Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells.

BACKGROUND: Extracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs. METHODS: Cell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins. RESULTS: We observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion. CONCLUSIONS: Our findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.

Small particles or vesicles released by cancer cells into their surroundings have the potential to stimulate the spread and growth of cancer cells. In this study, we focused on two specific molecules presented by these cancer cell-derived vesicles that could play a role in promoting the dissemination of cancer cells: a protein related to blood clotting and a protein on the cell surface.We found that large vesicles from bladder cancer cells that have the ability to spread had higher levels of these proteins than vesicles from nonspreading cancer cells. We also found that the former could make cancer cells move about more, produce more of a substance that helps cancer cells spread, and invade other tissues.To counteract the cancer-promoting actions of these vesicles, we examined the impact of combining a naturally occurring anticlotting protein that can be released by medications derived from heparin with an inhibitor targeting the cancer cell surface protein. We found that this combination stopped the vesicles from helping cancer cells move about more, produce more of the spreading substance, and invade other tissues.This approach of simultaneously targeting the two protein molecules present on cancer cell-derived vesicles might be a new way to treat bladder cancer.

White-brown adipose tissue interplay in polycystic ovary syndrome: Therapeutic avenues.

This study highlights the therapeutic potential of activating brown adipose tissue (BAT) for managing polycystic ovary syndrome (PCOS), a prevalent endocrine disorder associated with metabolic and reproductive abnormalities. BAT plays a crucial role in regulating energy expenditure and systemic insulin sensitivity, making it an attractive target for the treatment of obesity and metabolic diseases. Recent research suggests that impaired BAT function and mass may contribute to the link between metabolic disturbances and reproductive issues in PCOS. Additionally, abnormal white adipose tissue (WAT) can exacerbate these conditions by releasing adipokines and nonesterified fatty acids. In this review, we explored the impact of WAT changes on BAT function in PCOS and discussed the potential of BAT activation as a therapeutic strategy to improve PCOS symptoms. We propose that BAT activation holds promise for managing PCOS; however, further research is needed to confirm its efficacy and to develop clinically feasible methods for BAT activation.

An update on the secretory functions of brown, white, and beige adipose tissue: Towards therapeutic applications.

Adipose tissue, including white adipose tissue (WAT), brown adipose tissue (BAT), and beige adipose tissue, is vital in modulating whole-body energy metabolism. While WAT primarily stores energy, BAT dissipates energy as heat for thermoregulation. Beige adipose tissue is a hybrid form of adipose tissue that shares characteristics with WAT and BAT. Dysregulation of adipose tissue metabolism is linked to various disorders, including obesity, type 2 diabetes, cardiovascular diseases, cancer, and infertility. Both brown and beige adipocytes secrete multiple molecules, such as batokines, packaged in extracellular vesicles or as soluble signaling molecules that play autocrine, paracrine, and endocrine roles. A greater understanding of the adipocyte secretome is essential for identifying novel molecular targets in treating metabolic disorders. Additionally, microRNAs show crucial roles in regulating adipose tissue differentiation and function, highlighting their potential as biomarkers for metabolic disorders. The browning of WAT has emerged as a promising therapeutic approach in treating obesity and associated metabolic disorders. Many browning agents have been identified, and nanotechnology-based drug delivery systems have been developed to enhance their efficacy. This review scrutinizes the characteristics of and differences between white, brown, and beige adipose tissues, the molecular mechanisms involved in the development of the adipocytes, the significant roles of batokines, and regulatory microRNAs active in different adipose tissues. Finally, the potential of WAT browning in treating obesity and atherosclerosis, the relationship of BAT with cancer and fertility disorders, and the crosstalk between adipose tissue with circadian system and circadian disorders are also investigated.

