RESUMO
Regorafenib, an oral small-molecule multi kinase inhibitor, is able to block Vascular Endothelial Growth Factor Receptors (VEGFR-1, 2, and 3), Platelet-Derived Growth Factor Receptors (PDGF), Fibroblast Growth Factor (FGF) receptor 1, Raf, TIE-2, and the kinases KIT, RET, and BRAF. Different studies have displayed its antitumor activity in several cancer models (both in vitro and in vivo), particularly in colorectal and gastrointestinal stromal cancers. The mechanism of resistance to regorafenib is largely unknown. In our investigation, we have generated regorafenib-resistant SW480 cells (Reg-R-SW480 cells) by culturing such cells with increasing concentration of regorafenib. Examination of intracellular signaling found that Akt signaling was activated in Reg-R-SW480 cells but not in wild-type SW480 cells, after regorafenib treatment as measured by Western Blot. The Notch pathway is a fundamental signaling system in the development and homeostasis of tissues since it regulates different cellular process such as proliferation, differentiation, and apoptosis and it can be a potential driver of resistance to a wide array of targeted therapies. In this study, we found that Notch-1 was significantly up-regulated in resistant tumor cells as well as HES1 and HEY. Additionally, inhibition of Notch-1 in resistant cells partially restored sensitivity to regorafenib treatment in vitro. Collectively, these data suggest a key role of Notch-1 in mediating the resistant effects of regorafenib in colorectal cancer cells, and also provide a rationale to improve the therapeutic efficacy of regorafenib.
Assuntos
Neoplasias do Colo/patologia , Compostos de Fenilureia/farmacologia , Piridinas/farmacologia , Receptores Notch/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Compostos de Fenilureia/química , Proteínas Proto-Oncogênicas c-kit/metabolismo , Piridinas/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
The limited availability of hepatic tissue suitable for the treatment of liver disease and drug research encourages the generation of hepatic-like cells from alternative sources as support for the regenerative medicine. Human blood derived stem cells (BDSCs) express surface markers and genes characteristic of pluripotent stem cells and have the ability to differentiate into different cell types, including tissues of endodermal origin (i.e., liver). Therefore they can represent a valuable source of hepatocytes for medicine. In this investigation, we exploited a fast hepatic differentiation protocol to generate hepatocyte-like cells from human BDSCs using only hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) as growth factors. The resulting cell population exhibited hepatic cell-like morphology and it was characterized with a variety of biological endpoint analyses. Here, we demonstrate how human BDSCs can be reprogrammed in hepatocyte-like cells by morphological, functional analysis, reverse transcriptase (RT)-PCR, and Western Blot assay. This study defines a fast and easy reprogramming strategy that facilitates the differentiation of human BDSCs along a hepatic lineage and provides a framework for a helpful source in the stem cells therapy and liver disorders.
Assuntos
Células-Tronco Adultas/fisiologia , Células Sanguíneas/fisiologia , Diferenciação Celular , Linhagem da Célula , Hepatócitos/fisiologia , Células-Tronco Adultas/metabolismo , Biomarcadores/metabolismo , Células Sanguíneas/metabolismo , Western Blotting , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células , Separação Celular/métodos , Forma Celular , Células Cultivadas , Fator 4 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Glicogênio/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/metabolismo , Humanos , Imuno-Histoquímica , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Ureia/metabolismoRESUMO
The following study demonstrated that, in in vitro differentiated neurons, SIRT1 silencing induced an increase of IGF-1 protein expression and secretion and of IGF-1R protein levels which, in turn, prolonged neuronal cell survival in presence of an apoptotic insult. On the contrary, SIRT1 overexpression increased cell death. In particular, IGF-1 and IGF-1R expression levels were negatively regulated by SIRT1. In SIRT1 silenced cells, the increase in IGF-1 and IGF-1R expression was associated to an increase in AKT and ERK1/2 phosphorylation. Moreover, neuronal differentiation was reduced in SIRT1 overexpressing cells and increased in SIRT1 silenced cells. We conclude that SIRT1 silenced neurons appear more committed to differentiation and more resistant to cell death through the activation of IGF-1 survival pathway.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Sobrevivência Celular , Regulação para Baixo/genética , Camundongos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores , RNA Interferente Pequeno/genética , Ratos , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
Stem cell technology has evoked considerable excitement among people interested in the welfare of animals, as it has suggested the potential availability of new tools for several pathologies, including eye disease, which in many cases is considered incurable. One such example is ulcerative keratitis, which is very frequent in horses. Because some of these corneal ulcers can be very severe, progress rapidly and, therefore, can be a possible cause of vision loss, it is important to diagnose them at an early stage and administer an appropriate treatment, which can be medical, surgical, or a combination of both. The therapeutic strategy should eradicate the infection in order to reduce or stop destruction of the cornea. In addition, it should support the corneal structures and control the uveal reaction, and the pain associated with it, in order to minimize scarring. In this study, we address how stem cells derived from peripheral blood can be used also in ophthalmological pathologies. Our results demonstrate that this treatment protocol improved eye disease in four horse cases, including corneal ulcers and one case of retinal detachment. In all cases, we detected a decrease in the intense inflammatory reaction as well as the restoration of the epithelial surface of the central cornea.
