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1.
Anticancer Res ; 41(10): 4725-4732, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34593421

RESUMO

BACKGROUND/AIM: We investigated the cytotoxic effects of plumbagin on metastatic retinoblastoma, using the highly metastatic cell line Y79. MATERIALS AND METHODS: Effect of plumbagin on cell growth was assessed with water-soluble tetrazolium 1 (WST-1) cell proliferation assay and automated hemocytometry with trypan blue-exclusion assay. Cell death was studied with acridine orange/ethidium bromide live-dead assay and annexin-V-fluorescein isothiocyanate/propidium iodide microscopy. Loss of mitochondrial membrane potential was studied with JC-10 dye and caspase activation was investigated using CellEvent Caspase-3/7 Green detection reagent. RESULTS: Plumbagin highly significantly reduced the growth of Y79 cells treated for 24 h with 2.5 µM or more. Plumbagin also induced significantly high levels of cell death which was associated with loss of mitochondrial membrane potential and caspase activation. CONCLUSION: At very low concentration (2.5 µM), plumbagin potently induced cytotoxicity in metastatic retinoblastoma cells via loss of mitochondrial membrane potential and caspase activation.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Caspases/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Naftoquinonas/farmacologia , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Mitocôndrias , Metástase Neoplásica
2.
Arch Insect Biochem Physiol ; 108(3): e21844, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34519097

RESUMO

Animals maintain homeostasis of cell numbers, constantly creating new cells and eliminating others. Programmed cell death, apoptosis, is a mechanism of cell elimination and it acts in many aspects of animal biology. Drawing on the biomedical background, several signals launch the apoptosis mechanisms, including prostaglandins (PGs). Based on this information, we posed the hypothesis that PGs similarly induce apoptosis in insect cell lines. We used three Spodoptera frugiperda cell lines, including two newly established, BCIRL-SfNS-0518B-YL derived from the central nervous system and BCIRL-Sf4FB-0614-SGS derived from fat body, and the commercially available Sf9 cells. Using a kinetic apoptosis kit, we found treating SfNS cells for 18 h with 15 or 20 µM PGA2 led to decreases in cell numbers, coupled with increased numbers of apoptotic and dead cells. Similar exposures to 10 µM PGA2 (24 h) led to substantial increases in apoptotic cells, confirmed by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay on a flow cytometer. The influence of PGA2 treatments increased with dosage, as we recorded about 20% apoptosis at 24 h post-PGA2 treatments (10 µM) and about 34% apoptosis at 24 h post-30 µM treatments. PGA2 treatments led to 10- to 30-fold increases in messenger RNAs (mRNAs) encoding apoptosis-specific caspases-1, -2, -3, and -5 at 12 h and 40- to 60-fold increases in mRNAs encoding caspases-1 and -2, 10-fold increases for caspases-3 and -5 at 24 h. These findings strongly support our hypothesis that PGs induce apoptosis in an insect cell line and confirm an additional PG action in insect biology.


Assuntos
Caspases , Prostaglandinas A/farmacologia , Células Sf9/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Caspases/efeitos dos fármacos , Caspases/metabolismo , Spodoptera/metabolismo
3.
Molecules ; 26(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34443605

RESUMO

Extracts derived from the Ceratonia siliqua L. (carob) tree have been widely studied for their ability to prevent many diseases mainly due to the presence of polyphenolic compounds. In this study, we explored, for the first time, the anti-cancer properties of Cypriot carobs. We produced extracts from ripe and unripe whole carobs, pulp and seeds using solvents with different polarities. We measured the ability of the extracts to inhibit proliferation and induce apoptosis in cancer and normal immortalized breast cells, using the MTT assay, cell cycle analysis and Western Blotting. The extracts' total polyphenol content and anti-oxidant action was evaluated using the Folin-Ciocalteu method and the DPPH assay. Finally, we used LC-MS analysis to identify and quantify polyphenols in the most effective extracts. Our results demonstrate that the anti-proliferative capacity of carob extracts varied with the stage of carob maturity and the extraction solvent. The Diethyl-ether and Ethyl acetate extracts derived from the ripe whole fruit had high Myricetin content and also displayed specific activity against cancer cells. Their mechanism of action involved caspase-dependent and independent apoptosis. Our results indicate that extracts from Cypriot carobs may have potential uses in the development of nutritional supplements and pharmaceuticals.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Fabaceae/química , Fenóis/química , Fenóis/farmacologia , Solventes/química , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Frutas/química , Humanos , Sementes/química
4.
Molecules ; 26(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34443427