Diagnostic Value of ATG5, Apo-Lipoprotein B-48, Thyroid Hormones, and Homocysteine in Patients with Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia. In this study, serum levels of autophagy-related 5 (ATG5), apolipoprotein B-48, thyroid hormones, and homocysteine were examined in patients with AD to determine their diagnostic and predictive value for early diagnosis and prevention of AD. MATERIALS: For this study, fifty serum samples were obtained from patients with AD and fifty serum samples from healthy controls. Serum levels of ATG 5, apo B48, thyroid hormones, homocysteine, vitamin B12, and folic acid were determined by ELISA. Spectrophotometry was used to determine serum lipid concentrations. RESULTS: The mean age of the case group was 69 ± 6.4 years and that of the control group was 67 ± 4.2 years. There were differences between the control and case groups in serum levels of homocysteine, apo B48, ATG5, hsCRP, LDL, HDL, cholesterol, and VitB12 (p < 0.05). According to the results of the ROC curve, measurements of serum levels of ATG5, homocysteine, and apo B48 have excellent performance in distinguishing patients with Alzheimer's disease from patients without AD. CONCLUSIONS: This study suggests that the measurement of serum levels of ATG5, homocysteine, and apo B48, along with other available biomarkers, can be helpful in the diagnosis and management of patients with AD in the early stages of their disease.

Fatty acids of type 2 diabetic serum decrease the stemness properties of human adipose-derived mesenchymal stem cells.

In type 2 diabetes, dyslipidemia and increased serum free fatty acids (FFAs) exacerbate the development of the disease through a negative effect on insulin secretion. Adipose-derived mesenchymal stem cells (AdMSCs) play a key role in regenerative medicine, and these cells can potentially be applied as novel therapeutic resources in the treatment of diabetes. In this study, AdMSCs were treated with diabetic or nondiabetic serum FFAs isolated from women of menopausal age. Serum FFAs were analyzed using gas-liquid chromatography. The expression level of the stemness markers CD49e and CD90 and the Wnt signaling target genes Axin-2 and c-Myc were evaluated using real-time PCR. The proliferation rate and colony formation were also assessed using a BrdU assay and crystal violet staining, respectively. The level of glutathione was assessed using cell fluorescence staining. Compared to nondiabetic serum, diabetic serum contained a higher percentage of oleate (1.5-fold, p < 0.01). In comparison with nondiabetic FFAs, diabetic FFAs demonstrated decreasing effects on the expression of CD90 (-51%, p < 0.001) and c-Myc (-48%, p < 0.05), and proliferation rate (-35%, p < 0.001), colony formation capacity (-50%, p < 0.01), and GSH levels (-62%, p < 0.05). The negative effect of the FFAs of diabetic serum on the stemness characteristics may impair the regenerative capabilities of AdMSCs.

The porcupine inhibitor WNT974 provokes ectodermal lineage differentiation of human embryonic stem cells.

Porcupine (Porcn) enzyme plays an essential role in Wnt signaling activation. Stearoyl-CoA desaturase-1 (SCD1) is required to provide Porcn substrates. The aim of this study was to determine the effect of a novel Porcn inhibitor on the fate of human embryonic stem cells (hESCs) and the reliance of Porcn on SCD1 activity. hESCs were cultured on a feeder layer or Matrigel-coated plates. Small molecules WNT974 (LGK-974) and CAY10566 were used to inhibit Porcn and SCD1 activity, respectively. We assessed the effect of Porcn inhibition on viability, expression of Wnt signaling targets, pluripotency markers, proliferation, differentiation, and protein fatty acylation. hESCs' conditioned medium (CM) containing secreted Wnt proteins were applied in rescue experiments. To examine the catalytic dependency of Porcn on SCD1, the results of combined inhibitor treatment were compared with the SCD1 inhibitor alone. LGK-974 at the selected concentrations showed mild effects on hESCs viability, but significantly reduced messenger RNA and protein expression of Wnt signaling targets (Axin-2 and c-Myc) and pluripotency markers (OCT-4 and SOX-2) (p < .05). Adding 1 µM of Porcn inhibitor reduced proliferation (p = .03) and enhanced differentiation capacity into ectodermal progenitors (p = .02), which were reverted by CM. Click chemistry reaction did not show significant alteration in protein fatty acylation upon LGK-974 treatment. Moreover, combined inhibitor treatment caused no further substantial reduction in Wnt signaling targets, pluripotency markers, and protein fatty acylation relative to CAY10566-treated cultures. The substrate availability for Porcn activity is regulated by SCD1 and targeting Porcn by LGK-974 prompts the transition of hESCs from self-renewal state to ectodermal lineage.

