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1.
Food Chem ; 367: 130767, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34391996

RESUMO

This study aimed to investigate the effect of caspase-3 inhibitor in mitochondrial apoptosis activation on structure protein degradation during postmortem storage. Mitochondrial dysfunction, apoptotic factors, structure protein degradation and the myofibrillar rupture index between the control and caspase-3 inhibitor groups were determined. The results show caspase-3 inhibitor repressed the mitochondrial membrane permeability and mitochondrial swelling, as well as increased mitochondrial membrane potential, causing a decrease in the release of cytochrome c from mitochondria to cytoplasm and caspase-9/3 activities (P < 0.05). Subsequently, small myofibrillar proteins (desmin and troponin-T) were susceptible to degradation, initiating texture deterioration. By contrast, giant structure proteins (titin and nebulin) were degraded during later postmortem storage, predominantly contributing to fish softening. The results further suggest that caspase-3 is involved in degradation of structure proteins during postmortem through mitochondrial apoptosis pathways.


Assuntos
Esocidae , Mitocôndrias , Animais , Apoptose , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/metabolismo , Esocidae/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo
2.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638988

RESUMO

Endometriosis is characterized by the formation and development of endometrial tissues outside the uterus, based on an imbalance between proliferation and cell death, leading to the uncontrolled growth of ectopic foci. The potential target for the regulation of these processes is the endocannabinoid system, which was found to be involved in the migration, proliferation, and survival of tumor cells. In this paper, we investigated the effect of endocannabinoid-like compounds from the N-acyl dopamine (NADA) family on the viability of stromal cells from ectopic and eutopic endometrium of patients with ovarian endometriosis. N-arachidonoyldopamine, N-docosahexaenoyldopamine, and N-oleoyldopamine have been shown to have a five-times-more-selective cytotoxic effect on endometrioid stromal cells. To study the mechanisms of the toxic effect, inhibitory analysis, measurements of caspase-3/9 activity, reactive oxygen species, and the mitochondrial membrane potential were performed. It was found that NADA induced apoptosis via an intrinsic pathway through the CB1 receptor and downstream serine palmitoyltransferase, NO synthase activation, increased ROS production, and mitochondrial dysfunction. The higher selectivity of NADA for endometriotic stromal cells and the current lack of effective drug treatment can be considered positive factors for further research of these compounds as possible therapeutic agents against endometriosis.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos Araquidônicos/farmacologia , Dopamina/análogos & derivados , Endometriose/metabolismo , Endométrio/metabolismo , Células Estromais/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dopamina/farmacologia , Endometriose/patologia , Endométrio/efeitos dos fármacos , Feminino , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos
3.
Biochemistry ; 60(37): 2824-2835, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34472839

RESUMO

Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4-P1' sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4-P1' sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.


Assuntos
Caspase 3/química , Caspase 6/química , Caspase 9/metabolismo , Sequência de Aminoácidos/genética , Apoptose/fisiologia , Caspase 3/metabolismo , Caspase 6/metabolismo , Caspase 8/metabolismo , Caspase 9/fisiologia , Caspases/metabolismo , Ativação Enzimática , Humanos , Transdução de Sinais/genética
4.
Int J Mol Sci ; 22(18)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34576232

RESUMO

Neuroblastoma, the most common extra-cranial solid tumor of early childhood, is one of the major therapeutic challenges in child oncology: it is highly heterogenic at a genetic, biological, and clinical level. The high-risk cases have one of the least favorable outcomes amongst pediatric tumors, and the mortality rate is still high, regardless of the use of intensive multimodality therapies. Here, we observed that neuroblastoma cells display an increased expression of Cockayne Syndrome group B (CSB), a pleiotropic protein involved in multiple functions such as DNA repair, transcription, mitochondrial homeostasis, and cell division, and were recently found to confer cell robustness when they are up-regulated. In this study, we demonstrated that RNAi-mediated suppression of CSB drastically impairs tumorigenicity of neuroblastoma cells by hampering their proliferative, clonogenic, and invasive capabilities. In particular, we observed that CSB ablation induces cytokinesis failure, leading to caspases 9 and 3 activation and, subsequently, to massive apoptotic cell death. Worthy of note, a new frontier in cancer treatment, already proved to be successful, is cytokinesis-failure-induced cell death. In this context, CSB ablation seems to be a new and promising anticancer strategy for neuroblastoma therapy.


