RESUMO
Ovarian cancer poses a significant threat to patients in its advanced stages, often with limited treatment options available. In such cases, palliative management becomes the primary approach to maintaining a reasonable quality of life. Therefore, the administration of any medication that can benefit patients without a curative option holds potential. Resveratrol, a natural compound known for its in vitro anticancer activities, has generated contrasting results in vivo and human studies. In this study, we aimed to assess the anticancer effects of resveratrol on ovarian cancer cells grown on the chorioallantoic membrane (CAM) of chicken embryos. Two ovarian cancer cell lines, OVCAR-8 and SKOV-3, were cultured in collagen scaffolds for four days before being implanted on the CAM of chicken embryos on day 7. Different doses of resveratrol were applied to the CAM every two days for six days. Subsequently, CAM tissues were excised, fixed, and subjected to histological analysis. Some CAM tumours were extracted to analyse proteins through Western blotting. Our findings indicate that specific doses of resveratrol significantly reduce angiogenic activities, pNF-κB levels, and SLUG protein levels by using immunohistochemistry. These results suggest that resveratrol may have the potential to impact the behaviour of ovarian cancer CAM tumours, thereby warranting further consideration as a complementary treatment option for women with incurable ovarian cancer.
Assuntos
Membrana Corioalantoide , Neoplasias Ovarianas , Resveratrol , Resveratrol/farmacologia , Membrana Corioalantoide/efeitos dos fármacos , Animais , Feminino , Embrião de Galinha , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Humanos , Linhagem Celular Tumoral , Fatores de Transcrição da Família Snail/metabolismo , Neovascularização Patológica/tratamento farmacológico , NF-kappa B/metabolismo , Antineoplásicos Fitogênicos/farmacologiaRESUMO
AIM: This review aimed to describe the potential for therapeutic targeting of the JAK/STAT signaling pathway by repurposing the clinically-approved JAK inhibitor ruxolitinib in the patients with epithelial ovarian cancer (OC) setting. METHODS: We reviewed publications that focus on the inhibition of the JAK/STAT pathway in hematological and solid malignancies including OC. RESULTS: Preclinical studies showed that ruxolitinib effectively reduces OC cell viability and metastasis and enhances the anti-tumor activity of chemotherapy drugs. There are a number of recent clinical trials exploring the role of JAK/STAT inhibition in solid cancers including OC. Early results have not adequately supported efficacy in solid tumors. However, there are preclinical data and clinical studies supporting the use of ruxolitinib in combination with both chemotherapy and other targeted drugs in OC setting. CONCLUSION: Inflammatory conditions and persistent activation of the JAK/STAT pathway are associated with tumourigenesis and chemoresistance, and therapeutic blockade of this pathway shows promising results. For women with OC, clinical investigation exploring the role of ruxolitinib in combination with chemotherapy agents or other targeted therapeutics is warranted.
Assuntos
Janus Quinases , Neoplasias Ovarianas , Humanos , Feminino , Janus Quinases/metabolismo , Janus Quinases/farmacologia , Reposicionamento de Medicamentos , Carcinoma Epitelial do Ovário/tratamento farmacológico , Fatores de Transcrição STAT/metabolismo , Fatores de Transcrição STAT/farmacologia , Transdução de Sinais , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
The roles of the extracellular biophysical environment in cancer are barely studied. This study considers the possibility that cell-like topography of a cancer cell environment may influence chemo-responses. Here, a novel bioimprinting technique was employed to produce cell-like topography on the polystyrene substrates used for cell culture. In this work, we have shown that extracellular biophysical cues generated from the topography alter the chemosensitivity of ovarian cancer cells. The three-dimensionality of the bioimprinted surface altered the cell-surface interaction, which consequently modulated intracellular signalling and chemoresponses. Sensitivity to platinum was altered more than that to paclitaxel. The effect was largely mediated through the integrin/focal adhesion system and the Rho/ROCK pathway. Moreover, the results provided evidence that biophysical cues also modulate MAPK signalling associated with chemo-resistance in ovarian cancer. Therefore, the novel findings from of this study revealed for the first time that the effects of the biophysical environment, such as substrate topography, influences ovarian cancer cell responses to clinical drugs. These observations suggest that a full clinical understanding of ovarian cancer will include biophysical aspects of tumour microenvironment.
