Flavonoids as regulators of TIMPs expression in cancer: Consequences, opportunities, and challenges.

Cancer is one of the leading causes of death in patients worldwide, where invasion and metastasis are directly responsible for this statement. Although cancer therapy has progressed in recent years, current therapeutic approaches are ineffective due to toxicity and chemoresistance. Therefore, it is essential to evaluate other treatment options, and natural products are a promising alternative as they show antitumor properties in different study models. This review describes the regulation of tissue inhibitors of metalloproteinases (TIMPs) expression and the role of flavonoids as molecules with the antitumor activity that targets TIMPs therapeutically. These inhibitors regulate tissue extracellular matrix (ECM) turnover; they inhibit matrix metalloproteinases (MMPs), cell migration, invasion, and angiogenesis and induce apoptosis in tumor cells. Data obtained in cell lines and in vivo models suggest that flavonoids are chemopreventive and cytotoxic against various types of cancer through several mechanisms. Flavonoids also regulate crucial signaling pathways such as focal adhesion kinase (FAK), phosphatidylinositol-3-kinase (PI3K)-Akt, signal transducer and activator of transcription 3 (STAT3), nuclear factor κB (NFκB), and mitogen-activated protein kinase (MAPK) involved in cancer cell migration, invasion, and metastasis. All these data reposition flavonoids as excellent candidates for use in cancer therapy.

Mutation of putative phosphorylation sites in the free fatty acid receptor 1: Effects on signaling, receptor phosphorylation, and internalization.

Free fatty acid receptor 1 phosphorylation sites were studied using mutants, including a) a mutant with T215V in the third intracellular loop (3IL), b) another with changes in the carboxyl terminus (C-term): T287V, T293V, S298A, and c) a mutant with all of these changes (3IL/C-term). Agonist-induced increases in intracellular calcium were similar between cells expressing wild-type or mutant receptors. In contrast, agonist-induced FFA1 receptor phosphorylation was reduced in mutants compared to wild type. Phorbol ester-induced FFA1 receptor phosphorylation was rapid and robust in cells expressing the wild-type receptor and essentially abolished in the mutants. Agonist-induced ERK 1/2 phosphorylation and receptor internalization were decreased in cells expressing the mutant receptors compared to those expressing the wild-type receptor. Our data suggest that the identified sites might participate in receptor phosphorylation, signaling, and internalization.

Effect of docosahexaenoic acid, phorbol myristate acetate, and insulin on the interaction of the FFA4 (short isoform) receptor with Rab proteins.

Human embryonic kidney (HEK) 293 cells were co-transfected with plasmids for the expression of mCherry fluorescent protein-tagged FFA4 receptors and the enhanced green fluorescent protein-tagged Rab proteins involved in retrograde transport and recycling, to study their possible interaction through Förster Resonance Energy Transfer (FRET), under the action of agents that induce FFA4 receptor phosphorylation and internalization through different processes, i.e., the agonist, docosahexaenoic acid, the protein kinase C activator phorbol myristate acetate, and insulin. Data indicate that FFA4 receptor internalization varied depending on the agent that induced the process. Agonist activation (docosahexaenoic acid) induced an association with early endosomes (as suggested by interaction with Rab5) and rapid recycling to the plasma membrane (as indicated by receptor interaction with Rab4). More prolonged agonist stimulation also appears to allow the FFA4 receptors to interact with late endosomes (interaction with Rab9), slow recycling (interaction with Rab 11), and target to degradation (Rab7). Phorbol myristate acetate, triggered a rapid association with early endosomes (Rab5), slow recycling to the plasma membrane (Rab11), and some receptor degradation (Rab7). Insulin-induced FFA4 receptor internalization appears to be associated with interaction with early endosomes (Rab5) and late endosomes (Rab9) and fast and slow recycling to the plasma membrane (Rab4, Rab11). Additionally, we observed that agonist- and PMA-induced FFA4 internalization was markedly reduced by paroxetine, which suggests a possible role of G protein-coupled receptor kinase 2.

Leptin induces cell migration and invasion in a FAK-Src-dependent manner in breast cancer cells.

Breast cancer is the most common invasive neoplasia, and the second leading cause of the cancer deaths in women worldwide. Mammary tumorigenesis is severely linked to obesity, one potential connection is leptin. Leptin is a hormone secreted by adipocytes, which contributes to the progression of breast cancer. Cell migration, metalloproteases secretion, and invasion are cellular processes associated with various stages of metastasis. These processes are regulated by the kinases FAK and Src. In this study, we utilized the breast cancer cell lines MCF7 and MDA-MB-231 to determine the effect of leptin on FAK and Src kinases activation, cell migration, metalloprotease secretion, and invasion. We found that leptin activates FAK and Src and induces the localization of FAK to the focal adhesions. Interestingly, leptin promotes the activation of FAK through a Src- and STAT3-dependent canonical pathway. Specific inhibitors of FAK, Src and STAT3 showed that the effect exerted by leptin in cell migration in breast cancer cells is dependent on these proteins. Moreover, we established that leptin promotes the secretion of the extracellular matrix remodelers, MMP-2 and MMP-9 and invasion in a FAK and Src-dependent manner. Our findings strongly suggest that leptin promotes the development of a more aggressive invasive phenotype in mammary cancer cells.

