed of 800 naturally-derived bioactive compounds to identify natural products that are able to sensitize KRAS-mutant PDAC cells to the MAPK inhibition. We discover that tetrandrine, a natural bisbenzylisoquinoline alkaloid, shows a synergistic effect with MAPK inhibitors in PDAC cells and xenograft models. Mechanistically, pharmacological inhibition of the MAPK pathway exhibits a double-edged impact on the TRAIL-death receptor axis, transcriptionally upregulating TRAIL yet downregulating its agonistic receptors DR4 and DR5, which may explain the limited therapeutic outcomes of MAPK inhibitors in KRAS-mutant PDAC. Of great interest, tetrandrine stabilizes DR4/DR5 protein via impairing ubiquitination-mediated protein degradation, thereby allowing a synergy with MAPK inhibition in inducing apoptosis in KRAS-mutant PDAC. Our findings identify a new combinatorial approach for treating KRAS-mutant PDAC and highlight the role of TRAIL-DR4/DR5 axis in dictating the therapeutic outcome in KRAS-mutant PDAC.
Assuntos
Benzilisoquinolinas , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Benzilisoquinolinas/farmacologia , Benzilisoquinolinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Morte Celular , Neoplasias PancreáticasRESUMO
Oxidative disruption of dopaminergic neurons is regarded as a crucial pathogenesis in Parkinson's disease (PD), eventually causing neurodegenerative progression. (-)-Clausenamide (Clau) is an alkaloid isolated from plant Clausena lansium (Lour.), which is well-known as a scavenger of lipid peroxide products and exhibiting neuroprotective activities both in vivo and in vitro, yet with the in-depth molecular mechanism unrevealed. In this study, we evaluated the protective effects and mechanisms of Clau on dopaminergic neuron. Our results showed that Clau directly interacted with the Ser663 of ALOX5, the PKCα-phosphorylation site, and thus prevented the nuclear translocation of ALOX5, which was essential for catalyzing the production of toxic lipids 5-HETE. LC-MS/MS-based phospholipidomics analysis demonstrated that the oxidized membrane lipids were involved in triggering ferroptotic death in dopaminergic neurons. Furthermore, the inhibition of ALOX5 was found to significantly improving behavioral defects in PD mouse model, which was confirmed associated with the effects of attenuating the accumulation of lipid peroxides and neuronal damages. Collectively, our findings provide an attractive strategy for PD therapy by targeting ALOX5 and preventing ferroptosis in dopaminergic neurons.
Assuntos
Ferroptose , Doença de Parkinson , Animais , Camundongos , Neurônios Dopaminérgicos , Cromatografia Líquida , Espectrometria de Massas em TandemRESUMO
Acute kidney injury (AKI) is a worldwide problem, and there is no effective drug to eliminate AKI. The death of renal cells is an important pathological basis of intrinsic AKI. At present, targeted therapy for TEC death is a research hotspot in AKI therapy. There are many ways of cell death involved in the occurrence and development of AKI, such as apoptosis, necrosis, ferroptosis, and pyroptosis. This article mainly focuses on the role of pyroptosis in AKI. The assembly and activation of NLRP3 inflammasome is a key event in the occurrence of pyroptosis, which is affected by many factors, such as the activation of the NF-κB signaling pathway, mitochondrial instability and excessive endoplasmic reticulum (ER) stress. The activation of NLRP3 inflammasome can trigger its downstream inflammatory cytokines, which will lead to pyroptosis and eventually induce AKI. In this paper, we reviewed the possible mechanism of pyroptosis in AKI and the potential effective inhibitors of various key targets in this process. It may provide potential therapeutic targets for novel intrinsic AKI therapies based on pyroptosis, so as to develop better therapeutic strategies.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Piroptose , Injúria Renal Aguda/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
Pyroptosis is a form of programmed cell death activated by various stimuli and is characterized by inflammasome assembly, membrane pore formation, and the secretion of inflammatory cytokines (IL-1ß and IL-18). Atherosclerosis-related risk factors, including oxidized low-density lipoprotein (ox-LDL) and cholesterol crystals, have been shown to promote pyroptosis through several mechanisms that involve ion flux, ROS, endoplasmic reticulum stress, mitochondrial dysfunction, lysosomal rupture, Golgi function, autophagy, noncoding RNAs, post-translational modifications, and the expression of related molecules. Pyroptosis of endothelial cells, macrophages, and smooth muscle cells in the vascular wall can induce plaque instability and accelerate atherosclerosis progression. In this review, we focus on the pathogenesis, influence, and therapy of pyroptosis in atherosclerosis and provide novel ideas for suppressing pyroptosis and the progression of atherosclerosis.