Assuntos
Antineoplásicos/farmacologia , Carboplatina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Adesão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Estrutura Molecular , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacosRESUMO
The importance of the biophysical cellular environment in cancer development has been increasingly recognised but so far has been only superficially studied. Here we investigated the effect of cell-like substrate topography on ovarian cancer cell behaviour and potential underlying signalling pathways. We observed changes in cell morphology in response to substrate topography, which implies modification of structure-function associations. Differences in focal adhesion signalling and Rho/ROCK activity suggested their involvement in the biomechanically-driven cellular responses. Cell-like topography was also shown to modulate the MAPK pathway and hence potentially regulate cell proliferation. The selective regulation of the cells by the mechanotransduction pathways that we noted has wide ranging implications for understanding cancer development. We established that the physical architecture of cell culture substrate is sufficient to influence cancer cell behaviour, independent of genetic composition or biochemical milieu.
Assuntos
Espaço Extracelular/química , Neoplasias Ovarianas/patologia , Actinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Forma Celular , Tamanho Celular , Citoesqueleto/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina beta1/metabolismo , Mecanotransdução Celular , Neoplasias Ovarianas/enzimologia , Fosforilação , Tropomiosina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Nanomedicine has advanced the biomedical field with the availability of multifunctional nanoparticles (NPs) systems that can target a disease site enabling drug delivery and helping to monitor the disease. In this paper, we synthesised the gold nanoparticles (AuNPs) with an average size 18, 40, 60 and 80 nm, and studied the effect of nanoparticles size, concentration and incubation time on ovarian cancer cells namely, OVCAR5, OVCAR8, and SKOV3. The size measured by transmission electron microscopy images was slightly smaller than the hydrodynamic diameter; measured size by ImageJ as 14.55, 38.13, 56.88 and 78.56 nm. The cellular uptake was significantly controlled by the AuNPs size, concentration, and the cell type. The nanoparticles uptake increased with increasing concentration, and 18 and 80 nm AuNPs showed higher uptake ranging from 1.3 to 5.4 µg depending upon the concentration and cell type. The AuNPs were associated with a temporary reduction in metabolic activity, but metabolic activity remained more than 60% for all sample types; NPs significantly affected the cell proliferation activity in first 12 h. The increase in nanoparticle size and concentration induced the production of reactive oxygen species in 24 h.
Assuntos
Endocitose , Ouro/toxicidade , Nanopartículas Metálicas/toxicidade , Neoplasias Ovarianas/patologia , Tamanho da Partícula , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA de Neoplasias/metabolismo , Feminino , Humanos , Nanopartículas Metálicas/ultraestrutura , Microscopia de Fluorescência , Neoplasias Ovarianas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Three-dimensional (3D) in vitro models have an invaluable role in understanding the behaviour of tumour cells in a well defined microenvironment. This is because some aspects of tumour characteristics cannot be fully recapitulated in a cell monolayer (2D). In the present study, we compared growth patterns, expression of signalling molecules, and metabolism-associated proteins of endometrial cancer cell lines in 3D and 2D cell cultures. Cancer cells formed spherical structures in 3D reconstituted basement membrane (3D rBM), and the morphological appearance was cell line dependent. Cell differentiation was observed after 8 days in the 3D rBM. There was reduced proliferation, detected by less expression of PCNA in 3D rBM than in 2D cell monolayers. The addition of exogenous epidermal growth factor (EGF) to cancer cells induced phosphorylation of EGFR and Akt in both cell culture conditions. The uptake of glucose was selectively altered in the 3D rBM, but there was a lack of association with Glut-1 expression. The secretion of vascular endothelial growth factor (VEGF) and prostaglandin E(2) (PGE(2)) was selectively altered in 3D rBM, and it was cell line dependent. Our data demonstrated that 3D rBM as an in vitro model can influence proliferation and metabolism of endometrial cancer cell behaviour compared to 2D cell monolayer. Changes are specific to individual cell types. The use of 3D rBM is, therefore, important in the in vitro study of targeted anticancer therapies.
Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Emerging multifunctional nanoparticulate formulations take advantage of nano-meter scale size and surface chemistry to work as a therapeutic delivery agent and a diagnostic tool for non-invasive real-time monitoring using imaging technologies. Here, we evaluate the selective uptake of 18 nm and 80 nm sized gold nanoparticles (AuNPs) by SKOV3 (4 times higher) ovarian cancer (OC) cells (compared to OVCAR5) in vitro, quantified by inductively coupled plasma (ICP) and MARS spectral photon-counting CT imaging (MARS SPCCT). Based on in vitro analysis, pristine AuNPs (18 nm) and surface modified AuNPs (18 nm) were chosen as a contrast agent for MARS SPCCT. The chemical analysis by FTIR spectroscopy confirmed the luteinizing hormone-releasing hormone (LHRH) conjugation to the AuNPs surface. For the first time, LHRH conjugated AuNPs were used for in vitro and selective in vivo OC targeting. The ICP-MS analysis confirmed preferential uptake of LHRH modified AuNPs by organs residing in the abdominal cavity with OC nodules (pancreas: 0.46 ng mg-1, mesentery: 0.89 ng mg-1, ovary: 1.43 ng mg-1, and abdominal wall: 2.12 ng mg-1) whereas the MARS SPCCT analysis suggested scattered accumulation of metal around the abdominal cavity. Therefore, the study showed the exciting potential of LHRH conjugated AuNPs to target ovarian cancer and also as a potential contrast agent for novel SPCCT imaging technology.
Assuntos
Nanopartículas Metálicas , Neoplasias Ovarianas , Humanos , Feminino , Ouro/química , Meios de Contraste/química , Nanopartículas Metálicas/química , Tomografia Computadorizada por Raios X , Hormônio Liberador de GonadotropinaRESUMO
Correction for 'LHRH conjugated gold nanoparticles assisted efficient ovarian cancer targeting evaluated via spectral photon-counting CT imaging: a proof-of-concept research' by Dhiraj Kumar et al., J. Mater. Chem. B, 2023, 11, 1916-1928, https://doi.org/10.1039/D2TB02416K.
RESUMO
To streamline the drug discovery pipeline, there is a pressing need for preclinical models which replicate the complexity and scale of native tumors. While there have been advancements in the formation of microscale tumor units, these models are cell-line dependent, time-consuming and have not improved clinical trial success rates. In this study, two methods for generating 3D tumor microenvironments are compared, rapidly fabricated hydrogel microspheres and traditional cell-dense spheroids. These modules are then bioassembled into 3D printed thermoplastic scaffolds, using an automated biofabrication process, to form tumor-scale models. Modules are formed with SKOV3 and HFF cells as monocultures and cocultures, and the fabrication efficiency, cell architecture, and drug response profiles are characterized, both as single modules and as multimodular constructs. Cell-encapsulated Gel-MA microspheres are fabricated with high-reproducibility and dimensions necessary for automated tumor-scale bioassembly regardless of cell type, however, only cocultured spheroids form compact modules suitable for bioassembly. Chemosensitivity assays demonstrate the reduced potency of doxorubicin in coculture bioassembled constructs and a ≈five-fold increase in drug resistance of cocultured cells in 3D modules compared with 2D monolayers. This bioassembly system is efficient and tailorable so that a variety of relevant-sized tumor constructs could be developed to study tumorigenesis and modernize drug discovery.
