Search

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.)

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.)

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.

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.

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.

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.)

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: 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.

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.)

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.

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.

Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000-2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100-200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Characteristic of the tumor microenvironment are fluctuating gradients of reduced nutrient levels and released lactate. A fundamental issue is how tumor cells modulate their metabolic activity when both glucose and glutamine levels become limiting in the presence of high exogenous lactate. For functional analyses, the activities of pyruvate kinase, lactate dehydrogenase (LDH) and plasma membrane NADH oxidase (NOX) as well as cell growth were measured in breast cancer MCF-7 cells cultured in medium containing various concentrations of these metabolites. After 3 days at glucose concentrations below 2.5 mM, cell number was higher with 0.1 mM than with 1.0 mM glutamine, indicating that the glucose/glutamine balance is important for growth. On the other hand, NOX activity increased with increasing glucose >2.5 mM, but only with low glutamine (0.1 mM). Pyruvate kinase activity also increased, with LDH activity remaining 2-3-fold lower. Here NOX could have a complementary role in reoxidizing NADH for glycolysis. Exogenous lactate supported cell survival at limiting concentrations of glucose and glutamine while increasing NOX and pyruvate kinase activities as well as NADH levels. It is proposed that lactate supports cell survival by fuelling gluconeogenesis and/or the TCA cycle in mitochondria, from where NADH could be shuttled to the cytosol and reoxidized by NOX. Cell survival and the metabolic phenotype are thus interrelated to the dynamics of NADH and plasma membrane NOX activity, which are regulated by the balance of glucose/glutamine levels, in conjunction with lactate in a precarious tumor microenvironment., (© 2014 Wiley Periodicals, Inc.)

Multifunctional nanoparticles that actively target-specific tissues are studied for cancer diagnosis and treatment. Magnetically and optically active particles are of particular interest because they enable multiple imaging modalities and physically modulated therapies, such as magnetic hyperthermia. Fe-Au nanorods are synthesized that have a long iron segment, coated with polyethylene glycol, and a short gold tip functionalized with heregulin (HRG), a known ligand of ErbB family of receptors. HRG-nanorods preferentially target MCF7 cells relative to MDA-MB-231 cells, as demonstrated in a novel microfluidics device. Targeting rates of these classical breast cancer cells correlate with their differential expression of ErbB2/3 receptors. HRG-nanorod binding stimulates the extracellular signal-regulated kinase 1/2 (ERK) phosphorylation in MCF7 cells. The increase in ERK phosphorylation is linked to "active zones," dynamic regions in the cell periphery, which exhibit higher rates of particle binding than the rest of the cell. Periodically stretching cells using magnetic tweezers further activates ERK, which leads to cell death in cells co-treated with B-Raf inhibitors, through ERK hyperactivation. Although to a lesser extent, cell death is also achieved through magnetic hyperthermia. These results demonstrate nanoscale targeting and localized mechanochemical treatment of specific cancer cell lines based on their receptor expression using multifunctional nanoparticles., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background/aims: Large-scale epidemiological studies support a correlation between obesity and breast cancer in postmenopausal women. Circulating leptin levels are increased in obese and it has been suggested to play a significant role in mammary tumor formation and progression. Moreover, regulation of oxidative stress is another important factor in both tumor development and responses to anticancer therapies. The aim of this study was to examine the relationship between oxidative stress and chronic leptin exposure., Methods: We treated MCF-7 breast cancer cells with 100 ng/mL leptin for 10 days and analyzed cell growth, ROS production and oxidative damage, as well as, some of the main antioxidant systems. Furthermore, since the hyperleptinemia has been associated with a worse pathology prognosis, we decided to test the influence of leptin in response to cisplatin anticancer treatment., Results: Leptin signalling increased cell proliferation but reduced ROS production, as well as, oxidative damage. We observed an upregulation of SIRT1 after leptin exposure, a key regulator of stress response and metabolism. Additionally, leptin counteracted cisplatin-induced cytotoxicity in tumor cells, showing a decrease in cell death., Conclusion: Chronic leptin could contribute to the effective regulation of endogenous and treatment-induced oxidative stress, and it contributes to explain in part its proliferative effects.