Assuntos
Aterosclerose/imunologia , Células Endoteliais/imunologia , Imunidade Celular/imunologia , Mediadores da Inflamação/imunologia , Piroptose/imunologia , Animais , Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Lipoproteínas LDL/imunologia , Lipoproteínas LDL/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismoRESUMO
The purpose of this study is to investigate the effectiveness of sphingosine-1-phosphate (S1P) and Z-VAD-FMK (Z-VAD) as anti-apoptotic agents to preserve ovarian function and prevent tissue damage during ovarian tissue cryopreservation and transplantation. This study consisted of two steps, in vitro and in vivo. In the first step, human ovarian tissues were cryopreserved using slow-freezing media alone, S1P, or Z-VAD (control, S1P, Z-VAD group); based on the outcomes in these groups, Z-VAD was selected for subsequent xenotransplantation. In the second step, human frozen/thawed ovarian tissues were grafted into fifty mice divided into three groups: slow-freezing/thawing and transplantation without an anti-apoptotic agent (Trans-control) and xenotransplantation with or without Z-VAD injection (Trans-Z-VAD-positive and Trams-Z-VAD-negative groups, respectively). In the first step, the Z-VAD group had a significantly higher primordial follicular count than the S1P (p = 0.005) and control groups (p = 0.04). Transplanted ovarian tissues were obtained 4 weeks after transplantation (second step). Angiogenesis was significantly increased in the Z-VAD-negative (p = 0.03) and -positive (p = 0.04) groups compared to the control group. This study demonstrated that slow-freezing and transplantation with Z-VAD is an effective method for preserving primordial follicle counts, decreasing double-strand DNA breaks, and increasing angiogenesis in a mouse model. Further molecular and clinical studies are needed to confirm these results.
Assuntos
Apoptose/efeitos dos fármacos , Xenoenxertos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Adolescente , Adulto , Clorometilcetonas de Aminoácidos/uso terapêutico , Animais , Criopreservação/métodos , Feminino , Congelamento , Humanos , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos SCID , Folículo Ovariano/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Transplante Heterólogo/métodos , Adulto JovemRESUMO
Despite the discovery of tyrosine kinase inhibitors (TKIs) for the treatment of breakpoint cluster region-Abelson (BCR-ABL)+ cancer types, patients with chronic myeloid leukemia (CML) treated with TKIs develop resistance and severe adverse effects. Combination treatment, especially with a histone deacetylase (HDAC) 6 inhibitor (HDAC6i), appears to be an attractive option to prevent TKI resistance, considering the potential capacity of an HDAC6i to diminish BCR-ABL expression. We first validated the in vivo anti-cancer potential of the compound 7b by significantly reducing the tumor burden of BALB/c mice xenografted with K-562 cells, without notable organ toxicity. Here, we hypothesize that the HDAC6i compound 7b can lead to BCR-ABL downregulation in CML cells and sensitize them to TKI treatment. The results showed that combination treatment with imatinib and 7b resulted in strong synergistic caspase-dependent apoptotic cell death and drastically reduced the proportion of leukemia stem cells, whereas this treatment only moderately affected healthy cells. Ultimately, the combination significantly decreased colony formation in a semisolid methylcellulose medium and tumor mass in xenografted zebrafish compared to each compound alone. Mechanistically, the combination induced BCR-ABL ubiquitination and downregulation followed by disturbance of key proteins in downstream pathways involved in CML proliferation and survival. Taken together, our results suggest that an HDAC6i potentiates the effect of imatinib and could overcome TKI resistance in CML cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ubiquitinação/efeitos dos fármacos , Animais , Caspases/efeitos dos fármacos , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Receptor-interacting protein 1 (RIP1, also known as RIPK1) is not only a tumor-promoting factor in several cancers but also mediates either apoptosis or necroptosis in certain circumstances. In this study we investigated what role RIP1 plays in human ovarian cancer cells. We showed that knockout (KO) of RIP1 substantially suppressed cell proliferation, accompanied by the G2/M checkpoint arrest in two human ovarian cancer cell lines SKOV3 and A2780. On the other hand, RIP1 KO remarkably attenuated cisplatin-induced cytotoxicity, which was associated with reduction of the apoptosis markers PARP cleavage and the necroptosis marker phospho-MLKL. We found that RIP1 KO suppressed cisplatin-induced ROS accumulation in both SKOV3 and A2780 cells. ROS scavenger BHA, apoptosis inhibitor Z-VAD or necroptosis inhibitor NSA could effectively suppress cisplatin's cytotoxicity in the control cells, suggesting that ROS-mediated apoptosis and necroptosis were involved in cisplatin-induced cell death. In addition, blocking necroptosis with MLKL siRNA effectively attenuated cisplatin-induced cytotoxicity. In human ovarian cancer A2780 cell line xenograft nude mice, RIP1 KO not only significantly suppressed the tumor growth but also greatly attenuated cisplatin's anticancer activity. Our results demonstrate a dual role of RIP1 in human ovarian cancer: it acts as either a tumor-promoting factor to promote cancer cell proliferation or a tumor-suppressing factor to facilitate anticancer effects of chemotherapeutics such as cisplatin.
Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Necroptose/fisiologia , Neoplasias Ovarianas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Necroptose/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
PURPOSE: To evaluate the efficiency of ovarian tissue treatment with Z-VAD-FMK, a broad-spectrum caspase inhibitor, to prevent follicle loss induced by ischemia/reperfusion injury after transplantation. METHODS: In vitro, granulosa cells were exposed to hypoxic conditions, reproducing early ischemia after ovarian tissue transplantation, and treated with Z-VAD-FMK (50 µM). In vivo, cryopreserved human ovarian fragments (n = 39) were embedded in a collagen matrix containing or not Z-VAD-FMK (50 µM) and xenotransplanted on SCID mice ovaries for 3 days or 3 weeks. RESULTS: In vitro, Z-VAD-FMK maintained the metabolic activity of granulosa cells, reduced HGL5 cell death, and decreased PARP cleavage. In vivo, no improvement of follicular pool and global tissue preservation was observed with Z-VAD-FMK in ovarian tissue recovered 3-days post-grafting. Conversely, after 3 weeks of transplantation, the primary follicular density was higher in fragments treated with Z-VAD-FMK. This improvement was associated with a decreased percentage of apoptosis in the tissue. CONCLUSIONS: In situ administration of Z-VAD-FMK slightly improves primary follicular preservation and reduces global apoptosis after 3 weeks of transplantation. Data presented herein will help to guide further researches towards a combined approach targeting multiple cell death pathways, angiogenesis stimulation, and follicular recruitment inhibition.
Assuntos
Clorometilcetonas de Aminoácidos/administração & dosagem , Apoptose/efeitos dos fármacos , Folículo Ovariano/transplante , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Inibidores de Caspase/administração & dosagem , Feminino , Células da Granulosa/efeitos dos fármacos , Humanos , Camundongos SCID , Folículo Ovariano/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Transplante Heterólogo/efeitos adversosRESUMO
Context: Caragana pruinosa Kom. (Fabaceae), a commonly used folk medicine, has been found to possess antitumor effects. However, the antiproliferative effect of 2,4-dihydroxy-3'-methoxy-4'-ethoxychalcone (DMEC) derived from C. pruinosa against multiple myeloma (MM) has never been investigated. Objective: This study systematically evaluates the antiproliferative effect of DMEC against MM cells. Materials and methods: The antiproliferative effect of DMEC (1, 2, 4, 8, 16, 32, and 64 µM) on MM cells lines, including RPMI8226, MM.1S, and U266, was examined using Cell counting kit-8 (CCK-8) assay after 24 h incubation. The proapoptotic effect of DMEC (20 µM) was determined using fluorescent microscope and flow cytometer, and its possible underlying mechanisms were further studied by using western blotting analysis. Results: The half maximal inhibitory concentrations (IC50) of DMEC on RPMI8226, MM.1S, and U266 cells were calculated as 25.97, 18.36, and 15.02 µM, respectively. The inhibitory effect of DMEC on MM cells was related to mitochondria-mediated apoptosis via upregulation of the cleaved-caspase-3 (C-3), cleaved-caspase-9 (C-9), Bad, and cytochrome C (Cyto C), but downregulation of the Bcl-2 and poly ADP-ribose polymerase (PARP). Furthermore, DMEC (5, 10, and 20 µM) reduced the expression of phosphatidylinositol-3-kinase (PI3K), phosphorylated (p)-protein kinase B (Akt), and p-mammalian target of rapamycin (p-mTOR), which were further evidenced by pretreatment with IGF-1, a PI3K activator. Conclusion: Collectively, our results indicate that the DMEC could be treated as a new candidate for treatment of multiple myeloma in the future. Also, an in vivo study is warranted in the future.