In Vitro Effects of Combining Genistein with Aromatase Inhibitors: Concerns Regarding Its Consumption during Breast Cancer Treatment.

INTRODUCTION: The third-generation of aromatase inhibitors (AIs)-Exemestane (Exe), Letrozole (Let), and Anastrozole (Ana)-is the main therapeutic approach applied for estrogen receptor-positive (ER+) breast cancer (BC), the most common neoplasm in women worldwide. Despite their success, the development of resistance limits their efficacy. Genistein (G), a phytoestrogen present in soybean, has promising anticancer properties in ER+ BC cells, even when combined with anticancer drugs. Thus, the potential beneficial effects of combining G with AIs were investigated in sensitive (MCF7-aro) and resistant (LTEDaro) BC cells. METHODS: The effects on cell proliferation and expression of aromatase, ERα/ERß, and AR receptors were evaluated. RESULTS: Unlike the combination of G with Ana or Let, which negatively affects the Ais' therapeutic efficacy, G enhanced the anticancer properties of the steroidal AI Exe, increasing the antiproliferative effect and apoptosis relative to Exe. The hormone targets studied were not affected by this combination when compared with Exe. CONCLUSIONS: This is the first in vitro study that highlights the potential benefit of G as an adjuvant therapy with Exe, emphasizing, however, that soy derivatives widely used in the diet or applied as auxiliary medicines may increase the risk of adverse interactions with nonsteroidal AIs used in therapy.

Antitumor effects of co-treatment of resveratrol with antitumor drugs in ER- and HER2-positive breast cancer cells are due to induction of apoptosis and modulation of estrogen receptor expression.

BACKGROUND: Resveratrol, a natural compound, may be an alternative to improving conventional breast cancer therapy. Thus, we assessed the capability of resveratrol at a low dose to enhance the in vitro effect of conventional theray in estrogen receptor (ER) and human epidermal growth factor receptor type 2 (HER2)-positive breast cancer cells. METHODS: Cell viability of breast cancer cells was measured with neutral red uptake assay. Apoptosis, autophagy, cell cycle progression and cell proliferation were detected through hypotonic fluorescent solution assay, formation of acidic vesicular organelles, flow cytometry, and bromodeoxyuridine assay, respectively. Western blotting was performed to study the expression of pro-apoptotic, anti-apoptotic and autophagic proteins, and estrogen receptors. RESULTS: Resveratrol combined with tamoxifen metabolites or trastuzumab reduced cell viability of ER- and HER2-positive breast cancer cells, respectively. This effect was mainly associated with induction of apoptosis due to a greater formation of hypodiploid nuclei, reduced protein expression of procaspase-7, Bcl-2, Bcl-xL, and PARP; and increased expression of cleaved PARP. Resveratrol decreased the expression of ERα and increased that of ERß, contributing to the reduced viability on breast cancer cells. Combined treatments induced autophagy, evidenced by increased levels of acidic vesicular organelles and degradation of p62/SQSTM1 protein. Nevertheless, on inhibiting autophagy with 3-methyladenine, cell viability was further reduced and apoptosis was induced, suggesting a pro-survival role of autophagy, impairing apoptosis. CONCLUSIONS: Resveratrol increasead the in vitro cytotoxic effect of conventional therapy in breast cancer cells. However, it was necessary to block resveratrol-induced autophagy to improve the therapeutic response.

Soybean extract modified by Aspergillus awamori stimulates a greater collagen-I synthesis in the intracellular matrix of human fibroblasts.

Aglycone isoflavones are estrogen-like bioactive compounds found in low amounts in soybean, which are increased by biotransformation processes. This study investigated two biotransformation processes of soybean extracts with Aspergillus awamori fungus, evaluating aglycone content and capability of stimulation of collagen-I deposition. Isoflavones were quantified via HPLC; cytotoxicity of biotransformed extracts toward mouse and human fibroblasts was evaluated via NRU and apoptosis/necrosis assays; and collagen-I deposition was measured through Western blot, immunofluorescence, and immunoassay. BSE-2 was the biotransformed soybean extract with the highest aglycone content and did not decrease viability or demonstrated cytotoxicity to either L929 or HDFa cells. BSE-2, at the optimal concentration of 1.33 µg/mL, increased substantially collagen-I amount in HDFa intracellular matrix compared to non-biotransformed soybean extract (NBSE) and immunoassay demonstrated that the extracellular deposition was mostly inhibited by BSE-2 concentrations, except at 1.33 µg/mL. Hence, biotransformed soybean extract by the enzymatic filtrate of Aspergillus awamori fungus demonstrated a high nutricosmetic potential, showing safeness and effective collagen-I augmentation.

The anti-cancer potential of crotoxin in estrogen receptor-positive breast cancer: Its effects and mechanism of action.

