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Bevacizumab (Bev) a humanized monoclonal antibody that fights vascular endothelial growth factor A (VEGF-A). It was the first specifically considered angiogenesis inhibitor and it has now become the normative first-line therapy for advanced non-small-cell lung cancer (NSCLC). In the current study, polyphenolic compounds were isolated from bee pollen (PCIBP) and encapsulated (EPCIBP) inside moieties of hybrid peptide-protein hydrogel nanoparticles in which bovine serum albumin (BSA) was combined with protamine-free sulfate and targeted with folic acid (FA). The apoptotic effects of PCIBP and its encapsulation (EPCIBP) were further investigated using A549 and MCF-7 cell lines, providing significant upregulation of Bax and caspase 3 genes and downregulation of Bcl2, HRAS, and MAPK as well. This effect was synergistically improved in combination with Bev. Our findings may contribute to the use of EPCIBP simultaneously with chemotherapy to strengthen the effectiveness and minimize the required dose.

Nine new flavonoids dimers, psocorylins R-Z (1-9), were isolated from the fruits of Psoralea corylifolia L. (Psoraleae Fructus), a traditional Chinese medicine. The structures of these compounds were elucidated via multiple spectroscopic techniques and X-ray diffraction. Psocorylins R (1) and S (2) were rare cyclobutane-containing chalcone dimers, and psocorylins T-Z (3-9) were established by CC or COC bond of two flavonoid monomers. The structural-types, flavonoids dimers, were isolated from the plant for the first time, enriching the chemical diversity. The cytotoxicity assay suggested that compounds 1, 2, 4, 5, 6 and 8 exhibited cytotoxic activities against MCF-7 cells. Furthermore, compounds 1 and 8 significantly increased intracellular ROS levels, decreased MMP and induced apoptosis of MCF-7 cells. They markedly upregulated the expression of Bax and cleaved caspase-3, and suppressed Bcl-2 and caspase-3 levels, indicating their mechanism of Bcl-2/Bax/Cleaved caspase-3 pathway. Hence, our findings not only promoted the chemical investigation of Psoraleae Fructus, but also provided potential bioactive natural products for anti-cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Polyamine levels decrease with menopause; however, little is known about the mechanisms regulated by menopause. In this study, we found that among the genes involved in the polyamine pathway, polyamine oxidase ( PAOX ) mRNA levels were the most significantly reduced by treatment with 17β-estradiol in estrogen receptor (ESR)-positive MCF-7 breast cancer cells. Treatment with 17β-estradiol also reduced the PAOX protein levels. Treatment with selective ESR antagonists and knockdown of ESR members revealed that estrogen receptor 2 (ESR2; also known as ERβ) was responsible for the repression of PAOX by 17β-estradiol. A luciferase reporter assay showed that 17β-estradiol downregulates PAOX promoter activity and that 17β-estradiol-dependent PAOX repression disappeared after deletions (-3126/-2730 and -1271/-1099 regions) or mutations of activator protein 1 (AP-1) binding sites in the PAOX promoter. Chromatin immunoprecipitation analysis showed that ESR2 interacts with AP-1 bound to each of the two AP-1 binding sites. These results demonstrate that 17β-estradiol represses PAOX transcription by the interaction of ESR2 with AP-1 bound to the PAOX promoter. This suggests that estrogen deficiency may upregulate PAOX expression and decrease polyamine levels.

Tissues are subjected to dynamic communication between cells and the extracellular matrix (ECM), resulting in ECM remodeling. One of the ECM components is elastin, which releases elastin-derived peptides (EDPs) during the aging process. Therefore, the aim of the present study was to evaluate the impact of the VGVAPG hexapeptide and elastin-like peptide VVGPGA (control) on certain metabolism parameters in human breast adenocarcinoma (MCF-7) and human lung carcinoma (A549) cell lines. The results did not show a significant effect of the peptides on metabolic activity and caspase-3 activity. However, more specific analysis revealed that VGVAPG and VVGPGA were able to increase KI67 protein expression in both tested cell lines after 24-h treatment. Moreover, the same correlation was observed at the KI67 gene level. VGVAPG also increased the P53, ATM and SHH gene expression in the A549 cells up to 19.08%, 20.74%, and 28.77%, respectively. Interestingly, the VGVAPG peptide exerted an effect on the expression of antioxidant enzymes SOD2 and CAT in the A549 and MCF-7 cells, especially after the 24-h treatment. Lastly, both peptides influenced the CAV1 and CLTC1 expression. Our results show that the tested EDPs have an effect on both A549 and MCF-7 cells at the cellular level. This may be correlated with the multidrug-resistance (MDR) phenotype in these cancer cells, which is an emerging problem in the current anticancer treatment. However, more research is needed in this field., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Bag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms (Bag-1S, M, or L) in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation., Competing Interests: The authors have declared that no competing interests exist.

Heterocycles containing thienopyrimidine moieties have attracted attention due to their interesting biological and pharmacological activities. In this research article, we reported the synthesis of a series of new hybrid molecules through merging the structural features of chalcones and pyridothienopyrimidinones. Our results indicated that the synthesis of chalcone-thienopyrimidine derivatives from the corresponding thienopyrimidine and chalcones proceeded in a relatively short reaction time with good yields and high purity. Most of these novel compounds exhibited moderate to robust cytotoxicity against HepG2 and MCF-7 cancer cells similar to that of 5-fluorouracil (5-FU). The results indicated that IC 50 of the two compounds ( 3b and 3g ) showed more potent anticancer activities against HepG2 and MCF-7 than 5-FU. An MTT assay and flow cytometry showed that only 3b and 3g had anticancer activity and antiproliferative activities at the G1 phase against MCF-7 cells, while six compounds ( 3a - e and 3g ) had cytotoxicity and cell cycle arrest at different phases against HepG2 cells. Their cytotoxicity was achieved through downregulation of Bcl-2 and upregulation of Bax, caspase-3, and caspase-9. Although all tested compounds increased oxidative stress via increment of MDA levels and decrement of glutathione reductase (GR) activities compared to control, the 3a , 3b , and 3g in HepG2 and 3b and 3g in MCF-7 achieved the target results. Moreover, there was a positive correlation between cytotoxic efficacy of the compound and apoptosis in both HepG2 ( R 2 = 0.531; P = 0.001) and MCF-7 ( R 2 = 0.219; P = 0.349) cell lines. The results of molecular docking analysis of 3a-g into the binding groove of Bcl-2 revealed relatively moderate binding free energies compared to the selective Bcl-2 inhibitor, DRO. Like venetoclax, compounds 3a-g showed 2 violations from Lipinski's rule. However, the results of the ADME study also revealed higher drug-likeness scores for compounds 3a-g than for venetoclax. In conclusion, the tested newly synthesized chalcone-pyridothienopyrimidinone derivatives showed promising antiproliferative and apoptotic effects. Mechanistically, the compounds increased ROS production with concomitant cell cycle arrest and apoptosis. Therefore, regulation of the cell cycle and apoptosis are possible targets for anticancer therapy. The tested compounds could be potent anticancer agents to be tested in future clinical trials after extensive pharmacodynamic, pharmacokinetic, and toxicity profile investigations., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Ghada M. Safwat et al.)