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Flavonoides/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Caragana/química , Cartilagem Articular , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Humanos , Fator de Crescimento Insulin-Like I , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Primary effusion lymphoma (PEL) is a lymphoma that shows malignant effusion in body cavities without contiguous tumor masses and has a very poor prognosis. We recently developed a novel drug screening system using patient-derived xenograft (PDX) cells that maintained the primary cell phenotype better than cell lines. This screening is expected to discover anti-tumor drugs that have been overlooked by conventional screening using cell lines. We herein performed this screening to identify new therapeutic agents for PEL. We screened 3518 compounds with known pharmaceutical activities based on cytotoxic effects on PDX cells of PEL and selected YM155, a possible survivin inhibitor. It exerted strong anti-tumor effects in PDX cells and three cell lines of PEL; the GI50 of YM155 was 1.2-7.9nM. We found that YM155 reduced myeloid cell leukemia-1 (MCL-1) protein levels prior to decreasing survivin levels, and this was inhibited by a proteasome inhibitor. The knockdown of MCL-1 by siRNA induced cell death in a PEL cell line, suggesting the involvement of decreased MCL-1 levels in YM155-induced cell death. YM155 also induced the phosphorylation of ERK1/2 and MCL-1, and a MEK1 inhibitor inhibited the phosphorylation of ERK1/2, degradation of MCL-1, and YM155-induced apoptosis. These results indicate that YM155 induces the proteasome-dependent degradation of MCL-1 through its phosphorylation by ERK1/2 and causes apoptosis in PEL cells. Furthermore, a treatment with YM155 significantly inhibited the development of ascites in PEL PDX mice. These results suggest the potential of YM155 as an anti-cancer agent for PEL.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Imidazóis/uso terapêutico , Linfoma de Efusão Primária/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Naftoquinonas/uso terapêutico , Proteólise/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Imidazóis/farmacologia , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/patologia , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Naftoquinonas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
A key to successfully generate the penaeid shrimp cell line is to find out how primary cells died. The most suitable period to culture Penaeus monodon haemocytes was in the first 48 h of culture because cells had normal morphology, high percent of viable cells (65.29 ± 5.43%), low percent of early (11.75 ± 1.30%) and late apoptotic cells (15.47 ± 11.71%) determined by Annexin V and TUNEL including constant IAP (0.06 ± 0.01-0.07 ± 0.01) and caspase-3 expression (0.30 ± 0.06-0.39 ± 0.10) by real-time PCR throughout the experiment. Moreover, adding 50 and 250 µM of the cell permeable pan caspase inhibitor Z-VAD-FMK produced some melanised cells since the 48(th) hour, while percent of viable cells was decreased since the 24(th) hour with no difference in percent of early and late apoptotic cells compared to control at each time point. No difference of IAP and caspase-3 expression level in both Z-VAD-FMK groups was found compared to control and vehicle groups at each time point, excluding caspase-3 in 250 µM Z-VAD-FMK at the 24(th) hour was higher than control and vehicle. Supplementing sodium fluoride (NaF) induced cell membrane damage and cellular shrinkage of primary haemocytes within 2 h. Even percent of viable cells was reduced down to zero and percent of late apoptotic cells was increased by 2 h of incubation in 25 and 50 mM NaF, IAP and caspase-3 in all NaF groups was not different from control. These results indicate that a number of primary haemocytes derived in this study die through the apoptotic process.