Assuntos
Oftalmopatias/terapia , Oftalmopatias/veterinária , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/veterinária , Doenças dos Cavalos/terapia , Animais , Mordeduras e Picadas/patologia , Mordeduras e Picadas/terapia , Mordeduras e Picadas/veterinária , Úlcera da Córnea/patologia , Úlcera da Córnea/terapia , Úlcera da Córnea/veterinária , Oftalmopatias/patologia , Feminino , Doenças dos Cavalos/patologia , Cavalos , Ceratite/patologia , Ceratite/terapia , Ceratite/veterinária , Masculino , Descolamento Retiniano/patologia , Descolamento Retiniano/terapia , Descolamento Retiniano/veterinária , Uveíte/parasitologia , Uveíte/terapia , Uveíte/veterináriaRESUMO
The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1α silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NF-κB) expression. In contrast, NF-κB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1α. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-κB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-κB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-κB as well as cell invasion without affecting HIF-1α stabilization. Once in the nucleus, NF-κB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damage-associated molecular patterns receptors by HIF-1α represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype.
Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Núcleo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Imunológicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Immunoblotting , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/genética , Fosforilação , Transporte Proteico , RNA Interferente Pequeno/genética , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Transdução de SinaisRESUMO
At the end of 2019, a new disease recognized such as severe acute respiratory syndrome (SARS), was reported in Wuhan, China. This disease was caused by an unknown SARS coronavirus 2 (SARS-CoV-2); a virus is characterized by high infectivity among humans. In some cases, this disease can be asymptomatic, while in other cases can induce flu-like symptoms or acute respiratory distress syndrome, pneumonia and death. For this reason, the World Health Organization and Public Health Emergency of International Concern declared a pandemic status in January 2020. Currently, numerous countries have been involved in the development of effective vaccines to protect humans against SARS-CoV-2 infection. The present review will discuss the four vaccines, AZD1222 (AstraZeneca or Vaxzevria), Janssen (Ad26.COV2.S), Moderna/mRNA-1273 and BioNTech/Fosun/Pfizer BNT162b1, that are currently in use worldwide to understand their efficacy, but also evaluate the difficulties and challenges of vaccine development. Although several questions should be addressed regarding these vaccines, the current review will examine the viral elements used in the coronavirus-19 vaccine that can play a crucial role in inducing a strong immune response, as well as the different adverse effects that they can cause to individuals.
RESUMO
Our previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases Bovine Herpesvirus 1 (BHV-1) replication through a dose-dependent increase in cytopathy and increased viral titer. Furthermore, TCDD was able to trigger BHV-1-induced apoptosis by up-regulating the activation of initiator caspases 8 and 9, as well as of effector caspase 3. Since TCDD activates caspase 3 after 4 h of infection, we have hypothesized an involvement of BHV-1 infected cell protein 0 (bICP0) in this process. Such protein, the major transcriptional regulatory protein of BHV-1, has been shown to indirectly induce caspase 3 activation and apoptosis. In order to elucidate the role of bICP0 in this apoptotic pathway, here we have analyzed the effects of TCDD on bICP0 expression. Following infection of bovine cells with BHV-1, we detected apoptotic features already at 12 h after infection, only in TCDD exposed groups. Furthermore, in the presence of different doses of TCDD, we observed a time-dependent modulation and increase of bICP0 gene expression levels, as revealed by RT-PCR analysis. Western blot analysis and immunocytochemistry revealed that TCDD induced an increase of bICP0 protein levels in a dose-dependent manner, compared to unexposed groups. Moreover, Western blot analysis of nuclear and cytosolic fractions of infected cells revealed that TCDD anticipated the presence of bICP0 protein in the cytoplasm. In conclusion, both the increase of replication of BHV-1 and anticipation of BHV-1-induced apoptosis could be the result of a relationship between TCDD and bICP0.