RESUMO

Pterostilbene, a natural metabolite of resveratrol, has been indicated as a potent anticancer molecule. Recently, several pterostilbene derivatives have been reported to exhibit better anticancer activities than that of the parent pterostilbene molecule. In the present study, a series of pterostilbene derivatives were designed and synthesized by the hybridization of pterostilbene, chalcone, and cinnamic acid. The cytotoxic effect of these hybrid molecules was determined using two oral cancer cell lines, HSC-3 and OECM-1. (E)-3-(2-((E)-4-Hydroxystyryl)-4,6-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (4d), with IC50 of 16.38 and 18.06 µM against OECM-1 and HSC-3, respectively, was selected for further anticancer mechanism studies. Results indicated that compound 4d effectively inhibited cell proliferation and induced G2/M cell cycle arrest via modulating p21, cyclin B1, and cyclin A2. Compound 4d ultimately induced cell apoptosis by reducing the expression of Bcl-2 and surviving. In addition, cleavage of PARP and caspase-3 were enhanced following the treatment of compound 4d with increased dose. To conclude, a number of pterostilbene derivatives were discovered to possess potent anticancer potentials. Among them, compound 4d was the most active, more active than the parent pterostilbene.


Assuntos
Antineoplásicos/farmacologia , Chalcona/farmacologia , Estilbenos/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chalcona/química , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Poli(ADP-Ribose) Polimerases/metabolismo , Estilbenos/química , Relação Estrutura-Atividade
5.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445633

RESUMO

Caspases, a family of cysteine-aspartic proteases, have an established role as critical components in the activation and initiation of apoptosis. Alongside this a variety of non-apoptotic caspase functions in proliferation, differentiation, cellular plasticity and cell migration have been reported. The activity level and context are important factors in determining caspase function. As a consequence of their critical role in apoptosis and beyond, caspases are uniquely situated to have pathological roles, including in cancer. Altered caspase function is a common trait in a variety of cancers, with apoptotic evasion defined as a "hallmark of cancer". However, the role that caspases play in cancer is much more complex, acting both to prevent and to promote tumourigenesis. This review focuses on the major findings in Drosophila on the dual role of caspases in tumourigenesis. This has major implications for cancer treatments, including chemotherapy and radiotherapy, with the activation of apoptosis being the end goal. However, such treatments may inadvertently have adverse effects on promoting tumour progression and acerbating the cancer. A comprehensive understanding of the dual role of caspases will aid in the development of successful cancer therapeutic approaches.


Assuntos
Apoptose , Carcinogênese , Caspases/metabolismo , Neoplasias/patologia , Animais , Drosophila , Humanos , Neoplasias/enzimologia , Neoplasias/etiologia , Neoplasias/prevenção & controle
7.
Biomolecules ; 11(7)2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34356678

RESUMO

Allograft kidney transplantation, which triggers host cellular- and antibody-mediated rejection of the kidney, is a major contributor to kidney damage during transplant. Here, we asked whether PrC-210 would suppress damage seen in allograft kidney transplant. Brown Norway (BN) rat kidneys were perfused in situ (UW Solution) with or without added 30 mM PrC-210, and then immediately transplanted into Lewis (LEW) rats. 20 h later, the transplanted BN kidneys and LEW rat plasma were analyzed. Kidney histology, and kidney/serum levels of several inflammation-associated cytokines, were measured to assess mismatch-related kidney pathology, and PrC-210 protective efficacy. Twenty hours after the allograft transplants: (i) significant histologic kidney tubule damage and mononuclear inflammatory cell infiltration were seen in allograft kidneys; (ii) kidney function metrics (creatinine and BUN) were significantly elevated; (iii) significant changes in key cytokines, i.e., TIMP-1, TNF-alpha and MIP-3A/CCL20, and kidney activated caspase levels were seen. In PrC-210-treated kidneys and recipient rats, (i) kidney histologic damage (Banff Scores) and mononuclear infiltration were reduced to untreated background levels; (ii) creatinine and BUN were significantly reduced; and (iii) activated caspase and cytokine changes were significantly reduced, some to background. In conclusion, the results suggest that PrC-210 could provide broadly applicable organ protection for many allograft transplantation conditions; it could protect transplanted kidneys during and after all stages of the transplantation process-from organ donation, through transportation, re-implantation and the post-operative inflammation-to minimize acute and chronic rejection.