Receptor tyrosine kinase activation induces free fatty acid 4 receptor phosphorylation, ß-arrestin interaction, and internalization.

FFA4 (Free Fatty Acid receptor 4, previously known as GPR120) is a G protein-coupled receptor that acts as a sensor of long-chain fatty acids, modulates metabolism, and whose dysfunction participates in endocrine disturbances. FFA4 is known to be phosphorylated and internalized in response to agonists and protein kinase C activation. In this paper report the modulation of this fatty acid receptor by activation of receptor tyrosine kinases. Cell-activation with growth factors (insulin, epidermal growth factor, insulin-like growth factor-I, and platelet-derived growth factor) increases FFA4 phosphorylation in a time- and concentration-dependent fashion. This effect was blocked by inhibitors of protein kinase C and phosphoinositide 3-kinase, suggesting the involvement of these kinases in it. FFA4 phosphorylation did not alter agonist-induced FFA4 calcium signaling, but was associated with decreased ERK 1/2 phosphorylation. In addition, insulin, insulin-like growth factor-I, epidermal growth factor, and to a lesser extent, platelet-derived growth factor, induce receptor internalization. This action of insulin, insulin-like growth factor I, and epidermal growth factor was blocked by inhibitors of protein kinase C and phosphoinositide 3-kinase. Additionally, cell treatment with these growth factors induced FFA4-ß-arrestin coimmunoprecipitation. Our results evidenced cross-talk between receptor tyrosine kinases and FFA4 and suggest roles of protein kinase C and phosphoinositide 3-kinase in such a functional interaction.

Free fatty acid receptor 4 agonists induce lysophosphatidic acid receptor 1 (LPA1 ) desensitization independent of LPA1 internalization and heterodimerization.

The crosstalk between the free fatty acid receptor FFA4 and the lysophosphatidic acid receptor LPA1 seems to be of pathophysiological importance. We explored this crosstalk employing co-expression of fluorescent protein-tagged receptors. FFA4 activation induces functional desensitization of LPA1 receptors and phosphorylation of both receptors. LPA1 activation induces phosphorylation of LPA1 , but not of FFA4, and induces internalization of both receptors into heterogeneous types of vesicles. Docosahexaenoic acid (DHA) induces internalization of FFA4 but not of LPA1 . Fatty acid-induced FFA4-LPA1 interaction was observed using Förster resonance energy transfer and co-immunoprecipitation. Such interaction took place after desensitization was already established. Data indicate that FFA4 activation induces LPA1 desensitization in an internalization-independent process and that complex cellular processes participate in the crosstalk of these receptors.

Arachidonic acid promotes migration and invasion through a PI3K/Akt-dependent pathway in MDA-MB-231 breast cancer cells.

Arachidonic acid (AA) is a common dietary n-6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, however it might be present in the extracellular microenvironment. AA and its metabolites mediate FAK activation, adhesion and migration in MDA-MB-231 breast cancer cells. However, it remains to be investigated whether AA promotes invasion and the signal transduction pathways involved in migration and invasion. Here, we demonstrate that AA induces Akt2 activation and invasion in MDA-MB-231 cells. Akt2 activation requires the activity of Src, EGFR, and PIK3, whereas migration and invasion require Akt, PI3K, EGFR and metalloproteinases activity. Moreover, AA also induces NFκB-DNA binding activity through a PI3K and Akt-dependent pathway. Our findings demonstrate, for the first time, that Akt/PI3K and EGFR pathways mediate migration and invasion induced by AA in MDA-MB-231 breast cancer cells.

Benzo-[a]-pyrene induces FAK activation and cell migration in MDA-MB-231 breast cancer cells.

Benzo-[a]-pyrene (B[a]P) is a family member of polycyclic aromatic hydrocarbons and a widespread environmental pollutant. It is a mammary carcinogen in rodents and contributes to the development of human breast cancer. However, the signal transduction pathways induced by B[a]P and its role in breast cancer progression have not been studied in detail. Here, we demonstrate that B[a]P induces cell migration through a lipoxygenase- and Src-dependent pathway, as well as the activation of focal adhesion kinase, Src, and the extracellular signal-regulated kinase 2 in MDA-MB-231 breast cancer cells. However, B[a]P is not able to promote migration in the mammary nontumorigenic epithelial cells MCF12A. Moreover, B[a]P promotes an increase of αvß3 integrin-cell surface levels and an increase of metalloproteinase (MMP)-2 and MMP-9 secretions. In summary, our findings demonstrate that B[a]P induces the activation of signal transduction pathways and biological processes involved in the invasion/metastasis process in MDA-MB-231 breast cancer cells.