Assuntos
Transporte Ativo do Núcleo Celular , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , Transativadores/genética , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Bovinos , Citosol , Infecções por Herpesviridae , Herpesvirus Bovino 1/química , Herpesvirus Bovino 1/fisiologia , Proteínas Imediatamente Precoces/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
In our study we found that pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs) mediated apoptosis in primary leukemia cells from 27 chronic myelogenous leukemia (CML) patients at onset through the activation of the caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP). The bax:bcl-2 ratio was increased as a consequence of down-regulation of bcl-2 and up-regulation of bax proteins in response to treatment with PBTDs. In addition, PBTDs were able to induce cell death in primary leukemia cells derived from 23 CML-chemoresistant patients. Furthermore, the effects of PBTDs on the Akt-mTOR (mammalian target of rapamycin) pathway were determined by Western blot. PBTDs possessed inhibitory activity against mTOR and also impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by rapamycin. The results presented in this study demonstrate that we have identified the PBTDs as restoring the apoptotic pathways both in primary leukemia cells derived from CML patients at onset and in primary leukemia cells derived from CML-chemoresistant patients, thus showing their ability to undergo apoptosis. These compounds constitute a promising therapeutic approach for patients with leukemia. They provide the basis for new strategies for an additional anticancer drug in leukemia therapies, especially when conventional ones fail.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adenosina Difosfato Ribose/metabolismo , Adenosina Difosfato Ribose/farmacologia , Apoptose/efeitos dos fármacos , Benzodiazepinas/farmacologia , Benzotiepinas/farmacologia , Caspase 9/metabolismo , Óxidos S-Cíclicos/farmacologia , Regulação para Baixo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/uso terapêutico , Masculino , Pessoa de Meia-Idade , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Serina-Treonina Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/farmacologiaRESUMO
BACKGROUND: A large body of evidence shows that a single bout of strenuous exercise induces oxidative stress in circulating human lymphocytes leading to lipid peroxidation, DNA damage, mitochondrial perturbations, and protein oxidation.In our research, we investigated the effect of physical load on the extent of apoptosis in primary cells derived from blood samples of sixteen healthy amateur runners after marathon (a.m.). RESULTS: Blood samples were collected from ten healthy amateur runners peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and bcl-2, bax, heat shock protein (HSP)70, Cu-Zn superoxide dismutase (SOD), Mn-SOD, inducible nitric oxide synthase (i-NOS), SIRT1, SIRT3 and SIRT4 (Sirtuins) RNA levels were determined by Northern Blot analysis. Strenuous physical load significantly increased HSP70, HSP32, Mn-SOD, Cu-Zn SOD, iNOS, GADD45, bcl-2, forkhead box O (FOXO3A) and SIRT1 expression after the marathon, while decreasing bax, SIRT3 and SIRT4 expression (P < 0.0001). CONCLUSION: These data suggest that the physiological load imposed in amateur runners during marathon attenuates the extent of apoptosis and may interfere with sirtuin expression.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Resistência Física/fisiologia , Corrida/fisiologia , Sirtuínas/genética , Proteínas de Ciclo Celular/genética , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Proteínas de Choque Térmico HSP70/genética , Heme Oxigenase-1/genética , Humanos , Peroxidação de Lipídeos/fisiologia , Linfócitos/fisiologia , Masculino , Proteínas Mitocondriais/genética , Óxido Nítrico Sintase Tipo II/genética , Proteínas Nucleares/genética , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Sirtuína 1/genética , Sirtuína 3/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Proteína X Associada a bcl-2/genéticaRESUMO
Kaempferol (3,4',5,7-tetrahydroxyflavone) is a flavonoid with anti- and pro-oxidant activity present in various natural sources. Kaempferol has been shown to posses anticancer properties through the induction of the apoptotic program. Here we report that treatment of the chronic myelogenous leukemia cell line K562 and promyelocitic human leukemia U937 with 50 microM kaempferol resulted in an increase of the antioxidant enzymes Mn and Cu/Zn superoxide dismutase (SOD). Kaempferol treatment induced apoptosis by decreasing the expression of Bcl-2 and increasing the expressions of Bax. There were also induction of mitochondrial release of cytochrome c into cytosol and significant activation of caspase-3, and -9 with PARP cleavage. Kaempferol treatment increased the expression and the mitochondria localization of the NAD-dependent deacetylase SIRT3. K562 cells stably overexpressing SIRT3 were more sensitive to kaempferol, whereas SIRT3 silencing did not increase the resistance of K562 cells to kaempferol. Inhibition of PI3K and de-phosphorylation of Akt at Ser473 and Thr308 was also observed after treating both K562 and U937 cells with kaempferol. In conclusion our study shows that the oxidative stress induced by kaempferol in K562 and U937 cell lines causes the inactivation of Akt and the activation of the mitochondrial phase of the apoptotic program with an increase of Bax and SIRT3, decrease of Bcl-2, release of cytochrome c, caspase-3 activation, and cell death.
