Hydroxytyrosol and Oleuropein Inhibit Migration and Invasion via Induction of Autophagy in ER-Positive Breast Cancer Cell Lines (MCF7 and T47D).

Hydroxytyrosol (HT) and oleuropein (OL), the most abundant of the phenolic compounds in olives, have anticancer properties against breast cancer (BC). However, little attention has been paid to the mechanism of HT or OL in BC cells. The objective of this study was to identify the underlying molecular mechanisms of these compounds. ER-positive BC MCF7 and T47D cells were treated with HT and OL in combination with hepatocyte growth factor (HGF), rapamycin (Rapa, an agonist of autophagy) or 3-methyladenine (3-MA, an inhibitor of autophagy). Cell viability, metastasis capability and autophagy-related proteins were evaluated by wound healing assays, Transwell assays and Western blot. HT and OL reduced the cell viability of MCF-7 and T47D cells in a dose-dependent manner. Both cells were more sensitive to HT than OL. In addition, Rapa significantly inhibited HGF-induced migration and invasion, indicating that metastases of both BC cells could be inhibited by suppression of autophagy. Moreover, HT and OL significantly blocked HGF- or 3-MA-induced cell migration and invasion by reversing LC3II/LC3I and Beclin-1 downregulation and p62 upregulation. These findings revealed that HT and OL could suppress migration and invasion by activating autophagy in ER-positive BC cells, which might be a promising therapeutic strategy.

[Determination of four fatty acid ethyl esters in olive oil by solid phase extraction-gas chromatography].

The fatty acid ethyl ester (FAEE) content of olive oil is an important indicator of its quality. At present, the international standard method used to detect FAEEs in olive oil is silica gel (Si) column chromatography-gas chromatography (GC); however, this technique presents a number of disadvantages, including complex operation, long analysis times, and high reagent consumption. In this study, a method based on Si solid phase extraction (SPE)-GC was established to determine four FAEEs in olive oil, namely, ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate. First, the effects of the carrier gas were investigated, and He gas was ultimately selected as the carrier gas. Next, several internal standards were screened, and ethyl heptadecenoate (cis-10) was determined as the optimal internal standard. The SPE conditions were also optimized, and the effects of different brands of Si SPE columns on the recoveries of analytes were compared. Finally, a pretreatment method in which 0.05 g of olive oil was extracted with n-hexane and purified through a Si SPE column (1 g/6 mL) was developed. A sample could be processed within approximately 2 h using a total reagent volume of about 23 mL. Validation of the optimized method revealed that the four FAEEs have good linearities within the range of 0.1-5.0 mg/L (coefficients of determination (R2)>0.999). The limits of detection (LODs) of the method were within 0.78-1.11 mg/kg, and its limits of quantification (LOQs) were in the range of 2.35-3.33 mg/kg. The recoveries ranged from 93.8% to 104.0% at all spiked levels tested (4, 8, and 20 mg/kg), and the relative standard deviations were 2.2%-7.6%. Fifteen olive oil samples were tested using the established method, and the total FAEEs of three extra-virgin olive oil samples were found to exceed 35 mg/kg. Compared with the international standard method, the proposed method has the advantages of simpler pretreatment process, shorter operation time, lower reagent consumption and detection cost, high precision, and good accuracy. The findings provide an effective theoretical and practical reference for improving olive oil detection standards.

BCL2 Ala43Thr is a functional variant associated with protection against azoospermia in a Han-Chinese population.

Apoptosis is very common during various stages of mammalian germ cell development and differentiation, and the BCL2 gene is one of the most important apoptotic regulators. Although its genetic variants are reported to be involved in cancers and autoimmune diseases, little information is available regarding BCL2 polymorphisms in male spermatogenesis. In the present study, single nucleotide polymorphisms (SNPs) in coding regions of the BCL2 gene were examined in a hospital-based, case-control study including 198 infertile patients with idiopathic azoospermia and 183 fertile controls. Subsequently, a functional study was conducted for comparison of paclitaxel-induced cytotoxicity and apoptosis between the BCL2 variant and the wild type in vitro. Three SNPs were found in exon 2--A21G (rs1801018), G127A (rs1800477), and C300T (rs61733416)--with the latter first reported in the Han-Chinese population. The frequency of G127A (GA+AA) genotype was significantly lower in azoospermic, infertile men compared to the age-matched controls (P = 0.01). This genotype may confer a lower risk of azoospermia (adjusted odd ratio [OR] = 0.448, 95% confidence interval = 0.226-0.889). In addition, HeLa cells expressing the BCL2 Ala43Thr (G127A), similar to the control cells, were more sensitive to paclitaxel-induced cytotoxicity and apoptosis than those expressing wild-type BCL2. Consistently, the cleaved PARP and p-BCL2 proteins were subsequently increased after paclitaxel treatment, as also predicted by the bioinformatics analysis. Considering the decreased antiapoptotic function of BCL2, these results suggest that the Ala43Thr variant is associated with protection against azoospermia in the Han-Chinese population.

