Metastatic Breast Cancer Cells Research Articles

Curcumin arrests G-quadruplex in the nuclear hyper-sensitive III1 element of c-MYC oncogene leading to apoptosis in metastatic breast cancer cells

c-MYC is deregulated in triple negative breast cancer (TNBC) pointing to be a promising biomarker for breast cancer treatment. Precise level of MYC expression is important in the control of cellular growth and proliferation. Designing of c-MYC-targeted antidotes to restore its basal level of cellular expression holds an optimistic approach towards anti-cancer treatment. MYC transcription is dominantly controlled by Nuclear Hypersensitive Element III-1 (NHEIII1) upstream of the promoter region possessing G-Quadruplex silencer element (Pu-27). We have investigated the selective binding-interaction profile of a natural phytophenolic compound Curcumin with native MYC G-quadruplex by conducting an array of biophysical experiments and in silico based Molecular Docking and Molecular Dynamic (MDs) simulation studies. Curcumin possesses immense anti-cancerous properties. We have observed significantly increased stability of MYC-G Quadruplex and thermodynamic spontaneity of Curcumin-MYC GQ binding with negative ΔG value. Transcription of MYC is tightly regulated by a complex mechanism involving promoters, enhancers and multiple transcription factors. We have used Curcumin as a model drug to understand the innate mechanism of controlling deregulated MYC back to its basal expression level. We have checked MYC-expression at transcriptional and translational level and proceeded for Chromatin Immuno-Precipitation assay (ChIP) to study the occupancy level of SP1, Heterogeneous nuclear ribonucleoprotein K (hnRNPK), Nucleoside Diphosphate Kinase 2 (NM23-H2) and Nucleolin at NHEIII1 upon Curcumin treatment of MDA-MB-231 cells. We have concluded that Curcumin binding tends to drive the equilibrium towards stable G-quadruplex formation repressing MYC back to its threshold-level. On retrospection of the synergistic effect of upregulated c-MYC and BCL-2 in cancer, we have also reported a new pathway [MYC-E2F-1-BCL-2-axis] through which Curcumin trigger apoptosis in cancer cells. Communicated by Ramaswamy H. Sarma

Proteomic Characterization of Cytoplasmic Lipid Droplets in Human Metastatic Breast Cancer Cells.

One of the characteristic features of metastatic breast cancer is increased cellular storage of neutral lipid in cytoplasmic lipid droplets (CLDs). CLD accumulation is associated with increased cancer aggressiveness, suggesting CLDs contribute to metastasis. However, how CLDs contribute to metastasis is not clear. CLDs are composed of a neutral lipid core, a phospholipid monolayer, and associated proteins. Proteins that associate with CLDs regulate both cellular and CLD metabolism; however, the proteome of CLDs in metastatic breast cancer and how these proteins may contribute to breast cancer progression is unknown. Therefore, the purpose of this study was to identify the proteome and assess the characteristics of CLDs in the MCF10CA1a human metastatic breast cancer cell line. Utilizing shotgun proteomics, we identified over 1500 proteins involved in a variety of cellular processes in the isolated CLD fraction. Interestingly, unlike other cell lines such as adipocytes or enterocytes, the most enriched protein categories were involved in cellular processes outside of lipid metabolism. For example, cell-cell adhesion was the most enriched category of proteins identified, and many of these proteins have been implicated in breast cancer metastasis. In addition, we characterized CLD size and area in MCF10CA1a cells using transmission electron microscopy. Our results provide a hypothesis-generating list of potential players in breast cancer progression and offers a new perspective on the role of CLDs in cancer.

IP-10 (CXCL10) Can Trigger Emergence of Dormant Breast Cancer Cells in a Metastatic Liver Microenvironment.

