Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 15.832
Filtrar
1.
Molecules ; 27(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36364165

RESUMO

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable, cell-permeable redox-cycling nitroxide water-soluble superoxide dismutase (SOD) mimetic agent. However, little is known about its cytotoxic effects on lung-related cells. Thus, the present study investigated the effects of Tempol on cell growth and death as well as changes in reactive oxygen species (ROS) and glutathione (GSH) levels in Calu-6 and A549 lung cancer cells, normal lung WI-38 VA-13 cells, and primary pulmonary fibroblast cells. Results showed that Tempol (0.5~4 mM) dose-dependently inhibited the growth of lung cancer and normal cells with an IC50 of approximately 1~2 mM at 48 h. Tempol induced apoptosis in lung cells with loss of mitochondrial membrane potential (MMP; ∆Ψm) and activation of caspase-3. There was no significant difference in susceptibility to Tempol between lung cancer and normal cells. Z-VAD, a pan-caspase inhibitor, significantly decreased the number of annexin V-positive cells in Tempol-treated Calu-6, A549, and WI-38 VA-13 cells. A 2 mM concentration of Tempol increased ROS levels, including O2•- in A549 and WI-38 VA-13 cells after 48 h, and specifically increased O2•- levels in Calu-6 cells. In addition, Tempol increased the number of GSH-depleted cells in Calu-6, A549, and WI-38 VA-13 cells at 48 h. Z-VAD partially downregulated O2•- levels and GSH depletion in Tempol-treated these cells. In conclusion, treatment with Tempol inhibited the growth of both lung cancer and normal cells via apoptosis and/or necrosis, which was correlated with increased O2•- levels and GSH depletion.


Assuntos
Apoptose , Neoplasias Pulmonares , Humanos , Espécies Reativas de Oxigênio/metabolismo , Glutationa/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Potencial da Membrana Mitocondrial , Proliferação de Células
2.
Cell Death Dis ; 13(11): 938, 2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36347842

RESUMO

Inhibition of the mitochondrial metabolism offers a promising therapeutic approach for the treatment of cancer. Here, we identify the mycotoxin viriditoxin (VDT), derived from the endophytic fungus Cladosporium cladosporioides, as an interesting candidate for leukemia and lymphoma treatment. VDT displayed a high cytotoxic potential and rapid kinetics of caspase activation in Jurkat leukemia and Ramos lymphoma cells in contrast to solid tumor cells that were affected to a much lesser extent. Most remarkably, human hematopoietic stem and progenitor cells and peripheral blood mononuclear cells derived from healthy donors were profoundly resilient to VDT-induced cytotoxicity. Likewise, the colony-forming capacity was affected only at very high concentrations, which provides a therapeutic window for cancer treatment. Intriguingly, VDT could directly activate the mitochondrial apoptosis pathway in leukemia cells in the presence of antiapoptotic Bcl-2 proteins. The mitochondrial toxicity of VDT was further confirmed by inhibition of mitochondrial respiration, breakdown of the mitochondrial membrane potential (ΔΨm), the release of mitochondrial cytochrome c, generation of reactive oxygen species (ROS), processing of the dynamin-like GTPase OPA1 and subsequent fission of mitochondria. Thus, VDT-mediated targeting of mitochondrial oxidative phosphorylation (OXPHOS) might represent a promising therapeutic approach for the treatment of leukemia and lymphoma without affecting hematopoietic stem and progenitor cells.


Assuntos
Leucemia , Linfoma , Micotoxinas , Humanos , Micotoxinas/metabolismo , Leucócitos Mononucleares/metabolismo , Apoptose , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Potencial da Membrana Mitocondrial
3.
Artigo em Inglês | MEDLINE | ID: mdl-36361152

RESUMO

Octachlorostyrene (OCS) is a ubiquitous persistent organic pollutant; however, information regarding the toxicological effects of OCS remains limited. In this study, we studied the toxicity mechanisms of OCS using human liver carcinoma (HepG2) cells. The results showed that OCS reduced cell viability in a time- and dose-dependent manner. Compared with that in the control, the level of reactive oxygen species (ROS) was significantly increased in all treated HepG2 cells. We also found that (1) OCS induced damage in the HepG2 cells via the apoptotic signaling pathway, (2) OCS increased intracellular free Ca2+ concentration (>180%), and (3) following exposure to 80 µM OCS, there was an increase in mitochondrial transmembrane potential (MMP, ~174%), as well as a decrease in ATP levels (<78%). In conclusion, OCS is cytotoxic and can induce apoptosis, in which ROS and mitochondrial dysfunction play important roles; however, the observed increase in MMP appears to indicate that HepG2 is resistant to the toxicity induced by OCS.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Células Hep G2 , Espécies Reativas de Oxigênio/metabolismo , Potencial da Membrana Mitocondrial , Apoptose
4.
Toxins (Basel) ; 14(10)2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-36287946