Assuntos
Apoptose/efeitos dos fármacos , Quempferóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirtuínas/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Linhagem Celular Tumoral , Citocromos c/efeitos dos fármacos , Humanos , Células K562 , Proteínas Mitocondriais/efeitos dos fármacos , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Sirtuína 3 , Sirtuínas/efeitos dos fármacos , Proteína X Associada a bcl-2/efeitos dos fármacosRESUMO
The aim of this study was to determine whether heat-shock pretreatment exerted a protective effect against sorbitol-induced apoptotic cell death in K562, U937 and HeLa cell lines and whether such protection was associated with a decreased cytochrome c release from mithocondria and a decreased activation of caspase-9 and -3. Following heat-shock pretreatment (42 +/- 0.3 degrees C for 1 hr), these cell lines were exposed to sorbitol for 1 hr. Apoptosis was evaluated by DNA fragmentation, whereas caspase-9,-3 activation, cytochrome c release and heat-shock protein70 (HSP70) were assayed by Western Blot. Sorbitol exposure-induced apoptosis in these different cell lines with a marked activation of caspase-9 and caspase-3, whereas heat-shock pretreatment before sorbitol exposure, induced expression of HSP70 and inhibited sorbitol-mediated cytochrome c release and subsequent activation of caspase-9 and caspase-3. Similarly, overexpression of HSP70 in the three cell lines studied prevented caspase-9 cleavage and activation as well as cell death. Furthermore, we showed that the mRNA expression of iNOS decreased during both the heat-shock treatment and heat-shock pretreatment before sorbitol exposure. By contrast, the expression of Cu-Zn superoxide dismutase (SOD) and Mn-SOD proteins increased during heat-shock pretreatment before sorbitol exposure. We conclude that, heat-shock pretreatment protects different cell lines against sorbitol-induced apoptosis through a mechanism that is likely to involve SOD family members.
Assuntos
Apoptose/efeitos dos fármacos , Temperatura Alta , Sorbitol/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Citocromos c/metabolismo , Fragmentação do DNA , Proteínas de Choque Térmico HSP70/biossíntese , Células HeLa , Humanos , Células K562 , Óxido Nítrico Sintase Tipo II/genética , Proteínas Proto-Oncogênicas c-bcl-2/análise , RNA Mensageiro/análise , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Células U937 , Proteína X Associada a bcl-2/análiseRESUMO
Exposure to environmental contaminants, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to an increased susceptibility to infectious agents. Infection of bovine cells (MDBK) with Bovine Herpesvirus 1 (BHV-1) anticipates virus-induced apoptosis, suggesting an involvement of TCDD in virus infection. Herein we analyzed the effects of TCDD on apoptotic pathway in MDBK cells infected with BHV-1. After 12 h of infection, TCDD induced a significant increase in apoptotic cells. TCDD caused a dose-dependent up-regulation and anticipated activation of caspases 3, 8 and 9, with respect to unexposed groups. TCDD anticipated cleavage of PARP, compared to controls. Furthermore TCDD increased Bax and Bid levels, and decreased Bcl-2 and Bcl-XL levels. Such events took place earlier in exposed than unexposed cells. These results showed that TCDD influences BHV-1 induced apoptosis through members of Bcl-2 family and up-regulating activation of caspases.