Hydroxytyrosol and Oleuropein Inhibit Migration and Invasion of MDA-MB-231 Triple-Negative Breast Cancer Cell via Induction of Autophagy.

BACKGROUND: Breast Cancer (BC) is the leading cause of cancer-related deaths among women. As such, novel chemotherapeutic agents are urgently needed, especially for Triple-Negative Breast Cancer (TNBC). Hydroxytyrosol (HT) and Oleuropein (OL) are rich in olive oil, which is associated with a low occurrence of BC. However, the effects and mechanisms of action of HT and OL in BC cells are still unclear. This study aimed to explore the molecular mechanisms underlying the antitumor effect of HT and OL in TNBC. METHODS: TNBC MDA-MB-231 cells were treated with HT and OL in combination with Hepatocyte Growth Factor (HGF), rapamycin (Rapa, an inducer of autophagy) or 3-methyladenine (3-MA, an inhibitor of autophagy). Cell viability, migration, invasion, and autophagy signaling were analyzed by scratch assays, transwell migration assays, and Western blot analysis. RESULTS: Treatment with HT or OL reduced MDA-MB-231 cell viability in a dose-dependent manner. MDAMB- 231 cells were more sensitive to HT treatment than OL treatment. Rapa treatment could significantly block HGF-induced MDA-MB-231 cell migration and invasion, suggesting that inhibition of autophagy could promote migration and invasion. Moreover, HT or OL treatment significantly suppressed HGF or 3-MA induced cell migration and invasion by reversing LC3-II/LC3-I and Beclin-1 downregulation and reversing p62 upregulation. CONCLUSION: These data indicated that HT and OL may inhibit migration and invasion of TNBC cells by activating autophagy. These findings provide potential therapeutic strategies that target autophagy to limit the pathogenesis and progression of BC.

Optimization of heme precursors for the expression of human cytochrome P450 2A13 and its co-expression with oxidoreductase in baculovirus/sf9 system.

Human cytochrome P450 2A13 (CYP2A13), mainly expressed in respiratory tract, is active towards numerous toxicants. To establish the metabolism in vitro, we expressed CYP2A13 and NADPH-CYP450 oxidoreductase (POR) in a baculovirus/sf9 system. Due to the deficiency of sf9 cells in heme incorporation, we investigated the effects of different heme precursors on the expression of CYP2A13, POR and their co-expression. The present results showed that both CYP2A13 and POR were presented the highest expression levels or activity with 0.2 mM δ-aminolaevulinic acid (5-ALA), 0.02 mM Fe(3+) and 0.5-1.0 µg/ml hemin. The combination of 0.2 mM 5-ALA and 0.02 mM Fe(3+) significantly improved CYP2A13 expression and content compared with heme precursors alone, so was POR activity. A multiplicity of infection (MOI) value of 5 pfu/cell for CYP2A13 baculovirus particles induced very high CYP2A13 expression. When co-infected with different POR MOI values, a viral ratio of 5 : 2 was associated with the highest CYP2A13 activity, whereas POR activity dose dependently increased with POR MOI. Furthermore, the expressed CYP2A13 in the optimized conduction could eliminate its substrate aflatoxin B1 at a significantly higher than those in other condition (P < 0.01). Our results provide an efficient approach for expressing functionally characterized, highly active and homogeneous CYP2A13 proteins.

Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells.

Cytochrome P450 (CYP) 2A13 is mainly expressed in the respiratory system and has the ability to metabolize aflatoxin B(1) (AFB(1)). However, the role of CYP2A13-mediated AFB(1) metabolism and its consequences in human lung epithelial cell is not clear. Therefore, the objectives of this study were to investigate the significance of CYP2A13 in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis. To achieve these objectives, CYP2A13 was stably over-expressed in immortalized human bronchial epithelial BEAS-2B cells (B-2A13) and its significance in AFB(1)-induced cytotoxicity, DNA adducts, and apoptosis was compared to cells with stably expression of CYP1A2 (B-1A2), the predominant AFB(1) metabolizing enzyme in liver, as well as CYP2A6 (B-2A6) as controls. AFB(1) induced B-2A13 cytotoxicity and apoptosis in a dose- and time-dependent manner. The cytotoxic and apoptotic effects of AFB(1) were significantly remarkable in B-2A13 cells than those of B-1A2 and B-2A6 cells. The increased expression of pro-apoptotic proteins, such as C-PARP, C-caspase-3, and Bax, and decreased expression of anti-apoptotic proteins, such as caspase-3, Bcl-2, and p-Bad further confirmed the data of AFB(1)-induced cytotoxicity and apoptosis. Furthermore, increased DNA adduct was observed in B-2A13 after AFB(1) treatment as compared to B-1A2 cells and B-2A6 cells. Finally, treatment with nicotine, a competitor of AFB(1), and 8-methoxypsoralen (8-MOP), an inhibitor of CYP enzyme, further confirm the critical role of CYP2A13 in AFB(1)-induced cytotoxicity and apoptosis. Collectively, these findings suggest adverse effects of AFB(1) in respiratory diseases mediated by CYP2A13.