RESUMO

Furanocoumarins, the secondary metabolites of plants, are considered to be natural insecticides and fungicides because they prevent the invasion of plant pathogenic microorganisms and the predation of herbivorous insects. In this study, novel 2-arylfuranocoumarin derivatives were designed to synthesize by condensation, esterification, bromination, and Wittig reaction. The results showed an excellent photosensitive activity of 2-thiophenylfuranocoumarin (I34). Cell Counting Kit-8 detected that I34 could inhibit the proliferation of Spodoptera frugiperda (Sf9) cells in a time- and concentration-dependent manner under ultraviolet A (UV-A) light for 3 min. The inverted microscope revealed that cells treated with I34 swelled, the membrane was ruptured, and apoptotic bodies appeared. The flow cytometry detected that I34 could induce apoptosis of Sf9 cells, increase the level of intracellular reactive oxygen species (ROS), decrease the mitochondrial membrane potential, and block cell cycle arrest in the G2/M phase. Transmission electron microscopy detected cell mitochondrial cristae damage, matrix degradation, and mitochondrial vacuolation. Further enzyme activity detection revealed that the enzyme activities of apoptosis-related proteins caspase-3 and caspase-9 increased significantly (p &lt; 0.05). Finally, Western blotting analysis detected that the phosphorylation level of Akt and Bad and the expression of the apoptosis inhibitor protein Bcl-XL were inhibited, cleaved-PARP and P53 were increased, and cytochrome C was released from the mitochondria into the cytoplasm. Moreover, under UV-A irradiation, I34 promoted the increase in ROS in Sf9 cells, activated the mitochondrial apoptotic signal transduction pathway, and finally, inhibited cell proliferation. Thus, novel furanocoumarins exhibit a potential application prospect as a biochemical pesticide.


Assuntos
Fungicidas Industriais , Furocumarinas , Inseticidas , Praguicidas , Animais , Caspase 9/metabolismo , Caspase 9/farmacologia , Spodoptera/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Citocromos c/metabolismo , Citocromos c/farmacologia , Caspase 3/metabolismo , Inseticidas/farmacologia , Inseticidas/metabolismo , Fungicidas Industriais/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Mitocôndrias , Potencial da Membrana Mitocondrial , Apoptose , Proliferação de Células , Furocumarinas/farmacologia
5.
Toxins (Basel) ; 14(10)2022 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-36287985

RESUMO

Scorpion-venom-derived peptides have become a promising anticancer agent due to their cytotoxicity against tumor cells via multiple mechanisms. The suppressive effect of the cationic antimicrobial peptide Smp24, which is derived from the venom of Scorpio Maurus palmatus, on the proliferation of the hepatoma cell line HepG2 has been reported earlier. However, its mode of action against HepG2 hepatoma cells remains unclear. In the current research, Smp24 was discovered to suppress the viability of HepG2 cells while having a minor effect on normal LO2 cells. Moreover, endocytosis and pore formation were demonstrated to be involved in the uptake of Smp24 into HepG2 cells, which subsequently interacted with the mitochondrial membrane and caused the decrease in its potential, cytoskeleton reorganization, ROS accumulation, mitochondrial dysfunction, and alteration of apoptosis- and autophagy-related signaling pathways. The protecting activity of Smp24 in the HepG2 xenograft mice model was also demonstrated. Therefore, our data suggest that the antitumor effect of Smp24 is closely related to the induction of cell apoptosis, cycle arrest, and autophagy via cell membrane disruption and mitochondrial dysfunction, suggesting a potential alternative in hepatocellular carcinoma treatment.


Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Venenos de Escorpião , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/metabolismo , Células Hep G2 , Escorpiões/metabolismo , Venenos de Escorpião/metabolismo , Espécies Reativas de Oxigênio , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Apoptose , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Peptídeos/metabolismo , Proliferação de Células , Potencial da Membrana Mitocondrial
6.
Toxicol Appl Pharmacol ; 456: 116256, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36208702