A small molecule modulating monounsaturated fatty acids and Wnt signaling confers maintenance to induced pluripotent stem cells against endodermal differentiation.

BACKGROUND: Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. Through the fatty acid acylation process, this enzyme orchestrates post-translational modifications to proteins involved in cell development and differentiation. In this study, we used biochemical methods, immunostaining, and covalent labeling to evaluate whether a small molecule modulating unsaturated fatty acids can influence the early endodermal differentiation of human-induced pluripotent stem cells (iPSCs). METHODS: The hiPSCs were cultured in an endoderm-inducing medium containing activin A and defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The cell cycles and the yields of the three germ layers (endoderm, mesoderm, and ectoderm) were assessed using flow cytometry. The expression of endoderm and pluripotency markers and the expressions of Wnt signaling pathway proteins were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction. RESULTS: When SCD1 was inhibited on the first day, the population of cells with endodermal features decreased at the end of differentiation. Moreover, early SCD1 inhibition preserved the properties of hiPSCs, preventing their shift toward mesodermal or ectodermal lineage. Also, first-day-only treatment of cells with the SCD1 inhibitor decreased ß-catenin gene expression and the intensity of fluorescent emission in the click chemistry assay. The cells were effectively rescued from these effects by cotreatment with oleate. Late treatment with the inhibitor in the two subsequent days of endoderm induction did not have any significant effects on endoderm-specific markers or fluorescent intensity. Reproducible results were also obtained with human embryonic stem cells. CONCLUSION: The small molecule SCD1 inhibitor attenuates the Wnt/ß-catenin signaling pathway, conferring the maintenance of hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in the endoderm commitment of pluripotent stem cells may be of importance in disorders of endoderm-derived organs and dysregulated metabolism. The schematic representation of the study design and main results. Activin A induces endoderm features through Smad2/3/4 and increases the expression of SCD1. SCD1 can produce MUFAs and subsequently modify the Wnt molecules. MUFA acylated/activated Wnts are secreted to interact with corresponding receptors on the target cells. ß-catenin accumulates in the cytoplasm and is translocated into the nucleus after the interaction of Wnt with the receptor. Then, ß-catenin increases the expression of the endoderm markers Sox17 and CXCR4.

Activation of toll-like receptor signaling in endothelial progenitor cells dictates angiogenic potential: from hypothesis to actual state.

Human endothelial progenitor cells (EPCs) were isolated from cord blood samples and enriched by magnetic activated cell sorting method based on the CD133 marker. Cells were incubated with different doses of bacterial lipopolysaccharide, ranging from 2, 5, 10, 50, 100, 200, 250, 500, to 1000 µg/ml, for 48 h. The cell survival rate was determined by using MTT assay. To confirm activation of the toll-like receptor signaling pathway, PCR array analysis was performed. Protein levels of ERK1/2, p-ERK1/2, NF-ƙB and TRIF proteins were measured using western blotting. The content of TNF-α and lipoprotein lipase activity were analyzed by immunofluorescence imaging. Flow cytometric analysis of CD31 was performed to assess the maturation rate. Cell migration was studied by the Transwell migration assay. The expression of genes related to exosome biogenesis was measured using real-time PCR analysis. In vivo gel plug angiogenesis assay was done in nude mice. Lipopolysaccharide changed endothelial progenitor cells' survival in a dose-dependent manner with maximum viable cells in groups treated with 2 µg/ml. PCR array analysis showed the activation of toll-like signaling pathways after exposure to LPS (p<0.05). Western blotting analysis indicated an induction of p-ERK1/2 and Erk1/2, NF-kB and TRIF in LPS-treated EPCs compared with the control (p<0.05). Immunofluorescence staining showed an elevation of TNF-α and lipoprotein lipase activity after lipopolysaccharide treatment (p<0.05). Lipopolysaccharide increased EPC migration and expression of exosome biogenesis-related genes (p<0.05). In vivo gel plug analysis revealed enhanced angiogenesis in cells exposed to bacterial lipopolysaccharide. Data highlighted the close relationship between the toll-like receptor signaling pathway and functional activity in EPCs.

