The Effects of αvß3 Integrin Blockage in Breast Tumor and Endothelial Cells under Hypoxia In Vitro.

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.

Synthetic Spirocyclohexadienones as New Anti-Migratory Compounds in Triple- Negative Breast Cancer Cell Migration.

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.

Inhibition of αvß3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles.

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.

Alternagin-C (ALT-C), a disintegrin-like protein, attenuates alpha2beta1 integrin and VEGF receptor 2 signaling resulting in angiogenesis inhibition.

Angiogenesis, a crucial process in tumor progression, is mainly regulated by vascular endothelial growth factor (VEGF) and its receptor, VEGFR2. Studies have shown the interaction between α2ß1 integrin, a collagen receptor, and VEGFR2 in VEGF-driven angiogenesis in vitro and in vivo. Alternagin-C (ALT-C), an ECD-disintegrin-like protein from Bothrops alternatus snake venom, has high affinity for α2ß1 integrin and shows antiangiogenic activity in concentrations higher than 100 nM. Despite previous results, its mechanism of action on angiogenic signaling pathways has not been addressed. Here we evaluate the antiangiogenic activity of ALT-C in human umbilical vein endothelial cells (HUVECs) associated or not with VEGF, as well as its interference in the α2ß1/VEGFR2 crosstalk. ALT-C (1000 nM) affected actin cytoskeleton, decreased the number of cell filopodia, and strongly inhibited HUVEC tube formation, adhesion to type I collagen and cell migration. Down-regulation of α2ß1/VEGFR2 crosstalk by ALT-C decreased the protein content and phosphorylation of VEGFR2 and ß1 integrin subunit, inhibited ERK 1/2 and PI3K signaling and regulated FAK/Src and paxillin pathways. Furthermore, ALT-C increased the content of the autophagic markers LC3B and Beclin-1 in the presence of VEGF, which is associated with decreased angiogenesis. In conclusion, we suggest that ALT-C, after binding to α2ß1 integrin, inhibits VEGF/VEGFR2 signaling, which results in impaired angiogenesis. These results demonstrate that ALT-C may be a potential candidate for the development of antiangiogenic therapies for tumor and metastasis treatment and help to understand the complexity and fundamental role of integrin inhibition in the tumor microenvironment.

Metastasis inhibition in breast cancer by targeting cancer cell extravasation.

The spread of cells from primary tumors toward distant tissues and organs, also known as metastasis, is responsible for most cancer-associated deaths. The metastasis cascade comprises a series of events, characterized by the displacement of tumor cells (TCs) from the primary tumor to distant organs by traveling through the bloodstream, and their subsequent colonization. The first step in metastasis involves loss of cell-cell and cell-matrix adhesions, increased invasiveness and migratory abilities, leading to intravasation of TCs into the blood or lymphatic vessels. Stationary TCs must undergo the process of epithelial-mesenchymal transition in order to achieve this migratory and invasive phenotype. Circulating tumor cells that have survived in the circulation and left the blood or lymphatic vessels will reach distant sites where they may stay dormant for many years or grow to form secondary tumors. To do this, cells need to go through the mesenchymal-epithelial transition to revert the phenotype in order to regain epithelial cell-to-cell junctions, grow and become a clinically relevant and detectable tumor mass. This work will review the main steps of the metastatic cascade and describe some strategies to inhibit metastasis by reducing cancer cell extravasation presenting recent studies in the context of breast cancer.

Recombinant RGD-disintegrin DisBa-01 blocks integrin αvß3 and impairs VEGF signaling in endothelial cells.

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.

Impact of chemotherapy on metabolic reprogramming: Characterization of the metabolic profile of breast cancer MDA-MB-231 cells using 1H HR-MAS NMR spectroscopy.

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.

Effects of Doxorubicin, Cisplatin, and Tamoxifen on the Metabolic Profile of Human Breast Cancer MCF-7 Cells As Determined by 1H High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance.

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.

Characterization of metabolic profile of intact non-tumor and tumor breast cells by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy.

(1)H high-resolution magic angle spinning nuclear magnetic resonance ((1)H HR-MAS NMR) spectroscopy was used to analyze the metabolic profile of an intact non-tumor breast cell line (MCF-10A) and intact breast tumor cell lines (MCF-7 and MDA-MB-231). In the spectra of MCF-10A cells, six metabolites were assigned, with glucose and ethanol in higher concentrations. Fifteen metabolites were assigned in MCF-7 and MDA-MB-231 (1)H HR-MAS NMR spectra. They did not show glucose and ethanol, and the major component in both tumor cells was phosphocholine (higher in MDA-MB-231 than in MCF-7), which can be considered as a tumor biomarker of breast cancer malignant transformation. These tumor cells also show acetone signal that was higher in MDA-MB-231 cells than in MCF-7 cells. The high acetone level may be an indication of high demand for energy in MDA-MB-231 to maintain cell proliferation. The higher acetone and phosphocholine levels in MDA-MB-231 cells indicate the higher malignance of the cell line. Therefore, HR-MAS is a rapid reproducible method to study the metabolic profile of intact breast cells, with minimal sample preparation and contamination, which are critical in the analyses of slow-growth cells.

N-(1'-naphthyl)-3,4,5-trimethoxybenzohydrazide as microtubule destabilizer: Synthesis, cytotoxicity, inhibition of cell migration and in vivo activity against acute lymphoblastic leukemia.

Tubulin-interacting agents, like vinca alkaloid and taxanes, play a fundamental role in cancer chemotherapy, making cellular microtubules (MT), one of the few validated anticancer targets. Cancer resistance to classical MT inhibitors has motivated the development of novel molecules with increased efficacy and lower toxicity. Aiming at designing structurally-simple inhibitors of MT assembly, we synthesized a series of thirty-one 3,4,5-trimethoxy-hydrazones and twenty-five derivatives or analogs. Docking simulations suggested that a representative N-acylhydrazone could adopt an appropriate stereochemistry inside the colchicine-binding domain of tubulin. Several of these compounds showed anti-leukemia effects in the nanomolar concentration range. Interference with MT polymerization was validated by the compounds' ability to inhibit MT assembly at the biochemical and cellular level. Selective toxicity investigations done with the most potent compound, a 3,4,5-trimethoxy-hydrazone with a 1-naphthyl group, showed remarkably selective toxicity against leukemia cells in comparison with stimulated normal lymphocytes, and no acute toxicity in vivo. Finally, this molecule was as active as vincristine in a murine model of human acute lymphoblastic leukemia at a weekly dose of 1 mg/kg.

Jatrophidin I, a cyclic peptide from Brazilian Jatropha curcas L.: isolation, characterization, conformational studies and biological activity.

A cyclic peptide, jatrophidin I, was isolated from the latex of Jatropha curcas L. Its structure was elucidated by extensive 2D NMR spectroscopic analysis, with additional conformational studies performed using Molecular Dynamics/Simulated Annealing (MD/SA). Jatrophidin I had moderate protease inhibition activity when compared with pepstatin A; however, the peptide was inactive in antimalarial, cytotoxic and antioxidant assays.

Cytotoxic 3,4,5-trimethoxychalcones as mitotic arresters and cell migration inhibitors.

Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand-tubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine acute lymphoblastic leukemia cells compared with a previously described parent compound, and inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in scratch wound and Boyden chamber assays.