RESUMO

Colorectal cancer (CRC) is estimated as the third most incident cancer and second in mortality worldwide. Moreover, CRC metastasis reduces patients' survival rates. Thus, the study and identification of new compounds with anticancer activity selectively to tumor cells are encouraged in the CRC treatment. Naphtoquinones are compounds with several pharmacologic activities, including antitumoral properties. Therefore, this study aimed to investigate the anticancer mechanism of synthetic 8-Hydroxy-2-(P-Nitrothiophenol)-1,4-Naphthoquinone (CNN16) in colon cancer cell line HCT-116. CNN16 showed an IC50 of 5.32 µM in HCT-116, and 9.36, 10.77, and 24.57 µM in the non-cancerous cells MRC-5, MNP-01, and PMBC, respectively, evaluated by the MTT assay. CNN16 showed an anticlonogenic effect in HCT-116 and induced cell fragmentation identified by flow cytometry analysis. Furthermore, we observed that CNN16 presented genotoxicity and induces reactive oxygen species (ROS) after 3 h of treatment visualized by alkaline comet assay and DCFH-DA dye fluorescence, respectively. Furthermore, CNN16 caused cellular membrane disruption, reduction in the mitochondrial membrane polarization, and the presence of apoptotic bodies and chromatin condensation was visualized by differential stained (HO/FD/PI) in fluorescent microscopy along with PARP1, TP53, BCL-2, and BAX analyzed by RT-qPCR. Results also evidenced inhibition in the migratory process analyzed by wound healing assay. Therefore, CNN16 can be considered as a potential new leader molecule for CRC treatment, although further studies are still necessary to comprehend the effects of CNN16 in in vivo models to evaluate the anti-migratory effect, and toxicology and assure compound safety and selectively.


Assuntos
Antineoplásicos , Neoplasias do Colo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular , Antineoplásicos/farmacologia , Apoptose , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Linhagem Celular , Dano ao DNA , Naftalenos/farmacologia , Linhagem Celular Tumoral , Potencial da Membrana Mitocondrial
7.
J Nat Prod ; 85(10): 2372-2384, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36215157

RESUMO

A new strategy for the semisynthesis of the aromatic cassane-type diterpene taepeenin F (6) is reported. The introduction of the methyl group at C-14, characteristic of the target compound, was achieved via dienone 13, easily prepared from abietic acid (10), the major compound in renewable rosin. Biological assays of selected compounds are reported. The antiproliferative activity against HT29, B16-F10, and HepG2 tumor cell lines has been investigated. Salicylaldehyde 21 was the most active compound (IC50 = 7.72 µM). Products 16 and 21 displayed apoptotic effects in B16-F10 cells, with total apoptosis rates of 46 and 38.4%, respectively. This apoptotic process involves a significant arrest of the B16-F10 cell cycle, blocking the G0/G1 phase. Dienone 16 did not cause any loss of the mitochondrial membrane potential (MMP), while salicylaldehyde 21 caused a partial loss of the MMP. The anti-inflammatory activity of the selected compounds was investigated with the LPS-stimulated RAW 264.7 macrophages. All compounds showed potent NO inhibition, with percentages between 80 and 99% at subcytotoxic concentrations. Dienone 16 inhibited LPS-induced differentiation of RAW 264.7 cells, by increasing the proportion of cells in the S phase. In addition, salicylaldehyde 21 had effects on the cell cycle, recovering the cells from the G0/G1 full arrest produced in response to LPS action.


Assuntos
Antineoplásicos , Diterpenos , Lipopolissacarídeos/farmacologia , Potencial da Membrana Mitocondrial , Apoptose , Linhagem Celular Tumoral , Diterpenos/farmacologia , Anti-Inflamatórios/farmacologia , Proliferação de Células , Antineoplásicos/farmacologia
8.
Pharm Biol ; 60(1): 1876-1883, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36200643

RESUMO

CONTEXT: Genistein is a multifunctional natural compound. OBJECTIVE: In this study, we demonstrate the activity of genistein on non-small lung cancer A549 and 95D cells. MATERIALS AND METHODS: A CCK8 assay was used to detect the cytotoxicity of genistein (0, 25, 50, 100, 150, 200 and 250 µM) on A549 and 95D cells for 48 h. AnnexinV-FITC/PI and TUNEL assay were performed to examine the apoptotic cell death induced by genistein (0, 50, 100 and 150 µM, 48 h). Intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential were measured by flow cytometry. Mitochondrial activity in A549 and 95D cells, treated with 0, 50, 100 and 150 µM genistein for 48 h was detected by MitoTracker Orange staining. Western blot analysis was performed to evaluate the expression of the mitochondrial apoptosis-related proteins. Meanwhile, the expression level of FOXO3a/PUMA signalling was measured by flow cytometry and Western blot assay. RESULTS: IC50 value of genistein against 95D cells and A549 cells was 32.96 ± 2.91 and 110.6 ± 2.41 µM, respectively. The percentage of apoptotic death cells was 20.03%, 29.26% and 27.14% in 95D cells, and 41.62%, 55.24% and 43.45% in A549 cells when treated with 50, 100 and 150 µM genistein, respectively. Our observations also revealed that genistein could elevate intracellular ROS generation, decrease mitochondrial membrane potential, decrease mitochondrial activity (MitoTracker Orange staining), and up-regulate the expression of mitochondrial apoptosis-related proteins. Further examinations revealed that the expression level of FOXO3a and PUMA in NSCLC was significantly increased by genistein. DISCUSSION AND CONCLUSIONS: Our data may provide basic information for further development of genistein as a promising lead compound targeting NSCLC by inducing mitochondrial apoptosis.