Assuntos
Esferoides Celulares , Microambiente Tumoral , Avaliação Pré-Clínica de Medicamentos , Reprodutibilidade dos Testes , Técnicas de CoculturaRESUMO
BACKGROUND: Advanced endometrial cancer often shows resistance to clinical chemotherapy although potencies of anticancer drugs in vitro are promising. The disparity suggests that in vivo microenvironments are not recapitulated by in vitro models used for preclinical testing. However, spheroids replicate some important properties of tumours in vivo. Therefore, for the first time, we compared effects of doxorubicin and cisplatin on 3D multicellular structures and 2D cell monolayers of endometrial cancer cells. METHODS: 3D multicellular structures were generated by culturing cancer cells on non-adherent surfaces; and for comparison cell monolayers were cultured on adherent culture plates. Ishikawa, RL95-2, and KLE cell lines were studied. Morphologies of 3D multicellular structures were examined. After 48 hours treatment with anticancer drugs, apoptosis, proliferation, glucose metabolism and vascular endothelial growth factor (VEGF) were analysed. Immunostaining of PCNA, Glut-1, p-Erk1/2, SOD-1 and p-Akt1/2/3 was also performed. RESULTS: Distinct 3D multicellular morphologies were formed by three different endometrial cancer cell lines. Doxorubicin induced less apoptosis in 3D multicellular structures of high grade cancer cells (RL95-2 and KLE cell lines) than in cell monolayers. Parallel alterations in Erk1/2 phosphorylation and cell proliferation might suggest they were linked and again doxorubicin had less effect on 3D multicellular structures than cell monolayers. On the other hand, there was no correlation between altered glucose metabolism and proliferation. The responses depended on cancer cell lines and were apparently not mediated by altered Glut-1 levels. The level of SOD-1 was high in 3D cell cultures. The effects on VEGF secretion were various and cancer cell line dependent. Importantly, both doxorubicin and cisplatin had selective paradoxical stimulatory effects on VEGF secretion. The microenvironment within 3D multicellular structures sustained Akt phosphorylation, consistent with it having a role in anchorage-independent pathways. CONCLUSIONS: The cancer cells responded to microenvironments in a distinctive manner. 3D multicellular structures exhibited greater resistance to the agents than 2D monolayers, and the differences between the culture formats were dependent on cancer cell lines. The effects of anticancer drugs on the intracellular mediators were not similar in 3D and 2D cultures. Therefore, using 3D cell models may have a significant impact on conclusions derived from screening drugs for endometrial carcinomas.
Assuntos
Cisplatino/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias do Endométrio/patologia , Espaço Intracelular/metabolismo , Técnicas de Cultura de Tecidos/métodos , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Densitometria , Desoxiglucose/análogos & derivados , Desoxiglucose/metabolismo , Neoplasias do Endométrio/classificação , Neoplasias do Endométrio/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Humanos , Integrina beta1/metabolismo , Espaço Intracelular/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Superóxido Dismutase/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The structure and function of membrane-wall attachment sites in walled cells, and how these relate to animal focal adhesions, is an area that is poorly understood. In view of this, we investigated how membrane-wall attachments that form upon plasmolysis, respond to peptides that disrupt animal focal adhesions. The degree of cytoplasmic disruption during plasmolysis was also investigated. Upon hyperosmotic challenge, the protoplast in hyphae of the oomycete Achlya bisexualis typically retracted incompletely due to membrane-wall attachments. The inclusion, in the plasmolysing solution, of peptides containing the sequence RGD disrupted these attachments in a dose-dependent manner. In some hyphae, protoplast retraction stopped temporarily at attachment points - upon resumption of retraction, material was left that traced the outline of the static protoplast. Staining of this material with fluorescence brightener indicated the presence of cellulose, which suggests that wall deposition was able to occur despite plasmolysis. The F-actin cytoskeleton was disrupted during plasmolysis; peripheral F-actin staining was observed, but there was no distinct F-actin cap; staining was more diffuse; and there were fewer plaques compared with nonplasmolysed hyphae. Our data indicate that membrane-wall attachment points are sensitive to RGD-containing peptides and that wall deposition continues despite protoplast retraction and F-actin disruption.