Assuntos
Citocinese/fisiologia , DNA Helicases/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Neuroblastoma/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Interferência de RNA , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Centrossomo , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fuso Acromático
5.
Biomolecules ; 11(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208033

RESUMO

Previous studies have reported that 4,6'-Anhydrooxysporidinone (SSF2-2), isolated from Fusarium lateritium SSF2, has neuroprotective effects on the HT-22 hippocampal neuronal cell line. However, the anti-cancer effect of SSF2-2 remains unclear. Here, we examined the viability of MCF-7 human breast cancer cells treated with SSF2-2 or left untreated using a cell viability assay kit. The underlying molecular mechanism was further investigated by Western blotting and immunocytochemistry studies. The results demonstrated that SSF2-2 inhibited the viability of MCF-7 cells. Treatment with SSF2-2 increased the levels of cleaved caspase-9, cleaved caspase-7, poly (ADP-ribose) polymerase (PARP), and LC3B. Additionally, SSF2-2 significantly increased the conversion of LC3-I to LC3II and LC3-positive puncta in MCF-7 cells.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Piridonas/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Caspase 7/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Fusarium/metabolismo , Humanos , Células MCF-7 , Proteínas Associadas aos Microtúbulos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Piridonas/química , Espécies Reativas de Oxigênio/metabolismo
6.
Chem Biol Interact ; 347: 109582, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34302802

RESUMO

Different aspects of reproductive functions are regulated by mitochondrial-controlled events. This study investigated the effect of plumbagin (PL) on testicular mitochondria with a view to unravelling the mechanism of the antifertility potential of plumbagin in testis of healthy rats. Thirty-two male Wistar strain albino rats were randomly allocated into four groups of eight animals each. The control or healthy group received orally 0.1 % DMSO while animals in the remaining three groups received 2.5 mg PL/kg bdwt, 5.0 mg PL/kg bdwt and 10 mg PL/kg bdwt, respectively, for 14 days. In study two, twenty-four male Wistar rats were randomly divided into three (3) groups and were orally administered 0.1% DMSO (control), 30 and 100 mg/kg PL, respectively once daily for 72 h. Rat testis mitochondria were isolated using differential centrifugation. The mitochondrial Permeability Transition (mPT) pore, mitochondrial ATPase (mATPase) activity and mitochondrial lipid peroxidation were assessed spectrophotometrically. Expression of apoptotic proteins (p53, Bax, Bcl-2) and the release of cytochrome c were determined by immunochemical technique. Reproductive receptors (FSH, PR), the expression of aromatase, Testis Specific Kinase-1 {TESK-1} were quantified by RT-PCR. The various doses of plumbagin (2.5, 5.0 and 10 mg/kg bdwt) induced opening of the testicular mPT pore by 2, 5 and 8 folds, respectively, after 14 days of oral administration. These doses of plumbagin also caused enhancement of mATPase activity, elevated generation of mLPO as well as increases in the concentrations of caspases 9 and 3. Sperm analysis revealed that these doses of PL also caused significant decreases in sperm count and motility and increased sperm abnormalities compared to control. Interestingly, these effects were accompanied by dose-dependent expressions of the Bak, p53 and cytochrome c release. Conversely, the abundance of anti-apoptotic Bcl-2 protein decreased relative to control. The levels of transcripts of FSH and progesterone receptors as well as TESK-1 and aromatase decreased significantly relative to control. Furthermore, PL strongly inhibited p53-MDM2 compared to control. Altogether, these findings show that plumbagin damages testicular cells through the activation of mitochondrial pathway involving the p53 protein network.