Assuntos
Apoptose/efeitos dos fármacos , Herpesvirus Bovino 1/fisiologia , Dibenzodioxinas Policloradas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Caspases/metabolismo , Bovinos , Contagem de Células , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Herpesvirus Bovino 1/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
The induction of cell death in human erythroleukemic cells (K562) by sorbitol shows the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation and nuclear membrane breakdown. Using a cytofluorimetric approach, we found that sorbitol induced production of reactive oxygen species (ROS) followed by DNA fragmentation in leukemic cells. In this study, we investigated effect of curcumin and rosmarinic acid on cell viability in three different cell lines: erythroleukemia K562, papillary NPA, and anaplastic ARO thyroid cancers. Curcumin was able to induce apoptosis in a concentration- and time dependent manner in three cell lines, while rosmarinic acid was less effective on this process. To examine this possibility in cellular system, this study evaluated the capacities of both compounds acting as antioxidant inhibiting sorbitol-induced apoptosis. K562, NPA and ARO cells were pre-incubated with 25 microM rosmarinic acid to allow the uptake and then the cell lines were treated with 1 M sorbitol. Afterwards, the cells were subjected to agarose gel electrophoresis to assess the DNA fragmentation. In conclusion, the antioxidant activity of rosmarinic acid is able to inhibit sorbitol-induced apoptosis.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Cinamatos/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Depsídeos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Curcumina/farmacologia , Humanos , Células K562 , Pressão Osmótica , Sorbitol/antagonistas & inibidores , Sorbitol/toxicidade , Ácido RosmarínicoRESUMO
BACKGROUND: The objective of this study was to gain insight into the molecular mechanism of induced cell death (apoptosis) by PYRROLO [1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) series compounds, using human (K562) cells as a model. METHODS: We focused our attention on some members of the PBTDs family to test their potential apoptotic activity in K562 cells. Important apoptotic activity was demonstrated, as evidenced by the concentration and percentage of cell death quantified by measuring PI-uptake by flow cytometry, and DNA fragmentation analyzed by agarose gel electrophoresis, generating a characteristic ladder pattern of discontinuous DNA fragments. The expression of Bcl-2 family was tested using western blotting and transfection method. RESULTS: PBTDs-mediated suppression of K562 cell proliferation was induced by apoptosis characterized by the appearance of DNA fragmentation and was associated with the poly(ADP-ribose)polymerase (PARP) cleavage. PBTD-1 and -3 treatment resulted in caspase-3 activation through down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we used K562/vector and K562/bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with K562/vector, K562/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 10 muM PBTD-1 and -3 for 24 h produced morphological features of apoptosis and DNA fragmentation in K562/vector cells, respectively. In contrast, PBTD-1 and -3-induced caspase-3 activation and apoptosis were inhibited in K562/Bcl-2. Furthermore, Bcl-2 overexpressing cells exhibited less cytocrome c release during PBTDs-induced apoptosis. CONCLUSION: These results indicate that PBTDs effectively induce apoptosis of K562 leukemia cells through the activation of caspase cascades. In addition, these findings indicate that Bcl-2 inhibits PBTD-1 and -3 induced-apoptosis via a mechanism that interferes with cytocrome c release, and the activity of caspase-3, which is involved in the execution of apoptosis.
Assuntos
Apoptose , Benzodiazepinas/farmacologia , Ciclo Celular/efeitos dos fármacos , Óxidos S-Cíclicos/farmacologia , Leucemia/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/efeitos dos fármacos , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Regulação para Baixo , Genes bcl-2/fisiologia , Humanos , Células K562 , Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
In this study, we found that production of both reactive oxygen (ROS) and nitrogen (RNS) species is a very early event related to treatment with hyperosmotic concentration of sorbitol. The production of nitric oxide (NO) was paralleled by the increase of the mRNA and protein level of the inducible form of the nitric oxide synthase (iNOS). ROS and RNS enhancement, process concomitant to the failure of mitochondrial trans-membrane potential (DeltaPsi), was necessary for the induction of apoptosis as demonstrated by the protection against sorbitol-mediated toxicity observed after treatment with ROS scavengers or NOS inhibitors. The synergistic action of ROS and RNS was finally demonstrated by pre-treatment with rosmarinic acid that, by powerfully buffering both these species, prevents impairment of DeltaPsi and cell death. Overall results suggest that the occurrence of apoptosis upon sorbitol treatment is an event mediated by oxidative/nitrosative stress rather than a canonical hyperosmotic shock.
Assuntos
Apoptose , Leucemia/patologia , Espécies Reativas de Nitrogênio , Espécies Reativas de Oxigênio , Sorbitol/farmacologia , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Cinamatos/farmacologia , Fragmentação do DNA , Depsídeos/farmacologia , Humanos , Células K562 , Potenciais da Membrana , Óxido Nítrico Sintase Tipo II/metabolismo , Fatores de Tempo , Ácido RosmarínicoRESUMO
Pyrrolo[1,2-b][1,2,5]benzothiadiazepine 5,5-dioxides (PBTDs) induced apoptosis in human BCR-ABL-expressing leukemia cells. The apoptotic activity was also observed in primary leukemic blasts, obtained from chronic myelogenous leukemia (CML) patients at onset or from patients in blast crisis and who were imatinib-resistant. Compounds 5 and 14 induced apoptosis before BCR-ABL protein expression and tyrosin phosphorylation were affected and activated different caspases in the apoptotic pathway. PBTDs are a new class of valid candidates for the treatment of CML.