Arg-Arg-Leu (RRL) is a potent tumor-homing tripeptide. However, the binding target is unclear. In this study, we intended to identify the binding target of RRL and evaluate the tumor targeting of 99m Tc-MAG 3 -RRL in vivo . Biotin-RRL, 5-TAMRA-RRL, and 99m Tc-MAG 3 -RRL were designed to trace the binding target and tumor lesion. Immunoprecipitation-mass spectrometry was conducted to identify the candidate proteins and determination of the subcellular localization was also performed. A pull-down assay was performed to demonstrate the immunoprecipitate. Fluorescence colocalization and cell uptake assays were performed to elucidate the correlation between the selected binding protein and RRL, and the internalization mechanism of RRL. Biodistribution and in vivo imaging were performed to evaluate the tumor accumulation and targeting of 99m Tc-MAG 3 -RRL. The target for RRL was screened to be heat shock protein 70 (HSP70). The prominent uptake distribution of RRL was concentrated in the membrane and cytoplasm. A pull-down assay demonstrated the existence of HSP70 in the biotin-RRL captured complex. Regarding fluorescence colocalization and cell uptake assays, RRL may interact with HSP70 at the nucleotide-binding domain (NBD). Clathrin-dependent endocytosis and macropinocytosis could be a vital internalization mechanism of RRL. In vivo imaging and biodistribution both demonstrated that 99m Tc-MAG 3 -RRL can trace tumors with satisfactory accumulation in hepatoma xenograft mice. The radioactive signals accumulated in tumor lesions can be blocked by VER-155008, which can bind to the NBD of HSP70. Our findings revealed that RRL may interact with HSP70 and that 99m Tc-MAG 3 -RRL could be a prospective probe for visualizing overexpressed HSP70 tumor sections.

In this article, it was examined whether the combined application of nanoparticle-based radiosensitizers with radiotherapy is beneficial. Nanoparticular or nanoparticle-based radiosensitizers appear promising in terms of feasibility, safety, and efficacy, as evidenced by preliminary results of ongoing studies. However, this method has its own advantages and disadvantages. According to current knowledge, it is impossible to mention that this method is the definitive treatment method because cancer is an individual disease and the treatments may differ from person to person. With the studies to be done, the application methods of optimal combinations with radiotherapy should be defined.

This work combines single cell Raman spectroscopy (RS) with group and basis restricted non-negative matrix factorisation (GBR-NMF) to identify individual biochemical changes associated with radiation exposure in three human cancer cell lines. The cell lines analysed were derived from lung (H460), breast (MCF7) and prostate (LNCaP) tissue and are known to display varying degrees of radio sensitivity due to the inherent properties of each cell type. The GBR-NMF approach involves the deconstruction of Raman spectra into component biochemical bases using a library of Raman spectra of known biochemicals present in the cells. Subsequently, scores are obtained on each of these bases which can be directly correlated with the contribution of each chemical to the overall Raman spectrum. We validated GBR-NMF through the correlation of GBR-NMF-derived glycogen scores with scores that were previously observed using principal component analysis (PCA). Phosphatidylcholine, glucose, arginine and asparagine showed a distinct differential score pattern between radio-resistant and radio-sensitive cell types. In summary, the GBR-NMF approach allows for the monitoring of individual biochemical radiation-response dynamics previously unattainable with more traditional PCA-based approaches.

We present a quantitative study of the metabolic activity of a single spheroid culture of human cancer cells. NMR (nuclear magnetic resonance) spectroscopy is an ideal tool for observation of live systems due to its non-invasive nature. However, limited sensitivity has so far hindered its application in microfluidic culture systems. We have used an optimised micro-NMR platform to observe metabolic changes from a single spheroid. NMR spectra were obtained by directly inserting microfluidic devices containing spheroids ranging from 150 [Formula: see text]m to 300 [Formula: see text]m in diameter in 2.5 [Formula: see text]L of culture medium into a dedicated NMR probe. Metabolite concentrations were found to change linearly with time, with rates approximately proportional to the number of cells in the spheroid. The results demonstrate that quantitative monitoring of a single spheroid with [Formula: see text] 2500 cells is possible. A change in spheroid size by 600 cells leads to a clearly detectable change in the L-Lactic acid production rate ([Formula: see text]). The consumption of D-Glucose and production of L-Lactic acid were approximately 2.5 times slower in spheroids compared to monolayer culture of the same number of cells. Moreover, while cells in monolayer culture were found to produce L-Alanine and L-Glutamine, spheroids showed slight consumption in both cases.

Background: Metabolic reprogramming within cancer cells has been recognized as a potential barrier to chemotherapy. Additionally, metabolic tumor heterogeneity is the one of factors behind discernible hallmarks such as drug resistance, relapse of the tumor and the formation of secondary tumors., Methods: In this paper, cell-based assays including PI/annexin V staining and immunoblot assay were performed to show the apoptotic cell death in MCF-7 cells treated with DOX. Further, MCF-7 cells were lysed in a hypotonic buffer and the whole cell lysate was purified by a novel and specifically designed metabolite (~ 100 to 1000 Da) fractionation system called vertical tube gel electrophoresis (VTGE). Further, purified intracellular metabolites were subjected to identification by LC-HRMS technique., Results: Cleaved PARP 1 in MCF-7 cells treated with DOX was observed in the present study. Concomitantly, data showed the absence of active caspase 3 in MCF-7 cells. Novel findings are to identify key intracellular metabolites assisted by VTGE system that include lipid (CDP-DG, phytosphingosine, dodecanamide), non-lipid (N-acetyl-D-glucosamine, N1-acetylspermidine and gamma-L-glutamyl-L-cysteine) and tripeptide metabolites in MCF-7 cells treated by DOX. Interestingly, we reported the first evidence of doxorubicinone, an aglycone form of DOX in MCF-7 cells that are potentially linked to the mechanism of cell death in MCF-7 cells., Conclusion: This paper reported novel methods and processes that involve VTGE system based purification of hypotonically lysed novel intracellular metabolites of MCF-7 cells treated by DOX. Here, these identified intracellular metabolites corroborate to caspase 3 independent and mitochondria induced apoptotic cell death in MCF-7 cells. Finally, these findings validate a proof of concept on the applications of novel VTGE assisted purification and analysis of intracellular metabolites from various cell culture models.

Mitochondrial dysfunction plays a significant role in the metabolic flexibility of cancer cells. This study aimed to investigate the metabolic alterations due to Coenzyme Q depletion in MCF-7 cells., Method: The Coenzyme Q depletion was induced by competitively inhibiting with 4-nitrobenzoate the coq2 enzyme, which catalyzes one of the final reactions in the biosynthetic pathway of CoQ. The bioenergetic and metabolic characteristics of control and coenzyme Q depleted cells were investigated using polarographic and spectroscopic assays. The effect of CoQ depletion on cell growth was analyzed in different metabolic conditions., Results: we showed that cancer cells could cope from energetic and oxidative stress due to mitochondrial dysfunction by reshaping their metabolism. In CoQ depleted cells, the glycolysis was upregulated together with increased glucose consumption, overexpression of GLUT1 and GLUT3, as well as activation of pyruvate kinase (PK). Moreover, the lactate secretion rate was reduced, suggesting that the pyruvate flux was redirected, toward anabolic pathways. Finally, we found a different expression pattern in enzymes involved in glutamine metabolism, and TCA cycle in CoQ depleted cells in comparison to controls., Conclusion: This work elucidated the metabolic alterations in CoQ-depleted cells and provided an insightful understanding of cancer metabolism targeting.

Two different cell signals often affect transcription of the same gene. In such cases, it is natural to ask how the combined transcriptional response compares to the individual responses. The most commonly used mechanistic models predict additive or multiplicative combined responses, but a systematic genome-wide evaluation of these predictions is not available. Here, we analyzed the transcriptional response of human MCF-7 cells to retinoic acid and TGF-β, applied individually and in combination. The combined transcriptional responses of induced genes exhibited a range of behaviors, but clearly favored both additive and multiplicative outcomes. We performed paired chromatin accessibility measurements and found that increases in accessibility were largely additive. There was some association between super-additivity of accessibility and multiplicative or super-multiplicative combined transcriptional responses, while sub-additivity of accessibility associated with additive transcriptional responses. Our findings suggest that mechanistic models of combined transcriptional regulation must be able to reproduce a range of behaviors., Competing Interests: ES, BE, AC No competing interests declared, AR Receives consulting income and royalties related to Stellaris RNA FISH probes., (© 2020, Sanford et al.)

