Oleuropein inhibits migration ability through suppression of epithelial-mesenchymal transition and synergistically enhances doxorubicin-mediated apoptosis in MCF-7 cells

Distinct metastasis is one of the main causes of breast cancer (BC)-related mortality and epithelial-mesenchymal transition (EMT) is a primary step in metastasis dissemination. On the other hand, doxorubicin (DOX) is an effective chemotherapeutic agent against BC; unfortunately, its clinical use is limited by dose-dependent side effects. Therefore, extensive efforts have been dedicated to suppressing metastasis of BC and also to overcome DOX side effects together with keeping its antitumor efficacy. Studies supported the role of oleuropein (OLEU) in reducing DOX-induced side effects besides its antitumor actions. In this study, the antimigratory effect of OLEU was assessed and real-time PCR (RT-PCR) was used to detect OLEU effect on the expression level of EMT markers, in MCF-7 cells. The cytotoxic effect of OLEU and DOX was assessed by MTT assay, whereas the ratio of apoptosis was investigated by flow cytometry. The results showed that migration ability of MCF-7 cells remarkably decreased in OLEU treated group and RT-PCR results showed that OLEU may exert its antimigratory action by suppressing EMT through downregulation of sirtuin1 (SIRT1). Also, the results indicated that both OLEU and DOX were cytotoxic to MCF-7 cells, whereas DOX-OLEU cotreatment led to additive cytotoxicity and apoptosis rate. This study provides evidence regarding the suppressive role of OLEU on MCF-7 cells migration ability through suppression of EMT, and for the first time, it was proposed that SIRT1 downregulation can be involved in the OLEU antimigratory effect. Also, the findings demonstrated that OLEU can reduce DOX-induced side effects by reducing its effective dose.     

Unbiased identification of cysteine S-nitrosylation sites on proteins

Covalent addition of nitric oxide (NO) to Cys-sulfur in proteins, or S-nitrosylation, plays pervasive roles in the physiological and pathophysiological modulation of mammalian protein functions. Knowledge of the specific protein Cys residues that undergo NO addition in different biological settings is fundamental to understanding NO-mediated signal transduction. Here, we describe in detail an MS-based proteomic protocol for facile, high-throughput and unbiased discovery of SNO-Cys residues in proteins from complex biological samples. The approach, termed SNOSID (SNO-Cys site identification), can be used to identify endogenous and chemically induced S-nitrosylation sites in proteins from tissues or cells. Identified SNO-Cys sites may provide insights into novel mechanisms and proteins that mediate NO bioactivities in health and disease. SNOSID builds on the biotin-switch method for covalent addition of disulfide-linked biotin at S-nitrosylation sites on proteins. Biotinylated proteins are then subjected to trypsinolysis and the resulting biotin-tagged peptides are affinity-captured on streptavidin-agarose. After selective elution with beta-mercaptoethanol, the peptides are sequenced using nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS). Validation that identified peptide ions as originating from authentic NO-Cys-containing precursor proteins can be provided by establishing that these peptide ions are absent from control samples where S-NO bonds were subjected to prior photolysis, using a UV transilluminator. The protocol requires approximately 2 days for sample processing, including the incubation time for proteolysis. An additional 1-2 days is needed for sample analysis by nLC-MS/MS and data analysis/interpretation.     

N-Acyl taurines trigger insulin secretion by increasing calcium flux in pancreatic β-cells

Pancreatic β-cells secrete insulin in response to various stimuli to control blood glucose levels. This insulin release is the result of a complex interplay between signaling, membrane potential and intracellular calcium levels. Various nutritional and hormonal factors are involved in regulating this process. N-Acyl taurines are a group of fatty acids which are amidated (or conjugated) to taurine and little is known about their physiological functions. In this study, treatment of pancreatic β-cell lines (HIT-T15) and rat islet cell lines (INS-1) with N-acyl taurines (N-arachidonoyl taurine and N-oleoyl taurine), induced a high frequency of calcium oscillations in these cells. Treatment with N-arachidonoyl taurine and N-oleoyl taurine also resulted in a significant increase in insulin secretion from pancreatic β-cell lines as determined by insulin release assay and immunofluorescence (p < 0.05). Our data also show that the transient receptor potential vanilloid 1 (TRPV1) channel is involved in insulin secretion in response to N-arachidonoyl taurine and N-oleoyl taurine treatment. However our data also suggest that receptors other than TRPV1 are involved in the insulin secretion response to treatment with N-oleoyl taurine.     

A new insight on serum microRNA expression as novel biomarkers in breast cancer patients

Breast cancer (BC) is one of the widespread lethal diseases affecting a large number of women worldwide. As such, employing and identifying significant markers for detecting BC in different stages can assist in better diagnosis and management of the disease. Several diverse markers have been introduced for diagnosis, but their limitations, including low specificity and sensitivity, reduce their application. microRNAs (miRNAs), as short noncoding RNAs, have been shown to significantly influence gene expression in different disease pathologies, especially BC. Clearly, among different samples used for detecting miRNA expressions, circulating miRNAs present as promising and useful biomarkers. Among different body fluid samples, serum serves as one of the most reliable samples, thanks to its high stability under various severe conditions and some unique features. Extensive research has suggested that BC-related miRNAs can remain stable in the serum. The objective of this review is to describe different samples used for detecting miRNAs in BC subjects with emphasis on serum miRNAs. So, this study highlights serum miRNAs with the potential of acting as biomarkers for different stages of BC. We reviewed the possible correlation between potential miRNAs and the risk of early breast cancer, metastatic breast cancer, response to chemotherapy, and relapse.     

Downregulation of HDAC2 and HDAC3 via oleuropein as a potent prevention and therapeutic agent in MCF-7 breast cancer cells

Breast cancer is the most common malignancy in the world with the highest rate of morbidity and mortality. Due to the several side effects of chemotherapy and radiotherapy, recent studies have focused on the use of herbal medicines. Epidemiological reports have shown the inverse relationship between breast cancer risk and intake of olive. Oleuropein (OLE) is a polyphenolic compound in virgin olive oil with antineoplastic properties and it is well tolerated by humans. Recent reports have shown that OLE has effects on the control of cancer by modulating epigenetics, such as histone deacetylase (HDAC) inhibition. However, the epigenetic mechanisms of OLE anticancer properties are yet to be properly investigated. Therefore, this study aimed to determine the therapeutic effects of OLE through the modulation of histone deacetylase 2 (HDAC2) and histone deacetylase 3 (HDAC3) expression in breast cancer cell line. MCF-7 cells were tested with and without OLE, and also the cell viability, apoptosis, and migration were examined. HDAC2 and HDAC3 expression genes were assessed by quantitative real-time polymerase chain reaction. It was found that OLE decreased the expression of both HDAC2 and HDAC3 (P < 0.05), induced apoptosis and retarded cell migration and cell invasion in a dose-dependent manner (P < 0.05). These results showed that OLE is a potential therapeutic and preventive agent for breast cancer.