Assuntos
Morte Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Naftoquinonas/farmacologia , Testículo/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 9/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/metabolismo , Proteína Supressora de Tumor p53/metabolismo
7.
Aging (Albany NY) ; 13(14): 18757-18768, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34324434

RESUMO

Both homoharringtonine (HHT) and curcumin exhibit anti-proliferative effects on lymphoma cells, but the effects of combined HHT and curcumin treatment remain unclear. Here, we investigated the effects of HHT/curcumin combination on the proliferation, apoptosis, and invasion in lymphoma cells. CCK-8, flow cytometry, and transwell assays were used to assess proliferation, apoptosis, and invasion of U937 and Raji cells. p-Smad3, E-cadherin, and N-cadherin expression were also measured in Raji cells using Western blot assays. Combination of HHT and curcumin synergistically inhibited U937 and Raji cell proliferation and invasion. In addition, the combination treatment markedly increased apoptosis of Raji cells as evidenced by increased Bax, cleaved caspase 3, and cleaved caspase 9 expression. Meanwhile, the combination treatment promoted anti-tumor mechanisms in Raji cells as indicated by decreases in p-Smad3 and N-cadherin and increases in E-cadherin. In vivo experiments showed that the combination treatment suppressed tumor growth in a mouse Raji xenograft model. Our findings indicate that combination of HHT and curcumin inhibited lymphoma cell growth by downregulating the TGF-ß/Smad3 pathway. These results suggest that HHT combined with curcumin might be a promising therapeutic approach for the treatment of lymphoma.


Assuntos
Antineoplásicos/farmacologia , Curcumina/farmacologia , Mepesuccinato de Omacetaxina/farmacologia , Linfoma/tratamento farmacológico , Extratos Vegetais/farmacologia , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Antineoplásicos/uso terapêutico , Apoptose , Caderinas/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Cephalotaxus/química , Curcuma/química , Curcumina/uso terapêutico , Quimioterapia Combinada , Mepesuccinato de Omacetaxina/uso terapêutico , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Fitoterapia , Extratos Vegetais/uso terapêutico , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/metabolismo
8.
Bioengineered ; 12(1): 4100-4110, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34288800

RESUMO

Preeclampsia (PE) is a potentially fatal pregnancy complication; however, its pathogenesis remains unclear. Long non-coding RNAs (lncRNAs) are associated with occurrence and progression of PE. Therefore, this study aimed to explore the function of the lncRNA prospero homeobox 1-antisense RNA 1 (PROX1-AS1) and elucidate its underlying molecular mechanism of action. We found that the expression levels of PROX1-AS1 were elevated in both the placental tissues and blood samples of the patients with PE. Moreover, the results of the flow cytometry and transwell assay showed that the knockdown of PROX1-AS1 inhibited the apoptosis and promoted the migration and invasion of HTR-8/SVneo cells. We also assessed the interactions between PROX1-AS1, caspase-9, and microRNA (miR)-211-5p via dual-luciferase reporter and RNA pull-down analyses. The data indicated that PROX1-AS1 acted as a sponge for miR-211-5p to regulate the expression of caspase-9. Moreover, the expression of miR-211-5p was reduced in PE and negatively related to PROX1-AS1, while that of caspase-9 was increased in PE and negatively regulated by miR-211-5p. Furthermore, inhibition of miR-211-5p rescued the facilitation of cell apoptosis, migration and invasion induced by the knockdown of PROX1-AS1. We also found that caspase-9 improved the apoptosis rate, and suppressed the cell migration and invasion induced by the overexpression of miR-211-5p. In conclusion, the knockdown of PROX1-AS1 promoted the cell morbidity of the trophoblast cells by modulating the miR-211-5p/caspase-9 axis, which may alleviate the progression of PE. This novel regulatory network may contribute to the pathogenesis and progression of PE.


Assuntos
Caspase 9/metabolismo , Movimento Celular/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Trofoblastos/citologia , Trofoblastos/metabolismo , Adulto , Sequência de Bases , Estudos de Casos e Controles , Linhagem Celular , Feminino , Humanos , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Gravidez , RNA Longo não Codificante/genética , Regulação para Cima/genética
9.
Biomed Res Int ; 2021: 6630232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34195274