Receptor change induced by ligand binding is a new issue to face in the field of targeted delivery. Receptor clustering, the main pattern of receptor changes, decreases the affinity between ligand and receptor due to the redistribution of receptor position. In an attempt to respond to such challenge, we designed and constructed three RGD-modified nanocarriers with different ligand intervals: stealth liposomes modified with the monomeric RGD (moRGD-LP), dimeric RGD (diRGD-LP) and a special dimeric RGD with a linker between two cyclic RGD motifs (P-diRGD-LP). The αvβ3-positive and -negative tumor cells (Melanoma B16 and MCF-7) were used as the cell models. As a result, P-diRGD-LP demonstrated strongest interaction with B16 cells in surface plasmon resonance study and highest cellular uptake in B16 cells in real-time confocal analysis. The enhanced endocytosis of P-diRGD-LP was found to be αvβ3-mediated and P-diRGD-LP increased the involvement of the clathrin-dependent pathway. Importantly, P-diRGD-LP demonstrated the best targeting effect in B16-tumor bearing mice in both in vivo and ex vivo near-infrared fluorescent images, about 2.4-fold that of moRGD-LP and 2.8-fold that of diRGD-LP at 3 h. Further, we validated integrin αvβ3 clustering on B16 cells via a single-molecule imaging by a total internal reflection fluorescence microscopy. Finally, the 3D models of αvβ3 clustering suggested a receptor interval within 41.916-65.779 Å, while the molecular computation revealed an RGD ligand interval of 20.944 Å, 42.753 Å and 78.196 Å for diRGD-LP, P-diRGD-LP and moRGD-LP, respectively, confirming the best matching between clustered αvβ3 and P-diRGD-LP. In conclusion, P-diRGD-LP could achieve higher targeting to αvβ3-positive tumor via the enhanced interaction based on the better ligand-receptor compatibility. The design of targeted nanocarriers against receptor clustering might provide new insight into the nanotechnology-based anticancer therapy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Adipose tissue is an attractive source of mesenchymal stem/stromal cells (MSCs) with potential applications in reconstructive plastic surgery and regenerative medicine. The aim of this study was to characterise human adipose tissue MSCs (ASCs) derived from healthy individuals and cancer patients and to compare their interactions with tumour cells. ASCs were isolated from adipose tissue of healthy donors, breast cancer-adjacent adipose tissue of breast cancer patients and tumour-adjacent adipose tissue of non-breast cancer patients. Their proliferation, differentiation, immunophenotype and gene expression were assessed and effects on the proliferation of human breast cancer cell line MCF-7 compared. ASCs from all sources exhibited similar morphology, proliferative and differentiation potential, showing the characteristic pattern of mesenchymal surface markers expression (CD90, CD105, CD44H, CD73) and the lack of HLA-DR and hematopoietic markers (CD11a, CD33, CD45, Glycophorin-CD235a), but uneven expression of CD34. ASCs also shared a common positive gene expression of HLA-DR, HLA-A, IL-6, TGF-β and HIF-1, but were negative for HLA-G, while the expression levels of Cox-2 and IDO-1 varied. All ASCs significantly stimulated the proliferation of MCF-7 tumour cells in direct mixed co-cultures and transwell system, although their conditioned media displayed antiproliferative activity. Data obtained showed that ASCs with similar characteristics are easily isolated from various donors and sites of origin, although ASCs could both suppress and favour tumour cells growth, emphasising the importance of cellular context within the microenvironment and pointing to the significance of safety studies to exclude any potential clinical risk of their application in regenerative medicine., (© 2013 International Federation for Cell Biology.)

Objective: To investigate the molecular effects of Nerium oleander leaf distillate on paclitaxel and vincristine resistant (MCF-7/Pac and MCF-7/Vinc) cells and sensitive (MCF-7/S) cell lines., Methods: Nerium oleander (N. oleander) leaf extract was obtained by hydrodistillation method. The toxicological effects of N. oleander distillate, previously suggested as medicinal food supplement, on drug resistant cells were evaluated by XTT tests. MDR modulation potential of the plant material was evaluated by flow cytometry and fluorescent microscopy. Paclitaxel and vincristine were applied to the sublines in combination with N. oleander distillate., Results: Fractional inhibitory indices show that N. oleander distillate did not increase the antiproliferative effects of anticancer drugs. N. oleander treatment in to MCF-7/Pac and MCF-7/Vinc did not inhibit P-gp activity and MDR1 gene expression level., Conclusions: As a result it may be suggested that although N. oleander distillate has some medicinal effects as food supplement it may not be suitable as an MDR modulator for drug resistant breast cancer cells.