Assuntos
Genisteína , Neoplasias Pulmonares , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Fluoresceína-5-Isotiocianato/metabolismo , Genisteína/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Biosensors (Basel) ; 12(10)2022 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-36290933

RESUMO

The mitochondrial membrane potential (MMP, ΔΨmito) provides the charge gradient required for mitochondrial functions and is a key indicator of cellular health. The changes in MMP are closely related to diseases and the monitoring of MMP is thus vital for pathological study and drug development. However, most of the current fluorescent probes for MMP rely solely on the cell fluorescence intensity and are thus restricted by poor photostability, rendering them not suitable for long-term dynamic monitoring of MMP. Herein, an MMP-responsive fluorescent probe pyrrolyl quinolinium (PQ) which is capable of reversible migration between mitochondria and nucleolus is developed and demonstrated for dynamic evaluation of MMP. The fluorescence of PQ translocates from mitochondria to nucleoli when MMP decreases due to the intrinsic RNA-specificity and more importantly, the translocation is reversible. The cytoplasm to nucleolus fluorescence intensity ratio is positively correlated with MMP so that this method avoids the negative influence of photostability and imaging parameters. Various situations of MMP can be monitored in real time even without controls. Additionally, long-term dynamic evaluation of MMP is demonstrated for HeLa cells using PQ in oxidative environment. This study is expected to give impetus to the development of mitochondria-related disease diagnosis and drug screening.


Assuntos
Corantes Fluorescentes , RNA , Humanos , Potencial da Membrana Mitocondrial , Células HeLa , Microscopia de Fluorescência/métodos
10.
Arch Biochem Biophys ; 731: 109448, 2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-36306919

RESUMO

Alzheimer's disease (AD), a common neurodegenerative disease, is characterised by the deposition of amyloid-ß (Aß) plaques and neurofibrillary tangles. An increasing number of studies have demonstrated that Aß causes neuronal damage and mitochondrial dysfunction. Herein, we evaluated the neuroprotective effect of sodium butyrate (NaB) against Aß induced neurotoxicity in PC12 cells. The results revealed that 3 mM of NaB promoted the expression of angiotensin-converting enzyme and brain-derived neurotrophic factor, which exert a neuroprotective effect by activating G protein-coupled receptors. Moreover, NaB could significantly improve mitochondrial dysfunction caused by Aß. In conclusion, NaB protected PC12 cells from Aß-induced cell damage, highlighting the potential of NaB in AD treatment.


Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Fármacos Neuroprotetores , Animais , Ratos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/metabolismo , Apoptose , Ácido Butírico/farmacologia , Sobrevivência Celular , Potencial da Membrana Mitocondrial , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Células PC12 , Fragmentos de Peptídeos/toxicidade , Fragmentos de Peptídeos/metabolismo
11.
J Neural Transm (Vienna) ; 129(12): 1435-1446, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36242655

RESUMO

Parkinson's disease (PD) is a neurodegenerative movement disorder, affecting 1-2% of the human population over 65. A previous study by our group identified a p.G849D variant in neurexin 2α (NRXN2) co-segregating with PD, prompting validation of its role using experimental methods. This novel variant had been found in a South African family with autosomal dominant PD. NRXN2α is an essential synaptic maintenance protein with multiple functional roles at the synaptic cleft. The aim of the present study was to investigate the potential role of the translated protein NRXN2α and the observed mutant in PD by performing functional studies in an in vitro model. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells to assess the effect of the mutant on cell viability and apoptosis [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay; ApoTox-Glo™ Triplex Assay)], mitochondrial membrane potential (MMP; MitoProbe™ JC-1 Assay), mitochondrial network analysis (MitoTracker®) and reactive oxygen species (ROS; ROS-Glo™ H2O2 Assay). Cells transfected with the mutant NRXN2α plasmid showed decreased cell viability and MMP. They also exhibited increased ROS production. However, these cells showed no changes in mitochondrial fragmentation. Our findings led us to speculate that the p.G849D variant may be involved in a toxic feedback loop leading to neuronal death in PD. Mitochondrial dysfunction and synaptic dysfunction have been linked to PD. Therefore, findings from this exploratory study are in line with previous studies connecting these two processes and warrants further investigation into the role of this variant in other cellular and animal models.