RESUMO

Background: Ischemia-reperfusion injury is one of the most critical phenomena in lung transplantation and causes primary graft failure. Its pathophysiology remains incompletely understood, although the inflammatory response and apoptosis play key roles. Lidocaine has anti-inflammatory properties. The aim of this research is to evaluate the effect of intravenous lidocaine on the inflammatory and apoptotic responses in lung ischemia-reperfusion injury. Methods: We studied the histological and immunohistochemical changes in an experimental model of lung transplantation in pigs. Twelve pigs underwent left pneumonectomy, cranial lobectomy, caudal lobe reimplantation, and 60 minutes of graft reperfusion. Six of the pigs made up the control group, while six other pigs received 1.5 mg/kg of intravenous lidocaine after induction and a 1.5 mg/kg/h intravenous lidocaine infusion during surgery. In addition, six more pigs underwent simulated surgery. Lung biopsies were collected from the left caudal lobe 60 minutes after reperfusion. We conducted a double study on these biopsies and assessed the degree of inflammation, predominant cell type (monocyte-macrophage, lymphocytes, or polymorphous), the degree of congestion, and tissue edema by hematoxylin and eosin stain. We also conducted an immunohistochemical analysis with antibodies against CD68 antigens, monocyte chemoattractant protein-1 (MCP-1), Bcl-2, and caspase-9. Results: The lungs subjected to ischemia-reperfusion injury exhibited a higher degree of inflammatory infiltration. The predominant cell type was monocyte-macrophage cells. Both findings were mitigated by intravenous lidocaine administration. Immunohistochemical detection of anti-CD68 and anti-MCP-1 showed higher infiltration in the lungs subjected to ischemia-reperfusion injury, while intravenous lidocaine decreased the expression. Ischemia-reperfusion induced apoptotic changes and decreased Bcl-2 expression. The group treated with lidocaine showed an increased number of Bcl-2-positive cells. No differences were observed in caspase-9 expression. Conclusions: In our animal model, intravenous lidocaine was associated with an attenuation of the histological markers of lung damage in the early stages of reperfusion.


Assuntos
Apoptose/efeitos dos fármacos , Inflamação/tratamento farmacológico , Injeções Intravenosas , Lidocaína/administração & dosagem , Pulmão/cirurgia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Anti-Inflamatórios/administração & dosagem , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biópsia , Caspase 9/metabolismo , Quimiocina CCL2/metabolismo , Hemodinâmica , Pulmão/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Suínos
10.
Dev Cell ; 56(13): 1900-1916.e5, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34197726

RESUMO

Stem cells (SCs) play a key role in homeostasis and repair. While many studies have focused on SC self-renewal and differentiation, little is known regarding the molecular mechanism regulating SC elimination and compensation upon loss. Here, we report that Caspase-9 deletion in hair follicle SCs (HFSCs) attenuates the apoptotic cascade, resulting in significant temporal delays. Surprisingly, Casp9-deficient HFSCs accumulate high levels of cleaved caspase-3 and are improperly cleared due to an essential caspase-3/caspase-9 feedforward loop. These SCs are retained in an apoptotic-engaged state, serving as mitogenic signaling centers by continuously releasing Wnt3 and instructing proliferation. Investigating the underlying mechanism, we reveal a caspase-3/Dusp8/p38 module responsible for Wnt3 induction, which operates in both normal and Casp9-deleted HFSCs. Notably, Casp9-deleted mice display accelerated wound repair and de novo hair follicle regeneration. Taken together, we demonstrate that apoptotic cells represent a dynamic SC niche, from which emanating signals drive SC proliferation and tissue regeneration.


Assuntos
Caspase 3/genética , Caspase 9/genética , Fosfatases de Especificidade Dupla/genética , Regeneração/genética , Proteína Wnt3/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Autorrenovação Celular/genética , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Nicho de Células-Tronco/genética , Células-Tronco/metabolismo , Cicatrização/genética
11.
Int J Biol Macromol ; 185: 813-820, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34186122

