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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.
Environ Toxicol Pharmacol ; 88: 103741, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34517121

RESUMO

Trichloropyridinol (TCP); 3, 5, 6-trichloro-2-pyridinol is the primary metabolites of the organophosphorus pesticide chlorpyrifos. It is more highly persistent than parent compounds in the environment and might represent serious risks to human health. In this study, we investigated the toxicological effects and mechanism of TCP on HepG2 cells. The results revealed that TCP induced DNA damage and apoptosis on HepG2 cells. Besides, up-regulating the expression level of Bax /Bcl-2, a reduction in mitochondrial membrane potential, caspase-9/-3 activation and the release of cytochrome-c are contributed to the toxicological effects of TCP on HepG2 cells. These data indicated that the cytotoxic effects of TCP might be associated with the activity of mitochondrial apoptotic pathways. In conclusion, the results demonstrated that TCP poses a potential threat to human health by inducing toxicological effects in the liver.


Assuntos
Piridonas/toxicidade , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Compostos Organofosforados/metabolismo , Praguicidas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Molecules ; 26(18)2021 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-34577000

RESUMO

Rutin has been well recognized for possessing numerous pharmacological and biological activities in several human cancer cells. This research has addressed the inhibitory potential of rutin against the Jab1 oncogene in SiHa cancer cells, which is known to inactivate various tumor suppressor proteins including p53 and p27. Further, the inhibitory efficacy of rutin via Jab1 expression modulation in cervical cancer has not been yet elucidated. Hence, we hypothesized that rutin could exhibit strong inhibitory efficacy against Jab1 and, thereby, induce significant growth arrest in SiHa cancer cells in a dose-dependent manner. In our study, the cytotoxic efficacy of rutin on the proliferation of a cervical cancer cell line (SiHa) was exhibited using MTT and LDH assays. The correlation between rutin and Jab1 mRNA expression was assessed by RT-PCR analysis and the associated events (a mechanism) with this downregulation were then explored via performing ROS assay, DAPI analysis, and expression analysis of apoptosis-associated signaling molecules such as Bax, Bcl-2, and Caspase-3 and -9 using qRT-PCR analysis. Results exhibit that rutin produces anticancer effects via inducing modulation in the expression of oncogenes as well as tumor suppressor genes. Further apoptosis induction, caspase activation, and ROS generation in rutin-treated SiHa cancer cells explain the cascade of events associated with Jab1 downregulation in SiHa cancer cells. Additionally, apoptosis induction was further confirmed by the FITC-Annexin V/PI double staining method. Altogether, our research supports the feasibility of developing rutin as one of the potent drug candidates in cervical cancer management via targeting one such crucial oncogene associated with cervical cancer progression.


Assuntos
Apoptose/efeitos dos fármacos , Complexo do Signalossomo COP9/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeo Hidrolases/metabolismo , Rutina/farmacologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Complexo do Signalossomo COP9/genética , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Peptídeo Hidrolases/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
6.
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
7.
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
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.
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
10.
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
11.
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
12.
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
13.
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
14.
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
15.
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
16.
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
17.
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
18.
Gene ; 787: 145638, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33848578

RESUMO

BACKGROUND: Green tea is a natural compound with anti-neoplastic properties. Paclitaxel (PTX) is a natural anti-tumor medication used to manage patients with advanced ovarian cancer. This manuscript evaluated the cytotoxic effects of green tea extract combined with PTX drug in two human ovarian cancer cell lines (p53-negative cell line, SKOV-3; and mutant type p53 cell line, OVCAR-3) and underlying mechanisms. METHODS: The human ovarian cancer cell lines were treated with green tea extract, PTX, and green tea plus PTX for 24 h, and cell viability was assessed using the MTT method. Flow cytometric analyses were carried out to detect apoptosis. For the apoptotic process, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis were applied to study pAkt, Bax, Bcl-2, Cytochrome C (Cyt-C), cleaved-caspase-3, and cleaved-caspase-9 levels after drug treatments. RESULTS: Our results pointed out that various green tea (25 and 50 µg/ml) concentrations combined with PTX (20 and 40 µg/ml) synergistically inhibited cell viability of cancer cells more than green tea or PTX alone after 24 h of treatment. Also, green tea and PTX combination induced apoptosis in ovarian cancer cells by blocking the phosphorylation of Akt and the expression of Bcl-2 while inducing Bax, Cyt-C, cleaved-caspase 3, and cleaved-caspase 9. CONCLUSION: Our results showed that the combination of green tea and PTX could be more potent than the individual drug to induce cytotoxicity and apoptosis in ovarian cancer cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Mitocôndrias/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Extratos Vegetais/farmacologia , Chá/química , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Humanos , Extratos Vegetais/química , Polifenóis/análise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
19.
Life Sci ; 277: 119471, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33811898