Assuntos
Membrana Celular/metabolismo , Parede Celular/metabolismo , Oligopeptídeos/metabolismo , Protoplastos/metabolismo , Achlya/química , Achlya/metabolismo , Actinas/metabolismo , Parede Celular/fisiologia , Citoplasma/metabolismo , Hifas/citologia , Hifas/metabolismoRESUMO
Most ovarian cancer patients are diagnosed with advanced stage disease, which becomes unresponsive to chemotherapeutic treatments. The PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK kinase signaling pathways are attractive targets for potential therapeutic inhibitors, due to the high frequency of mutations to PTEN, PIK3CA, KRAS and BRAF in several ovarian cancer subtypes. However, monotherapies targeting one of these pathways have shown modest effects in clinical trials. This limited efficacy of the agents could be due to upregulation and increased signaling via the adjacent alternative pathway. In this study, the efficacy of combined PI3K/mTOR (BEZ235) and ERK inhibition (SCH772984) was investigated in four human ovarian cancer cell lines, grown as monolayer and three-dimensional cell aggregates. The inhibitor combination reduced cellular proliferation in a synergistic manner in OV-90 and OVCAR8 monolayers and in OV-90, OVCAR5 and SKOV3 aggregates. Sensitivity to the inhibitors was reduced in three-dimensional cell aggregates in comparison to monolayers. OV-90 cells cultured in large spheroids were sensitive to the inhibitors and displayed a robust synergistic antiproliferative response to the inhibitor combination. In contrast, OVCAR8 spheroids were resistant to the inhibitors. These findings suggest that combined PI3K/mTOR and ERK inhibition could be a useful strategy for overcoming treatment resistance in ovarian cancer and warrants further preclinical investigation. Additionally, in some cell lines the use of different three-dimensional models can influence cell line sensitivity to PI3K/mTOR and RAS/RAF/MEK/ERK pathway inhibitors.
RESUMO
As a principal matrisomal protein, collagen is involved in the regulation of the structural framework of extracellular matrix (ECM) and therefore is potentially crucial in determining the biophysical character of the ECM. It has been suggested that collagen architecture plays a role in ovarian cancer development, progression and therapeutic responses which led us to examine the collagen morphology in normal and cancerous ovarian tissue. Also, the behaviour of ovarian cancer cells cultured in four qualitatively different collagen gels was investigated. The results here provide evidence that collagen I morphology in the cancerous ovary is distinct from that in the normal ovary. Tumour-associated collagen I showed streams or channels of thick elongated collagen I fibrils. Moreover, fibril alignment was significantly more prevalent in endometrioid and clear cell cancers than other ovarian cancer subtypes. In this work, for the first-time collagen I architecture profiling (CAP) was introduced using histochemical staining, which distinguished between the collagen I morphologies of ovarian cancer subtypes. Immunohistochemical examination of ovarian normal and cancerous tissues also supported the notion that focal adhesion and Rho signalling are upregulated in ovarian cancers, especially in the high-grade serous tumours, as indicated by higher expression of p-FAK and p190RhoGEF. The results also support the concept that collagen I architecture, which might be collagen I concentration-dependent, influences proliferation in ovarian cancer cells. The study provides evidence that modification of collagen I architecture integrity is associated with ovarian cancer development and therapeutic responses.
