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1.
Mol Med Rep ; 27(2)2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36601752

RESUMO

The cell­killing potential of most chemotherapeutic agents is enhanced by a temperature elevation. Isofraxidin (IF) is a coumarin compound widely found in plants, such as the Umbelliferae or Chloranthaceae families. IF induces anticancer effects in lung and colorectal cancer. To the best of our knowledge, the combined effects of hyperthermia (HT) and IF on heat­induced apoptosis have not been reported. Acute monocytic leukemia U937 cells were exposed to HT with or without IF pre­treatment. Apoptosis was measured by Annexin V­FITC/PI double staining assay using flow cytometry and cell viability was observed by cell counting kit assay, DNA fragmentation. The mechanism involved in the combination was explored by measuring changes in the mitochondrial membrane potential, (MMP), intracellular ROS generation, expression of apoptosis related protein, and intracellular calcium ion level. It was demonstrated that IF enhanced HT­induced apoptosis in U937 cells. The results demonstrated that combined treatment enhanced mitochondrial membrane potential loss and transient superoxide generation increased protein expression levels of caspase­3, caspase­8 and phosphorylated­JNK and intracellular calcium levels. Moreover, the role of caspases and JNK was confirmed using a pan caspase inhibitor (zVAD­FMK) and JNK inhibitor (SP600125) in U937 cells. Collectively, the data demonstrated that IF enhanced HT­induced apoptosis via a reactive oxygen species mediated mitochondria/caspase­dependent pathway in U937 cells.


Assuntos
Hipertermia Induzida , Leucemia Monocítica Aguda , Humanos , Células U937 , Cálcio/metabolismo , Apoptose , Cumarínicos/farmacologia , Caspases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oxirredução , Potencial da Membrana Mitocondrial
2.
Drug Deliv ; 30(1): 108-120, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36533874

RESUMO

Along with its wide range of potential applications, human exposure to mesoporous tantalum oxide nanomaterials (PEG@mTa2O5) has substantially risen. Accumulative toxic investigations have shown the PEG@mTa2O5 intake and cardiovascular diseases (CVD). Endothelial cell death is crucial in the onset and development of atherosclerosis. Still, the molecular mechanism connecting PEG@mTa2O5 and endothelium apoptosis remains unclear. Herein, we studied the absorption and toxic action of mesoporous tantalum oxide (mTa2O5) nanomaterials with polyethylene glycol (PEG) utilizing human cardio microvascular endothelial cells (HCMECs). We also showed that PEG@mTa2O5 promoted apoptosis in endothelial cells using flow cytometry and AO-EB staining. In conjunction with the ultrastructure modifications, PEG@mTa2O5 prompted mitochondrial ROS production, cytosolic Ca2+ overload, ΔΨm collapse, and ER stress verified by elevated ER-Tracker staining, upregulated XBP1 and GRP78/BiP splicing. Remarkably, the systemic toxicity and blood compatibility profile of PEG@mTa2O5 can greatly improve successive therapeutic outcomes of NMs while reducing their adverse side effects. Overall, our findings suggested that PEG@mTa2O5-induced endothelium apoptosis was partially mediated by the activation of the endoplasmic reticulum stress-mitochondrial cascade.


Assuntos
Células Endoteliais , Nanoestruturas , Humanos , Apoptose , Estresse do Retículo Endoplasmático , Células Endoteliais/metabolismo , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismo
3.
Eur J Pharmacol ; 939: 175470, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36543287

RESUMO

Mitochondria in tumor cells are functionally different from those in normal cells and could be targeted to develop new anticancer agents. We showed recently that the aryl-ureido fatty acid CTU is the prototype of a new class of mitochondrion-targeted agents that kill cancer cells by increasing the production of reactive oxygen species (ROS), activating endoplasmic reticulum (ER)-stress and promoting apoptosis. However, prolonged treatment with high doses of CTU were required for in vivo anti-tumor activity. Thus, new strategies are now required to produce agents that have enhanced anticancer activity over CTU. In the present study we prepared a novel aryl-urea termed 3-thiaCTU, that contained an in-chain sulfur heteroatom, for evaluation in tumor cell lines and in mice carrying tumor xenografts. The principal finding to emerge was that 3-thiaCTU was several-fold more active than CTU in the activation of aryl-urea mechanisms that promoted cancer cell killing. Thus, in in vitro studies 3-thiaCTU disrupted the mitochondrial membrane potential, increased ROS production, activated ER-stress and promoted tumor cell apoptosis more effectively than CTU. 3-ThiaCTU was also significantly more active than CTUin vivo in mice that carried MDA-MB-231 cell xenografts. Compared to CTU, 3-thiaCTU prevented tumor growth more effectively and at much lower doses. These findings indicate that, in comparison to CTU, 3-thiaCTU is an aryl-urea with markedly enhanced activity that could now be suitable for development as a novel anticancer agent.