Assuntos
Neuroblastoma , Doença de Parkinson , Animais , Humanos , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo , Linhagem Celular Tumoral , Apoptose
12.
Int J Mol Sci ; 23(19)2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36232954

RESUMO

Since cancer treatment by radio- and chemotherapy has been linked to safety concerns, there is a need for new and alternative anticancer drugs; as such, compounds isolated from plants represent promising candidates. The current study investigates the anticancer features of halimane (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid (HAL) and the labdane diterpenes 1α,6ß-diacetoxy-8α,13R*-epoxy-14-labden-11-one (PLEC) and forskolin-like 1:1 mixture of 1,6-di-O-acetylforskolin and 1,6-di-O-acetyl-9-deoxyforskolin (MRC) isolated from Plectranthus ornatus in MCF7 and FaDu cancer cell lines. Cytotoxicity was assessed by MTT assay, ROS production by Di-chloro-dihydro-fluorescein diacetate assay (DCFH) or Red Mitochondrial Superoxide Indicator (MitoSOX) and Mitochondrial Membrane Potential (MMP) by fluorescent probe JC-1 (5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide). In addition, the relative amounts of mitochondrial DNA (mtDNA) were determined using quantitative Real-Time-PCR (qRT-PCR) and damage to mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) by semi-long run quantitative Real-Time-PCR (SLR-qRT-PCR). Gene expression was determined using Reverse-Transcription-qPCR. Caspase-3/7 activity by fluorescence was assessed. Assessment of General In Vivo Toxicity has been determined by Brine Shrimp Lethality Bioassay. The studied HAL and PLEC were found to have a cytotoxic effect in MCF7 with IC50 = 13.61 µg/mL and IC50 = 17.49 µg/mL and in FaDu with IC50 = 15.12 µg/mL and IC50 = 32.66 µg/mL cancer cell lines. In the two tested cancer cell lines, the phytochemicals increased ROS production and mitochondrial damage in the ND1 and ND5 gene regions and reduced MMP (ΔΨm) and mitochondrial copy numbers. They also changed the expression of pro- and anti-apoptotic genes (Bax, Bcl-2, TP53, Cas-3, Cas-8, Cas-9, Apaf-1 and MCL-1). Studies demonstrated increase in caspase 3/7 activity in tested cancer cell lines. In addition, we showed no toxic effect in in vivo test for the compounds tested. The potential mechanism of action may have been associated with the induction of apoptosis in MCF7 and FaDu cancer cells via the mitochondrial pathway; however, further in vivo research is needed to understand the mechanisms of action and potential of these compounds.


Assuntos
Antineoplásicos , Diterpenos , Plectranthus , Antineoplásicos/farmacologia , Apoptose , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Colforsina/farmacologia , DNA Mitocondrial/metabolismo , Diterpenos/farmacologia , Corantes Fluorescentes/farmacologia , Iodetos , Potencial da Membrana Mitocondrial , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Compostos Fitoquímicos/metabolismo , Compostos Fitoquímicos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos , Proteína X Associada a bcl-2/metabolismo
13.
Cell Calcium ; 107: 102654, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36166935

RESUMO

The mitochondrial Ca2+ uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca2+ signaling, is mediated via the calcium uniporter complex (MCUC). In animal cells the MCUC is regulated by the mitochondrial calcium uptake 1 and 2 dimer (MICU1/MICU2), which has been proposed to act as gatekeeper preventing mitochondrial Ca2+ overload at low cytosolic Ca2+ levels. In contrast to animal cells, knockout of either MICU1 or MICU2 in Trypanosoma cruzi, the etiologic agent of Chagas disease, did not allow Ca2+ uptake at low extramitochondrial Ca2+ concentrations ([Ca2+]ext) and it was though that in the absence of one MICU the other would replace its role. However, previous attempts to knockout both genes were unsuccessful. Here, we designed a strategy to generate TcMICU1/TcMICU2 double knockout cell lines using CRISPR/Cas9 genome editing. Ablation of both genes was confirmed by PCR and Southern blot analyses. The absence of both proteins did not allow Ca2+ uptake at low [Ca2+]ext, significantly decreased the mitochondrial Ca2+ uptake at different [Ca2+]ext, without dissipation of the mitochondrial membrane potential, and increased the [Ca2+]ext set point needed for Ca2+ uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg2+ was found to be a negative regulator of MCUC-mediated mitochondrial Ca2+ uptake at different [Ca2+]ext. Occlusion of the MCUC pore by Mg2+ could partially explain the lack of mitochondrial Ca2+ uptake at low [Ca2+]ext in TcMICU1/TcMICU2-KO cells. In addition, TcMICU1/TcMICU2-KO epimastigotes had a lower growth rate, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes.