Assuntos
Colágeno Tipo I/biossíntese , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/sangue , Neoplasias Ovarianas/metabolismo , Linhagem Celular Tumoral , Colágeno Tipo I/genética , Feminino , Humanos , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologiaRESUMO
It has been long known that the oncogenic extracellular environment plays an indispensable role in developing and nurturing cancer cell progression and in resistance to standard treatments. However, by how much the biophysical components of tumour extracellular environment contribute to these processes is uncertain. In particular, the topographic environment is scarcely explored. The biophysical modulation of cell behaviour is primarily facilitated through mechanotransduction-associated mechanisms, including focal adhesion and Rho/ROCK signalling. Dysregulation of these pathways is commonly observed in ovarian cancer and, therefore, biophysical modulation of these mechanisms may be of great importance to ovarian cancer development and progression. In this work, aspects of the biophysical environment were explored using a bioimprinting technique. The study showed that topography-mediated substrate sensing delayed cell attachment, however, cell-cell interactions overrode the effect of topography in some cell lines, such as OVCAR-5. Also, 3D topographical cues were shown to modulate the inhibition of focal adhesion and Rho signalling, which resulted in higher MAPK activity in cells on the bioimprints. It was revealed that c-Src is vital to the biophysical modulation of cell proliferation and inhibition of c-Src could downregulate biophysically modulated MAPK activity. This study provides evidence that the biophysical extracellular environment affects key intracellular mechanisms associated with tumourigenicity in ovarian cancer cells.
Assuntos
Adesão Celular/fisiologia , Transdução de Sinais/fisiologia , Microambiente Tumoral , Proteína Tirosina Quinase CSK/antagonistas & inibidores , Proteína Tirosina Quinase CSK/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Humanos , MAP Quinase Quinase Quinases/metabolismo , Mecanotransdução Celular , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Helicobacter pylori bacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pylori is considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA(+) strain and OMV from a VacA(-) isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA(+) strain was inhibited by H. pylori LPS (58% inhibition with 50 µg/ml LPS), while uptake of OMV from the VacA(-) mutant strain was less affected (25% inhibition with 50 µg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA(-) mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 µg/ml chlorpromazine), while uptake of OMV from the VacA(+) strain was less affected (25% inhibition with 15 µg/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Linhagem Celular , Colesterol/metabolismo , Colesterol/fisiologia , Endocitose/fisiologia , Células Epiteliais/metabolismo , Citometria de Fluxo , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Microscopia de FluorescênciaRESUMO
Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.
Assuntos
Transformação Celular Neoplásica , Helicobacter pylori/patogenicidade , Neoplasias Gástricas/virologia , Vesículas Transportadoras/virologia , Proteínas de Bactérias , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/virologia , Imunofluorescência , Glutationa/metabolismo , Humanos , Ferro/metabolismo , Micronúcleos com Defeito Cromossômico , Estresse Oxidativo/fisiologia , Carbonilação Proteica/fisiologia , Neoplasias Gástricas/patologia , Vesículas Transportadoras/metabolismoRESUMO
Ovarian cancer patients in the advanced stages of the disease show clinical ascites, which is associated with a poor prognosis. There is limited understanding of the effect of ascitic fluid on ovarian cancer cells and their response to anticancer drugs. We investigated the antitumour effects of EGFR/Her-2 (canertinib) and c-Met (PHA665752) inhibitors in a 3D cell model of three ovarian cancer lines. Single and combined inhibitor treatments affected cell growth of OVCAR-5 and SKOV-3â¯cell lines but not OV-90â¯cell line. Growth reduction was correlated with the down expression of PCNA, EGFR, HER-2, c-MET, ERK and AKT and their phosphorylation status in cells in growth factor supplemented media. However, these effects were not re-producible in OVCAR-5 and SKOV-3â¯cell lines when they were exposed to ascitic fluid obtained from three ovarian cancer patients. Serum albumin and protein components in the ascitic fluids may reduce the cellular uptake of the inhibitors.