Assuntos
Antineoplásicos , Ácidos Graxos , Humanos , Animais , Camundongos , Ácidos Graxos/farmacologia , Ácidos Graxos/metabolismo , Ureia/farmacologia , Ureia/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias , Apoptose , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Potencial da Membrana Mitocondrial
4.
J Biol Chem ; 299(1): 102780, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36496071

RESUMO

Ischemia and reperfusion affect multiple elements of cardiomyocyte electrophysiology, especially within the mitochondria. We previously showed that in cardiac monolayers, upon reperfusion after coverslip-induced ischemia, mitochondrial inner membrane potential (ΔΨ) unstably oscillates between polarized and depolarized states, and ΔΨ instability corresponds with arrhythmias. Here, through confocal microscopy of compartment-specific molecular probes, we investigate the mechanisms underlying the postischemic ΔΨ oscillations, focusing on the role of Ca2+ and oxidative stress. During reperfusion, transient ΔΨ depolarizations occurred concurrently with periods of increased mitochondrial oxidative stress (5.07 ± 1.71 oscillations/15 min, N = 100). Supplementing the antioxidant system with GSH monoethyl ester suppressed ΔΨ oscillations (1.84 ± 1.07 oscillations/15 min, N = 119, t test p = 0.027) with 37% of mitochondrial clusters showing no ΔΨ oscillations (versus 4% in control, odds ratio = 14.08, Fisher's exact test p < 0.001). We found that limiting the production of reactive oxygen species using cyanide inhibited postischemic ΔΨ oscillations (N = 15, t test p < 10-5). Furthermore, ΔΨ oscillations were not associated with any discernable pattern in cell-wide oxidative stress or with the changes in cytosolic or mitochondrial Ca2+. Sustained ΔΨ depolarization followed cytosolic and mitochondrial Ca2+ increase and was associated with increased cell-wide oxidative stress. Collectively, these findings suggest that transient bouts of increased mitochondrial oxidative stress underlie postischemic ΔΨ oscillations, regardless of Ca2+ dynamics.


Assuntos
Mitocôndrias , Estresse Oxidativo , Humanos , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Isquemia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reperfusão , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/metabolismo , Cálcio/metabolismo
5.
Biochim Biophys Acta Biomembr ; 1864(1): 183776, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34547253

RESUMO

Alpha-synuclein (α-syn) aggregation and mitochondrial dysfunction are considered as two of the main factors associated with Parkinson's disease (PD). In the present investigation, the effectiveness of the amyloid fibrils obtained from α-syn with those of hen egg white lysozyme (HEWL), as disease-related and-unrelated proteins, to damage rat brain and rat liver mitochondria have been investigated. This was extended by looking at SH-SY5Y human neuroblastoma cells and erythrocytes, thereby investigating the significance of structural characteristics of amyloid fibrils related to their interactions with biomembranes obtained from various sources. Results presented clearly demonstrate substantial differences in the response of tested biomembranes to toxicity induced by α-syn/HEWL amyloid fibrils, highlighting a structure-function relationship. We found that fibrillar aggregates of α-syn, but not HEWL, caused a significant increase in mitochondrial ROS, loss of membrane potential, and mitochondrial swelling, in a dose-dependent manner. Toxicity was found to be more pronounced in brain mitochondria, as compared to liver mitochondria. For SH-SY5Y cells and erythrocytes, however, both α-syn and HEWL amyloid fibrils showed the capacity to induce toxicity. Taken together, these results may suggest selective toxicity of α-syn amyloid fibrils to mitochondria mediated likely by their direct interaction with the outer mitochondrial membrane, indicating a correlation between specific structural characteristics of α-syn fibrils and an organelle strongly implicated in PD pathology.