Steroid-depleted endometriosis serum improves oocyte maturation in IVM systems.

In vitro maturation (IVM) is a novel approach to overcome the adverse effects of human in vitro fertilization (IVF). The aim of the present study is to evaluate the effect of total and steroid-depleted serum obtained from patients with endometriosis on IVM outcome as supplementation for this system. To this purpose, patients with endometriosis were selected according to in/excluding criteria. Germinal vesicles (GVs) and cumulus cells were treated with 10% of each serum. The expression levels of stearoyl CoA desaturase 1 (SCD 1) and cyclooxygenase-2 (COX-2) genes were evaluated by RT-qPCR. Gas-liquid chromatography and flow cytometry were performed to analyze fatty acids composition and apoptosis. The mRNA expression levels of SCD1 (2.47 fold) and COX-2 (6.4 fold), and also the synthesis of oleate, linoleate, and arachidonate were increased (1.19, 1.06, and 2.37 folds, respectively) in cumulus cells treated with steroid-depleted serum (p < .05). The synthesis of palmitate, palmitoleate, and stearate (0.995, 0.67, and 0.7 folds, respectively) and also the rate of apoptosis were significantly decreased in these cells (p < .05). Moreover, GVs cultured in steroid-depleted group showed a significantly higher rate of maturation (p < .001). Overall, our findings imply a new insight into the expansion of IVM system in oocytes development.

Fatty acids of follicular fluid phospholipids and triglycerides display distinct association with IVF outcomes.

RESEARCH QUESTION: Are triglyceride fatty acids in the follicular fluid associated with either follicular fluid phospholipid fatty acids or IVF outcomes and, if so, how are they associated? DESIGN: In a prospective cross-sectional study, 70 women undergoing intracytoplasmic sperm injection were recruited. Follicular fluid phospholipids and triglycerides were separated by thin-layer chromatography. Fatty acids were measured using gas-liquid chromatography and flame ionization detection system. RESULTS: Significant differences in fatty acid composition were observed between follicular fluid phospholipid and triglyceride fractions. Phospholipid stearic acid and n-3 polyunsaturated fatty acids, particularly alpha-linolenic acid, were negatively associated with the number of mature oocytes and cleaved embryos, whereas arachidonic acid was in direct correlation with cleavage rate per IVF cycle (ß = 0.325, P = 0.022). In the case of triglyceride fraction, total monounsaturated fatty acids, oleic acid in particular, displayed significantly positive associations with the number of oocytes (ß = 0.261, P = 0.043) and embryos (ß = 0.310, P = 0.018). Furthermore, cleavage rate correlated inversely with palmitic acid (ß = -0.359, P = 0.007) and directly with pentadecanoic acid (ß = 0.378, P = 0.005). Most of these associations, however, were not independent of predictive fatty acids belonging to phospholipid fraction, according to multivariate analysis. CONCLUSIONS: Fatty acid compositions of phospholipid and triglyceride fractions from human follicular fluid differentially correlate with IVF cycle parameters.

A mechanical non-enzymatic method for isolation of mouse embryonic fibroblasts.