Assuntos
Líquido Ascítico/metabolismo , Resistencia a Medicamentos Antineoplásicos , Indóis/farmacologia , Morfolinas/farmacologia , Neoplasias Ovarianas/complicações , Inibidores de Proteínas Quinases/farmacologia , Sulfonas/farmacologia , Técnicas de Cultura de Células , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Modelos Biológicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor ErbB-2/metabolismo , Células Tumorais CultivadasRESUMO
INTRODUCTION: Advanced ovarian cancer is often a fatal disease as chemotherapeutic drugs have limited effectiveness. Better targeted therapy is needed to improve the survival and quality of life for these women. Receptor tyrosine kinases including EGFR, Her-2 and c-Met are associated with a poor prognosis in ovarian cancer. Therefore, the co-activation of these receptors may be crucial for growth promoting activity. In this study, we explored the effect of combining two small molecule inhibitors that target the EGFR/Her-2 and c-Met receptor tyrosine kinases in two ovarian cancer cell lines. The aim of this study was to investigate the combined inhibition activity of a dual EGFR/Her-2 inhibitor (canertinib) and a c-Met inhibitor (PHA665752) in ovarian cancer cell lines in 3D cell aggregates. METHODS: OVCAR-5 and SKOV-3 ovarian cancer cell lines were cultured on a non-adherent surface to produce 3D cell clusters and aggregates. Cells were exposed to canertinib and PHA665752, both individually and in combination, for 48 h. The effect on growth, metabolism and the expression/phosphorylation of selective signaling proteins associated with EGFR, Her-2 and c-Met were investigated. RESULTS: The single drug treatments significantly decreased cell growth and altered the expression of signaling proteins in OVCAR-5 and SKOV-3 cell lines. The combination treatment showed greater reduction of cell numbers for both cell lines. Total expression and phosphorylation of signaling proteins were further reduced in the combination drug treatments, compared to the single inhibitor treatments. CONCLUSION: Our findings suggest that the concurrent targeting of more than one receptor tyrosine kinase may be useful in developing more effective targeted drug regimens for patients, who have EGFR, Her-2 and c-Met positive ovarian cancer cells.
RESUMO
BACKGROUND: Key features of advanced ovarian cancer include metastasis via cell clusters in the abdominal cavity and increased chemoresistance. Resveratrol and derivatives of resveratrol have been shown to have antitumour properties. The purpose of this study was to investigate the effect of resveratrol and acetyl-resveratrol on 3D cell aggregates of ovarian cancer, and establish if NF-κB signalling may be a potential target. METHODS: Poly-HEMA coated wells were used to produce 3D aggregates of two ovarian cancer cell lines, SKOV-3 and OVCAR-5. The aggregates were exposed to 10, 20 or 30 µM resveratrol or acetyl-resveratrol for 2, 4 or 6 days. Cell growth and metabolism were measured then ELISA, western blot and immunofluorescence were utilised to evaluate VEGF, IL-8 and NF-κB levels. RESULTS: Resveratrol and acetyl-resveratrol reduced cell growth and metabolism of SKOV-3 aggregates in a dose- and time-dependent manner. After 6 days all three doses of both compounds inhibited cell growth. This growth inhibition correlated with the attenuated secretion of VEGF and a decrease of NF-κB protein levels. Conversely, the secretion of IL-8 increased with treatment. The effects of the compounds were limited in OVCAR-5 cell clusters. CONCLUSIONS: The results suggest that resveratrol and its derivative acetyl-resveratrol may inhibit in vitro 3D cell growth of certain subtypes of ovarian cancer, and growth restriction may be associated with the secretion of VEGF under the control of the NF-κB protein.
Assuntos
Agregação Celular/efeitos dos fármacos , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Neoplasias Ovarianas/metabolismo , Estilbenos/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Células Tumorais CultivadasRESUMO
The structural properties of amyloid fibrils combined with their highly functional surface chemistry make them an attractive new food ingredient, for example as highly effective gelling agents. However, the toxic role of amyloid fibrils in disease may cause some concern about their food safety because it has not been established unequivocally if consumption of food fibrils poses a health risk to consumers. Here we present a study of amyloid-like fibrils from whey, kidney bean, soy bean, and egg white to partially address this concern. Fibrils showed varied resistance to proteolytic digestion in vitro by either Proteinase K, pepsin or pancreatin. The toxicity of mature fibrils was measured in vitro and compared to native protein, early-stage-fibrillar protein, and sonicated fibrils in two immortalised human cancer cell lines, Caco-2 and Hec-1a. There was no reduction in the viability of either Caco-2 or Hec-1a cells after treatment with a fibril concentration of up to 0.25 mg/mL.