Assuntos
Diaminas/farmacologia , Inflamação/tratamento farmacológico , Transplante de Rim/efeitos adversos , Rim/efeitos dos fármacos , Compostos de Sulfidrila/farmacologia , Adenosina , Aloenxertos , Alopurinol , Animais , Caspases/metabolismo , Creatinina/sangue , Citocinas/metabolismo , Diaminas/administração & dosagem , Sequestradores de Radicais Livres/farmacologia , Glutationa , Inflamação/patologia , Insulina , Rim/patologia , Transplante de Rim/métodos , Masculino , Mitocôndrias/efeitos dos fármacos , Soluções para Preservação de Órgãos , Rafinose , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Compostos de Sulfidrila/administração & dosagem
8.
FEBS Lett ; 595(17): 2237-2247, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34318487

RESUMO

Plant metacaspases type I (MCA-Is), the closest structural homologs of caspases, are key proteases in stress-induced regulated cell death processes in plants. However, no plant MCA-Is have been characterized in vitro to date. Here, we show that only plant MCA-Is contain a highly hydrophobic loop within the C terminus of their p10 domain. When removed, soluble and proteolytically active plant MCA-Is can be designed and recombinantly produced. We show that the activity of MCA-I depends on calcium ions and that removal of the hydrophobic loop does not affect cleavage and covalent binding to its inhibitor SERPIN. This novel approach will finally allow the development of tools to detect and manipulate the activity of these cysteine proteases in vivo and in planta.


Assuntos
Caspases/química , Caspases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Cálcio/metabolismo , Caspases/genética , Chlamydomonas reinhardtii/enzimologia , Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/genética , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serpinas/metabolismo
9.
FASEB J ; 35(8): e21757, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34233045

RESUMO

Pyroptosis and intrinsic apoptosis are two forms of regulated cell death driven by active caspases where plasma membrane permeabilization is induced by gasdermin pores. Caspase-1 induces gasdermin D pore formation during pyroptosis, whereas caspase-3 promotes gasdermin E pore formation during apoptosis. These two types of cell death are accompanied by mitochondrial outer membrane permeabilization due to BAK/BAX pore formation in the external membrane of mitochondria, and to some extent, this complex also affects the inner mitochondrial membrane facilitating mitochondrial DNA relocalization from the matrix to the cytosol. However, the detailed mechanism responsible for this process has not been investigated. Herein, we reported that gasdermin processing is required to induce mitochondrial DNA release from cells during pyroptosis and apoptosis. Gasdermin targeted at the plasma membrane promotes a fast mitochondrial collapse along with the initial accumulation of mitochondrial DNA in the cytosol and then facilitates the DNA's release from the cell when the plasma membrane ruptures. These findings demonstrate that gasdermin action has a critical effect on the plasma membrane and facilitates the release of mitochondrial DNA as a damage-associated molecular pattern.


Assuntos
Apoptose/fisiologia , DNA Mitocondrial/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Piroptose/fisiologia , Animais , Caspases/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/genética , Pirina/metabolismo , Receptores de Estrogênio/fisiologia
10.
Cancer Sci ; 112(7): 2803-2820, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34109710

