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A relevant challenge for the treatment of patients with neoplasia is the development of resistance to chemo-, immune-, and radiotherapies. Although the causes of therapy resistance are poorly understood, evidence suggests it relies on compensatory mechanisms that cells develop to replace specific intracellular signaling that should be inactive after pharmacological inhibition. One such mechanism involves integrins, membrane receptors that connect cells to the extracellular matrix and have a crucial role in cell migration. The blockage of one specific type of integrin is frequently compensated by the overexpression of another integrin dimer, generally supporting cell adhesion and migration. In particular, integrin αvß3 is a key receptor involved in tumor resistance to treatments with tyrosine kinase inhibitors, immune checkpoint inhibitors, and radiotherapy; however, the specific inhibition of the αvß3 integrin is not enough to avoid tumor relapse. Here, we review the role of integrin αvß3 in tumor resistance to therapy and the mechanisms that have been proposed thus far. Despite our focus on the αvß3 integrin, it is important to note that other integrins have also been implicated in drug resistance and that the collaborative action between these receptors should not be neglected.
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The fabrication of customized implants by additive manufacturing has allowed continued development of the personalized medicine field. Herein, a 3D-printed bioabsorbable poly (lactic acid) (PLA)- ß-tricalcium phosphate (TCP) (10 wt %) composite has been modified with CeO2 nanoparticles (CeNPs) (1, 5 and 10 wt %) for bone repair. The filaments were prepared by melt extrusion and used to print porous scaffolds. The nanocomposite scaffolds possessed precise structure with fine print resolution, a homogenous distribution of TCP and CeNP components, and mechanical properties appropriate for bone tissue engineering applications. Cell proliferation assays using osteoblast cultures confirmed the cytocompatibility of the composites. In addition, the presence of CeNPs enhanced the proliferation and differentiation of mesenchymal stem cells; thereby, increasing alkaline phosphatase (ALP) activity, calcium deposition and bone-related gene expression. Results from this study have shown that the 3D printed PLA-TCP-10%CeO2 composite scaffold could be used as an alternative polymeric implant for bone tissue engineering applications: avoiding additional/revision surgeries and accelerating the regenerative process.
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BACKGROUND: Poly (lactic acid) (PLA) is a biodegradable polyester that has been exploited for a variety of biomedical applications, including tissue engineering. The incorporation of ß-tricalcium phosphate (TCP) into PLA has imparted bioactivity to the polymeric matrix. METHODS: We have modified a 90%PLA-10%TCP composite with SiO2 and MgO (1, 5 and 10 wt%), separately, to further enhance the material bioactivity. Filaments were prepared by extrusion, and scaffolds were fabricated using 3D printing technology associated with fused filament fabrication. RESULTS: The PLA-TCP-SiO2 composites presented similar structural, thermal, and rheological properties to control PLA and PLA-TCP. In contrast, the PLA-TCP-MgO composites displayed absence of crystallinity, lower polymeric molecular weight, accelerated degradation ratio, and decreased viscosity within the 3D printing shear rate range. SiO2 and MgO particles were homogeneously dispersed within the PLA and their incorporation increased the roughness and protein adsorption of the scaffold, compared to a PLA-TCP scaffold. This favorable surface modification promoted cell proliferation, suggesting that SiO2 and MgO may have potential for enhancing the bio-integration of scaffolds in tissue engineering applications. However, high loads of MgO accelerated the polymeric degradation, leading to an acid environment that imparted the composite biocompatibility. The presence of SiO2 stimulated mesenchymal stem cells differentiation towards osteoblast; enhancing extracellular matrix mineralization, alkaline phosphatase (ALP) activity, and bone-related genes expression. CONCLUSION: The PLA-10%TCP-10%SiO2 composite presented the most promising results, especially for bone tissue regeneration, due to its intense osteogenic behavior. PLA-10%TCP-10%SiO2 could be used as an alternative implant for bone tissue engineering application.