Mouse embryonic fibroblasts (MEFs) accessibility coupled with their simple generation make them as a typical embryonic cell model and feeder layer for in vitro expansion of pluripotent stem cells (PSCs). In this study, a mechanical isolation technique was adopted to isolate MEFs and the efficiency of this technique was compared with enzymatic digestion method. The suspended MEFs were prepared either by mechanical method or 0.25% trypsin enzymatic digestion. The effect of tissue processing on cell apoptosis/necrosis, morphology, viable cell yield, population doubling time, surface marker expression, and the capacity to support PSCs were determined. The mechanical method yielded a significantly higher number of viable cells. However, it showed similar morphology and proliferation characteristics as compared to enzymatic digestion. The mechanical method induced slight apoptosis in MEFs; however, it did not exert the necrotic effect of trypsinization. Treatment of tissue slurry with trypsin solution caused cell lysis and subsequently cell clump formation. Mechanically isolated cells exhibited a higher expression of the MEF surface antigens Sca1, CD106, and CD105. The PSCs on mechanically isolated MEFs displayed a higher expression of pluripotency genes, and formed more compact colonies with a stronger tendency to crowding compared with those cultured on cells isolated by enzymatic digestion. The mechanical method based on tissue inter-syringe processing is relatively a rapid and simple method for MEF isolation. Compared to the enzymatic digestion, the cells obtained from this method show higher expression of embryonic fibroblasts markers and a more functional capacity in supporting PSCs culture.

Fractalkine and apoptotic/anti-apoptotic markers in granulosa cells of women with polycystic ovarian syndrome.

Owing to the role of fractalkine in regulating cellular apoptosis/proliferation, we investigated fractalkine effects on apoptosis/proliferation signaling of granulosa cells in polycystic ovarian syndrome (PCOS) patients through in vitro and in vivo experiments. In vivo, granulosa cells were collected from 40 women undergoing oocyte retrieval (20 controls and 20 PCOS). The expression levels of fractalkine, BAX, Bcl2, Bcl2-XL, Bad, and TNF-α were assessed using RT-PCR. In vitro, we determined the effect of different doses of fractalkine on the expression of the above mentioned genes in GCs of both groups. We found that the expression levels of fractalkine and Bcl-2 were significantly lower in the GCs of PCOS patients compared to the control group (p < 0.05). In contrast, the expression levels of TNF-α and BAX were higher in the patient's group than in the control group. The results suggested that expression levels of fractalkine were negatively and positively correlated with the number of oocytes and fertilized oocytes respectively. Moreover, fractalkine could dose-dependently increase fractalkine and decrease BAD, BAX, Bcl-xl, and TNF-α expressions in the control GCs. In contrast, GCs collected from PCOS patients revealed an increase in expression of BAD, BAX, and Bcl-xl following fractalkine treatment. Our findings indicated that insufficient expression of fractalkine in PCOS patients is related with elevated apoptotic and inflammatory markers and reduced anti-apoptotic genes in the GCs.

Effect of flaxseed oil supplementation on the erythrocyte membrane fatty acid composition and endocannabinoid system modulation in patients with coronary artery disease: a double-blind randomized controlled trial.

BACKGROUND: The endocannabinoid system (ECS) overactivation, associated with increased inflammatory process, may act as a risk factor for coronary artery disease (CAD). Dietary fat may influence the ECS tone. The aim of the present study was to investigate the effect of flaxseed oil on the erythrocyte membrane fatty acid profile and ECS activity by the measurement of serum N-arachydonoil ethanolamine (AEA) and cannabinoid receptor type-1 (CB1), cannabinoid receptor type-2 (CB2), and fatty acid amide hydrolase (FAAH) mRNA expression. METHODS: This clinical trial was performed on 44 patients with CAD. The intervention group received 1.5% fat milk supplemented with flaxseed oil (containing 2.5 g α-linolenic acid or ALA), while the placebo group received 1.5% fat milk for 10 weeks. The fatty acid profile of erythrocyte membrane phospholipids was measured by gas chromatography. The AEA level was determined using an ELISA kit, and real-time PCR was performed to measure CB1, CB2, and FAAH mRNA expression pre- and post-intervention. RESULTS: Flaxseed oil supplementation resulted in a significant increase in the ALA content and a significant reduction in linoleic acid (LA) content of membrane phospholipids, compared to the placebo group (MD = - 0.35 and 2.89, respectively; P < 0.05). The within group analysis showed that flaxseed oil supplementation caused a significant reduction in both LA and arachidonic acid (MD = - 4.84 and - 4.03, respectively; P < 0.05) and an elevation in the ALA (MD = 0.37, P < 0.001) content of membrane phospholipids compared with the baseline. In the intervention group, a marked reduction was observed in the serum AEA level after 10 weeks of intervention, compared with the placebo group (MD = 0.64, P = 0.016). Changes in CB2 mRNA expression in the flaxseed oil group were significant (fold change = 1.30, P = 0.003), compared with the placebo group. CONCLUSION: Flaxseed oil supplementation could attenuate the ECS tone by decreasing the AEA level and increasing CB2 mRNA expression. Therefore, flaxseed oil may be considered a promising agent with cardioprotective properties.

