All-trans-retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells. METHODS: MCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(ß) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student's t-test or one-way ANOVA was used to analyze data from replicates. RESULTS: Our results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(ß) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(ß) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression. CONCLUSIONS: To the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.

Shoe configuration effects on third phalanx and capsule motion of unaffected and laminitic equine hooves in-situ.

Equine shoes provide hoof protection and support weakened or damaged hoof tissues. Two hypotheses were tested in this study: 1) motion of the third phalanx (P3) and hoof wall deformation are greater in laminitic versus unaffected hooves regardless of shoe type; 2) P3 displacement and hoof wall deformation are greatest while unshod (US), less with open-heel (OH), then egg-bar (EB) shoes, and least with heart-bar (HB) shoes for both hoof conditions. Distal forelimbs (8/condition) were subjected to compressive forces (1.0x102-5.5x103 N) while a real-time motion detection system recorded markers on P3 and the hoof wall coronary band, vertical midpoint, and solar margin. Magnitude and direction of P3 displacement and changes in proximal and distal hemi-circumference, quarter and heel height and proximal and distal heel width were quantified. Hoof condition and shoe effects were assessed with 2-way ANOVA (p<0.05). P3 displacement was greater in laminitic hooves when US or with OH, and EB and HB reduced P3 displacement in laminitic hooves. P3 displacement was similar among shoes in unaffected hooves and greatest in laminitic hooves with OH, then US, EB and HB. EB and HB increased P3 displacement from the dorsal wall in unaffected hooves and decreased it in laminitic hooves. OH and EB increased P3 motion from the coronary band in laminitic hooves, and HB decreased P3 motion toward the solar margin in unaffected and laminitic hooves. In laminitic hooves, HB reduced distal hemi-circumference and quarter deformation and increased heel deformation and expansion. Proximal hemi-circumference constriction was inversely related to proximal heel expansion with and without shoes. Overall, shoe configuration alters hoof deformation distinctly between unaffected and laminitic hooves, and HB provided the greatest P3 stability in laminitic hooves. These unique results about P3 motion and hoof deformation in laminitic and unaffected hooves inform shoe selection and design.

Enhancing cancer treatment and understanding through clustering of gene responses to categorical stressors.

Cancer cells have a unique metabolic activity in the glycolysis pathway compared to normal cells, which allows them to maintain their growth and proliferation. Therefore, inhibition of glycolytic pathways may be a promising therapeutic approach for cancer treatment. In this novel study, we analyzed the genetic responses of cancer cells to stressors, particularly to drugs that target the glycolysis pathway. Gene expression data for experiments on different cancer cell types were extracted from the Gene Expression Omnibus and the expression fold change was then clustered after dimensionality reduction. We identified four groups of responses: the first and third were most affected by anti-glycolytic drugs, especially those acting on multiple pathways at once, and consisted mainly of squamous and mesenchymal tissues, showing higher mitotic inhibition and apoptosis. The second and fourth groups were relatively unaffected by treatment, comprising mainly gynecologic and hormone-sensitive groups, succumbing least to glycolysis inhibitors. Hexokinase-targeted drugs mainly showed this blunted effect on cancer cells. This study highlights the importance of analyzing the molecular states of cancer cells to identify potential targets for personalized cancer therapies and to improve our understanding of the disease.

Microarray analysis of breast cancer gene expression profiling in response to 2-deoxyglucose, metformin, and glucose starvation.

BACKGROUND: Breast cancer (BC) is the most frequently diagnosed cancer in women. Altering glucose metabolism and its effects on cancer progression and treatment resistance is an emerging interest in BC research. For instance, combining chemotherapy with glucose-lowering drugs (2-deoxyglucose (2-DG), metformin (MET)) or glucose starvation (GS) has shown better outcomes than with chemotherapy alone. However, the genes and molecular mechanisms that govern the action of these glucose deprivation conditions have not been fully elucidated. Here, we investigated the differentially expressed genes in MCF-7 and MDA-MB-231 BC cell lines upon treatment with glucose-lowering drugs (2-DG, MET) and GS using microarray analysis to study the difference in biological functions between the glucose challenges and their effect on the vulnerability of BC cells. METHODS: MDA-MB-231 and MCF-7 cells were treated with 20 mM MET or 4 mM 2-DG for 48 h. GS was performed by gradually decreasing the glucose concentration in the culture medium to 0 g/L, in which the cells remained with fetal bovine serum for one week. Expression profiling was carried out using Affymetrix Human Clariom S microarrays. Differentially expressed genes were obtained from the Transcriptome Analysis Console and enriched using DAVID and R packages. RESULTS: Our results showed that MDA-MB-231 cells were more responsive to glucose deprivation than MCF-7 cells. Endoplasmic reticulum stress response and cell cycle inhibition were detected after all three glucose deprivations in MDA-MB-231 cells and only under the metformin and GS conditions in MCF-7 cells. Induction of apoptosis and inhibition of DNA replication were observed with all three treatments in MDA-MB-231 cells and metformin-treated MCF-7 cells. Upregulation of cellular response to reactive oxygen species and inhibition of DNA repair mechanisms resulted after metformin and GS administration in MDA-MB-231 cell lines and metformin-treated MCF-7 cells. Autophagy was induced after 2-DG treatment in MDA-MB-231 cells and after metformin in MCF-7 cells. Finally, inhibition of DNA methylation were observed only with GS in MDA-MB-231 cells. CONCLUSION: The procedure used to process cancer cells and analyze their expression data distinguishes our study from others. GS had the greatest effect on breast cancer cells compared to 2-DG and MET. Combining MET and GS could restrain both cell lines, making them more vulnerable to conventional chemotherapy.