RESUMO

The stability of IFN-γ as a therapeutic protein can play a key role on its anticancer effects. Herein, we explored the thermodynamic parameters and conformational stability of IFN-γ in the presence of calycosin, the main active compound of Radix astragali, by different biophysical and theoretical analysis. Afterwards, the improved anticancer effects of IFN-γ-calycosin interaction relative to IFN-γ alone were assessed on hepatocellular carcinoma (HepG2) cell line by MTT and caspase assays. ITC data indicated that upon interaction of calycosin with IFN-γ the binding and thermodynamic parameters were as follows: Kd = 1.9 µM, ΔG° = -32.45 kJ/mol, ΔH° = -11.91 kJ/mol, and TΔS° = 20.54 kJ/mol. ANS/synchronous fluorescence, CD and UV-Vis spectroscopy studies indicated that the interaction between calycosin and IFN-γ caused the folding of the IFN-γ backbone in to a more packed structure with enhanced α-helix content and higher melting temperature (Tm) value. The spectroscopic outcomes were then verified by molecular docking and molecular dynamic analysis. It was also shown that after incubation of the IFN-γ samples at 50 °C for 60 min in the presence of calycosin (5 µM), the IFN-γ-calycosin system showed a significant antiproliferative effects against hepatocellular carcinoma (HepG2) cells through caspase-9/3 activation and this anticancer effect was more pronounced than free IFN-γ. This data may provide useful information about the development of IFN-γ-based therapeutic platforms.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Interferon gama/farmacologia , Isoflavonas/química , Neoplasias Hepáticas/metabolismo , Antineoplásicos/química , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estabilidade de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Interferon gama/química , Neoplasias Hepáticas/tratamento farmacológico , Dobramento de Proteína/efeitos dos fármacos , Termodinâmica
12.
AAPS PharmSciTech ; 22(5): 158, 2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34009603

RESUMO

The present study was aimed to enhance the mitochondrial function in oxidative stress-induced diabetes. To achieve this, Ficus religiosa L. extract loaded solid lipid nanoparticles (ETNPs) were prepared and functionalized by using triphenylphosphonium. Developed nanoparticles demonstrated desired quality attributes with sustained release for up to 24 h and excellent storage stability for up to 180 days at 40 ± 2°C and 75 ± 5% relative humidity. In vitro cytotoxicity assessment showed no toxicity of ETNPs. Interestingly, oral administration of ETNPs to diabetic rats demonstrated improved mitochondrial function by normalizing the mitochondrial morphology, intracellular calcium ion concentration, complexes I, II, IV, and V activity, mitochondrial membrane potential, and antioxidant levels. Further, reduction in apoptotic markers viz. cytochrome-C, caspase-3, and caspase-9 was observed following the ETNP treatment. Moreover, significant reduction in blood glucose and glycosylated hemoglobin while significant improvement in plasma insulin was observed as compared to the diabetic group following the treatment with developed formulation. Furthermore, histopathology studies confirmed the safety of the developed formulation and thus, data in hand collectively suggest that proposed strategy can be effectively used to improve the mitochondrial function in oxidative stress-induced diabetes along with better control over blood glucose and glycosylated hemoglobin.


Assuntos
Antioxidantes/farmacologia , Ficus/química , Nanopartículas , Compostos Organofosforados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Citocromos c/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Compostos Organofosforados/administração & dosagem , Compostos Organofosforados/isolamento & purificação , Extratos Vegetais/administração & dosagem , Ratos , Ratos Wistar
13.
Mol Biol Rep ; 48(4): 3439-3449, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33999319

RESUMO

Heat shock protein 90 (Hsp90) is a key chaperone that is abnormally expressed in cancer cells, and therefore, designing novel compounds to inhibit chaperone activities of the Hsp90 is a promising therapeutic approach for cancer drug discovery. Debio-0932 is a second-generation Hsp90 inhibitor that exhibited promising anticancer activity against a wide variety of cancer types with a strong binding affinity for Hsp90 and high oral bioavailability. Anticancer activities of the Debio-0932 were tested in MCF-7 and MDA-MB-231 cell lines. Molecular docking results indicated that Debio-0932 was selectively bound to the ATP binding pocket of the Hsp90 with an estimated free energy of binding - 7.24 kcal/mol. Antiproliferative activity of Debio-0932 was determined by XTT assay and Debio-0932 exhibited a cytotoxic effect on MCF-7 and MDA-MB-231 cells in a time and dose-depended manner. Apoptosis inducer role of Debio-0932 was evaluated in MCF-7 and MDA-MB-231 cells with fluorometric apoptosis/necrosis detection kit. Treatment with Debio-0932 stimulated apoptosis in both breast cancer cell lines. mRNA and protein expression levels of Bax, Bcl-2 and Casp-9 were determined in MCF-7 and MDA-MB-231 cells by RT-PCR and Western blotting respectively. Debio-0932 stimulated the down-regulation of anti-apoptotic protein Bcl-2 and the up-regulation of apoptotic protein Bax and cleavage of Casp-9 in cancer cells. Moreover, the anti-invasive potential of Debio-0932 was evaluated in endothelial cells (HUVEC) by wound-healing assay. Debio-0932 decreased the migration of HUVEC cells as compared to the control group. These results indicate that Debio-0932 is a promising compound to treat triple-negative breast cancer and hormone receptor-positive breast cancer, and their metastases.