Application of porcupine inhibitors in stem cell fate determination.

Porcupine (Porcn), a membrane-bound O-acyltransferase, is an endoplasmic reticulum-located protein that has catalytic activity. Porcn is involved in post-translational lipid modification of wingless-Int (Wnt) proteins and serves as an indispensable step in the Wnt proper secretion and signaling. Small-molecule inhibitors targeting Porcn catalytic function in vitro and in vivo are of great interest not only for treating cancer and fibrotic disorders but also in the field of regenerative medicine. Although a number of studies have been conducted, the exact role of Porcn in stem cell fate is not entirely clear. In some cases, Porcn inhibition declined differentiation rate, and in others, it induced stem cell differentiation toward specific lineages. In this review, we first elaborated the Porcn catalytic activity and its inhibitors. Then, we discussed about the recently reported results of Porcn inhibitors in stem cells self-renewal and differentiation.

Arachidonic acid alleviates the detrimental effects of acetylsalicylic acid on human granulosa cells performance in vitro.

Here, we investigated the biological effects of arachidonic acid (AA) in human cumulus granulosa cells (CGCs) after exposure to ASA. Cells were isolated from the follicular fluid and incubated with 0.5 mM acetylsalicylic acid (ASA) and 50 µM AA. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. E2 and P4 levels were measured by chemiluminescence assay. Expression of genes including CYP19A1, FACN, and SCD1 was measured by real-time polymerase chain reaction assay. Oxidative status was analyzed by monitoring glutathione peroxidase activity. The fatty acid profile was analyzed by the gas chromatography technique. Enzyme-linked immunosorbent assay was used to measure prostaglandin E2 (PGE2 ) in CGCs after exposure to ASA and AA. Protein levels of the estrogen receptor were studied by immunofluorescence staining. Ultrastructural changes were evaluated by transmission electron microscopy imaging. ASA treatment reduced E2 production, Cyp19a1 expression, glutathione peroxidase (GPx) activity, and estradiol receptor expression in CGCs. The addition of AA prevented the ASA-induced E2 reduction (p < .05) and expression of Cyp19a1. Moreover, AA increased the antioxidant capacity of CGCs exposed to ASA by promoting GPx activity (p < .05). AA increased monounsaturated fatty acid/saturated fatty acid ratio compared with the ASA group (p < .05). AA supplementation triggered the synthesis and secretion of PGE2 in ASA-treated CGCS (p < .05). Cytoplasmic vacuolation observed in the ASA group and treatment with AA intensified vacuolation rate. The expression of the estrogen receptor was increased after AA supplementation. Data demonstrated that AA decreased the detrimental effects of ASA on human CGCs after 72 hr.

Autophagy modulation altered differentiation capacity of CD146+ cells toward endothelial cells, pericytes, and cardiomyocytes.