Assuntos
Proteínas de Artrópodes/genética , Caspase 3/genética , Morte Celular , Proteínas Inibidoras de Apoptose/genética , Penaeidae/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose , Proteínas de Artrópodes/metabolismo , Caspase 3/metabolismo , Técnicas de Cultura de Células , Feminino , Hemócitos/efeitos dos fármacos , Hemócitos/fisiologia , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Penaeidae/genéticaRESUMO
It has been shown that necroptosis-caspase-independent programmed necrotic cell death-can be induced by treatment with tumor necrosis factor (TNF) in the L929 murine fibrosarcoma cell line, even in the absence of a caspase inhibitor. Although it was reported that necrostatin-1-a specific inhibitor of necroptosis-inhibited TNF-induced necroptosis in L929 cells, it has not been elucidated whether the cells eventually die by apoptosis in the presence of necrostatin-1. In this paper, induction of apoptosis was demonstrated in TNF-treated L929 cells in the presence of necrostatin-1. Co-treatment with cycloheximide expedited apoptosis induction in necrostatin-1/TNF-treated L929 cells: typical apoptotic morphological changes, including membrane blebbing and nuclear fragmentation, induction of caspase-3 activity, proteolytic activation of caspases-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase (PARP) (a well-known substrate of caspase-3) were observed. Moreover, co-treatment with Z-VAD-fmk (a pan-caspase inhibitor) inhibited apoptosis by completely inhibiting caspases, resulting in a shift from apoptosis to necroptosis. In contrast, co-treatment with Z-Asp-CH2-DCB (a caspase inhibitor preferential to caspase-3) inhibited apoptosis without expediting necroptosis. These results indicate that apoptosis can be induced in TNF-treated L929 cells when the cells are protected from necroptosis, and support the notion that partial activation of caspase-8 in the presence of a caspase inhibitor preferential to caspase-3 suppresses both apoptosis and necroptosis.
Assuntos
Apoptose/efeitos dos fármacos , Imidazóis/toxicidade , Indóis/toxicidade , Fator de Necrose Tumoral alfa/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Caspase 3/química , Caspase 3/metabolismo , Caspase 8/química , Caspase 8/metabolismo , Caspase 9/química , Caspase 9/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Cicloeximida/toxicidade , Camundongos , Necrose , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
N-Glycanase 1, encoded by NGLY1, catalyzes the deglycosylation of misfolded N-linked glycoproteins retrotranslocated into the cytosol. We identified nine cases with mutations in NGLY1. The patients show developmental delay, seizures, peripheral neuropathy, abnormal liver function and alacrima (absence of tears). The mutations in NGLY1 resulted in the absence of N-glycanase 1 protein in patient-derived fibroblasts. Applying a recently established cellular deglycosylation-dependent Venus fluorescence assay, we found that patient fibroblasts had dramatically reduced fluorescence, indicating a pronounced reduction in N-glycanase enzymatic activity. Using this assay, we could find no evidence of other related activities. Our findings reveal that NGLY1 mutations destroy both N-glycanase 1 protein and enzymatic activity.
Assuntos
Deficiências do Desenvolvimento/genética , Oftalmopatias Hereditárias/genética , Insuficiência Hepática/genética , Doenças do Aparelho Lacrimal/genética , Mutação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Doenças do Sistema Nervoso Periférico/genética , Convulsões/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deficiências do Desenvolvimento/patologia , Ensaios Enzimáticos , Éxons , Fibroblastos/enzimologia , Fibroblastos/patologia , Expressão Gênica , Genes Reporter , Insuficiência Hepática/congênito , Humanos , Doenças do Aparelho Lacrimal/congênito , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Doenças do Sistema Nervoso Periférico/congênito , Cultura Primária de Células , Convulsões/congênitoRESUMO
PURPOSE: Because ovarian granulosa cells are essential for oocyte survival, we examined three human granulosa cell lines as models to evaluate the ability of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) to prevent primordial follicle loss after ovarian tissue transplantation. METHODS: To validate the efficacy of Z-VAD-FMK, three human granulosa cell lines (GC1a, HGL5, COV434) were treated for 48 h with etoposide (50 µg/ml) and/or Z-VAD-FMK (50 µM) under normoxic conditions. To mimic the ischemic phase that occurs after ovarian fragment transplantation, cells were cultured without serum under hypoxia (1 % O(2)) and treated with Z-VAD-FMK. The metabolic activity of the cells was evaluated by WST-1 assay. Cell viability was determined by FACS analyses. The expression of apoptosis-related molecules was assessed by RT-qPCR and Western blot analyses. RESULTS: Our assessment of metabolic activity and FACS analyses in the normoxic experiments indicate that Z-VAD-FMK protects granulosa cells from etoposide-induced cell death. When cells are exposed to hypoxia and serum starvation, their metabolic activity is reduced. However, Z-VAD-FMK does not provide a protective effect. In the hypoxic experiments, the number of viable cells was not modulated, and we did not observe any modifications in the expressions of apoptosis-related molecules (p53, Bax, Bcl-xl, and poly (ADP-ribose) polymerase (PARP)). CONCLUSION: The death of granulosa cell lines was not induced in our ischemic model. Therefore, a protective effect of Z-VAD-FMK in vitro for further use in ovarian tissue transplantation could not be directly confirmed. It will be of interest to potentially use Z-VAD-FMK in vivo in xenograft models.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Ovário/transplante , Inibidores de Caspase/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Etoposídeo/farmacologia , Feminino , Células da Granulosa/citologia , Células da Granulosa/fisiologia , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