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the most chemoresistant cancers. An understanding of the molecular mechanism by which PDAC cells have a high chemoresistant potential is important for improvement of the poor prognosis of patients with PDAC. Here we show for the first time that disruption of heat shock protein 47 (HSP47) enhances the efficacy of the therapeutic agent gemcitabine for PDAC cells and that the efficacy is suppressed by reconstituting HSP47 expression. HSP47 interacts with calreticulin (CALR) and the unfolded protein response transducer IRE1α in PDAC cells. Ablation of HSP47 promotes both the interaction of CALR with sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase 2 and interaction of IRE1α with inositol 1,4,5-triphosphate receptor, which generates a condition in which an increase in intracellular Ca2+ level is prone to be induced by oxidative stimuli. Disruption of HSP47 enhances NADPH oxidase-induced generation of intracellular reactive oxygen species (ROS) and subsequent increase in intracellular Ca2+ level in PDAC cells after treatment with gemcitabine, resulting in the death of PDAC cells by activation of the Ca2+ /caspases axis. Ablation of HSP47 promotes gemcitabine-induced suppression of tumor growth in PDAC cell-bearing mice. Overall, these results indicated that HSP47 confers chemoresistance on PDAC cells and suggested that disruption of HSP47 may improve the efficacy of chemotherapy for patients with PDAC.


Assuntos
Calreticulina/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Resistencia a Medicamentos Antineoplásicos , Endorribonucleases/metabolismo , Proteínas de Choque Térmico HSP47/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Cálcio/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Caspases/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Técnicas de Inativação de Genes , Inativação Gênica , Proteínas de Choque Térmico HSP47/genética , Xenoenxertos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , NADPH Oxidases/metabolismo , Transplante de Neoplasias , Neoplasias Pancreáticas/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Resposta a Proteínas não Dobradas
11.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066632

RESUMO

Ethanol has been shown to exhibit therapeutic properties as an ablative agent alone and in combination with thermal ablation. Ethanol may also increase sensitivity of cancer cells to certain physical and chemical antitumoral agents. The aim of our study was to assess the potential influence of nontoxic concentrations of ethanol on hyperthermia therapy, an antitumoral modality that is continuously growing and that can be combined with classical chemotherapy and radiotherapy to improve their efficiency. Human leukemia cells were included as a model in the study. The results indicated that ethanol augments the cytotoxicity of hyperthermia against U937 and HL60 cells. The therapeutic benefit of the hyperthermia/ethanol combination was associated with an increase in the percentage of apoptotic cells and activation of caspases-3, -8 and -9. Apoptosis triggered either by hyperthermia or hyperthermia/ethanol was almost completely abolished by a caspase-8 specific inhibitor, indicating that this caspase plays a main role in both conditions. The role of caspase-9 in hyperthermia treated cells acquired significance whether ethanol was present during hyperthermia since the alcohol enhanced Bid cleavage, translocation of Bax from cytosol to mitochondria, release of mitochondrial apoptogenic factors, and decreased of the levels of the anti-apoptotic factor myeloid cell leukemia-1 (Mcl-1). The enhancement effect of ethanol on hyperthermia-activated cell death was associated with a reduction in the expression of HSP70, a protein known to interfere in the activation of apoptosis at different stages. Collectively, our findings suggest that ethanol could be useful as an adjuvant in hyperthermia therapy for cancer.


Assuntos
Etanol/farmacologia , Hipertermia Induzida , Leucemia Mieloide/patologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células U937
12.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067020

RESUMO

Current available therapies for pancreatic ductal adenocarcinoma (PDAC) provide minimal overall survival benefits and cause severe adverse effects. We have identified a novel molecule AS-10, a selenazolidine-bis-aspirinyl derivative, that was two to three orders of magnitude more potent than aspirin and at least one to two orders of magnitude more potent than gemcitabine in inhibiting PDAC cancer cell growth/viability against three PDAC cell lines while sparing mouse embryonic fibroblasts in the same exposure range. In Panc-1 cells, AS-10 induced apoptosis without necrosis, principally through caspase-3/7 cascade and reactive oxygen species, in addition to an induction of G1 cell cycle block. Transcriptomic profiling with RNA-seq indicated the top responses to AS-10 exposure as CDKN1A (P21Cip1), CCND1, and nuclear transcription factor-kappa B (NF-κB) complex and the top functions as cell cycle, cell death, and survival without inducing the DNA damage gene signature. AS-10 pretreatment (6 h) decreased cytokine tumor necrosis factor-alpha (TNF-α)-stimulated NF-κB nuclear translocation, DNA binding activity, and degradation of cytosolic inhibitor of κB (IκB) protein. As NF-κB activation in PDAC cells confers resistance to gemcitabine, the AS-10 combination with gemcitabine increased the in vitro cytotoxicity more than the additivity of both compounds. Overall, our results suggest AS-10 may be a promising drug lead for PDAC, both as a single agent and in combination therapy.