Background: Breast cancer (BC) is the second most common type of cancer worldwide. Among targeted therapies for Hormone Receptor-positive (HR + ) and Human Epidermal growth factor Receptor 2-negative (HER2 - ) BC, the Cyclin-Dependent Kinases (CDK4/6) are targeted by inhibitors such as Ribociclib (Rib); however, resistance to CDK4/6 inhibitors frequently develops. The aim of this work is to assess in vitro activity of Rib and Everolimus (Eve) in HR + HER2 - MCF-7 and HR - HER2 - BT-549 BC cell lines., Methods: HR + HER2 - MCF-7 and HR - HER2 - BT-549 BC cell lines were treated with increasing concentration of Rib and Eve (up to 80 μg/mL) for 48-72 h. Subsequently, HR + HER2 - MCF-7 cells were silenced for Retinoblastoma (Rb) gene, and thus, the effect of Rib in sequential or concurrent schedule with Eve for the treatment of both Rb wild type or Rb knock-down MCF-7 in vitro was evaluated. Cell viability of HR + HER2 - MCF-7cells treated with sequential and concurrent dosing schedule was analyzed by MTT assay. Moreover, cell cycle phases, cell death and senescence were evaluated by cytofluorimetric analysis after treatment with Rib or Eve alone or in combination., Results: The sequential treatment didn't produce a significant increase of cytotoxicity, compared to Rib alone. Instead, the cotreatment synergized to increase the cytotoxicity compared to Rib alone. The cotreatment reduced the percentage of cells in S and G2/M phases and induced apoptosis. Rib triggered senescence and Eve completely reversed this effect in Rb wild type BC cells. Rib also showed Rb-independent effects as shown by results in Rb knock-down MCF-7., Conclusion: Overall, the Rib/Eve concurrent therapy augmented the in vitro cytotoxic effect, compared to Rib/Eve sequential therapy or single treatments.

Biogenic synthesis of nanoparticles provides many advantages over synthetic nanoparticles including clean and non-toxic approaches. Nanoparticle-based application for the development of diagnostics and therapeutics is a promising field that requires further enrichment and investigation. The use of biological systems for the generation of gold nanoparticles (AuNPs) has been extensively studied. The search for a biocompatibility approach for the development of nanoparticles is of great interest since it can provide more targeting and less toxicity. Here, we reported a bio-reductive approach of gold to AuNPs using metabolites extracted from mammalian cells, which provided a simple and efficient way for the synthesis of nanomaterials. AuNPs were more efficiently synthesized by the metabolites extracted from breast cancer (MCF7) and normal fibroblasts (F180) cells when compared to metabolites extracted from cell-free supernatants. The metabolites involved in biogenic synthesis are mainly alcohols and acids. Spectroscopic characterization using UV-visible spectra, morphological characterization using electron microscopy and structural characterization using X-ray diffraction (XRD) confirmed the AuNPs synthesis from mammalian cells metabolites. AuNPs generated from MCF7 cells metabolites showed significant anticancer activities against MCF7 and low toxicity when compared to those generated from F180 cells metabolites. The results reflected the cytotoxic activities of the parent metabolites extracted from MCF7 versus those extracted from F180. Comparative metabolomics analysis indicated that MCF7-generated AuNPs harbored tetratetracontane, octacosane, and cyclotetradecane while those generated from F180 harbored a high percentage of stearic, palmitic, heptadecanoic acid. We related the variation in cytotoxic activities between cell types to the differences in AuNPs-harboring metabolites. The process used in this study to develop the nanoparticles is novel and should have useful future anticancer applications mainly because of proper specific targeting to cancer cells., Competing Interests: The authors have declared that no competing interests exist.

Background/aims: Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells., Methods: The model of MCF-7 cells with prolidase overexpression (MCF-7 PL ) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry., Results: Prolidase overexpression in MCF-7 PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7 PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7 PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7 PL cells. The effects of studied compounds in MCF-7 PL cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9., Conclusion: The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Prunus cerasoides (PC) products contain relatively high levels of flavones and isoflavones and may be potential sources of phytoestrogens for postmenopausal symptom relief. We assessed the PC extract (PCE) and its representative constituents in vitro with assays for estrogen receptor alpha binding, estrogen response element transcriptional activity, cell proliferation, and gene expression changes for pS2 in MCF-7 cells. PCE and its compounds showed strong estrogen receptor binding affinities and estrogen response element induction. A previously undescribed compound (designated as compound 18), now identified as being gentisic acid, 5-O-β-D-(6'-O-trans-4-coumaroyl)-glucopyranoside, also showed potent estrogenic properties and induced proliferation of MCF-7 cells. PCE was evaluated for its in vivo uterotrophic effects in immature female rats as well as for its lipid lowering effects in estrogen-deprived animals. For ovariectomized rats and aged female mice, PCE-treated groups had lower plasma triglyceride levels compared with control and, for the same comparison, had reduced serum levels of liver stress/damage markers. Our results point to strong estrogenic activities and beneficial metabolic effects for PCE, with properties that put PC and its extracts as promising sources of phytoestrogens for symptom relief in menopausal and postmenopausal cases., (© 2020 John Wiley & Sons, Ltd.)

Background: In recent times, coordination complexes of iron in various oxidation states along with variety of ligand systems have been designed and developed for effective treatment of cancer cells without adversely affecting the normal cell and tissues of various organs., Methods: In this study, we have evaluated the mechanism of action of a Fe(II) Schiff base complex in the crop plant Trigonella foenum-graecum L. (Fenugreek) as the screening system by using morphological, cytological, biochemical and molecular approaches. Further functional characterization was performed using MCF-7 cell line and solid tumour model for the assessment of anti-tumour activity of the complex., Results: Our results indicate efficiency of the Fe(II) Schiff base complex in the induction of double strand breaks in DNA. Complex treatment clearly induced cytotoxic and genotoxic damage in Trigonella seedlings. The Fe-complex treatment caused cell cycle arrest via the activation of ATM-ATR kinase mediated DNA damage response pathway with the compromised expression of CDK1, CDK2 and CyclinB1 protein in Trigonella seedlings. In cultured MCF-7 cells, the complex induces cytotoxicity and DNA fragmentation through intracellular ROS generation. Fe-complex treatment inhibited tumour growth in solid tumour model with no additional side effects., Conclusion: The growth inhibitory and cytotoxic effects of the complex result from activation of DNA damage response along with oxidative stress and cell cycle arrest., General Significance: Overall, our results have provided comprehensive information on the mechanism of action and efficacy of a Fe(II) Schiff base complex in higher eukaryotic genomes and indicated its future implications as potential therapeutic agent., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background/aim: The study aimed to test the potential for increasing the antiproliferative activity of 5-fluorouracil against breast cancer cells of various molecular subtypes by vitamin D receptor (VDR) agonists, calcitriol and tacalcitol, used at a low concentration of 10 nM., Materials and Methods: Calcitriol and tacalcitol were used to increase the antiproliferative effect of 5-fluorouracil against the following human breast cancer cell lines: MCF-7, T47D, BT-474 (luminal); JIMT-1, SKBR-3 (HER2-enriched); MDA-MB-231 (triple-negative/basal-B), and non-malignant MCF-10A breast epithelial cells., Results: Both calcitriol and tacalcitol significantly increased the sensitivity of MCF-7 and BT-474 cells to the antiproliferative effect of 5-fluorouracil, while no increase in the sensitivity of MDA-MB-231 cells to 5-fluorouracil treatment was observed., Conclusion: The VDR agonist used at the relatively low concentration of 10 nM may increase the sensitivity of breast cancer cells, at least of the luminal subtype, to the antiproliferative effect of 5-fluorouracil., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Although Taxol has improved the survival of cancer patients as a first-line chemotherapeutic agent, an increasing number of patients develop resistance to Taxol after prolonged treatment. The potential mechanisms of cancer cell resistance to Taxol are not completely clear. It has been reported that microRNAs (miRNAs) are involved in regulating the sensitivity of cancer cells to various chemotherapeutic agents. In this study, we aimed to explore the role of miR-129-5p in regulating the sensitivity of breast cancer cells to Taxol. Cell apoptosis and autophagy, and the sensitivity of MCF-7 cells to Taxol were assessed with a series of in vitro assays. Our results showed that the inhibition of autophagy increased the Taxol-induced apoptosis and the sensitivity of MCF-7 cells to Taxol. Up-regulation of miR-129-5p also inhibited autophagy and induced apoptosis. Furthermore, miR-129-5p overexpression increased the sensitivity of MCF-7 cells to Taxol. High mobility group box 1 (HMGB1), a target gene of miR-129-5p and a regulator of autophagy, was negatively regulated by miR-129-5p. We found that interference of HMGB1 enhanced the chemosensitivity of Taxol by inhibiting autophagy and inducing apoptosis in MCF-7 cells. Taken together, our findings suggested that miR-129-5p increased the chemosensitivity of MCF-7 cells to Taxol through suppressing autophagy and enhancing apoptosis by inhibiting HMGB1. Using miR-129-5p/HMGB1/autophagy-based therapeutic strategies may be a potential treatment for overcoming Taxol resistance in breast cancer.