The caspase inhibitor benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) has recently been shown to inhibit T cell proliferation without blocking caspase-8 and caspase-3 activation in primary T cells. We showed in this study that z-VAD-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-mediated T cell proliferation induced by z-VAD-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and l-cysteine, whereas d-cysteine which cannot be metabolised to GSH has no effect. These results suggest that the depletion of intracellular GSH is the underlying cause of z-VAD-FMK-mediated inhibition of T cell activation and proliferation. The presence of exogenous GSH also attenuated the inhibition of anti-CD3-induced CD25 and CD69 expression mediated by z-VAD-FMK. However, none of the low molecular weight thiols were able to restore the caspase-inhibitory properties of z-VAD-FMK in activated T cells where caspase-8 and caspase-3 remain activated and processed into their respective subunits in the presence of the caspase inhibitor. This suggests that the inhibition of T cell proliferation can be uncoupled from the caspase-inhibitory properties of z-VAD-FMK. Taken together, the immunosuppressive effects in primary T cells mediated by z-VAD-FMK are due to oxidative stress via the depletion of GSH.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase/farmacologia , Proliferação de Células , Inibidores do Crescimento/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta Imunológica , Glutationa/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Ativação Linfocitária/fisiologia , Estresse Oxidativo/imunologia , Espécies Reativas de Oxigênio/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologiaRESUMO
Over-expression of σ receptors by many tumor cell lines makes ligands for these receptors attractive as potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential σ-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the σ1 and σ2 receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of σ receptors, both compounds inhibited cell proliferation with IC50 values in the low µM range. No chiral differentiation between either the σ receptor binding affinity or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as the expression of bcl2 family members bax, bad and bid. However, apoptosis appeared to be caspase independent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However, further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotective. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the lipid antioxidant α-tocopherol attenuated LPO. Interestingly, α-tocopherol reduced significantly both apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autophagy in RPMI 8226 cells.
Assuntos
Autofagia/efeitos dos fármacos , Mieloma Múltiplo/metabolismo , Apoptose , Proliferação de Células , Humanos , Ligantes , Peroxidação de Lipídeos , Mieloma Múltiplo/genética , Transdução de SinaisRESUMO
Background and Objectives: Smoking can lead to airway inflammation and mucus secretion through the nucleotide-binding domain-like receptor protein 3/caspase-1 pathway. In this study, z-VaD-Ala-Asp-fluoromethyl ketone(Z-VAD), a pan-caspase inhibitor, and acetyl-Asp-Glu-Val-Asp-aldehyde(Ac-DEVD), a caspase-3 inhibitor, were used to investigate the effect of caspase inhibitors on the expression of interleukin(IL)-1ß and IL-8, airway inflammation, and mucus secretion in mice exposed to cigarette smoke(CS). Methods: Thirty-two C57BL/6J male mice were divided into a control group, Smoke group, Z-VAD group, and Ac-DEVD group. Except for the control group, the animals were all exposed to CS for three months. After the experiment, lung function was measured and hematoxylin and eosin staining and periodic acid-Schiff staining were performed. The levels of IL-1ß, IL-8, and mucin 5ac(Muc5ac) in serum and bronchoalveolar lavage fluid(BALF) were determined by enzyme-linked immunosorbent assay. Results: Compared with the control group, the lung function of mice exposed to smoke was poorer, with a large number of inflammatory cells infiltrating around the airway, collapse of alveoli, expansion and fusion of distal alveoli, and formation of emphysema. The Z-VAD group was relieved compared with the smoke group. Airway inflammation was also reduced in the Ac-DEVD group compared with the Smoke group, but the degree of emphysema was not significantly improved. Although Z-VAD relieved airway inflammation and emphysema, Ac-DEVD only relieved inflammation. Z-VAD and Ac-DEVD decreased serum IL-1ß and IL-8 levels. In BALF, IL-1ß was decreased in Z-VAD group and IL-8 was highest in Smoke +Ac-DEVD group compared with control group and Ac-DEVD group. There was no significant difference in the expression of Muc5ac in serum. However, in BALF, levels of Muc5ac were higher in the smoking group and the lowest in the Ac-DEVD group. Conclusion: Mice exposed to smoke had decreased lung function and significant cilia lodging, epithelial cell shedding, and inflammatory cell infiltration, with significant emphysema formation. The pan-caspase inhibitor, Z-VAD, improved airway inflammation and emphysema lesions in the mice exposed to smoke and reduced IL-1ß and IL-8 levels in serum. The caspase-3 inhibitor, Ac-DEVD, reduced airway inflammation, serum IL-1ß and IL-8 levels, and Muc5ac levels in BALF, but it did not improve emphysema.