BACKGROUND: To date, many attempts are employed to increase the regenerative potential of stem cells. In this study, we evaluated the hypothesis of whether an autophagy modulation could alter differentiation potency of CD146+ cells into mature pericyte, endothelial, and cardiomyocyte lineage. METHODS: In this study, CD146+cells were enriched from the human bone marrow aspirates and trans-differentiated into mature endothelial cells, pericytes, and cardiomyocytes after exposure to autophagy stimulator (50-µM Met)/inhibitor (15-µM HCQ). The protein levels of autophagy proteins were monitored by western blotting. NO content was measured using the Griess assay. Using real-time PCR assay and western blotting, we monitored the lineage protein and gene levels. Pro-inflammatory cytokine and angiocrine factors were measured by ELISA. The fatty acid change was determined by gas chromatography. We also measured exosome secretion capacity by measuring AChE activity and real-time PCR assay. RESULT: Data revealed the modulation of autophagy factors, Beclin-1, P62, and LC3 II/I ratio in differentiating CD146+ cells after exposure to Met and HCQ (p < 0.05). The inhibition of autophagy increased NO content compared to the Met-treated cells (p < 0.05). Real-time PCR analysis showed that the treatment of CD146+ cells with autophagy modulators altered the expression of VE-cadherin, cTnI, and α-SMA (p < 0.05). Met increased the expression of VE-cadherin, α-SMA, and cTnI compared to the HCQ-treated cells (p < 0.05) while western blotting revealed the protein synthesis of all lineage-specific proteins under the stimulation and inhibition of autophagy. None statistically significant differences were found in the levels of Tie-1, Tie-2, VEGFR-1, and VEGFR-2 after autophagy modulation. Fatty acid profile analysis revealed the increase of unsaturated fatty acids after exposure to HCQ (p < 0.05). The treatment of cells with HCQ increased the levels of TNF-α and IL-6 compared to the Met-treated cells. Data revealed the increase of exosome biogenesis and secretion to the supernatant in cells treated with HCQ compared to the Met groups (p < 0.05). CONCLUSIONS: In summary, autophagy modulation could alter differentiation potency of CD146+cells which is important in cardiac regeneration.

Synergistic Beneficial Effect of Docosahexaenoic Acid (DHA) and Docetaxel on the Expression Level of Matrix Metalloproteinase-2 (MMP-2) and MicroRNA-106b in Gastric Cancer.

BACKGROUND: Gastric cancer (GC) is one of the most common cancers with the majority of patients recognized in advanced stages. The efficacy of using docosahexaenoic acid (DHA) as a supplementary agent has been suggested in treatment along with chemotherapeutics including docetaxel. However, the molecular signatures of such beneficial effects are not well-understood. OBJECTIVE(S): We aimed to evaluate the effects of DHA and docetaxel on the expression level of metastasis-related genes, including MMP-2 and talin-2, and their controlling miRNAs, miR-106b and miR-194, in metastatic GC cell line, MKN45. METHOD(S): GC cell line, MKN45, was cultured, and determination of IC50 of DHA was done by MTT test. Cells were treated with docetaxel, DHA, and their combination for 24 h, and then total RNA was extracted and cDNA synthesis was done using standard protocols. The expression level of target genes, MMP-2 and talin-2, and miR-106b and miR-194 were determined by using quantitative real-time PCR. RESULTS: The expression level of MMP-2 was decreased significantly in all treated cells. However, talin-2 showed significant downregulation only after treatment with docetaxel. In contrary to increased expression after treatment with docetaxel, DHA led to a significant under-expression of miR-106b. The similar effect was seen for miR-194. CONCLUSION(S): Combination of docetaxel and DHA led to the significant downregulation of MMP-2. Also, DHA lowered the docetaxel-mediated upregulation of miR-106b oncomiR. In conclusion, supplementation of docetaxel therapy with DHA in GC patients would be considered as a beneficial approach in cancer treatment.

DHA Abolishes the Detrimental Effect of Docetaxel on Downregulation of the MICA via Decreasing the Expression Level of MicroRNA-20a in Gastric Cancer.