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Fosfatos de Cálcio , Óxido de Magnésio , Alicerces Teciduais , Óxido de Magnésio/farmacologia , Óxido de Magnésio/química , Alicerces Teciduais/química , Dióxido de Silício , Teste de Materiais , Poliésteres , Polímeros/química , Ácido Láctico/química , Impressão TridimensionalRESUMO
Human Hsp70-escort protein 1 (hHep1) is a cochaperone that assists in the function and stability of mitochondrial HSPA9. Similar to HSPA9, hHep1 is located outside the mitochondria and can interact with liposomes. In this study, we further investigated the structural and thermodynamic behavior of interactions between hHep1 and negatively charged liposomes, as well as interactions with cellular membranes. Our results showed that hHep1 interacts peripherally with liposomes formed by phosphatidylserine and cardiolipin and remains partially structured, exhibiting similar affinities for both. In addition, after being added to the cell membrane, recombinant hHep1 was incorporated by cells in a dose-dependent manner. Interestingly, the association of HSPA9 with hHep1 improved the incorporation of these proteins into the lipid bilayer. These results demonstrated that hHep1 can interact with lipids also present in the plasma membrane, indicating roles for this cochaperone outside of mitochondria.
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Bicamadas Lipídicas , Lipossomos , Humanos , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismoRESUMO
Several techniques, such as additive manufacturing, have been used for the manufacture of polymer-ceramic composite scaffolds for bone tissue engineering. A new extruder head recently developed for improving the manufacturing process is an experimental 3D printer Fab@CTI that enables the use of ceramic powders in the processing of composite materials or polymer blends. Still, the manufacturing process needs improvement to promote the dispersion of ceramic particles in the polymer matrix. This article addresses the manufacture of scaffolds by 3D printing from mixtures of poly(ϵ-caprolactone) (PCL) and a glass powder of same composition of 45S5Bioglass®, labeled as synthesized bioglass (SBG), according to two different methods that investigated the efficiency of the new extruder head. The first one is a single extrusion process in a Fab@CTI 3D printer, and the other consists in the pre-processing of the PCL-SBG mixture in a mono-screw extruder with a Maddock® element, followed by direct extrusion in the experimental Fab@CTI 3D printer. The morphological characterization of the extruded samples by scanning electron microscope showed an architecture of 0°/90° interconnected struts and suitable porosity for bone tissue engineering applications. Scaffolds fabricated by two methods shows compressive modulus ranging from 54.4 ± 14.2 to 155.9 ± 20.4 MPa, results that are compatible to use in bone tissue engineering. Cytotoxicity assays showed non-toxic effects and viability forin vitroMG-63 cell proliferation. Alizarin Red staining test showed calcium deposition in all scaffolds, which suggests PCL/SBG composites promising candidates for use in bone tissue engineering. Results of cell morphology suggest more cell growth and adhesion for scaffolds fabricated using the pre-processing in a mono-screw extruder.
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Engenharia Tecidual , Alicerces Teciduais , Cerâmica , Poliésteres , Polímeros , Porosidade , Impressão Tridimensional , Engenharia Tecidual/métodosRESUMO
Breast cancer is characterized by a hypoxic microenvironment inside the tumor mass, contributing to cell metastatic behavior. Hypoxia induces the expression of hypoxia-inducible factor (HIF-1α), a transcription factor for genes involved in angiogenesis and metastatic behavior, including the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and integrins. Integrin receptors play a key role in cell adhesion and migration, being considered targets for metastasis prevention. We investigated the migratory behavior of hypoxia-cultured triple-negative breast cancer cells (TNBC) and endothelial cells (HUVEC) upon αvß3 integrin blocking with DisBa-01, an RGD disintegrin with high affinity to this integrin. Boyden chamber, HUVEC transmigration, and wound healing assays in the presence of DisBa-01 were performed in hypoxic conditions. DisBa-01 produced similar effects in the two oxygen conditions in the Boyden chamber and transmigration assays. In the wound healing assay, hypoxia abolished DisBa-01's inhibitory effect on cell motility and decreased the MMP-9 activity of conditioned media. These results indicate that αvß3 integrin function in cell motility depends on the assay and oxygen levels, and higher inhibitor concentrations may be necessary to achieve the same inhibitory effect as in normoxia. These versatile responses add more complexity to the role of the αvß3 integrin during tumor progression.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células Endoteliais/metabolismo , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Hipóxia Tumoral , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Meios de Cultivo Condicionados/farmacologia , Desintegrinas/farmacologia , Células Endoteliais/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Oxigênio , Subunidades Proteicas/metabolismo , Hipóxia Tumoral/efeitos dos fármacosRESUMO
Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2ß1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2ß1 in the context of bone metastasis. In this study, we aimed to understand the role of α2ß1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2ß1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2ß1 expression and bone-metastatic potential. Inhibiting α2ß1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.