Estrogen receptor-positive (ER+) breast cancer is the most diagnosed subtype of breast cancer. Currently, aromatase inhibitors (AIs) are used as first-line treatment option in this type of tumors, however they cause several side effects, which is why new therapeutic approaches are demanding. The South American rattlesnake Crotalus durissus terrificus produces a venom enriched in several bioactive substances, like phospholipases A2 (PLA2). One of those is crotoxin, a ß-neurotoxin, that has already been reported for its anti-cancer properties in different cancers. Recently, its clinical interest has emerged and, in fact, a clinical trial in patients with advanced cancer is underway. Considering this, in this work, we studied the biological mechanisms behind the anti-cancer effects of crotoxin B (CTX) in an ER+ aromatase-overexpressing breast cancer cell line (MCF-7aro cells). Results revealed that CTX impairs MCF-7aro cells growth, through a cell cycle arrest at G2/M phase, inhibition of ERK1/2 pathway and by apoptosis through activation of caspase-8. In addition, it can be considered a safe natural compound as did not affect non-cancerous cells and only showed anti-growth effects in breast cancer cells. Therefore, this study represents an important landmark to better understand the effects and mechanisms of action of crotoxin in ER+ breast cancer.

Bothrops moojeni venom and BmooLAAO-I downmodulate CXCL8/IL-8 and CCL2/MCP-1 production and oxidative burst response, and upregulate CD11b expression in human neutrophils.

Bothrops snake venoms contain biologically active components, including L-amino acid oxidases (LAAO) that induce significant leukocyte accumulation at inflammatory sites characterized by early neutrophil infiltration. As it remains unclear how snake venoms modulate neutrophil activation and chemokine production, here we examined whether Bothrops moojeni crude venom (BmV) and its LAAO (BmooLAAO-I) affect expression of the surface activation markers CD11b and CD66b, production of the chemokines CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL-10/IP-10, and activation of oxidative burst in human neutrophils. Cell viability, expression of activation markers, and chemokine production were assessed by flow cytometry, while the oxidative burst response was measured by chemiluminescence. BmV at 50 and 75 µg/mL reduced CXCL8/IL-8 (p < 0.001 and p < 0.01, respectively) and CCL2/MCP-1 production (p < 0.05), while BmooLAAO-I at the same concentrations reduced only CCL2/MCP-1 production (p < 0.01). These effects were accompanied by CD11b upregulation (p < 0.05 for 50 and 75 µg/mL BmV; p < 0.01 for 50 and 75 µg/mL BmooLAAO-I) and CD66b downregulation (p < 0.05 for 50 and 75 µg/mL BmV). Both BmV and BmooLAAO-I at concentrations ranging from 0.625 to 5 µg/mL suppressed the oxidative burst of neutrophils stimulated with phorbol 12-myristate 13-acetate, while BmooLAAO-I at 2.5 and 5 µg/mL also suppressed the neutrophil response stimulated with opsonized zymosan. Considering that neutrophils participate in the pathogenesis of autoimmune and inflammatory diseases, the findings reported herein indicate that BmV and BmooLAAO-I are potential immunomodulating agents.

Effects of crotoxin, a neurotoxin from Crotalus durissus terrificus snake venom, on human endothelial cells.

Vascular endothelium plays an important modulatory role due to the production of molecules that mediate vasomotricity, inflammation, and leukocyte adhesion and rolling. Here we addressed whether crotoxin (25-200 µg/mL) - the main component of Crotalus durissus terrificus snake venom - interferes with cell viability, apotosis/necrosis, and cell response to oxidative stress in human umbilical vein endothelial cells (HUVEC) in vitro. We also examined whether crotoxin alters the levels of interleukins, adhesion molecules, and endothelial vasoactive factors in HUVEC cells treated or not with lipopolysaccharide (LPS; 1 µg/mL; 24 h). Crotoxin was not cytotoxic towards HUVEC cells, and downregulated the LPS-induced production of adhesion molecules (VCAM-1, ICAM-1, and E-selectin), vasoactive factors (endothelin-1 and prostaglandin I2), and interleukins (IL-6, IL-8, and IL1ß), as well as protected cells against H2O2-induced oxidative stress. Hence, crotoxin played anti-inflammatory, antioxidant, immunomodulating, and vasoactive actions on HUVEC cells, in vitro. Considering that the initial stages of atherosclerosis is characterized by vasoconstriction, increased levels of adhesion molecules, inflammatory cytokines, and oxidative stress in the vascular endothelium; and crotoxin downmodulated all these events, our findings indicate that the actions of crotoxin here demonstrated suggest that it may have an anti-atherogenic action in vivo, which deserves to be tested in future studies.

Crotoxin, a neurotoxin from Crotalus durissus terrificus snake venom, as a potential tool against thrombosis development.