Adseverin (Ads) is a Ca 2+ -dependent actin-capping and severing protein that is highly expressed in gastric, prostate and bladder cancer cells. Currently it is unknown whether Ads contributes to the subcortical actin remodeling associated with the formation of cell extensions and matrix invasion in cancer. We compared cell extension formation and matrix degradation in Ads wildtype and Ads-null MCF7 breast cancer cells generated by CRISPR/Cas9. Compared with wildtype, Ads-null cells plated on fibronectin or collagen exhibited a more circular morphology with shorter cell extensions (37% reduction on fibronectin; p < 0.001). Reconstitution of Ads in Ads-null cells restored the formation of cell extensions (p < 0.05). While cell migration on two-dimensional matrices was unchanged by Ads deletion, the formation of cell extensions across Transwell membranes was reduced (~40% reduction, p < 0.05). When plated on fibrillar collagen, compared with wildtype, Ads-null cells showed reduced expression of MT1-MMP, collagen degradation (p < 0.05) and phagocytosis of collagen-coated beads (25% reduction; p = 0.001). We conclude that Ads is involved in the formation of cell extensions and collagen degradation in MCF7 cells, which may in turn affect matrix invasion and metastasis., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Identification of primary targets associated with phenotypes can facilitate exploration of the underlying molecular mechanisms of compounds and optimization of the structures of promising drugs. However, the literature reports limited effort to identify the target major isoform of a single known target gene. The majority of genes generate multiple transcripts that are translated into proteins that may carry out distinct and even opposing biological functions through alternative splicing. In addition, isoform expression is dynamic and varies depending on the developmental stage and cell type. To identify target major isoforms, we integrated a breast cancer type-specific isoform coexpression network with gene perturbation signatures in the MCF7 cell line in the Connectivity Map database using the 'shortest path' drug target prioritization method. We used a leukemia cancer network and differential expression data for drugs in the HL-60 cell line to test the robustness of the detection algorithm for target major isoforms. We further analyzed the properties of target major isoforms for each multi-isoform gene using pharmacogenomic datasets, proteomic data and the principal isoforms defined by the APPRIS and STRING datasets. Then, we tested our predictions for the most promising target major protein isoforms of DNMT1, MGEA5 and P4HB4 based on expression data and topological features in the coexpression network. Interestingly, these isoforms are not annotated as principal isoforms in APPRIS. Lastly, we tested the affinity of the target major isoform of MGEA5 for streptozocin through in silico docking. Our findings will pave the way for more effective and targeted therapies via studies of drug targets at the isoform level.

Hypoxia is a ubiquitous feature of cancers, encouraging glycolytic metabolism, proliferation, and resistance to therapy. Nonetheless, hypoxia is a poorly defined term with confounding features described in the literature. Redox biology provides an important link between the external cellular microenvironment and the cell's response to changing oxygen pressures. In this paper, we demonstrate a correlation between intracellular redox potential (measured using optical nanosensors) and the concentrations of microRNAs (miRNAs) involved in the cell's response to changes in oxygen pressure. The correlations were established using surprisal analysis (an approach derived from thermodynamics and information theory). We found that measured redox potential changes reflect changes in the free energy computed by surprisal analysis of miRNAs. Furthermore, surprisal analysis identified groups of miRNAs, functionally related to changes in proliferation and metastatic potential that played the most significant role in the cell's response to changing oxygen pressure., Competing Interests: The authors declare no conflict of interest.

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434-440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca 2+ . The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca 2+ concentrations., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Breast cancer which is the most commonly diagnosed cancer among women; have been known as a serious threat for health and life around the world. So development of an approach for early-stage diagnosis of breast cancer is vital. In this study, we designed a double aptamer-nanoparticle conjugates-based (DANP) complex for specific detection and visualization of MCF-7 cells using Mucin 1 (MUC 1) aptamer-conjugated gold nanoparticles (MUC1 apt - GNPs) and adenosine triphosphate (ATP) aptamer-conjugated CdTe quantum dots (ATP apt-QDs). The ATP apt-QDs was attached onto MUC1 apt - GNPs surface through Van der Waals forces and electrostatic interactions between ATP aptamer and GNPs leading to the formation of DANP complex. Atomic force microscopy asserted DANP complex formation. The imaging process was based on the recognition of MUC1 protein on the surface of MCF-7 cells by MUC1 aptamer and specific internalization of DANP complex into target cells (MCF-7). Existence of abundant amounts of ATP in lysosome led to release of ATP apt-QDs from the MUC1 apt-GNPs surface resulting in strong fluorescence emission. The flow cytometry analysis and fluorescence microscopy confirmed significant internalization of DANP complex into MCF-7 cells (target) in comparison with CHO cells (non-target). Based on the obtained results, the DANP complex possesses high potency for efficient detection and monitoring of breast cancer cells (MCF-7)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: The aim of this study was to explore the mechanism of miR-98-5p in influencing the malignant proliferation and metastasis capacities of breast cancer cells., Patients and Methods: Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression level of miR-98-5p and GRB2-associated-binding protein 2 (Gab2) in breast cancer samples and cells. On-line target gene prediction software and Dual-Luciferase reporter assay were used to predict and verify the target genes of miR-98-5p, respectively. Cell proliferation was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Meanwhile, migration and invasion abilities, as well as the changes of epithelial-mesenchymal transition (EMT) after transfection were detected by transwell assay and Western blot assay, respectively., Results: Compared with adjacent non-tumor tissues and MCF-10A cells, the expression level of miR-98-5p in tumor tissues and MCF-7 cells was significantly declined, whereas Gab2 was markedly up-regulated. Besides, Gab2 was predicted as a target gene of miR-98-5p. Subsequent experiments indicated that the proliferation, migration, invasion and EMT of MCF-7 cells transfected with miR-98-5p were significantly inhibited. However, up-regulation of Gab2 attenuated the biological function of miR-98-5p on malignant abilities of breast cancer cells., Conclusions: We showed that miR-98-5p served as anti-oncogene in breast cancer, which might provide a new therapeutic target for its treatment.

Objective: To investigate the expression of zinc finger transcription factors-Snail and E-cadherin in adriamycin-resistant human breast cancer MCF-7/ADM cells and non-resistant MCF-7 cells., Study Design: An experimental study., Place and Duration of Study: The Affiliated Cancer Hospital of Nanjing Medical University and Jiangsu Cancer Hospital, and Jiangsu Institute of Cancer Research, China, from April 2017 to March 2018., Methodology: Real-time quantitative PCR technology was used to detect the expression levels of Snail mRNA and E-cadherin mRNA in normal breast cells, adriamycin-resistant human breast cancer MCF-7/ADM cells and non-resistant MCF-7 cells. Western blot was used to detect the expression levels of proteins of Snail and E-cadherin in normal breast cells, adriamycin-resistant human breast cancer MCF-7/ADM cells and non-resistant MCF-7 cells., Results: The expression of Snail mRNA and protein in adriamycin-resistant human breast cancer MCF-7/ADM cells was significantly higher than that in normal breast cells (p<0.001) and non-resistant MCF-7 cells (p<0.001). The expression of E-cadherin mRNA and protein in adriamycin-resistant human breast cancer MCF-7/ADM cells was significantly lower than that in normal breast cells (p<0.001) and non-resistant MCF-7 cells (p<0.001)., Conclusion: Adriamycin-resistant human breast cancer MCF-7/ADM cell strains had high expression of Snail and low expression of E-cadherin. This points out to a new research direction for the targeted therapy of drug-resistant breast cancer cells, and provides clinical guidance for breast cancer therapy and prognosis evaluation.