Assuntos
Apoptose , Benzodioxóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Imidazóis/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzodioxóis/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Caspase 9/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Imidazóis/uso terapêutico , Células MCF-7 , Simulação de Acoplamento Molecular , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genética
14.
Acta Chim Slov ; 68(1): 151-158, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34057526

RESUMO

Phytoalexins are substances with antimicrobial properties produced by plants after being attacked by microorganisms, especially phytopathogenic fungi and viruses. They are also currently being studied for their antitumor effect. We aimed to study the apoptosis-stimulating effect of homobrassinin and thiazino[6,5-b]indol in human ovarian adenocarcinoma A2780 and A2780cis cells via flow cytometric analysis of annexin V/PI, caspase 3 and 9 activity, cytochrome C release, and smac-diablo accumulation. Using the western blot technique, we also monitored the effect of both indoles on the response of heat shock proteins in these cells. Thiazino[6,5-b]indol showed more pronounced sensitizing and/or pro-apoptotic effect compared to homobrassinin accompanied by increased smac-diablo accumulation at earlier time intervals and pronounced externalization of phosphatidylserine at 72 h in A2780cis compared to A2780 cells. The apoptosis stimulating effect of thiazino[6,5-b]indol in A2780cis cells was associated with significant irreversible downregulation of HSP70 and HSP90 and partly with a decrease of HSP40. On the other hand, cisplatin-induced the apoptosis of sensitive A2780 cells with reversible downregulation of HSP40 and HSP57. In conclusion, the effect of thiazino[6,5-b]indol on resistant A2780cis cells could have a great utility in both the potential prevention and the treatment of other cisplatin-resistant tumor cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Indóis/farmacologia , Tiazinas/farmacologia , Tiocarbamatos/farmacologia , Anexina A5/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo
15.
Front Immunol ; 12: 658048, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953720

RESUMO

B cell activation by Tfh cells, i.e., through CD154 engagement of CD40 and IL-21, and survival within GCs are crucial for the T-dependent Ab response. LUBAC, composed of HOIP, SHARPIN, and HOIL-1, catalyzes linear ubiquitination (Linear M1-Ub) to mediate NF-κB activation and cell survival induced by TNF receptor superfamily members, which include CD40. As shown in this study, B cells expressing the Sharpin null mutation cpdm (Sharpincpdm ) could undergo proliferation, CSR, and SHM in response to immunization by a T-dependent Ag, but were defective in survival within GCs, enrichment of a mutation enhancing the BCR affinity, and production of specific Abs. Sharpincpdm B cells stimulated in vitro with CD154 displayed normal proliferation and differentiation, marginally impaired NF-κB activation and survival, but markedly exacerbated death triggered by IL-21. While activating the mitochondria-dependent apoptosis pathway in both Sharpin+/+ and Sharpincpdm B cells, IL-21 induced Sharpincpdm B cells to undergo sustained activation of caspase 9 and caspase 8 of the mitochondria-dependent and independent pathway, respectively, and ultimately caspase 3 in effecting apoptosis. These were associated with loss of the caspase 8 inhibitor cFLIP and reduction in cFLIP Linear M1-Ub, which interferes with cFLIP poly-ubiquitination at Lys48 and degradation. Finally, the viability of Sharpincpdm B cells was rescued by caspase inhibitors but virtually abrogated - together with Linear M1-Ub and cFLIP levels - by a small molecule HOIP inhibitor. Thus, LUBAC controls the cFLIP expression and inhibits the effects of caspase 8 and IL-21-activated caspase 9, thereby suppressing apoptosis of CD40 and IL-21-activated B cells and promoting GC B cell survival.