Assuntos
Antineoplásicos/síntese química , Apoptose , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Pirróis/síntese química , Tiazepinas/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzamidas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Proteínas de Fusão bcr-abl/biossíntese , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirróis/química , Pirróis/farmacologia , Tiazepinas/química , Tiazepinas/farmacologia , Células Tumorais CultivadasRESUMO
Colorectal cancer is among four most common malignancies and the second leading cause of cancer death in the western world. Epidermal Growth Factor Receptor (EGFR) and Vascular Endothelial Growth Factor (VEGF) are often overexpressed in colorectal cancer and are associated with inferior outcomes. More recently, further improvements in survival have occurred due to the use of novel targeted therapies such EGFR Tyrosine Kinase Inibitors (EGFR-TKIs), EGFR monoclonal antibodies (EGFR-mAb), and VEGF antibodies. Despite the initial clinical efficacy of these inhibitors in such cancer, resistance invariably develops, typically within 1 to 2 years. Over the past several years, multiple molecular mechanisms of resistance have been identified, and some common themes have emerged. One is the development of resistance mutations in the drug target and another it is activation of alternative signaling of key downstream pathways despite sustained inhibition of the original drug target. In this mini-review, we summarize the concepts underlying EGFR- and VEGF-mediated resistance, the specific examples known to date, and the challenges of applying this knowledge to develop improved therapeutic strategies to prevent or overcome resistance.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
The region of Campania (particularly Naples and Caserta) were subjected to extensive illegal dumping operations of toxic and radioactive wastes since the 1980s. The highly toxic wastes (HTW) dumping operations that have taken place both along the coast and the hinterland, have extremely adverse effects on health, livelihoods and the future prospect of sustainable development of the local population. The toxic wastes dumping in Campania is real and it has compromised (irreversibly) the human health, natural environment, food security and the long-term development prospects of the affected population. To reverse this tragic trend, it is necessery the identification, isolation and reclamation of the polluted sites and full assessment of the nature and the scale of the polluting chemicals and other hazardous wastes. The purpose of this review is to contribute significantly to the available evidence of the long-running toxic waste dumping in Campania and its negative impact on the health of population.
Assuntos
Resíduos Perigosos , Neoplasias/epidemiologia , Animais , Humanos , Itália/epidemiologia , Neoplasias/induzido quimicamente , Instalações de Eliminação de ResíduosRESUMO
Chronic myeloid leukemia (CML) is characterized by the accumulation of Philadelphia chromosome-positive (Ph+) myeloid cells. Ph+ cells occur via a reciprocal translocation between the long arms of chromosomes 9 and 22 resulting in constitutively active BCR-ABL fusion protein. Tyrosine kinase inhibitors (TKIs) are used against the kinase activity of BCR-ABL protein for the effective treatment of CML. However, the development of drug resistance, caused by different genetic mechanisms, is the major issue in the clinical application of TKIs. These mechanisms include changes in expression levels of microRNAs (miRNAs). miRNAs are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. In the present review, we highlight the roles of miRNAs both in the progression and chemotherapy-resistance of CML. Our understanding of these mechanisms may lead to the use of this knowledge not only in the treatment of patients with CML, but also in other type of cancers.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/genética , Regulação Neoplásica da Expressão Gênica/genética , HumanosRESUMO
Sphingosine kinases (Sphk1 and 2) regulate the prodution of sphingosine-1-phosphate (S1P), that is key molecule in cancer development. SphK1, which is commonly overexpressed in malignant tumours, significantly contributes to the pathogenesis of various types of cancer as well as to resistance to different Tyrosine Kinase inibitors (TKIs). Even, SphK2 may promote apoptosis and inhibit cell growth but its role has not yet been fully understood in pathologic conditions. Different growth factorsinduced activation of receptor tyrosine kinases (RTKs) results in production of Sphk1 which catalyzes the phosphorylation of sphingosine. Such enzyme, in turn, is involved in many cellular processes by its five receptors. These are able to transactivate RTKs through amplification of a positive-feedback signaling loop. In conclusion, development of pharmacological inhibitors of SphK1 has been limited by the lack of completely understanding of the enzymatic activation mechanisms of SphK1.