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Neoplasias Ósseas/secundário , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Expressão Gênica , Integrina alfa2beta1/genética , Animais , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Invasividade Neoplásica , Osteólise/genética , Osteólise/metabolismo , FenótipoRESUMO
BACKGROUND: Triple-negative BC is the most aggressive type of breast cancer and its lack of responsiveness to conventional therapies requires screening of new chemical entities. Anti-migratory compounds are promising to treat metastatic cancer since they inhibit one of the main steps of the metastatic cascade. Spirocyclic compounds are non-conventional structures used as building blocks for the synthesis of biologically active molecules and considered interesting structures in the search for new targets in cancer research. OBJECTIVE: Here, we evaluated the potential of eight synthetic spirocyclohexadienones as cell migration inhibitors. METHODS: The anti-migratory ability of compounds was tested by wound healing and Boyden chamber approaches. Experiments in tubulin were performed by fluorescence and tubulin polymerization techniques. Finally, compounds were submitted to cell proliferation inhibition and flow cytometry assays to explore the mechanism by which they inhibit cell migration. RESULTS: Four compounds inhibited cell migration significantly. Analogs containing the 3,4,5-trimethoxyphenil ring at R1 position were the most potent and, thus, selected for additional experiments. Tubulin polymerization and fluorescence assays highlighted a possible binding of spirocyclohexadienones in the colchicine binding site; however, these compounds did not affect the cell cycle to the same extent as colchicine. Cell proliferation was affected and, notably, the most potent analogs induced apoptosis of tumor cells, suggesting a different mechanism by which they inhibit cell migration. CONCLUSION: We presented, for the first time, a series of eight synthetic spirocyclohexadienones with the ability to inhibit TNBC cell migration. These compounds represent a new category to be explored as anticancer agents.
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Antineoplásicos/farmacologia , Cicloexenos/farmacologia , Compostos de Espiro/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cicloexenos/síntese química , Cicloexenos/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Polimerização/efeitos dos fármacos , Compostos de Espiro/síntese química , Compostos de Espiro/química , Neoplasias de Mama Triplo Negativas/patologia , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/químicaRESUMO
BACKGROUND: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. METHODS: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvß3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. RESULTS: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvß3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvß3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. CONCLUSION: In summary, our findings demonstrate for the first time a key role of αvß3 integrin in cell-cell communication through SEVs. Video Abstract.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Vesículas Extracelulares/metabolismo , Integrina alfaVbeta3/metabolismo , Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Vesículas Extracelulares/ultraestrutura , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Biológicos , Ligação ProteicaRESUMO
BACKGROUND: Integrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between αvß3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. DisBa-01, a recombinant His-tag fusion, RGD-disintegrin from Bothrops alternatus snake venom, binds to αvß3 integrin with nanomolar affinity blocking cell adhesion to the extracellular matrix. Here we present in vitro evidence of a direct interference of DisBa-01 with αvß3/VEGFR2 cross-talk and its downstream pathways. METHODS: Human umbilical vein (HUVECs) were cultured in plates coated with fibronectin (FN) or vitronectin (VN) and tested for migration, invasion and proliferation assays in the presence of VEGF, DisBa-01 (1000 nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of αvß3/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy. RESULTS: DisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of αvß3/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and ß3 integrin subunit, regulates FAK/SrC/Paxillin downstream signals, and inhibits ERK1/2 and PI3K pathways. These events result in actin re-organization and inhibition of HUVEC migration and adhesion. Labelled-DisBa-01 colocalizes with αvß3 integrin and VEGFR2 in treated cells. CONCLUSIONS: Disintegrin inhibition of αvß3 integrin blocks VEGFR2 signalling, even in the presence of VEGF, which impairs the angiogenic mechanism. These results improve our understanding concerning the mechanisms of pharmacological inhibition of angiogenesis.