Assuntos
Adenocarcinoma/patologia , Apoptose , Aspirina/farmacologia , Carcinoma Ductal Pancreático/patologia , Desoxicitidina/análogos & derivados , Pontos de Checagem da Fase G1 do Ciclo Celular , NF-kappa B/metabolismo , Neoplasias Pancreáticas/patologia , Acetilcisteína/farmacologia , Adenocarcinoma/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Aspirina/química , Carcinoma Ductal Pancreático/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Desoxicitidina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Neoplasias Pancreáticas/genética , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
13.
Biochem Biophys Res Commun ; 560: 119-125, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33989902

RESUMO

Amounting evidence suggested that long non coding RNAs (lncRNAs) played vital roles in the progression of various cancers. The aim of this study is to examine the biological roles and underlying mechanisms of lncRNA MAFG-AS1 in the tumorigenesis of breast cancer (BC) cells. Here we showed that downregulation of MAFG-AS1 inhibited the viability, migration, and invasion of BC cells. Mechanism investigation showed that inhibition of MAFG-AS1 induced apoptosis via the intrinsic apoptotic pathway and overexpression of Bcl-2 could inhibited it. Further, MAFG-AS1 acts as a sponge of miR-574-5p which directly binds to SOD2 mRNA. Re-expression of SOD2 using a 3'-UTR mutant SOD2 reversed the effects of silencing of MAFG-AS1 on BC cells. Finally, downregulation of MAFG-AS1 inhibited the growth of tumour in vivo. Together, MAFG-AS1 acts as an oncogene via regulation of miR-574-5p/SOD2 axis in BC cells.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Superóxido Dismutase/genética , Animais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Feminino , Humanos , Camundongos SCID , Invasividade Neoplásica , Superóxido Dismutase/metabolismo
14.
Biomed Pharmacother ; 139: 111628, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33940508

RESUMO

Pinus kesiya Royle ex Gordon (PK), widely found in Southeast Asia, has been traditionally used for the treatment of several illnesses. Our previous studies showed that PK was highly cytotoxicity against liver cancer cells. The detailed mechanism of anticancer action of 50% hydro-ethanolic extract of PK's twig was, therefore, investigated in hepatocellular carcinoma HepG2 cells. Cytotoxicity of PK was determined by using NR assay, followed by determination of the mode of cell death by flow cytometry. The apoptosis-inducing effect was determined based on caspases activity, mitochondria membrane potential change, and expression of proteins related to apoptosis by western blot. The biomolecular alteration in the PK-treated HepG2 cells was investigated by FTIR microspectroscopy. Inhibition of topoisomerase I enzyme was determined by using DNA relaxation assay. Results showed that PK displayed high selective cytotoxicity and induced apoptosis against HepG2. FTIR microspectroscopy indicated that PK altered major biomolecules in HepG2 different from melphalan (a positive control), indicating a different mechanism of anticancer action. PK induced apoptotic cell death through the intrinsic pathway by increasing caspases 9 and 3/7 activity, increasing Bax, and decreasing Bcl-2 expression leading to mitochondrial membrane potential changes. PK also inhibited Top I and PARP activity that triggered an intrinsic apoptotic pathway. The phytochemical test presented terpenoids (i.e., α-pinene confirmed by GC-MS), alkaloids, steroids, xanthone, reducing sugar, and saponin. α-Pinene exhibited low cytotoxicity against HepG2, therefore, several terpene derivatives may work synergistically for inducing apoptosis. Our data demonstrated that PK has the potential for further study with chemotherapeutic purposes.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , DNA Topoisomerases Tipo I/efeitos dos fármacos , Pinus/química , Extratos Vegetais/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Caspases/metabolismo , DNA Topoisomerases Tipo I/genética , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Extratos Vegetais/química , Espectroscopia de Infravermelho com Transformada de Fourier
15.
Cell Physiol Biochem ; 55(S1): 161-170, 2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-33961353