Assuntos
Trypanosoma cruzi , Animais , Trypanosoma cruzi/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Transporte Biológico , Potencial da Membrana Mitocondrial , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo
14.
Biomed Pharmacother ; 153: 113475, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36076500

RESUMO

BACKGROUND: Cancer remains one of the leading causalities of several morbidity and mortality with negative impact on global economy due to low workforce and management/treatment cost. A number of conventional therapies have been explored in the management/treatment of cancer including chemotherapeutic intervention, radiotherapy, and surgery. Among these treatment modalities, chemotherapy remains the most popular first line of intervention in management/treatment of cancer, and natural products have been implicated as the main source of antineoplastic agents with phenomenal efficacy. However, current antineoplastic agents suffer from lack of selectivity and specificity necessitating the need for further research in the search for novel anticancer drug molecules. METHODS: In this present study, the anticancer activity of Hoslundia opposita leaves extracts were tested against a number of cell lines including human hepatoma cell line (HepG2), human breast cancer cell lines (MDA-MB-231), intestinal epithelial cell lines (Caco-2), and human keratinocyte HACAT cell lines. A bio-guided fractionation assay and the structural elucidation of the pure isolate (hoslundin) was conducted by 1D and 2D NMR spectroscopy. The cell viability, colony formation, and apoptotic activities were investigated using MTT assay, clonogenic assay, and caspase - 3 and - 7 kits respectively. Flow cytometry was employed in assessing the altered cell cycle expression. The production of the intracellular reactive oxygen species (ROS) levels and the reduction of the mitochondrial membrane potential (MMP) was determined at the cellular level using fluorescent probe dyes dihydro-fluorescin diacetate (DCFH-DA) and tetramethylrhodamin (TMRE), respectively. RESULTS: The H. opposita fractions and its pure isolate (hoslundin) demonstrated a potent cytocidal activity against the tumorigenic cells (HepG2, MDA-MB-231, Caco-2) at concentration ranging from 25 to 100 µg/mL. The inhibition of the colony formation was significantly observed in HepG2 cell lines. More so, the cellular viability of the normal cells (HaCaT) was relatively unchanged in the presence of H. opposita fractions and its isolate proving the selectivity of the compounds towards tumourigenic cells. The H. opposita fractions and hoslundin exerted their anticancer activity via cell cycle arrest with the accumulation of the DNA content at the S-phase, activation of apoptosis in the caspase 3,7 activities and depolarized mitochondrial membrane potential mediated by mitochondrial-dependent ROS generation in the treated tumor cells. CONCLUSION: The anticancer activities of Hoslundia opposita Vahl and hoslundin exhibited significant efficacy against tumor cells and well tolerated in the presence of normal cells making them a potential antineoplastic agent.


Assuntos
Antineoplásicos Fitogênicos , Lamiaceae , Neoplasias , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Células CACO-2 , Linhagem Celular Tumoral , Humanos , Potencial da Membrana Mitocondrial , Neoplasias/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo
15.
Chem Biol Interact ; 366: 110129, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36067825

RESUMO

In the present work, dehydrodieugenol B (1) and its methyl ether (2), isolated from Nectandra leucantha twigs, were used as starting material for the preparation of two new derivatives (1a and 2a) containing an additional methoxycarbonyl unit on allyl side chains. Compounds 1a and 2a demonstrated activity against trypomastigotes (EC50 values of 13.5 and 23.0 µM, respectively) and against intracellular amastigotes (EC50 values of 10.2 and 6.1 µM, respectively). Additionally, compound 2a demonstrated no mammalian cytotoxicity up to 200 µM whereas compound 1a exhibited a CC50 value of 139.8 µM. The mechanism of action studies of compounds 1a and 2a demonstrated a significant depolarization of the plasma membrane potential in trypomastigotes, followed by a mitochondrial membrane potential collapse. Neither calcium level nor reactive oxygen species alterations were observed after a short-time incubation. Considering the potential of compound 2a against T. cruzi and its simple preparation from the natural product 2, isolated from N. leucantha, this compound could be considered a new hit for future drug design studies in Chagas disease.


Assuntos
Produtos Biológicos , Doença de Chagas , Trypanosoma cruzi , Anisóis/metabolismo , Produtos Biológicos/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/metabolismo
16.
Mar Drugs ; 20(9)2022 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-36135771