Assuntos
Amiloide/química , Encéfalo/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , alfa-Sinucleína/química , Amiloide/farmacologia , Animais , Encéfalo/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Galinhas , Clara de Ovo/química , Eritrócitos/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Muramidase/química , Muramidase/farmacologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ratos , Relação Estrutura-Atividade , alfa-Sinucleína/genética
6.
Mikrobiyol Bul ; 56(4): 692-705, 2022 Oct.
Artigo em Turco | MEDLINE | ID: mdl-36458715

RESUMO

Leishmania parasites, which are reported to be endemic in 98 countries around the world, infect humans as well as wild and domestic carnivores and small mammals, and are transmitted by sand flies (Phlebotomus, dwarf sandflies). It is reported that 350 million people are at risk and two million new cases are seen in the world every year. It has been reported that different drugs (topical paromomycin, oral miltefosine, ketoconazole, rifampin, and zinc) have been tried in studies especially in endemic regions in the treatment of cutaneous leishmaniasis, and response to treatment has been obtained at different rates. Today, the search for alternative treatments continues and many studies have been carried out for this purpose. For centuries, olive leaf extracts have been used to maintain health. Oleuropein has numerous health benefits, including antioxidant, antimicrobial, anti-inflammatory, antiatherogenic, anticarcinogenic, antiviral activities, cardio- and neuroprotective, hepatoprotective effects. The aim of this study was to determine and understand the mode of action of oleuropein, the cell death mechanisms caused by oleuropein in L.tropica promastigotes. In this study, the phenolic and flavonoid content of oleuropein was determined by HPLC method. The antioxidant capacity and the amount of oleuropein were determined. Afterwards, morphological and physiological (mitochondrial membrane potential, formation of reactive oxygen species, Annexin V binding) changes triggered by oleuropein in L.tropica promastigotes were investigated using flow cytometry. Our studies revealed that apoptotic properties such as mitochondrial dysfunction, production of reactive oxygen species, flip-flop action of phosphatidylserine could induce cell death in L.tropica promastigotes. It has been observed that oleuropein induced typical apoptotic morphological features in L.tropica promastigotes. Total phenolic content and total flavonoid content values of oleuropein extract were determined as 33 mg/g and 229 mg/g. The radical removal method was used to investigate the antioxidant capacity of methanol extracts against free radicals. Total antioxidant content of oleuropein extract was determined as 87%. In addition, the amount of oleuropein in the oleuropein extract was determined as 21. 1% by HPLC. The oleuropein dose that killed 50% of L.tropica promastigotes, that is the IC50 value, was detected as 46.6 µg/mL after 24 hours. It was observed that the parasites in the control group preserved their typical morphological features with a single nucleus, flagella, kinetoplast and narrow cell body at both 24 and 48 hours. It was observed that as oleuropein concentrations increased, the and kinetoplasts of L.tropica promastigotes could not be distinguished from each other, they moved away from the narrow cell body structure, they lost their flagella and turned into a round form, and they moved away from the typical form of the parasite. The percentage of Annexin V+ apoptotic cells was found to be 2.9 ± 0.4% in the untreated control group, and 38.1 ± 6.9% in the oleuropein-treated group. Polarization in the mitochondrial membrane of healthy promastigotes caused an approximately 1.7-fold change in the direction of depolarization in oleuropein-treated promastigotes. According to these findings, oleuropein triggered mitochondria-related death in L.tropica promastigotes. Moreover, 1.4 ± 0.2 fold increase in reactive oxygen species production was detected in oleuropein-treated promastigotes compared to the untreated control group. Comparisons between groups were made using the independent sample t test method. In conclusion, phenolic compounds of olive leaf extract oleuropein induced apoptotic cell death in L.tropica promastigotes. Our results support that olive products such as oleuropein may have anti-parasitic effects.


Assuntos
Leishmania tropica , Espécies Reativas de Oxigênio , Anexina A5 , Antioxidantes/farmacologia , Flavonoides/farmacologia , Leishmania tropica/efeitos dos fármacos , Potencial da Membrana Mitocondrial , Fenóis , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Glucosídeos Iridoides/farmacologia
7.
Adv Exp Med Biol ; 1395: 367-372, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36527664

RESUMO

In intact mitochondria, the transport of electrons, respiration and generation of proton gradients across the inner membrane (proton motive force) are mutually coupled, according to Peter Mitchell's hypothesis on oxidative phosphorylation. Thus, the inhibition of electron transport at either respiratory complex III or IV in the electron transport chain leads to failure in producing proton motive force along with the abolition of respiration. Here, we determined the mitochondrial membrane potential (MMP), as a measure of proton motive force, and cellular respiration in various cultured cells and demonstrated that inhibition of complex IV by KCN abolished mitochondrial respiration while MMP was sustained. These results are unexpected and appear incompatible with Mitchell's chemiosmotic hypothesis.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Força Próton-Motriz , Potencial da Membrana Mitocondrial , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fosforilação Oxidativa , Membranas Mitocondriais/metabolismo , Trifosfato de Adenosina/metabolismo
8.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi ; 40(11): 807-812, 2022 Nov 20.
Artigo em Chinês | MEDLINE | ID: mdl-36510713