Endothelium plays an important modulatory role due to the synthesis and secretion of molecules that act on hemostasis and fibrinolysis. As there are no literature data about the effect of crotoxin (CTX) - the main component of Crotalus durissus terrificus snake venom - on human endothelial cells, the present study examined how CTX (25-200 µg/mL) affects the endothelial production of the hemostatic factors antithrombin III, protein C, protein S, plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (t-PA), and vWF in human umbilical vein endothelial cells (HUVEC) in vitro. Production of hemostatic factors was assessed in HUVEC cells treated with CTX (25-200 µg/mL) in the presence or absence of lypopolysaccharide (LPS; 1 µg/mL; 24 h). We found that CTX alone did not exert pro- or anticoagulant effect on hemostasis, and pro- or antifibrinolytic effect on fibrinolysis. LPS alone had procoagulant (increased vWF and reduced protein C and S levels) and antifibrinolytic action (reduced t-PA and increased PAI-1 levels). However, CTX exerted anticoagulant and profibrinolytic action in the presence of LPS by lowering the levels of vWF and t-PA and elevating the levels of protein C and PAI-1. Therefore can be used as a potential tool against thrombosis development.

BthTX-I from Bothrops jararacussu induces apoptosis in human breast cancer cell lines and decreases cancer stem cell subpopulation.

BACKGROUND: Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide. Given the known snake venom cytotoxicity towards several tumor types, we evaluated the effects of BthTX-I from Bothrops jararacussu on MCF7, SKBR3, and MDAMB231 breast cancer cell lines. METHODS: BthTX-I cytotoxicity was determined via MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay. Cell death was measured by a hypotonic fluorescent solution method, annexin-V-FITC/propidium iodide staining and by apoptotic/autophagic protein expression. Cancer stem cells (CSCs) were quantified by flow cytometry using anti-CD24-FITC and anti-CD44-APC antibodies and propidium iodide. RESULTS: BthTX-I at 102 µg/mL induced cell death in all cell lines. The toxin induced apoptosis in MCF7, SKBR3, and MDAMB231 in a dose-dependent manner, as confirmed by the increasing number of hypodiploid nuclei. Expression of pro-caspase 3, pro-caspase 8 and Beclin-1 proteins were increased, while the level of the antiapoptotic protein Bcl-2 was diminished in MCF7 cells. BthTX-I changed the staining pattern of CSCs in MDAMB231 cells by increasing expression of CD24 receptors, which mediated cell death. CONCLUSIONS: BthTX-I induces apoptosis and autophagy in all breast cancer cell lines tested and also reduces CSCs subpopulation, which makes it a promising therapeutic alternative for breast cancer.

The L-amino acid oxidase from Calloselasma rhodostoma snake venom modulates apoptomiRs expression in Bcr-Abl-positive cell lines.

Anti-apoptotic genes and apoptomiRs deregulated expression contribute to apoptosis resistance in chronic myeloid leukemia (CML) Bcr-Abl(+) cells. Here, the L-amino acid oxidase from Calloselasma rhodostoma (CR-LAAO) venom altered the apoptotic machinery regulation by modulating the expression of the miR-145, miR-26a, miR-142-3p, miR-21, miR-130a, and miR-146a, and of the apoptosis-related proteins Bid, Bim, Bcl-2, Ciap-2, c-Flip, and Mcl-1 in Bcr-Abl(+) cells. CR-LAAO is a potential tool to instigate apoptomiRs regulation that contributes to drive CML therapy.

BthTX-I from Bothrops jararacussu induces apoptosis in human breast cancer cell lines and decreases cancer stem cell subpopulation

Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide. Given the known snake venom cytotoxicity towards several tumor types, we evaluated the effects of BthTX-I from Bothrops jararacussu on MCF7, SKBR3, and MDAMB231 breast cancer cell lines. Methods: BthTX-I cytotoxicity was determined via MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay. Cell death was measured by a hypotonic fluorescent solution method, annexin-V-FITC/propidium iodide staining and by apoptotic/autophagic protein expression. Cancer stem cells (CSCs) were quantified by flow cytometry using anti-CD24-FITC and anti-CD44-APC antibodies and propidium iodide. Results: BthTX-I at 102 µg/mL induced cell death in all cell lines. The toxin induced apoptosis in MCF7, SKBR3, and MDAMB231 in a dose-dependent manner, as confirmed by the increasing number of hypodiploid nuclei. Expression of pro-caspase 3, pro-caspase 8 and Beclin-1 proteins were increased, while the level of the antiapoptotic protein Bcl-2 was diminished in MCF7 cells. BthTX-I changed the staining pattern of CSCs in MDAMB231 cells by increasing expression of CD24 receptors, which mediated cell death. Conclusions: BthTX-I induces apoptosis and autophagy in all breast cancer cell lines tested and also reduces CSCs subpopulation, which makes it a promising therapeutic alternative for breast cancer.(AU)