Assuntos
Fumar Cigarros , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Camundongos , Masculino , Animais , Doença Pulmonar Obstrutiva Crônica/metabolismo , Caspase 3/metabolismo , Interleucina-8/metabolismo , Camundongos Endogâmicos C57BL , Inflamação/induzido quimicamente , Inflamação/metabolismo , Enfisema Pulmonar/metabolismo , Muco/metabolismoRESUMO
In the present study, 24 rabbits were firstly used to evaluate the apoptosis index and matrix degeneration after untreated adult meniscal tears. Vertical tears (0.25 cm in length) were prepared in the avascular zone of the anterior horn. Specimens were harvested at 1, 3, 6, 12 weeks postoperatively. The apoptosis index around tear sites stayed at a high level throughout the whole follow-up period. The depletion of glycosaminoglycans (GAG) and aggrecan at the tear site was observed, while the deposition of COL I and COL II was not affected, even at the last follow-up of 12 weeks after operation. The expression of SOX9 decreased significantly; no cellularity was observed at the wound interface at all timepoints. Secondly, another 20 rabbits were included to evaluate the effects of anti-apoptosis therapy on rescuing meniscal cells and enhancing meniscus repair. Longitudinal vertical tears (0.5 cm in length) were made in the meniscal avascular body. Tears were repaired by the inside-out suture technique, or repaired with sutures in addition to fibrin gel and blank silica nanoparticles, or silica nanoparticles encapsulating apoptosis inhibitors (z-vad-fmk). Samples were harvested at 12 months postoperatively. We found the locally administered z-vad-fmk agent at the wound interface significantly alleviated meniscal cell apoptosis and matrix degradation, and enhanced meniscal repair in the avascular zone at 12 months after operation. Thus, local administration of caspase inhibitors (z-vad-fmk) is a promising therapeutic strategy for alleviating meniscal cell loss and enhancing meniscal repair after adult meniscal tears in the avascular zone.
RESUMO
Ferroptosis is a regulated cell death induced by iron-dependent lipid peroxidation. The heme-responsive transcription factor BTB and CNC homology 1 (BACH1) promotes ferroptosis by repressing the transcription of genes involved in glutathione (GSH) synthesis and intracellular labile iron metabolism, which are key regulatory pathways in ferroptosis. We found that BACH1 re-expression in Bach1-/- immortalized mouse embryonic fibroblasts (iMEFs) can induce ferroptosis upon 2-mercaptoethanol removal, without any ferroptosis inducers. In these iMEFs, GSH synthesis was reduced, and intracellular labile iron levels were increased upon BACH1 re-expression. We used this system to investigate whether the major ferroptosis regulators glutathione peroxidase 4 (Gpx4) and apoptosis-inducing factor mitochondria-associated 2 (Aifm2), the gene for ferroptosis suppressor protein 1, are target genes of BACH1. Neither Gpx4 nor Aifm2 was regulated by BACH1 in the iMEFs. However, we found that BACH1 represses AIFM2 transcription in human pancreatic cancer cells. These results suggest that the ferroptosis regulators targeted by BACH1 may vary across different cell types and animal species. Furthermore, we confirmed that the ferroptosis induced by BACH1 re-expression exhibited a propagating effect. BACH1 re-expression represents a new strategy for inducing ferroptosis after GPX4 or system Xc- suppression and is expected to contribute to future ferroptosis research.