BACKGROUND: MHC class I chain-related protein A (MICA) is a membrane glycoprotein expressed abnormally on some malignant cells including gastric cancer (GC) cell and elicits anti-tumor immune responses. Downregulation of MICA expression could lead to immune-evasion of cancer cells. OBJECTIVE(S): In this study, we aimed to investigate the effect of docosahexaenoic acid (DHA) and docetaxel alone or in combination on the expression level of MICA and its regulating microRNA (miRNA), miR-20a in MKN45 GC cell line. METHOD(S): MKN45 GC cell line was cultured and MTT assay was performed to determine IC50 of docetaxel. Cells were treated by 18.5 µM docetaxel and 100 µM DHA. After that, RNA extraction and cDNA synthesis were done and the expression level of MICA and miR-20a were determined by quantitative real-time PCR for both treated and untreated cell lines. RESULTS: Our findings showed less downregulation of the expression level of MICA by the combination of docetaxel/DHA (5.34-fold) compared with docetaxel (45.45-fold) and DHA (55.55-fold). Consistently, combination therapy led to the more downregulation of the expression level of the miR-20a (5.20-fold) in comparison to docetaxel (2.38-fold) and DHA (1.60-fold). CONCLUSION(S): As an unwanted effect of docetaxel therapy in GC, downregulation of MICA expression could lead to weak anti-tumor immune responses. By increasing the expression level of MICA, combination therapy of docetaxel with DHA would be useful to overcome this side effect.

Cord blood stem cell derived CD16+ NK cells eradicated acute lymphoblastic leukemia cells using with anti-CD47 antibody.

Acute lymphoblastic leukemia (ALL) is an aggressive cancer in children and adults which possess higher CD47 expression than normal cells. ALL chemotherapy has a lot of side effects and in most cases is ineffective. However arrival of Natural killer (NK) cell immunotherapy raised hopes for successful treatment of cancers, tailoring NK cells to meet clinical requirements is still under investigation. Of note, CD16+ (FCγIIIa) NK cells eliminate tumor cells with antibody dependent cell cytotoxicity (ADCC) mechanism. Therefore, we evaluated ADCC effect of cord blood stem cell derived CD16+ NK cells with using anti CD47 blocking antibody. CD16+ NK cells generated efficiently from CD34 positive cord blood cells in vitro using IL-2, IL-15 and IL-21 cytokines, although it was not dose dependent. CD16+ cells derived from CD34+ cells in day 14 of culture efficiently increased apoptosis in ALL cells, produced INFγ and increased CD107-a expression when used anti CD47 antibody (increased around 30-40%). Interestingly, CD16+ NK cell cytotoxicity slightly increased in combination with macrophages against ALL cells (around 10%). Taken together, our findings induced this hope that cord blood stem cell derived CD16+ NK cells exploit antitumor immune response in cancer therapy with using anti-CD47 antibody.

Early oleate deficiency leads to severe defects in fetal rat liver development.

OBJECTIVES: Oleate can be produced through de novo synthesis, which contributes to biological processes and signaling pathways. However, the role of this non-essential fatty acid in hepatic development remains unclear. The current study aimed to evaluate the influence of early oleate deficiency induced by the inhibitor of de novo oleate synthesis MF-438 on fetal rat liver development. MATERIALS AND METHODS: Female Wistar rats with an average weight of 200±20 g were subjected to this study. After mating, pregnant rats were divided into three groups and gavaged with the vehicle, MF 438 or MF-438 plus oleate from day 3 of pregnancy for five days. Obtained fetuses were sacrificed and the liver tissues were retrieved. Hepatic morphological index, biochemical markers, and gene expression of hepatic development markers were analyzed using Hematoxylin-Eosine, spectrometry, and real-time PCR techniques, respectively. RESULTS: Relatively, deficient morphological indices and hepatic maturation markers were observed in fetus livers of the inhibitor-treated group. In comparison to the other two groups, total hepatic protein and glycogen content were increased with treatment of MF-438 plus oleate. Hepatocyte nuclear factor 1α, alpha fetoprotein, albumin, and cytochrome P450 gene expression were also significantly increased in the group treated with both MF-438 and oleate. CONCLUSION: Our data indicate that oleate availability during early embryo development is linked with fetal rat liver development.