RESUMO

Dental pulp stem cells (DPSCs) possess the ability of multi-lineage differentiation, and are excellent sources of tissue engineering and regenerative medicine. Oxygen concentration and inflammation are two critical environmental factors that affect the osteogenic differentiation of DPSCs. We aimed to study the role of the antimalarial drug artemisinin on the osteogenic differentiation of human DPSCs under the hypoxia and inflammation conditions. We demonstrated that hypoxia (5% O2) and inflammation (20 ng/mL TNF-α), alone or in combination, significantly diminished in vitro cell survival and increased apoptotic rates. Notably, hypoxia and TNF-α exerted accumulative effect in suppressing the osteogenic differentiation of DPSCs, as evidenced by reduced expression levels of osteogenesis-associated genes including ALP, RUNX2 and OCN in osteogenic condition, as well as reduced mineral nodules formation as indicated by alizarin red staining. Artemisinin at the dose of 40 µM markedly reversed the suppression in cell survival caused by hypoxia or inflammation, and reduced apoptotic rates and the expressions of pro-apoptotic proteins. Additionally, artemisinin restored osteogenic differentiation of DPSCs under the hypoxia or/and inflammation conditions. Moreover, the beneficial effect of artemisinin was dependent on upregulated expression of CA9 and CA9-mediated antioxidant responses, as CA9 knockdown abolished the protective role of artemisinin on DPSC osteogenesis. Furthermore, while hypoxia or/and inflammation significantly inactivated the Wnt/ß-catenin signaling in DPSCs, additional exposure to artemisinin re-activated this pathway to promote osteogenic differentiation of DPSCs. Our results provide novel insight on the link between artemisinin and DPSC osteogenesis, and suggest promising artemisinin-based strategies for better dentin/pulp tissue engineering.


Assuntos
Artemisininas/farmacologia , Polpa Dentária/metabolismo , Células-Tronco/efeitos dos fármacos , Artemisininas/metabolismo , Caspase 9/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Polpa Dentária/citologia , Humanos , Hipóxia/metabolismo , Osteogênese/efeitos dos fármacos , Células-Tronco/metabolismo , Engenharia Tecidual , Fator de Necrose Tumoral alfa/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos
20.
FEBS J ; 288(22): 6476-6491, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33899329

RESUMO

Necroptosis is a regulated necrotic-like cell death modality which has come into the focus of attention since it is known to contribute to the pathogenesis of many inflammatory and degenerative diseases as well as to tumor regulation. Based on current data, necroptosis serves as a backup mechanism when death receptor-induced apoptosis is inhibited or absent. However, the necroptotic role of the proteins involved in mitochondrial apoptosis has not been investigated. Here, we demonstrated that the stimulation of several death and pattern recognition receptors induced necroptosis under caspase-compromised conditions in wild-type, but not in caspase-9-negative human Jurkat and murine MEF cells. Cerulein-induced pancreatitis was significantly reduced in mice with acinar cell-restricted caspase-9 gene knockout. The absence of caspase-9 led to impaired association of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 and resulted in decreased phosphorylation of RIP kinases, but the overexpression of RIPK1 or RIPK3 rescued the effect of caspase-9 deficiency. Inhibition of either Aurora kinase A (AURKA) or its known substrate, glycogen synthase kinase 3ß (GSK3ß) restored necroptosis sensitivity of caspase-9-deficient cells, indicating an interplay between caspase-9 and AURKA-mediated pathways to regulate necroptosis. Our findings suggest that caspase-9 acts as a newly identified regulator of necroptosis, and thus, caspase-9 provides a promising therapeutic target to manipulate the immunological outcome of cell death.


Assuntos
Caspase 9/metabolismo , Necrose/metabolismo , Animais , Morte Celular , Linhagem Celular , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos , Pancreatite/metabolismo
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