Objective: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway., Materials and Methods: The cell line was treated with T3 at a physiological dose (10-9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%., Results: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA., Conclusion: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.

Objective: Easily oxidizable GC-rich DNA (GC-DNA) fragments accumulate in the cell-free DNA (cfDNA) of patients with various diseases. The human oxidized DNA penetrates the MCF7 breast cancer cells and significantly changes their physiology. It can be assumed that readily oxidizable GC-DNA fragments can penetrate the cancer cells and be expressed., Methods: MCF7 cells were cultured in the presence of two types of GC-DNA probes: (1) vectors pBR322 and pEGFP and (2) plasmids carrying inserted human rDNA (pBR322-rDNA and pEGFP-rDNA). pEGFP and pEGFP-rDNA contained a CMV promoter and a fluorescent protein gene EGFP. ROS generation rate, accumulation of the DNA probes in MCF7, 8-oxodG content, expression of EGFP and NOX4 , and localization of EGFP, NOX4, and 8-oxodG in MCF7 were explored. The applied methods were qPCR, fluorescent microscopy (FM), immunoassay, and flow cytometry (FCA)., Results: When GC-DNA is added to the cell culture medium, it interacts with the cell surface. At the site of GC-DNA contact with the cell, NOX4 is expressed, and ROS level increases. The ROS oxidize the GC-DNA. When using the plasmids pEGFP and pEGFP-rDNA, an increase in the amount of the DNA EGFP , RNA EGFP , and EGFP proteins was detected in the cells. These facts suggest that GC-DNA penetrates the cells and the EGFP gene is expressed. Insertions of the rDNA significantly increase the GC-DNA oxidation degree as well as the rate of plasmid transfection into the cells and the EGFP expression level. In the nucleus, the oxidized GC-rDNA fragments, but not the vectors, are localized within the nucleolus., Conclusions: GC-rich cfDNA fragments that are prone to oxidation can easily penetrate the cancer cells and be expressed. The cfDNA should become a target for the antitumor therapy.

Purpose: Autologous transplantation of adipose tissue into the breast is commonly performed in clinical practice, but its oncological safety has not been established., Methods: We conducted an in vitro study to assess the influence of factors released by adipose-derived stem cells (ASCs), from multiple source tissues and harvested using different techniques, on proliferation and invasiveness of two breast cancer cell lines., Results: Fat specimens of 66 donors (57 female, 9 male) were collected and 44 ASC cultures were established. ASC conditioning of the medium (CM) increased the proliferation of MCF-7 cells (178.4 ± 62.8%; P < 0.001), whereas MDA-MB321 proliferation was decreased (87.3 ± 15.3%; P = 0.032). We observed increased cell migration (174.0 ± 62.8%; P = 0.002), but not cell invasion (1.28 ± 0.51; P = 0.14) in MDA-MB231. Migration and invasion of MCF-7 cells were not affected by exposure to ASC-CM. For MCF-7 cell migration, lower BMI (< 25 kg/m 2 ) was associated with increased migration, both in univariate (P = 0.015) and multivariate (P = 0.039) analyses. Regarding the cytokine secretome, proliferation of MCF-7 was positively correlated with levels of eotaxin 1 and insulin-like growth factor-binding protein 3 in the CM, and inversely correlated with levels of interleukin 1β and transforming growth factor β-3. In case of MDA-MB231, granulocyte colony-stimulating factor, angiogenin, eotaxin 1 and 3, neutrophil activating peptide 2, and neurotrophin-3 were positively correlated with proliferation., Conclusions: We conclude that fat tissue transplantation increases proliferation and migration, but not invasion, of breast cancer cells. These findings are consistent with clinical data regarding the safety of autologous fat transplantation in breast cancer patients.

Exosomes are nano-sized vesicles composed of lipids, proteins, and nucleic acids. Their molecular landscape is diverse, and exosomes derived from different cell types have distinct biological activities. Since exosomes are now being utilized as delivery vehicles for exogenous therapeutic cargoes, their intrinsic properties and biological effects must be understood. We performed miRNA profiling and found substantial differences in the miRNA landscape of prostate cancer (PC3) and human embryonic kidney (HEK) 293 exosomes with little correlation in abundance of common miRNAs (R 2  = 0.16). Using a systems-level bioinformatics approach, the most abundant miRNAs in PC3 exosomes but not HEK exosomes were predicted to significantly modulate integrin signaling, with integrin-β3 loss inducing macrophage M2 polarization. PC3 but not HEK exosomes downregulated integrin-β3 expression levels by 70%. There was a dose-dependent polarization of RAW 264.7 macrophages toward an M2 phenotype when treated with PC3-derived exosomes but not HEK-derived exosomes. Conversely, HEK exosomes, widely utilized as delivery vehicles, were predicted to target cadherin signaling, with experimental validation showing a significant increase in the migratory potential of MCF7 breast cancer cells treated with HEK exosomes. Even widely utilized exosomes are unlikely to be inert, and their intrinsic activity ought to be assessed before therapeutic deployment.

Background and objectives: Cell culture is one of the mainstays in the research of breast cancer biology, although the extent to which this approach allows to preserve the original characteristics of originating tumor and implications of cell culture findings to real life situations have been widely debated in the literature. The aim of this study was to determine the role of three cell culture media on transcriptional expression of breast cancer markers in three breast cancer reference cell lines (MCF7, SkBr3 and MDA-MB-436). Materials and methods : Cell lines were conditioned in three studied media (all containing 5% fetal bovine serum (FBS) + hormones/growth factors; different composition of basal media) for four passages. Population growth was characterized by cumulative population doubling levels, average generation time, cell yield and viability at the fourth passage. Transcriptional expression of breast cancer differentiation markers and regulatory transcriptional programs was measured by qPCR. Results: Differences in the composition of growth media significantly influenced the growth of studied cell lines and the expression of mammary lineage governing transcriptional programs and luminal/basal markers. Effects of media on transcriptional expression were more pronounced in luminal cell lines (MCF7, SkBr3), than in the basal cell line (MDA-MB-436). Changes in growth media in terms of supplementation and basal medium delayed growth of cells, but improved cell yields. Conclusions: The expression of breast cancer cell differentiation phenotypic markers depends on the composition of cell growth medium, therefore cell culture as a tool in phenotypic studies should be used considering this effect. The findings of such studies should always be interpreted with caution. The formulation of cell growth media has greater effect on the expression of phenotypic markers in luminal, rather than basal cell lines. Media containing mitogens and higher vitamin content improved efficacy of cell culture in terms of cell yields, although greatly increased growth times., Competing Interests: The authors declare no conflict of interest.