Dietary fructose and its association with the metabolic syndrome in Lebanese healthy adults: a cross-sectional study.

BACKGROUND: Epidemiological studies investigating the association between dietary fructose intake and the metabolic syndrome (MetS) are scarce and have produced controversial findings. This study aimed at (1) assessing total dietary fructose intake in a sample of Lebanese healthy adults, and determining the intake levels of natural vs. added fructose; (2) investigating the association of dietary fructose with MetS; and (3) identifying the socioeconomic and lifestyle factors associated with high fructose intake. METHODS: A cross-sectional survey was conducted on a representative sample of adults living in Beirut, Lebanon (n = 283). Anthropometric and biochemical data were collected, and dietary intake was assessed using a food frequency questionnaire. Intakes of naturally-occurring fructose from fructose-containing food sources, such as fruits, vegetables, honey, were considered as "natural fructose". Acknowledging that the most common form of added sugar in commodities is sucrose or High Fructose Corn Syrup (HFCS), 50% of added sugar in food products was considered as added fructose. Total dietary fructose intake was calculated by summing up natural and added fructose intakes. Logistic regression analyses were conducted to investigate the association of total, added and natural fructose intakes with the MetS and to identify the socioeconomic predictors of high fructose intake. RESULTS: Mean intake of total fructose was estimated at 51.42 ± 35.54 g/day, representing 6.58 ± 3.71% of energy intakes (EI). Natural and added fructose intakes were estimated at 12.29 ± 8.57 and 39.12 ± 34.10 g/day (1.78 ± 1.41% EI and 4.80 ± 3.56% EI), respectively. Participants in the highest quartile of total and added fructose intakes had higher odds of MetS (OR = 2.84, 95%CI: 1.01, 7.94 and OR = 3.18, 95%CI: 1.06, 9.49, respectively). In contrast, natural fructose intake was not associated with MetS. Age, gender and crowding index were identified as factors that may modulate dietary fructose intakes. CONCLUSIONS: The observed association between high added fructose intake and the MetS highlights the need for public health strategies aimed at limiting sugar intake from industrialized foods and promoting healthier dietary patterns in Lebanon.

5 ns electric pulses induce Ca2+-dependent exocytotic release of catecholamine from adrenal chromaffin cells.

Previously we reported that adrenal chromaffin cells exposed to a 5 ns, 5 MV/m pulse release the catecholamines norepinephrine (NE) and epinephrine (EPI) in a Ca2+-dependent manner. Here we determined that NE and EPI release increased with pulse number (one versus five and ten pulses at 1 Hz), established that release occurs by exocytosis, and characterized the exocytotic response in real-time. Evidence of an exocytotic mechanism was the appearance of dopamine-ß-hydroxylase on the plasma membrane, and the demonstration by total internal reflection fluorescence microscopy studies that a train of five or ten pulses at 1 Hz triggered the release of the fluorescent dye acridine orange from secretory granules. Release events were Ca2+-dependent, longer-lived relative to those evoked by nicotinic receptor stimulation, and occurred with a delay of several seconds despite an immediate rise in Ca2+. In complementary studies, cells labeled with the plasma membrane fluorescent dye FM 1-43 and exposed to a train of ten pulses at 1 Hz underwent Ca2+-dependent increases in FM 1-43 fluorescence indicative of granule fusion with the plasma membrane due to exocytosis. These results demonstrate the effectiveness of ultrashort electric pulses for stimulating catecholamine release, signifying their promise as a novel electrostimulation modality for neurosecretion.

DKK1 promotes hepatocellular carcinoma inflammation, migration and invasion: Implication of TGF-ß1.

Dickkopf-1 (DKK1), an inhibitor of the most frequently impaired signaling pathway in hepatocellular carcinoma (HCC), the Wnt/beta-catenin pathway, seems to fulfill contradictory functions in the process of tumorigenesis, acting either as an oncogenic promoter of metastasis or as a tumor suppressor. Elevated serum levels of DKK1 have been reported in HCC; however, little is known about its functional significance. In the current study, we treated HepG2/C3A and PLC/PRF/5 with the recombinant protein DKK1. Cytotoxicity was first determined by the WST-8 assay. AFP expression was measured at both the mRNA and protein levels. Expression of the oncogenes MYC, CCND1, hTERT, and MDM2 and the tumor suppressor genes TP53, P21 and RB was assessed. Western blot analysis of non-phosphorylated ẞ-catenin and Sanger sequencing were performed to explain the functional differences between the two cell lines. Subsequently, inflammation, migration and invasion were evaluated by qPCR, ELISA, the Boyden chamber assay, zymography, and MMP-2 and MMP-9 western blot analysis. Knockdown of DKK1 and TGF-ß1 were also performed. Our results suggest that DKK1 exerts an oncogenic effect on HepG2/C3A cell line by upregulating the expression of oncogenes and downregulating that of tumor suppressor genes, whereas the opposite effect was demonstrated in PLC/PRF/5 cells. This differential impact of DKK1 can be explained by the mutations that affect the canonical Wnt pathway that were detected in exon 3 of the CTNNB1 gene in the HepG2 cell line. We further confirmed that DKK1 promotes inflammation, tumor invasion and migration in both cell types. The canonical pathway was not responsible for the DKK1 proinvasive effect, as indicated by the active ẞ-catenin levels in the two cell lines upon DKK1 treatment. Interestingly, knockdown of TGF-ß1 negatively affected the DKK1 proinvasive effect. Taken together, DKK1 appears to facilitate tumor invasion and migration through TGF- ß1 by remodeling the tumor microenvironment and inducing inflammation. This finding endorses the relevance of TGF-ß1 as a therapeutic target.