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Movimento Celular/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Desintegrinas/farmacologia , Células Endoteliais da Veia Umbilical Humana , Integrina alfaVbeta3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adesão Celular , Células Cultivadas , Quinase 1 de Adesão Focal/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Paxilina/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Quinases da Família src/metabolismoRESUMO
Accumulation of connective tissue, particularly extracellular matrix (ECM) proteins, has been observed in skeletal muscles with advancing age. Resistance training (RT) has been widely recommended to attenuate age-induced sarcopenia, even though its effects on the components that control ECM turnover in skeletal muscles remain to be elucidated. Thus, the aim of this study was to determine the effects of RT on connective tissue content and gene expression of key components of ECM in the skeletal muscles of aged rats. Young (3 mo.) and older (21 mo.) adult male Wistar rats were submitted to a RT protocol (ladder climbing with 65, 85, 95, and 100% load), 3 times a week for 12 weeks. Forty-eight hours post-training, the soleus (SOL) and gastrocnemius (GAS) muscles were dissected for histological and mRNA analysis. RT mitigated the age-associated increase of connective tissue content in both muscles, even though mRNA levels of COL-1 and-3 were elevated in older trained rats. Overall, RT significantly elevated the gene expression of key components of connective tissue deposition (TGFß and CTGF; MMP-2 and-9; TIMP-1 and-2) in the GAS and SOL muscles of older rats. In conclusion, RT blunted the age-induced accumulation of connective tissue concomitant to the upregulation of genes related to synthesis and degradation of the ECM network in the SOL and GAS muscles of older rats. Although our findings indicate that RT plays a crucial role reducing connective tissue accumulation in aged hindlimb muscles, key components of ECM turnover were paradoxically elevated. The phenotypic responses induced by RT were not accompanied by the gene expression of those components related to ECM turnover.
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There is increasing interest in the use of non-toxic natural products for the treatment of various pathologies, including cancer. In particular, biologically active constituents of the ginger oleoresin (Zingiber officinale Roscoe) have been shown to mediate anti-tumour activity and to contribute to the anti-inflammatory, antioxidant, antimicrobial, and antiemetic properties of ginger. Here we report on the inhibitory properties of [10]-gingerol against metastatic triple negative breast cancer (TNBC) in vitro and in vivo. We show that [10]-gingerol concentration-dependently induces apoptotic death in mouse and human TNBC cell lines in vitro. In addition, [10]-gingerol is well tolerated in vivo, induces a marked increase in caspase-3 activation and inhibits orthotopic tumour growth in a syngeneic mouse model of spontaneous breast cancer metastasis. Importantly, using both spontaneous and experimental metastasis assays, we show for the first time that [10]-gingerol significantly inhibits metastasis to multiple organs including lung, bone and brain. Remarkably, inhibition of brain metastasis was observed even when treatment was initiated after surgical removal of the primary tumour. Taken together, these results indicate that [10]-gingerol may be a safe and useful complementary therapy for the treatment of metastatic breast cancer and warrant further investigation of its efficacy, either alone or in combination with standard systemic therapies, in pre-clinical models of metastatic breast cancer and in patients.
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Adipose tissue development is associated with modifications involving extracellular matrix remodelling, and metalloproteinases play a significant role in this process. Reduced circulating sexual hormones cause impacts on the size, morphology and functions of the adipose tissue, increasing susceptibility to diseases. This study investigated whether exercise training may be an alternative strategy to combat the effects promoted by estrogen decay through modulation in gene expression patterns in the extracellular matrix (ECM) of visceral adipose tissue of ovariectomized rats. Nulliparous rats (n = 40) were randomly distributed into four groups (n = 10/group): sham sedentary (Sh-S), sham resistance training (Sh-Rt), ovariectomized sedentary (Ovx-S) and ovariectomized resistance training (Ovx-Rt). The Sh-S animals did not have any type of training. The body mass and food intake, ECM gene expression, gelatinase MMP-2 activity and adipocyte area were measured. A lack of estrogen promoted an increase in body mass, food intake and the visceral, parametrial and subcutaneous adipocyte areas. The ovariectomy upregulated the expression of MMP-2, MMP-9, TGF-ß, CTGF, VEGF-A and MMP-2 activity. On the other hand, resistance training decreased the body mass, food intake and the adipocyte area of the three fat depots analysed; upregulated TIMP-1, VEGF-A and MMP-2 gene expression; downregulated MMP-9, TGF-ß and CTGF gene expression; and decreased the MMP-2 activity. We speculate that resistance training on a vertical ladder could play an important role in maintaining and remodelling ECM by modulation in the ECM gene expression and MMP-2 activity, avoiding its destabilization which is impaired by the lack of estrogen.