Elevated concentration of microvesicles isolated from peripheral blood in breast cancer patients.

BACKGROUND AND AIMS: Breast cancer is the most common cancer and the main cause of cancer deaths in women worldwide. Microvesicles (MVs) are fragments of the plasma membrane secreted from cytoplasmic membrane compartments by normal and malignant cells. An increase in MV number has been found in peripheral blood of patients with several diseases including cancer. We hypothesized that MV number and the relative amount of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) proteins in plasma fractions enriched in MVs and deprived of platelet-derived MVs are related to the presence of breast cancer. METHODS: Plasma fractions enriched in MVs and deprived of platelet-derived MVs were obtained by differential centrifugation of blood samples. MV number was evaluated by BD TruCOUNT Tubes (BD Biosciences). FAK and EGFR proteins were analyzed by Western blot. RESULTS: MV number in plasma fractions enriched with MVs and deprived of platelet-derived MVs is higher in breast cancer patients with stages I-IV as well as with T2-T4 tumors, in comparison to control group. In addition, plasma fractions enriched in MVs present FAK and EGFR proteins and their amount is increased in some stages of breast cancer in comparison to control group. CONCLUSIONS: Our findings strongly suggest that MV number and the amount of FAK and EGFR in plasma fractions enriched in MVs are associated with some stages of breast cancer.

Role of arachidonic acid metabolism in Stat5 activation induced by oleic acid in MDA-MB-231 breast cancer cells.

Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of breast cancer. In breast cancer cells, the free fatty acid oleic acid (OLA) induces proliferation, migration, invasion and an increase of MMP-9 secretion. However, the role of OLA on Stat5 activation and the participation of COX-2 and LOXs activity in Stat5 activation induced by OLA remain to be investigated. We demonstrate here that stimulation of MDA-MB-231 breast cancer cells with 100 µM OLA induces Stat5 phosphorylation at Tyr-694 and an increase of Stat5-DNA complex formation. The Stat5 DNA-binding activity requires COX-2, LOXs, metalloproteinases and Src activities. In addition, OLA induces cell migration through a Stat5-dependent pathway. In summary, our findings establish that OLA induces cell migration through a Stat5-dependent pathway and that Stat5 activation requires AA metabolites in MDA-MB-231 breast cancer cells.

Role of LOXs and COX-2 on FAK activation and cell migration induced by linoleic acid in MDA-MB-231 breast cancer cells.

BACKGROUND: Epidemiological studies and animal models suggest a link between high levels of dietary fat intake and an increased risk of developing breast cancer. Particularly, free fatty acids (FFAs) are involved in several processes, including proliferation, migration and invasion, in breast cancer cells. Linoleic acid (LA) is a dietary n-6 polyunsaturated fatty acid that is known to induce proliferation and invasion in breast cancer cells. So far, however, the contribution of LA to focal adhesion kinase (FAK) activation and cell migration in breast cancer cells has not been studied. RESULTS: Here, we show that LA promotes FAK and Src activation, as well as cell migration, in MDA-MB-231 breast cancer cells. FAK activation and cell migration require Src, Gi/Go, COX-2 and LOXs activities, whereas both are independent of Δ6 desaturase activity. In addition, we show that cell migration requires FAK activity, whereas FAK activation requires Src activity, thus suggesting a reciprocal catalytic activation mechanism of FAK and Src. CONCLUSIONS: In summary, our findings show that LA induces FAK activation and cell migration in MDA-MB-231 breast cancer cells.

Arachidonic acid induces an increase of ß-1,4-galactosyltransferase I expression in MDA-MB-231 breast cancer cells.

Arachidonic acid (AA) is a common dietary n-6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, and it might be present in the extracellular microenvironment. AA and its metabolites are implicated in FAK activation and cell migration in MDA-MB-231 breast cancer cells, and an epithelial-to-mesenchymal-like transition process in mammary non-tumorigenic epithelial cells MCF10A. During malignant transformation is present an altered expression of glycosiltransferases, which promote changes on the glycosilation of cell-surface proteins. The ß-1,4-galactosyltransferase I (GalT I) is an enzyme that participates in a variety of biological functions including cell growth, migration, and spreading. However, the participation of AA in the regulation of GalT I expression and the role of this enzyme in the cell adhesion process in breast cancer cells remains to be investigated. In the present study, we demonstrate that AA induces an increase of GalT I expression through a PLA2α, Src, ERK1/2, and LOXs activities-dependent pathway in MDA-MB-231 breast cancer cells. Moreover, MDA-MB-231 cells adhere to laminin via GalT I expression and pretreatment of cells with AA induces an increase of cell adhesion to laminin. In conclusion, our findings demonstrate, for the first time, that AA promotes an increase of GalT I expression through an AA metabolism, Src and ERK1/2 activities-dependent pathway, and that GalT I plays a pivotal role in cell adhesion to laminin in MDA-MB-231 breast cancer cells.