RESUMO

Ilimaquinone (IQ), a metabolite found in marine sponges, has been reported to have a number of biological properties, including potential anticancer activity against colon cancer. However, no clear understanding of the precise mechanism involved is known. The aim of this study was to examine the molecular mechanism by which IQ acts on HCT-116 cells. The anticancer activity of IQ was investigated by means of a cell viability assay followed by the determination of induction of apoptosis by means of the use of acridine orange-ethidium bromide (AO/EB) staining, Annexin V/PI double staining, DNA fragmentation assays, and TUNEL assays. The mitochondrial membrane potential (ΔΨm) was detected using the JC-1 staining technique, and the apoptosis-associated proteins were analyzed using real-time qRT-PCR. A molecular docking study of IQ with apoptosis-associated proteins was also conducted in order to assess the interaction between IQ and them. Our results suggest that IQ significantly suppressed the viability of HCT-116 cells in a dose-dependent manner. Fluorescent microscopy, flow cytometry, DNA fragmentation and the TUNEL assay in treated cells demonstrated apoptotic death mode. As an additional confirmation of apoptosis, the increased level of caspase-3 and caspase-9 expression and the downregulation of Bcl-2 and mitochondrial dysfunction were observed in HCT-116 cells after treatment with IQ, which was accompanied by a decrease in mitochondrial membrane potential (ΔΨm). Overall, the results of our studies demonstrate that IQ could trigger mitochondria-mediated apoptosis as demonstrated by a decrease in ΔΨm, activation of caspase-9/-3, damage of DNA and a decrease in the proportion of Bcl-2 through the mitochondrial-mediated apoptosis pathway.


Assuntos
Neoplasias Colorretais , Poríferos , Laranja de Acridina , Animais , Anexina A5/metabolismo , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , DNA/metabolismo , Etídio , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial , Simulação de Acoplamento Molecular , Poríferos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinonas , Sesquiterpenos
17.
Molecules ; 27(17)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36080487

RESUMO

Chlorfenapyr (CHL) is a type of insecticide with a wide range of insecticidal activities and unique targets. The extensive use of pesticides has caused an increase in potential risks to the environment and human health. However, the potential toxicity of CHL and its mechanisms of action on humans remain unclear. Therefore, human liver cells (HepG2) were used to investigate the cytotoxic effect and mechanism of toxicity of CHL at the cellular level. The results showed that CHL induced cellular toxicity in HepG2 cells and induced mitochondrial damage associated with reactive oxygen species (ROS) accumulation and mitochondrial calcium overload, ultimately leading to apoptosis and autophagy in HepG2 cells. Typical apoptotic changes occurred, including a decline in the mitochondrial membrane potential, the promotion of Bax/Bcl-2 expression causing the release of cyt-c into the cytosol, the activation of cas-9/-3, and the cleavage of PARP. The autophagic effects included the formation of autophagic vacuoles, accumulation of Beclin-1, transformation of LC3-II, and downregulation of p62. Additionally, DNA damage and cell cycle arrest were detected in CHL-treated cells. These results show that CHL induced cytotoxicity associated with mitochondria-mediated programmed cell death (PCD) and DNA damage in HepG2 cells.


Assuntos
Apoptose , Mitocôndrias , Autofagia , Dano ao DNA , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Piretrinas , Espécies Reativas de Oxigênio/metabolismo
18.
Curr Protoc ; 2(9): e531, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36066206

RESUMO

Mitochondria are fundamental for human spermatozoa motility and fertilizing ability. Mitochondria participate not only in ATP production, but also in reactive oxygen species production, redox equilibrium, and calcium regulation, all of which are central for human spermatozoa motility, capacitation, acrosome reaction, and ultimately, oocyte fertilization. Mitochondrial membrane potential is a key indicator of mitochondrial health and activity. Most commonly used methods for the study of mitochondrial membrane potential, however, cannot be applied to human spermatozoa due to their unique characteristics, including high motility and time-dependent decay of quality, limiting the study of this important parameter in these cells. Here, we describe an easy, fast, and cheap protocol for the quantitative evaluation of human spermatozoa mitochondrial membrane potential, using the fluorescent cationic dye 5,5,6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1). JC-1 is a sensitive marker for mitochondrial membrane potential, exhibiting a potential-dependent accumulation in the mitochondria. At high mitochondrial membrane potential, JC-1 forms J-aggregates, which emit red fluorescence, whereas at low mitochondrial membrane potential, JC-1 remains at its monomer state, which emits green fluorescence. We first describe how to evaluate human spermatozoa mitochondrial membrane potential using JC-1 and a fluorescence plate reader, for high-throughput studies. The calculation of the JC-1 ratio (indicative of the J-aggregates/monomers ratio) is then used to quantitatively evaluate mitochondrial health and activity. In addition, we describe an imaging protocol for the qualitative evaluation of human spermatozoa mitochondrial membrane potential using a fluorescence microscope. This allows for a visual analysis of the results that can complement the quantitative data. These protocols can be used to study the effects of spermatozoa exposure to compounds of interest, and alterations due to diseases or different conditions. While these protocols are illustrated with human spermatozoa, they can be adapted and used on spermatozoa of different species. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Quantitative evaluation of human spermatozoa mitochondrial membrane potential using the JC-1 dye and a fluorescence plate reader Basic Protocol 2: Qualitative evaluation of human spermatozoa mitochondrial membrane potential using the JC-1 dye and fluorescence microscopy Support Protocol: Preparation of the JC-1 working solution.