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Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Estrogênios/metabolismo , Matriz Extracelular/metabolismo , Animais , Matriz Extracelular/genética , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Ovariectomia/métodos , Condicionamento Físico Animal/métodos , Ratos Sprague-Dawley , Treinamento Resistido/métodos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Doxorubicin, cisplatin, and tamoxifen are part of many chemotherapeutic regimens. However, studies investigating the effect of chemotherapy on the metabolism of breast cancer cells are still limited. We used 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to study the metabolic profile of human breast cancer MDA-MB-231 cells either untreated (control) or treated with tamoxifen, cisplatin, and doxorubicin. 1H HR-MAS NMR single pulse spectra evidenced signals from all mobile cell compounds, including fatty acids (membranes), water-soluble proteins, and metabolites. NMR spectra showed that phosphocholine (i.e., a biomarker of breast cancer malignant transformation) signals were stronger in control than in treated cells, but significantly decreased upon treatment with tamoxifen/cisplatin. NMR spectra acquired with Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence were interpreted only qualitatively because signal areas were attenuated according to their transverse relaxation times (T2). The CPMG method was used to identify soluble metabolites such as organic acids, amino acids, choline and derivatives, taurine, guanidine acetate, tyrosine, and phenylalanine. The fatty acid variations observed by single pulse as well as the lactate, acetate, glycine, and phosphocholine variations observed through CPMG 1H HR-MAS NMR have potential to characterize both responder and non-responder tumors in a molecular level. Additionally, we emphasized that comparable tumors (i.e., with the same origin, in this case breast cancer) may respond totally differently to chemotherapy. Our observations reinforce the theory that alterations in cellular metabolism may contribute to the development of a malignant phenotype and cell resistance.
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Neoplasias da Mama/metabolismo , Aminoácidos/metabolismo , Biomarcadores/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Colina/metabolismo , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Feminino , Humanos , Ácido Láctico/metabolismo , Metaboloma , Fosforilcolina/metabolismo , Espectroscopia de Prótons por Ressonância Magnética/métodos , Tamoxifeno/farmacologiaRESUMO
Doxorubicin (Doxo), cisplatin (Cis), and tamoxifen (Tamo) are part of many chemotherapeutic regimens. However, there have been limited studies of the way metabolism in breast cancer is affected by chemotherapy. We studied, through 1H high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy, the metabolic profile of human breast cancer MCF-7 control (Con) cells as well as MCF-7 cells treated with Tamo, Cis, and Doxo. 1H HR-MAS NMR single-pulse spectra evidenced signals from the cell compounds, including fatty acids (membranes), water-soluble proteins, and metabolites. The spectra showed that phosphocholine (i.e., biomarker of breast cancer malignant transformation) signals were stronger in Con than in treated cells. Betaine (i.e., the major osmolyte in cells) was observed at similar concentrations in MCF-7 control and treated cells but was absent in nontumor MCF-10A cells. The NMR spectra acquired with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence were used only in qualitative analyses because the signal areas were attenuated according to their transverse relaxation time (T2). The CPMG method was used to identify soluble metabolites such as organic acids, amino acids, choline and its derivatives, taurine, and guanidino acetate. 1H HR-MAS NMR spectroscopy efficiently demonstrated the effects of Tamo, Cis, and Doxo on the metabolic profile of MCF-7 cells. The fatty acid, phosphocholine, and choline variations observed by single-pulse HR-MAS NMR have the potential to characterize both responder and nonresponder tumors at a molecular level.
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Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Espectroscopia de Prótons por Ressonância Magnética/métodos , Tamoxifeno/farmacologia , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7RESUMO
Integrin αvß3 is most likely the foremost modulator of angiogenesis among all known integrins. Recombinant disintegrin DisBa-01, originally obtained from snake venom glands, binds to αvß3, thereby significantly inhibiting adhesion and generating in vivo anti-metastatic ability. However, its function in mediator production is not clear. Here, we observed that the mediators VEGF-A, IL-8, and TGF-ß are not produced by human umbilical vein endothelial cells (HUVEC cell line) or monocyte/macrophage cells (SC cell line) when cells adhered to vitronectin. However, when exposed to DisBa-01, HUVECs produced higher levels of TGF-ß, and SC cells produced higher levels of VEGF-A. Nonetheless, HUVECs also showed an enhancement of apoptosis after losing adherence when exposed to disintegrin, which is a characteristic of anoikis. We propose that disintegrin DisBa-01 could be used to modulate integrin αvß3 functions.