Assuntos
Apoptose , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Interleucinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Formação de Anticorpos/imunologia , Apoptose/efeitos dos fármacos , Biomarcadores , Caspase 8/metabolismo , Caspase 9 , Comunicação Celular , Sobrevivência Celular , Camundongos , Camundongos Transgênicos , Mutação , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ubiquitinas/metabolismo
16.
Acta Biochim Pol ; 68(2): 309-315, 2021 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-34022786

RESUMO

OBJECTIVE: The current study was to explore whether meisoindigo was effective in suppressing proliferation and inducing apoptosis of human glioblastoma multiforme U87 cells and to explore its possible mechanisms. METHOD: Morphological changes were observed by light microscopy. Cell counting kit-8 (CCK-8) assay was performed to detect cellular proliferation. Apoptosis was monitored by flow cytometry. Akt, phospho-Akt, PI3K, p65, phospho-p65 and apoptosis-related proteins caspase-3 and caspase-9 were examined by Western blotting assays. Immunofluorescence was used to evaluate level of P65 expression in cells. RESULT: Meisoindigo inhibited the proliferation of U87 cells, and the inhibitory effect increased in a dose dependent manner. Moreover, meisoindigo exposure triggered an increase in the level of caspase-3 and caspase-9, supporting its role in the activation of apoptosis. Furthermore, meisoindigo reduced the expression of PI3K, Akt, phospho-Akt, NF-κB, p65 and phospho-p65 in U87 cells, and displacement of p65 from the nucleus to the cytoplasm. CONCLUSION: Meisoindigo inhibits proliferation and induces apoptosis of U87 cells, probably through down-regulating the PI3K/Akt pathway and reducing nuclear translocation of NF-κB p65.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Indóis/metabolismo , Indóis/farmacologia , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo
17.
Eur J Pharmacol ; 904: 174138, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33933463

RESUMO

Neuroblastoma is the most common solid malignant tumor in infants and young children. Its origin is the incompletely committed precursor cells from the autonomic nervous system. Neuroblastoma cells are multipotent cells with a high potency of differentiation into the neural cell types. Neural differentiation leads to the treatment of neuroblastoma by halting the cell and tumor growth and consequently its expansion. Caspases are a family of proteins involved in apoptosis and differentiation. The present study aimed to investigate the potential role of caspase-9 activation on the differentiation of the human neuroblastoma SH-SY5Y cells. Here we investigated the caspase-9 and 3/7 activity during 1,25-dihydroxycholecalciferol (D3)-mediated differentiation of SH-SY5Y cells and took advantage of the inducible caspase-9 system in putting out the differentiation of the neuroblastoma cells. D3-induced differentiation of the cells could lead to activation of caspase-9 and caspase-3/7, astrocyte-like morphology, and increased expression of Glial fibrillary acidic protein (GFAP). By using the inducible caspase-9 system, we showed differentiation of SH-SY5Y cells to astrocyte-like morphology and increased level of GFAP expression. Furthered studies using a specific caspase-9 inhibitor showed inhibition of differentiation mediated by D3 or caspase-9 to astrocyte-like cells. These results show the potency of caspase-9 to direct differentiation of the human neuroblastoma SH-SY5Y cells into cells showing an astrocyte-like morphology.


Assuntos
Caspase 9/genética , Caspase 9/metabolismo , Diferenciação Celular/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Calcitriol/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Inibidores de Caspase/farmacologia , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Neuroblastoma/patologia , Compostos Orgânicos
18.
Methods Mol Biol ; 2255: 1-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033089

RESUMO

Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptosis is executed by a class of cysteine proteases called caspases. Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. The described methods can be applied to any particular protein of interest.