RESUMO

Objective: To study the underlying mechanism of cadmium-induced apoptosis of mouse spermatocytes (GC-2 spd) . Methods: In March 2021, GC-2 spd cells were exposed to different concentrations of CdCl(2) for 24 hours, namely 5 µmol/L CdCl(2) (low-dose) group and 10 µmol/L CdCl(2) (high-dose) group, and unexposed GC-2 spd cells were used as control group. Mitochondrial morphology was observed in the cells stained with Mito-Track Red CMXRos fluorescent probes by confocal microscopy and the mitochrondrial membrane potential was measured by flow cytometry with JC-1 fluorescent probes. Mitochrondrial proteins, cytosolic proteins and total cellular proteins of GC-2 spd cells were extracted using cell mitochondria isolation kit and RIPA buffer, respectively. The expression of mitochondrial homeostasis regulatory proteins (FIS1 and OPA1), and apoptosis-related proteins (Cytochrome c and cleaved Caspase-3) were examined by Western blot. Results: Compared with the cells in the control group, the relative ratio of JC-1 red/green fluorescence signal in the cells of the low-dose and high-dose CdCl(2) groups decreased significantly (0.740±0.071, 0.570±0.028), with a statistically significant difference (P=0.017, 0.004) ; The morphology of mitochondria changed from long tube to point, and the proportion of cells containing point mitochondria increased significantly (45.1%±3.7% and 25.7%±4.9%), the difference was statistically significant (P=0.005, 0.001) ; The relative expression level of mitochondrial FIS1 in cells of low and high dose CdCl(2) groups was significantly higher (1.271±0.120, 1.693±0.155), the difference was statistically significant (P=0.046, 0.000) ; The relative expression level of OPA1 decreased significantly (0.838±0.050, 0.682±0.040), and the difference was statistically significant (P=0.049, 0.001). Compared with the control group, the relative expression level of cytochrome c protein in the cytoplasm of cells in the low dose group of CdCl(2) was not significantly increased (1.249±0.151), and the difference was not statistically significant (P=0.075). However, the relative expression level in the cytoplasm of cells in the high dose group of CdCl(2) was significantly increased (2.355±0.110), and the difference was statistically significant (P=0.000) ; The relative expression level of Cytochrome c in mitochondria of low and high dose CdCl(2) groups decreased significantly (0.681±0.043, 0.619±0.114), with a statistically significant difference (P=0.004, 0.001) ; Moreover, the level of cleaved Caspase-3 protein in cells gradually increased (5.486±0.544, 11.493±1.739), the difference was statistically significant (P=0.004, 0.000) . Conclusion: Cadmium induced cleaved Caspase-3 mediated apoptosis of GC-2 spd cells via promoting mitochrondrial fission and the release of Cytochrome c from the mitochrondria to the cytosol.


Assuntos
Cádmio , Dinâmica Mitocondrial , Masculino , Camundongos , Animais , Caspase 3/metabolismo , Cádmio/toxicidade , Citocromos c/metabolismo , Corantes Fluorescentes , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Potencial da Membrana Mitocondrial
9.
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
10.
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
11.
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
12.
Vet Microbiol ; 275: 109596, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36335841

RESUMO

Porcine epidemic diarrhea (PED) is a highly contagious and virulent intestinal infectious disease characterized by diarrhea, vomiting and dehydration. Although PEDV-induced apoptosis has been characterized in vitro and vivo, the functional proteins related to this event and the mechanism still need further research. Here, we firstly demonstrated that PEDV epidemic strain JS2013 could trigger apoptosis in a dose- and time-dependent manner. Then, PEDV 3CLpro was further identified as a crucial inducer of PEDV-triggered apoptosis. In addition, using site-directed mutagenesis to disrupt the protease activity of 3CLpro by His41 and Cys144 mutations, we found that 3CLpro-induced apoptosis and mitochondrial damage significantly reduced, suggesting that the protease activity of 3CLpro was essential for apoptosis and mitochondrial damage. Furthermore, PEDV 3CLpro could synergistically promote MAVS-mediated apoptosis and MAVS was involved in the signaling pathway of 3CLpro-induced apoptosis, but no direct interaction between PEDV 3CLpro and MAVS was detected by immunoprecipitation assays. Our findings provide important insights into the role of 3CLpro in the pathogenicity of PEDV.


Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Suínos , Animais , Potencial da Membrana Mitocondrial , Apoptose , Transdução de Sinais , Peptídeo Hidrolases/metabolismo , Diarreia/veterinária , Infecções por Coronavirus/veterinária
13.
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
14.
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
15.
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
16.
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
17.
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
18.
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
19.
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
20.
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
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