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Inibidores da Angiogênese/farmacologia , Citocinas/biossíntese , Desintegrinas/farmacologia , Integrina alfaVbeta3/metabolismo , Apoptose/efeitos dos fármacos , Adesão Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Proteínas Recombinantes/farmacologia , Vitronectina/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) and αvß3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvß3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvß3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.
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Adesão Celular/efeitos dos fármacos , Desintegrinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Integrina alfaVbeta3 , Venenos de Serpentes/farmacologia , Fator A de Crescimento do Endotélio Vascular , Animais , Bothrops , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Desintegrinas/química , Desintegrinas/genética , Células Endoteliais/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Neovascularização Fisiológica , Peptídeos/química , Peptídeos/genética , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Venenos de Serpentes/química , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Frutalin is a homotetrameric alpha-d-galactose (d-Gal)-binding lectin that activates natural killer cells in vitro and promotes leukocyte migration in vivo. Because lectins are potent lymphocyte stimulators, understanding the interactions that occur between them and cell surfaces can help to the action mechanisms involved in this process. In this paper, we present a detailed investigation of the interactions of frutalin with phospho- and glycolipids using Langmuir monolayers as biomembrane models. The results confirm the specificity of frutalin for d-Gal attached to a biomembrane. Adsorption of frutalin was more efficient for the galactose polar head lipids, in contrast to the one for sulfated galactose, in which a lag time is observed, indicating a rearrangement of the monolayer to incorporate the protein. Regarding ganglioside GM1 monolayers, lower quantities of the protein were adsorbed, probably due to the farther apart position of d-galactose from the interface. Binary mixtures containing galactocerebroside revealed small domains formed at high lipid packing in the presence of frutalin, suggesting that lectin induces the clusterization and the forming of domains in vitro, which may be a form of receptor internalization. This is the first experimental evidence of such lectin effect, and it may be useful to understand the mechanism of action of lectins at the molecular level.
Assuntos
Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Adsorção , Artocarpus/química , Sítios de Ligação , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Elasticidade , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/metabolismo , Galactose/metabolismo , Humanos , Técnicas In Vitro , Fluidez de Membrana , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Membranas Artificiais , Modelos Biológicos , Ligação ProteicaRESUMO
A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 degrees C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aalpha-chain, apparently not degrading the Bbeta and gamma-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 degrees C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Trombina/química , Venenos de Víboras/enzimologia , Albuminas/química , Sequência de Aminoácidos , Animais , Coagulação Sanguínea/efeitos dos fármacos , Caseínas/química , Venenos de Crotalídeos/farmacologia , DNA Complementar/biossíntese , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Glândulas Exócrinas/química , Fibrinogênio/química , Fibrinolíticos/farmacologia , Biblioteca Gênica , Masculino , Camundongos , Dados de Sequência Molecular , Peso Molecular , Músculo Esquelético/patologia , Necrose/induzido quimicamente , Necrose/patologia , Peptídeo Hidrolases/química , Proteínas/química , Serina Proteases/farmacologia , Venenos de Víboras/farmacologiaRESUMO
The synthesis and characterization of ruthenium compounds of the type [RuCl(2)(NO)(dppp)(L)]PF(6) [dppp=1,3-bis(diphenylphosphino)propane; L=pyridine, 4-methylpyridine, 4-phenylpyridine and dimethyl sulfoxide] are described. The complexes were characterized by elemental analysis, UV/Vis and infrared spectroscopy, cyclic voltammetry, and X-ray crystallography for the complexes with the pyridine and 4-methylpyridine ligands. In vitro evaluation of these nitrosyl complexes revealed cytotoxic activity from 7.1 to 19.0 microM against the MDA-MB-231 breast tumor cells and showed that, in this case, they are more active than the reference metallodrug cisplatin. The 1,3-bis(diphenylphosphino)propane and the N-heterocyclic ligands alone failed to show cytotoxic activities at the concentrations tested (maximum concentration utilized=200 microM).