The use of cell-based assays is essential in reducing the number of vertebrates used in the investigation of chemical toxicities and in regulatory toxicology assessment. An important factor in obtaining meaningful results that can be accurately extrapolated is the use of biologically appropriate cell lines. In the present preliminary study, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy with multivariate analysis was used to assess the fundamental biomolecular differences between a commonly used cell line, MCF-7 cells, and an environmentally relevant cell line derived from mallard (Anas platyrhynchos) dermal fibroblasts. To better understand differences in basic cell biochemistry, the cells were analyzed in the untreated state or post exposure to polychlorinated biphenyl (PCB) and polybrominated diphenyl ester (PBDE) congeners. The main spectral peaks in spectra from both cell types were associated with cellular macromolecules, particularly proteins and lipids, but the spectra also revealed some cell-specific differences. Spectra from untreated mallard fibroblasts spectra contained a large peak associated with lipids. The cell-related differences in lipids and deoxyribonucleic acid (DNA) were also identified as regions of spectral alteration induced by PBDE and PCB exposure. Although lipid alterations were observed in post treatment spectra from both cell types, these may be of more significance to mallard fibroblasts, which may be the result of increased intracellular lipid as determined by Nile red staining. Untreated MCF-7 cell spectra contained unique peaks related to DNA and nucleic acids. The DNA-associated spectral regions were also identified as areas of considerable alteration in MCF-7 cells exposed to some congeners including PBDE 47 and PCB 153. The findings indicate that in their native state, MCF-7 and mallard cells have unique biochemical differences, which can be identified using ATR-FTIR spectroscopy. Such differences in biochemical composition may influence cell susceptibility to environmental contaminants and therefore influence the choice of cell type used in toxicology experiments. To our knowledge, the present study is the first study to analyze the biochemistry of a mallard dermal fibroblast cell line and to use ATR-FTIR spectroscopy for this purpose. Thus ATR-FTIR spectroscopy is demonstrated to be a useful tool for exploration of biomolecular variation at the cellular level and with further development, it could be used as part of a panel of cell-based assays to indicate when different results might be seen in environmental species compared with currently used cell lines. Environ Toxicol Chem 2017;36:3127-3137. © 2017 SETAC., (© 2017 SETAC.)

The use of gene expression signatures to classify compounds, identify efficacy or toxicity, and differentiate close analogs relies on the sensitivity of the method to identify modulated genes. We used a novel ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq®, to determine whether previously unreported compound-responsive genes could be identified and incorporated into a broad but specific compound signature. TempO-Seq exhibits 99.6% specificity, single cell sensitivity, and excellent correlation with fold differences measured by RNA-Seq (R2 = 0.9) for 20,629 targets. Unlike many expression assays, TempO-Seq does not require RNA purification, cDNA synthesis, or capture of targeted RNA, and lacks a 3' end bias. To investigate the sensitivity of the TempO-Seq assay to identify significantly modulated compound-responsive genes, we derived whole transcriptome profiles from MCF-7 cells treated with the histone deacetylase inhibitor Trichostatin A (TSA) and identified more than 9,000 differentially expressed genes. The TSA profile for MCF-7 cells overlapped those for HL-60 and PC-3 cells in the Connectivity Map (cMAP) database, suggesting a common TSA-specific expression profile independent of baseline gene expression. A 43-gene cell-independent TSA signature was extracted from cMAP and confirmed in TempO-Seq MCF-7 data. Additional genes that were not previously reported to be TSA responsive in the cMAP database were also identified. TSA treatment of 5 cell types revealed 1,136 differentially expressed genes in common, including 785 genes not previously reported to be TSA responsive. We conclude that TSA induces a specific expression signature that is consistent across widely different cell types, that this signature contains genes not previously associated with TSA responses, and that TempO-Seq provides the sensitive differential expression detection needed to define such compound-specific, cell-independent, changes in expression.

Sirtuins regulate several processes associated with tumor development. Resveratrol was shown to stimulate sirtuin 1 and 3 (SIRT1/3) activities and to result in cytotoxicity for some tumor types. The relationship between modulation of sirtuin activities, cellular metabolic remodeling and resveratrol cytotoxicity mechanism on breast cancer cells is still an open question. Here, we evaluated whether sirtuin 1 and 3 are involved in resveratrol toxicity and whether resveratrol leads to a metabolic remodeling and cell differentiation. Results using the Extracellular Flux Analyzer indicated that resveratrol inhibits mitochondrial respiration in breast cancer cells. We also demonstrated here for the first time that resveratrol cytotoxic effects on breast cancer cells were modulated by SIRT1 and also involved mitochondrial complex I inhibition. Importantly, we also demonstrated that resveratrol reduced the pool of breast cancer cells with stemness markers through a SIRT1-dependent mechanism. Our data highlights the role of SIRT1 in regulating resveratrol induced differentiation and/or toxicity in breast cancer cells.

Mammalian lignans or enterolignans are metabolites of plant lignans, an important category of phytochemicals. Although they are known to be associated with estrogenic activity, cell signaling pathways leading to specific cell functions, and especially the differences among lignans, have not been explored. We examined the estrogenic activity of enterolignans and their precursor plant lignans and cell signaling pathways for some cell functions, cell cycle and chemokine secretion. We used DNA microarray-based gene expression profiling in human breast cancer MCF-7 cells to examine the similarities, as well as the differences, among enterolignans, enterolactone and enterodiol, and their precursors, matairesinol, pinoresinol and sesamin. The profiles showed moderate to high levels of correlation (R values: 0.44 to 0.81) with that of estrogen (17β-estradiol or E2). Significant correlations were observed among lignans (R values: 0.77 to 0.97), and the correlations were higher for cell functions related to enzymes, signaling, proliferation and transport. All the enterolignans/precursors examined showed activation of the Erk1/2 and PI3K/Akt pathways, indicating the involvement of rapid signaling through the non-genomic estrogen signaling pathway. However, when their effects on specific cell functions, cell cycle progression and chemokine (MCP-1) secretion were examined, positive effects were observed only for enterolactone, suggesting that signals are given in certain directions at a position closer to cell functions. We hypothesized that, while estrogen signaling is initiated by the enterolignans/precursors examined, their signals are differentially and directionally modulated later in the pathways, resulting in the differences at the cell function level., Competing Interests: The authors have declared that no competing interests exist to Scinet Company and other funders relating to employment, consultancy, patents, products in development, or marketed products, etc., and that they do not alter our adherence to PLOS ONE policies on sharing data and materials.

Macrophage colony-stimulating factor is a vital factor in maintaining the biological function of monocyte-macrophage lineage. It is expressed in many tumor tissues and cancer cells. Recent findings indicate that macrophage colony-stimulating factor might contribute to chemoresistance, but the precise mechanisms are unclear. This study was to explore the effect of macrophage colony-stimulating factor on doxorubicin resistance in MCF-7 breast cancer cells and the possible mechanism. In the study, the human breast cancer cells, MCF-7, were transfected with macrophage colony-stimulating factor. We document that cytoplasmic macrophage colony-stimulating factor induces doxorubicin resistance and inhibits apoptosis in MCF-7 cells. Further studies demonstrated that cytoplasmic macrophage colony-stimulating factor-mediated apoptosis inhibition was dependent on the activation of PI3K/Akt/Survivin pathway. More importantly, we found that macrophage colony-stimulating factor-induced autophagic cell death in doxorubicin-treated MCF-7 cells. Taken together, we show for the first time that macrophage colony-stimulating factor-induced doxorubicin resistance is associated with the changes in cell death response with defective apoptosis and promotion of autophagic cell death.

Hereinwe investigated the effect of elderflower extracts (EFE) and of enterolactone/enterodiol on hormone production and proliferation of trophoblast tumor cell lines JEG-3 and BeWo, as well as MCF7 breast cancer cells. The EFE was analyzed by mass spectrometry. Cells were incubated with various concentrations of EFE. Untreated cells served as controls. Supernatants were tested for estradiol production with an ELISA method. Furthermore, the effect of the EFE on ER/ER/PR expression was assessed by immunocytochemistry. EFE contains a substantial amount of lignans. Estradiol production was inhibited in all cells in a concentration-dependent manner. EFE upregulated ER in JEG-3 cell lines. In MCF7 cells, a significant ER downregulation and PR upregulation were observed. The control substances enterolactone and enterodiol in contrast inhibited the expression of both ER and of PR in MCF7 cells. In addition, the production of estradiol was upregulated in BeWo and MCF7 cells in a concentration dependent manner. The downregulating effect of EFE on ER expression and the upregulation of the PR expression in MFC-7 cells are promising results. Therefore, additional unknown substances might be responsible for ER downregulation and PR upregulation. These findings suggest potential use of EFE in breast cancer prevention and/or treatment and warrant further investigation., Competing Interests: The authors declare no conflict of interest.