Assuntos
Apoptose , Western Blotting/métodos , Caspase 8/metabolismo , Caspase 9/metabolismo , Neoplasias Ovarianas/patologia , Ativação Enzimática , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
19.
Food Chem ; 358: 129910, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33957602

RESUMO

Sodium metabisulfite (SMB), an antioxidant agent, is extensively used as a preservative in food industry. The current study was aimed to clarify its potential toxic effects on human fetal foreskin fibroblasts (HFFF2) cells, in vitro. Subsequently, MTT results illustrated that exposure to SMB significantly (p < 0.0001) decreased HFFF2 cell viability in a dose-dependent manner, and the concentration of 25 µM reduced cell survival rates to 50% as the half-maximal inhibitory concentration of SMB. It was further shown that SMB exerted this cytotoxic effect on HFFF2 cells through apoptosis induction. qRT-PCR and western blotting results showed that treatment of HFFF2 cells with this food additive led to significant upregulation of Bax, caspase 8, and caspase 9 pro-apoptotic genes and downregulation of Bcl-2 expression as a pro-survival agent. Furthermore, SMB remarkably increased caspase 3 levels and promoted its activation through cleavage in treated cells. Besides, exposure to SMB increased ROS levels and activated autophagy in treated cells, which are considered as the other indicators for cell damage. Taken together, our findings suggested that SMB could exert remarkable toxic effects on human normal cells through multiple mechanisms, including apoptosis activation, and its widespread usage in food safety should be reconsidered.


Assuntos
Apoptose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Aditivos Alimentares/toxicidade , Sulfitos/toxicidade , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Caspase 3/genética , Caspase 8/genética , Caspase 9/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/fisiologia , Aditivos Alimentares/administração & dosagem , Prepúcio do Pênis/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Sulfitos/administração & dosagem , Proteína X Associada a bcl-2/genética
20.
Tumour Biol ; 43(1): 37-55, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33935122

RESUMO

BACKGROUND: Green synthesized nanoparticles have been earmarked for use in nanomedicine including for the development of better anticancer drugs. OBJECTIVE: The aim of this study was to undertake biochemical evaluation of anticancer activities of green synthesized silver nanoparticles (AgNPs) from ethanolic extracts of fruits (AgNPs-F) and leaves (AgNPs-L) of Annona muricata. METHODS: Previously synthesized silver nanoparticles were used for the study. The effects of the AgNPs and 5-Fluorouracil were studied on PC3, HeLa and PNT1A cells. The resazurin, migration and colonogenic assays as well as qRT-PCR were employed. RESULTS: The AgNPs-F displayed significant antiproliferative effects against HeLa cells with an IC50 of 38.58µg/ml and PC3 cells with an IC50 of 48.17µg/ml but selectively spared normal PNT1A cells (selectivity index of 7.8), in comparison with first line drug 5FU and AgNPs-L whose selectivity index were 3.56 and 2.26 respectively. The migration assay revealed potential inhibition of the metastatic activity of the cells by the AgNPs-F while the colonogenic assay indicated the permanent effect of the AgNPs-F on the cancer cells yet being reversible on the normal cells in contrast with 5FU and AgNPs-L. CASP9 was significantly over expressed in all HeLa cells treated with the AgNPs-F (1.53-fold), AgNPs-L (1.52-fold) and 5FU (4.30-fold). CXCL1 was under expressed in HeLa cells treated with AgNPs-F (0.69-fold) and AgNPs-L (0.58-fold) and over expressed in cells treated with 5FU (4.95-fold), but the difference was not statistically significant. CXCR2 was significantly over expressed in HeLa cells treated with 5FU (8.66-fold) and AgNPs-F (1.12-fold) but under expressed in cells treated with AgNPs-L (0.76-fold). CONCLUSIONS: Here we show that biosynthesized AgNPs especially AgNPs-F can be used in the development of novel and better anticancer drugs. The mechanism of action of the AgNPs involves activation of the intrinsic apoptosis pathway through upregulation of CASP9 and concerted down regulation of the CXCL1/ CXCR2 gene axis.


Assuntos
Annona/química , Antineoplásicos/farmacologia , Caspase 9/genética , Quimiocina CXCL1/genética , Nanopartículas Metálicas , Receptores de Interleucina-8B/genética , Prata/farmacologia , Adenocarcinoma/patologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Química Verde , Humanos , Masculino , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Neoplasias da Próstata/patologia , Neoplasias do Colo do Útero/patologia
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