RESUMO

Apoptosis is a programmed form of cell death culminating in packing cell content and corpse dismantling into membrane sealed vesicles called apoptotic bodies (ABs). Apoptotic bodies are engulfed and disposed by neighboring and immune system cells without eliciting a noxious inflammatory response, thus preventing sterile tissue damage. AB formation requires a total surface area larger than the apparent, initial cell's surface area. Apoptotic volume decrease (AVD) is a two-stage process leading to an isotonic, osmotic reduction in cell water content driven by net K+ and Cl- extrusion. In this article, the role of AVD is presented from a geometric point of view through the process of AB formation. AVD decisively contributes to (i) endowing the cell with the appropriate electrolytic environment for apoptotic execution; (ii) increasing the membrane surface area-to-volume ratio, along with the mobilization of membrane reservoirs (cell rounding, membrane folds and endosomal membranes), so that the cell corpse can be dismantled into ABs; and (iii) reducing plasmalemmal stretch, tension and stiffness, thus facilitating membrane bulging, blebbing and vesicle expansion ultimately leading to separation and release.


Assuntos
Vesículas Extracelulares/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Caspases/genética , Caspases/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Vesículas Extracelulares/genética , Humanos , Osmose
16.
J Theor Biol ; 525: 110765, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34019850

RESUMO

Apoptosis has been extensively characterized by both experimental approaches and model simulations. However, it is still not fully understood how the regulation occurs, especially in the intrinsic pathway, which can be activated by a great variety of signals. In addition, the conditions in which a point of no return could be reached remain elusive. In this work, we use differential equations models to approach these issues. Our starting point was the model for caspase activation of Legewie et al. (Legewie S, et al., PLoS Computational Biology 2006, 2(9): e120), which exhibits irreversible bistability. We added an activation module to this model, with the main events related to mitochondrial outer membrane permeabilization, which includes cytochrome C release by the mitochondria and its effects on caspase activation and respiratory chain disruption. This "Extended Legewie Model" (ELM) uses BAK as the apoptotic stimulus and active caspase 3 as a measure of apoptosis activation. Unexpectedly, in the extended model, BAK cannot trigger apoptosis activation using physiologically sound initial values of the variables, due to limitations in apoptosome concentration increase. Therefore, the next step was to find a regulatory mechanism, allowing apoptosis activation in the ELM, starting from physiological initial concentrations. For this aim, we performed a sensitivity analysis on the 61 parameters of the system, finding that those producing the most relevant changes in the qualitative behaviour were the rates of synthesis of caspase 3, caspase 9 and XIAP. Based on these results, the transcription factor E2F was included in the ELM because it directly regulates the rate of synthesis of caspase 3 and 9. Depending on the concentration of E2F, the ELM shows different qualitative behaviours. On one hand, for low E2F apoptosis is impossible and for high E2F apoptosis is inevitable. Therefore, if E2F is sufficiently increased, the point of no return is crossed. On the other hand, for intermediate values of E2F there is a bistable region where the fate of the system also depends on the concentration of BAK and other signalling species.


Assuntos
Apoptose , Caspases , Caspases/metabolismo , Citocromos c/metabolismo , Mitocôndrias , Membranas Mitocondriais/metabolismo
17.
Int J Nanomedicine ; 16: 3385-3405, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34040370