Zinc (Zn) is an essential trace elements, its deficiency is associated with increased incidence of human breast cancer. We aimed to study the effect of Zn on human breast cancer MCF-7 cells cultured in Zn depleted and Zn adequate medium. We found increased cancer cell growth in zinc depleted condition, further Zn supplementation inhibits the viability of breast cancer MCF-7 cell cultured in Zn deficient condition and the IC 25, IC 50 value for Zn is 6.2μM, 15μM, respectively after 48h. Zn markedly induced apoptosis through the characteristic apoptotic morphological changes and DNA fragmentation after 48h. In addition, Zn deficient cells significantly triggered intracellular ROS level and develop oxidative stress induced DNA damage; it was confirmed by elevated expression of CYP1A, GPX, GSK3β and TNF-α gene. Zinc depleted MCF-7 cells expressed significantly (p≤0.001) decreased levels of CDKN2A, pRb1, p53 and increased the level of mdm2 expression. Zn supplementation (IC 50 =15μM), increased significantly CDKN2A, pRB1 & p53 and markedly reduced mdm2 expression; also protein expression levels of CDKN2A and pRb1 was significantly increased. In addition, intrinsic apoptotic pathway related genes such as Bax, caspase-3, 8, 9 & p21 expression was enhanced and finally induced cell apoptosis. In conclusion, physiological level of zinc is important to prevent DNA damage and MCF-7 cell proliferation via regulation of tumor suppressor gene., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Faster biodegradation, immunogenicity and lack of cell penetrative capabilities are hurdles in development of peptidyl drugs for cancer therapy. Polymeric carriers can be used to overcome these problems. The present study is focused on the use of polyhydroxybutyrate as a potential nanovehicle for the delivery of anticancer peptides. PHB (72kDa) was produced by thermal treatment of high molecular weight PHB (300kDa) under melt conditions and then conjugated with PEG (4kDa) by Steglich esterification reaction. Anticancer peptide NuBCP-9 (FSRSLHSLL) encapsulated PHB(72K)-PEG(4K) NPs were prepared by double emulsion solvent evaporation method. PHB(72K)-PEG(4K) NPs showed encapsulation efficiency of 61% and exhibited sustained release of peptide over a period of 26days at physiological pH. NuBCP-9 loaded PHB(72K)-PEG(4K) NPs showed an IC50 value of 2.2μM & 1.6μM in MCF-7 cells in 48h and 72h respectively. Confocal laser microscopy confirmed efficient cellular uptake and induction of apoptosis by peptide loaded NPs in a time dependent manner. In vivo intraperitonial administration of 20mg/kg NuBCP-9/NPs twice a week for three weeks triggered 90% tumor regression in Ehrlich syngeneic mouse model. Our results illustrated the potential of PHB(72K)-PEG(4K) based nanoformulation as a tool for targeting intracellular proteins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Reactive oxygen species (ROS) are key players in the apoptotic effects induced by short-term tamoxifen treatment of breast cancer cells, but also in acquired resistance following long-term treatment. Whereas the use of the selective estrogen receptor down-regulator fulvestrant is promising, especially in patients who develop tamoxifen resistance, only few studies addressed its implication in the modulation of cellular redox status., Methods: The regulation of (pro/anti)-oxidant players were first investigated at the mRNA level in a MCF-7-derived cell line after short-term (24 h) estradiol treatment. Long-term anti-estrogen treated MCF-7 derived cell lines were also developed: 3 months of 4-hydroxytamoxifen alone (MCF7L-OHTLT) or followed by 3 months of fulvestrant (MCF7L-ICILT). Growth properties, hormone sensitivity, receptor content, ROS production and relative mRNA expression of pro or antioxidant enzymes were evaluated in these long-term treated cell lines., Results: Short-term estradiol treatment showed a hormone sensitivity of Nox2, GPx1, GPx2 and SOD1 mRNA levels. The long-term fulvestrant treatment (3 months) of MCF7L-OHTLT led to a reduced level of ROS production accompanied with a drastic drop of the accessory protein p22(phox) mRNA. This ROS reduction, although not clearly related to antioxidant enzymes level, seems to be involved in fulvestrant sensitivity of long-term anti-estrogen treated cells, as suggested by the effects of antiradical tempol treatment., Conclusion: When compared to long-term 4-hydroxytamoxifen-treated breast cancer cells, addition of fulvestrant treatment was able to diminish ROS production and p22(phox) mRNA level, and made cells more sensitive to growth inhibition induced by tempol. These effects may be a valuable asset of the fulvestrant treatment.

Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells.

Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane-disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals., (© 2016 Herrero et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Despite tamoxifen (TAM) is beneficial in treating a significant proportion of patients with breast cancer, many women still relapse after long-term therapy. Caffeic acid phenethyl ester (CAPE) is a component of honeybee propolis, with a plethora of important biological actions including anticancer activity. This study aimed to explore the cytotoxicity, the type of drugs interaction as well as the apoptotic and autophagic pathways of the combined treatment of TAM and CAPE in MCF-7 cells. Their antitumor activity and effect on survival of mice bearing Ehrlich tumor were also analyzed. The results showed synergistic cytotoxic effects, manifested by significant activation of apoptotic machinery, along with downregulation of protein levels of Bcl-2 and beclin-1, upon using the combination regimen. However, the ratio between microtubule-associated protein light chain 3-II and -I was not altered. Moreover, a decrease in vascular endothelial growth factor level was detected. Similarly, TAM + CAPE increased the life span of tumor-bearing animals and caused a marked regression in their tumor size and weight compared with those treated with either TAM or CAPE alone. In conclusion, CAPE relatively improved the anticancer activity of TAM in both in vitro and in vivo models via its apoptotic and angiostatic potentials.

Combretastatin A4 disodium phosphate (CA4P) is a fluorescent, water-soluble prodrug able to induce vascular shutdown within tumors at doses less than one-tenth of the maximum tolerated dose. As a continued effort to develop efficient liposomal CA4P to treat solid tumor, we herein investigate the physical and spectroscopic properties of CA4P in aqueous solution and the mechanism of CA4P release from archaeal tetraether liposomes (archaeosomes). We found that cis-CA4P can be photoisomerized to trans-CA4P. This photoisomerization results in an increase in fluorescence intensity. Both cis- and trans-CA4P undergo fluorescence intensity self-quenching after they reach a critical concentration Cq (∼0.15-0.25 mM). Moreover, both cis- and trans-CA4P in buffer exhibit a red shift in their excitation spectrum and an increase in excitation spectrum band sharpness with increasing concentration, which can be attributed to the formation of J-aggregates. The onset of the dramatic change in excitation maximum occurs at concentrations close to Cq, suggesting that the self-quenching arises from extensive J-aggregate formation and that, when CA4P concentration exceeds Cq, J-aggregate formation begins to increase sharply. Our data also suggest that the extent of J-aggregate formation plays a critical role in CA4P release from tetraether archaeosomes and in the subsequent cytotoxicity on cultured human breast cancer MCF-7 cells. The drug leakage and cytotoxicity rate constants vary with the initial CA4P concentration entrapped inside archaeosomes in a biphasic manner, reaching a local maximum at 0.25-0.50 mM. A mechanism based on the concept of J-aggregate formation has been proposed to explain the biphasic changes in drug release and cytotoxicity with increasing drug concentration. Tetraether archaeosomes are extraordinarily stable and relatively nontoxic to animals; thus, they are promising nano drug carriers. The results obtained from this study pave the way for future development of archaeosomal CA4P to treat solid tumors.