Functionalization is an important technique to increase the solubility and biocompatibility of single-wall carbon nanotubes (SWCNTs). In this study, we investigated the cytotoxicity of four types of SWCNTs functionalized with hydroxyl, amino, carboxyl and polyethyleneglycol on MCF7 cells. These functionalized SWCNTs (f-SWCNTs) have insignificant effects on mitochondrial activity and ROS production in MCF7 cells at all test concentrations. However, explicit results revealed that all the tested f-SWCNTs could cause changes of cell morphology, induce cell membrane damage, decrease cell adhesion, and increase cell apoptosis. Therefore, this study shows the potential side effects of f-SWCNTs accompanying with the increase of dispersibility and stability in environment or serum (to prevent their aggregation), and highlights the need for further research to examine the potential toxicity of f-SWCNTs before they are used in the environmental and biomedical fields., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Heregulin (HRG) β1 signaling promotes scattering of MCF7 cells by inducing breakdown of adherens and tight junctions. Here, we show that stimulation with HRG-β1 causes the F-actin backbone of junctions to destabilize prior to the loss of adherent proteins and scattering of the cells. The adherent proteins dissociate and translocate from cell-cell junctions to the cytosol. Moreover, using inhibitors we show that the MEK1 pathway is required for the disappearance of F-actin from junctions and p38 MAP kinase activity is essential for scattering of the cells. Upon treatment with a p38 MAP kinase inhibitor, adherens junction complexes immediately reassemble, most likely in the cytoplasm, and move to the plasma membrane in cells dissociated by HRG-β1 stimulation. Subsequently, tight junction complexes form, most likely in the cytoplasm, and move to the plasma membrane. Thus, the p38 MAP kinase inhibitor causes a re-aggregation of scattered cells, even in the presence of HRG-β1. These results suggest that p38 MAP kinase signaling to adherens junction proteins regulates cell aggregation, providing a novel understanding of the regulation of cell-cell adhesion.

In this work, we have prepared three types of aptamer-conjugated Rubpy-doped silica nanoparticles for Human breast carcinoma MCF-7 cells labeling. Probe A is prepared through covalent conjugation between amine-labeled MUC-1 aptamer and carboxyl-modified Rubpy-doped NPs (NPs-aptamer). Probe B is prepared based on the interaction between biotin-labeled MUC-1 aptamer and avidin-conjugated Rubpy-doped NPs (NPs-avidin-biotin-aptamer). For Probe C, there is a PEG with flexible long chain as the bridge between avidin and the NPs (NPs-PEG-avidin-biotin-aptamer). In addition, we further investigate the practical number of MUC-1 aptamers on an NP of each probe using hoechst33258 dye. The binding efficiency of MUC-1 aptamer on the three types of probes as follows: Probe A < Probe B < Probe C. In addition, microscopic fluorescence imaging shows that Probe C containing the PEG molecules can be effectively applied for the recognition of MUC-1 protein in human breast carcinoma MCF-7 cells thus demonstrates that the PEG with flexible long chain as the bridge between the aptamer and NP can greatly enhances the freedom of MUC-1 aptamer. Compared with common organic dyes, the dye-doped silica nanoparticles serve as a stable bioprobe because of their facile conjugation with the desirable biomolecules, and have exhibited great potential in bioanalysis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Serine 7 of centromere protein A (CENP-A) is a very important mitosis-specific phosphorylation site. In this study, we demonstrate the subcellular distribution of Ser7 phosphorylated CENP-A during mitosis in MCF-7 cells. The Ser7 phosphorylation of CENP-A was observed beginning at prophase at centromeres. Upon progression of mitosis, the fluorescence signals emerged in the central region of the metaphase plate and were maintained until anaphase at centromeres. At late anaphase, the fluorescence signals moved to the midzone gradually and transferred from the centromere to the midbody completely at telophase. They were compacted into the centre of the midbody in a thin cylinder consisting of a sandglass-like "mitotic machine" with microtubules and condensed chromosome. We also found that Ser10 phosphorylated H3 and Thr11 phosphorylated H3 were co-localized at the midbody in two bell-like symmetrical bodies with Ser7 phosphorylated CENP-A during the terminal stage of cytokinesis. Midbody isolation and immunoblotting experiments also indicated that Ser7 phosphorylated CENP-A are components of the midbody. These findings suggest that Ser7 phosphorylated CENP-A acts as a chromosomal passenger protein and may play an important role in cytokinesis.