RESUMO

Background: Hepatocellular carcinoma (HCC) is one of the main causes of cancer-related death. Sorafenib, which is the first-line therapy for this disease, is associated with reduced therapeutic efficacy that could potentially be overcome by combination with selumetinib. In this context, the main goal of this work was to develop a new nanosystem, composed of a polymeric core coated by a lipid bilayer containing the targeting ligand GalNAc, to specifically and efficiently co-deliver both drugs into HCC cells, in order to significantly increase their therapeutic efficacy. Methods: The physicochemical characterization of hybrid nanosystems (HNP) and their components was performed by dynamic light scattering, zeta potential, matrix-assisted laser desorption ionization - time of flight mass spectroscopy, and transmission electron microscopy. Cellular binding, uptake and specificity of HNP were evaluated through flow cytometry and confocal microscopy. The therapeutic activity was evaluated namely through: cell viability by the Alamar Blue assay; cell death by flow cytometry using FITC-Annexin V; caspases activity by luminescence; mitochondrial membrane potential by flow cytometry; and molecular target levels by Western blot. Results: The obtained data show that these hybrid nanosystems present high stability and loading capacity of both drugs, and suitable physicochemical properties, namely in terms of size and surface charge. Moreover, the generated formulation allows to circumvent drug resistance and presents high specificity, promoting great cell death levels in HCC cells, but not in non-tumor cells. This potentiation of the antitumor effect of co-loaded drugs was carried out by an increased programmed cell death, being associated with a strong reduction in the mitochondrial membrane potential, a significant increase in the activity of caspases 3/7 and caspase 9, and much greater number of annexin V-positive cells. Conclusion: The developed formulation resulted in a high and synergistic antitumor effect, revealing a translational potential to improve therapeutic approaches against HCC.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Nanomedicina/métodos , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico
18.
Methods Mol Biol ; 2255: 21-26, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033091

RESUMO

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.


Assuntos
Apoptose , Caspases/metabolismo , Citocromos c/metabolismo , Citoplasma/metabolismo , Mitocôndrias/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Western Blotting , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
19.
Methods Mol Biol ; 2255: 27-42, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033092

RESUMO

Cellular signals to resist apoptosis have been attributed as one of the mechanisms of tumorigenesis. Hence, apoptosis is a cardinal target for drug development in cancers, and several antitumor drugs have been designed to induce apoptosis in tumor cells. Recently, venetoclax, a Bcl2 inhibitor that induces apoptosis, has been approved by the FDA for the treatment of CLL and SLL patients. Proapoptotic antitumor drugs have been traditionally developed and tested, targeting apoptosis in tumor cells. The mechanism of such drug actions has been functionally connected to the mechanism of apoptosis. The identification of apoptosis in a tumor cell takes into account different characteristics in several steps of apoptosis. Thus, it is understandable that modes of identification of apoptosis observed in tumor cells in a laboratory have also been tuned to different characteristics in several parameters of apoptosis. Here, we present a detailed methodology for a triple-parameter-based co-fluorescence imaging to identify apoptosis in live tumor cells. The procedure involves co-fluorescence staining specific for three cardinal features of apoptosis in live cells. The procedure is simple, time-sensitive, and can be performed successfully in a laboratory-friendly manner.


Assuntos
Apoptose , Neoplasias da Mama/patologia , Fluorescência , Laboratórios/estatística & dados numéricos , Mitocôndrias/metabolismo , Imagem Óptica/métodos , Neoplasias Ovarianas/patologia , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Membrana Celular/metabolismo , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias Ovarianas/metabolismo , Fosfatidilserinas/metabolismo , Células Tumorais Cultivadas
20.
Methods Mol Biol ; 2255: 69-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033095

RESUMO

Anoikis is a type of programmed cell death triggered by the loss of cellular interaction with the extracellular matrix (ECM) and culminates in the activation of caspases. Specific interaction between cellular receptors such as integrins and the ECM is important to maintain cellular homeostasis in normal tissues through multiple cascades. This interaction provides not only physical attachment, but more importantly, vital interaction with the actin cytoskeleton and growth factors. Normal epithelial and endothelial cells require this interaction with ECM to survive. In cancer, the acquisition of anoikis resistance is a hallmark of malignant transformation and is required in the process of metastasis formation. As such, strategies to inhibit and/or counteract anoikis resistance are important in controlling cancer progression. In this chapter, we describe the method for detecting anoikis using cell viability and caspase activity assays.


Assuntos
Anoikis , Caspases/metabolismo , Corantes Fluorescentes/química , Leucemia Monocítica Aguda/patologia , Neoplasias Ovarianas/patologia , Sobrevivência Celular , Feminino , Humanos , Leucemia Monocítica Aguda/metabolismo , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
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