Omega 3 (n3) and Omega 6 (n6) polyunsaturated fatty acids (PUFAs) have been reported to exhibit opposing roles in cancer progression. Our objective was to determine whether different ratios of n6/n3 (AA/EPA+DHA) FAs could modulate the cell viability, lipid peroxidation, total cellular fatty acid composition and expression of tumor regulatory Matrix Attachment Region binding proteins (MARBPs) in breast cancer cell lines and in non-cancerous, MCF10A cells. Low ratios of n6/n3 (1:2.5, 1:4, 1:5, 1:10) FA decreased the viability and growth of MDA-MB-231 and MCF7 significantly compared to the non-cancerous cells (MCF10A). Contrarily, higher n6/n3 FA (2.5:1, 4:1, 5:1, 10:1) decreased the survival of both the cancerous and non-cancerous cell types. Lower ratios of n6/n3 selectively induced LPO in the breast cancer cells whereas the higher ratios induced in both cancerous and non-cancerous cell types. Interestingly, compared to higher n6/n3 FA ratios, lower ratios increased the expression of tumor suppressor MARBP, SMAR1 and decreased the expression of tumor activator Cux/CDP in both breast cancer and non-cancerous, MCF10A cells. Low n6/n3 FAs significantly increased SMAR1 expression which resulted into activation of p21WAF1/CIP1 in MDA-MB-231 and MCF7, the increase being ratio dependent in MDA-MB-231. These results suggest that increased intake of n3 fatty acids in our diet could help both in the prevention as well as management of breast cancer.

Introduction: Metformin is proposed as adjuvant therapy in cancer treatment because of its ability to limit cancer incidence by negatively modulating the PI3K/AKT/mTOR pathway. In vitro, in addition to inhibiting cancer cell proliferation, metformin can also induce apoptosis. The molecular mechanism underlying this second effect is still poorly characterized and published data are often contrasting. We investigated how nutrient availability can modulate metformin-induced apoptosis in three breast cancer cell lines., Material and Methods: MCF7, SKBR3 and MDA-MB-231 cells were plated in MEM medium supplemented with increasing glucose concentrations or in DMEM medium and treated with 10 mM metformin. Cell viability was monitored by Trypan Blue assay and treatment effects on Akt/mTOR pathway and on apoptosis were analysed by Western Blot. Moreover, we determined the level of expression of pyruvate kinase M2 (PKM2), a well-known glycolytic enzyme expressed in cancer cells., Results: Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium. Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation. Finally, we showed that, in nutrient-poor conditions, metformin was able to modulate the intracellular glycolytic equilibrium by downregulating PKM2 expression and that this mechanism was mediated by AMPK activation., Conclusion: We demonstrated that metformin induces breast cancer cell apoptosis and PKM2 downregulation only in nutrient-poor conditions. Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect. These data demonstrate that the reduction of nutrient supply in tumors can increase metformin efficacy and that modulation of PKM2 expression/activity could be a promising strategy to boost metformin anti-cancer effect.

Objective: We examined the phytoestrogenic effects of palmiwon on breast carcinoma, lipid accumulation in methyl-β-cyclodextrin-induced HepG2 cells, and lipid-related diseases in a rat model of menopausal hyperlipidemia., Methods: E-Screen assay was used to screen for phytoestrogens, especially those with antiestrogenic activity, in MCF-7 cells. Oil Red O staining and intracellular cholesterol analyses were used to quantify cellular cholesterol levels. 3-Hydroxy-3-methyl glutaryl coenzyme A reductase assay was used to measure enzyme activity. The levels of phosphorylated adenosine monophosphate-activated protein kinases and products of genes involved in cholesterol synthesis were measured by Western blot analysis. Thirty rats were either ovariectomized or sham-operated and randomly assigned to four groups (n = 5)-Sham, OVX, OVX-SV, or OVX-PMW (50, 150, or 450 mg/kg) group-for 8 weeks. A number of targets associated with lipid-related diseases were examined to confirm the estrogenic effects of palmiwon., Results: Palmiwon showed antiestrogenic activity in MCF-7 cells. Palmiwon decreased lipid accumulation, total cholesterol levels, and low-density lipoprotein/very-low-density lipoprotein levels in HepG2 cells. Moreover, palmiwon reversed the effects of methyl-β-cyclodextrin on cholesterol synthesis regulators and inhibited the activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase. Phosphorylation of adenosine monophosphate-activated protein kinase was stimulated by palmiwon. In ovariectomized rats, palmiwon reduced retroperitoneal and perirenal fat accumulation, serum lipids, atherogenic index, cardiac risk factor score, intima-media thickness, and nonalcoholic steatohepatitis scores., Conclusions: These results indicate that palmiwon inhibits lipid accumulation without estrogenic activity in the breast. Therefore, palmiwon may have potential as a therapeutic agent for the treatment of hyperlipidemia in postmenopausal women.

Bag-1, Bcl-2 associated athanogene-1, is a multifunctional protein that can regulate a wide variety of cellular processes: proliferation, cell survival, transcription, apoptosis and motility. Bag-1 interacts with various targets in the modulation of these pathways; yet molecular details of Bag-1's involvement in each cellular event are still unclear. We first showed that forced Bag-1 expression promotes cell survival and prevents drug-induced apoptosis in MCF-7 breast cancer cells. Increased mRNA expressions of c-myc protooncogene and ornithine decarboxylase (ODC), biosynthetic enzyme of polyamines, were detected in Bag-1L+ cells, and western blots against the protein product of c-Myc and ODC confirmed these findings. Once ODC, a c-Myc target, gets activated, polyamine biosynthesis increases. We observed enhanced polyamine content in the Bag-1L+ cells. On the contrary, when polyamine catabolic mechanisms were investigated, Bag-1 silencing suppressed biosynthesis of polyamines because of the downregulation of ODC and upregulation of PAO. Exposure of cells to apoptotic inducers enhances the cell death mechanism by producing toxic products such as H2 O2 and aldehydes. Bag-1L+ cells prevented drug-induced PAO activation leading to a decrease in H2 O2 production following cisplatin or paclitaxel treatment. In this line, our results suggested that Bag-1 indirectly affects cell survival through c-Myc activated signalling that causes elevation of ODC levels, leading to an increase of the polyamine content., (Copyright © 2015 John Wiley & Sons, Ltd.)

Epidemiologic studies link several factors related to estrogen production in women to an increased risk of breast cancer. These include early menarche, late menopause, obesity, use of post-menopausal hormone therapy, and plasma estradiol levels. Two possible mechanisms have been proposed to explain the increased risk: (1) estrogen receptor (ER) mediated stimulation of breast cell proliferation with a concomitant enhanced rate of mutations and (2) metabolism of estradiol to genotoxic metabolites with a resulting increase in DNA mutations. The metabolism of estradiol can cause DNA damage in two ways: (a) formation of estradiol-adenine - guanine adducts which are released from the DNA backbone leaving depurinated sites which undergo error prone DNA repair and mutations and (b) generation of oxygen free radicals resulting from redox cycling of 4-OH estradiol to the 3-4 estradiol quinone and back conversion to 4-OH estradiol. If one or both pathways are operative, sufficient numbers of mutations accumulate over a long period of time to induce neoplastic transformation. Our studies are based on the hypothesis that both receptor-mediated and genotoxic pathways contribute to breast cancer. We initially demonstrated that MCF-7 breast cancer cells and normal breast tissue in aromatase transfected mice contain the enzymes necessary to convert estradiol to the estradiol DNA adducts. We then utilized a highly reductionist model to separately analyze the effect of estrogen receptor alpha (ER) on tumor formation and the effects of estrogen depletion by castration in ER knock out/Wnt-1 (ERKO/Wnt) transgenic animals to assess the effects of estradiol in the absence of an ER. Estradiol was added back in castrate ERKO/Wnt animals to determine if Koch's postulates could be fulfilled to increase the incidence of cancer with administration of exogenous estradiol. Finally, we assessed the effects of an aromatase inhibitor on tumor incidence in non-castrate, ERKO/Wnt animals. The studies demonstrated the conversion of estradiol to genotoxic metabolites in breast tissue. In addition, knockout of ERα caused a reduction in incidence of tumor formation and a delay in the occurrence of those that formed. Oophorectomy further reduced the incidence of tumors and delayed their onset whereas estradiol add-back returned the incidence rate to that observed before oophorectomy. The aromatase inhibitor, letrozole, delayed the onset of tumor formation. Taken together, these data support a role for estradiol metabolism as one of the components in the development of experimental breast cancer., (Copyright © 2014. Published by Elsevier Inc.)