Assuntos
Benzimidazóis , Espermatozoides , Benzimidazóis/metabolismo , Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Masculino , Potencial da Membrana Mitocondrial , Espermatozoides/metabolismo
19.
Eur J Med Chem ; 243: 114777, 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36174412

RESUMO

A set of fifteen triterpenoid pyrazines and pyridines was prepared from parent triterpenoid 3-oxoderivatives (betulonic acid, dihydrobetulonic acid, oleanonic acid, moronic acid, ursonic acid, heterobetulonic acid, and allobetulone). Cytotoxicity of all compounds was tested in eight cancer and two non-cancer cell lines. Evaluation of the structure-activity relationships revealed that the triterpenoid core determined whether the final molecule is active or not, while the heterocycle is able to increase the activity and modulate the specificity. Five compounds (1b, 1c, 2b, 2c, and 8) were found to be preferentially and highly cytotoxic (IC50 ≈ 1 µM) against leukemic cancer cell lines (CCRF-CEM, K562, CEM-DNR, or K562-TAX). Surprisingly, compounds 1c, 2b, and 2c are 10-fold more active in multidrug-resistant leukemia cells (CEM-DNR and K562-TAX) than in their non-resistant analogs (CCRF-CEM and K562). Pharmacological parameters were measured for the most promising candidates and two types of prodrugs were synthesized: 1) Sugar-containing conjugates, most of which had improved cell penetration and retained high cytotoxicity in the CCRF-CEM cell line, unfortunately, they lost the selectivity against resistant cells. 2) Medoxomil derivatives, among which compounds 26-28 gained activities of IC50 0.026-0.043 µM against K562 cells. Compounds 1b, 8, 21, 22, 23, and 24 were selected for the evaluation of the mechanism of action based on their highest cytotoxicity against CCRF-CEM cell line. Several experiments showed that the majority of them cause apoptosis via the mitochondrial pathway. Compounds 1b, 8, and 21 inhibit growth and disintegrate spheroid cultures of HCT116 and HeLa cells, which would be important for the treatment of solid tumors. In summary, compounds 1b, 1c, 2b, 2c, 24, and 26-28 are highly and selectively cytotoxic against cancer cell lines and were selected for future in vivo tests and further development of anticancer drugs.


Assuntos
Antineoplásicos Fitogênicos , Antineoplásicos , Pró-Fármacos , Triterpenos , Humanos , Pró-Fármacos/farmacologia , Pirazinas/farmacologia , Potencial da Membrana Mitocondrial , Antineoplásicos Fitogênicos/farmacologia , Células HeLa , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular Tumoral , Triterpenos/farmacologia , Antineoplásicos/farmacologia , Piridinas/farmacologia
20.
Bioorg Chem ; 129: 106159, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36155091

RESUMO

BACKGROUND: The damage of podocytes is a primary hallmark of lupus nephritis (LN). Therefore, finding an effective way to inhibit the podocyte injury is important for improving the survival and development of patients with LN. Eucalyptus robusta exhibits anti-inflammatory properties. However, whether Formyl phloroglucinol meroterpenoids (FPMs), which are specialized metabolites of the genus Eucalyptus, is an anti-inflammatory active ingredient of E. robusta remains to be determined. PURPOSE: This study asimed to identify novel FPMs from E. robusta and investigated their anti-inflammatory effects. METHODS: Various separation methods were used to isolate and identify the compounds in the PE extract of E. robusta. The structures of the isolates were determined using 1D/2D NMR data and electron circular dichroism (ECD) calculations. The level of mitochondrial reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) of the podocyte cell line, MPC-5, were assessed using a multifunctional microplate reader combined with flow cytometry and fluorescence microscopy. RESULTS: Eight novel FPMs (1-8, Eucarbwenstols A-H, Fig. 1) and 15 known FPMs (9-23) were purified from the PE extract of E. robusta. It is noteworthy that compound 1 possesses an unprecedented FPM carbon skeleton. Among these compounds, compounds 1, 2, 4 and 5 showed the most promising potential for protecting MPC-5 cells because pretreatment with pro-inflammatory cytokines TGF-ß, IFN-α and IL-6 decreased ROS production and ameliorated the mitochondrial state. CONCLUSIONS: Our research contributes to the characterization of E. robusta constituents and highlights the anti-inflammatory effects of FPMs.


Assuntos
Eucalyptus , Humanos , Eucalyptus/química , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismo , Floroglucinol/química , Extratos Vegetais/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...