Metastatic breast cancer remains a largely incurable and fatal disease with liver involvement bearing the worst prognosis. The danger is compounded by a subset of disseminated tumor cells that may lie dormant for years to decades before re-emerging as clinically detectable metastases. Pathophysiological signals can drive these tumor cells to emerge. Prior studies indicated CXCR3 ligands as being the predominant signals synergistically and significantly unregulated during inflammation in the gut-liver axis. Of the CXCR3 ligands, IP-10 (CXCL10) was the most abundant, correlated significantly with shortened survival of human breast cancer patients with metastatic disease and was highest in those with triple negative (TNBC) disease. Using a complex ex vivo all-human liver microphysiological (MPS) model of dormant-emergent metastatic progression, CXCR3 ligands were found to be elevated in actively growing populations of metastatic TNBC breast cancer cells whereas they remained similar to the tumor-free hepatic niche in those with dormant breast cancer cells. Subsequent stimulation of dormant breast cancer cells in the ex vivo metastatic liver MPS model with IP-10 triggered their emergence in a dose-dependent manner. Emergence was indicated to occur indirectly possibly via activation of the resident liver cells in the surrounding metastatic microenvironment, as stimulation of breast cancer cells with exogenous IP-10 did not significantly change their migratory, invasive or proliferative behavior. The findings reveal that IP-10 is capable of triggering the emergence of dormant breast cancer cells within the liver metastatic niche and identifies the IP-10/CXCR3 as a candidate targetable pathway for rational approaches aimed at maintaining dormancy.

Combinatorial targeting of microRNA-26b and microRNA-101 exerts a synergistic inhibition on cyclooxygenase-2 in brain metastatic triple-negative breast cancer cells.

Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells. Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE. We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA. These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.

Protonectin peptides target lipids, act at the interface and selectively kill metastatic breast cancer cells while preserving morphological integrity

Despite the need for innovative compounds as antimicrobial and anticancer agents, natural sources of peptides remain underexplored. Protonectin (PTN), a cationic dodecapeptide of pharmacological interest, presents large hydrophobicity that is associated with the tendency to aggregate and supposedly influences bioactivity. A disaggregating role was assigned to PTN’ N-terminal fragment (PTN1-6), which enhances the bioactivity of PTN in a 1:1 mixture (PTN/PTN1-6). Spectroscopic techniques and model membranes (phospholipid bilayers and SDS micelles) revealed that environment-dependent aggregation is reduced for PTN/PTN1-6, but cytotoxicity of PTNs on MDA-MB-231 breast cancer showed the same CC50 values around 16 µM and on MCF-10A epithelial breast cells 6 to 5-fold higher values, revealing a selective interaction. Since PTN1-6 lacks activity on breast cells, its presence should differently affect PTN activity, suggesting that aggregation could modulate activity depending on the membrane characteristics. Indeed, increased partitioning and lytic activity of PTN/PTN1-6 were found in model membranes independently of charge density, but affected by the curvature tendency. PTN and PTN/PTN1-6 do not alter morphology and roughness of cancer cells, indicating a superficial interaction with membranes and consistent with results obtained in NMR experiments. Our results indicate that aggregation of PTNs depends on the membrane characteristics and modulates the activity of the peptides.

Dysregulation of tumour microenvironment driven by circ-TPGS2/miR-7/TRAF6/NF-κB axis facilitates breast cancer cell motility

Tumour microenvironment (TME) is frequently remodelled and deregulated in cancer development and progression. The underlying mechanisms are complex, multifactorial and largely unknown. Circular RNA (circRNA) is an endogenous RNA with a covalently closed loop that plays critical roles in the pathological and physiological processes of the organism. Here, we identified a TME-associated circRNA, circ-TPGS2, which promoted breast cancer (BC) cell dissemination via altering TME. High circ-TPGS2 was observed in metastatic BC tissues and cell lines in comparison to respective normal controls, which was linked to poor overall and recurrence-free survival. Overexpression of circ-TPGS2 notably promoted cell migration, while silencing of circ-TPGS2 resulted in an opposite trend. Moreover, circ-TPGS2 increased pro-inflammatory chemokine production and evoked tumour-associated inflammation by recruiting neutrophils via autocrine and paracrine manners. In terms of mechanism, circ-TPGS2 acted as a sponge of miR-7 and elevated TRAF6, leading to p65 phosphorylation and nuclear translocation, ultimately activating NF-κB signalling. In addition, p65 was abundantly occupied on circ-TPGS2 promoter, activating circ-TPGS2 transcription, thus, forming a positive feedback loop that amplified the pro-metastasis effect of circ-TPGS2. Taken together, our data for the first time reveal the biological implication of circ-TPGS2 in BC, it triggers TME reshaping that facilitates BC metastasis through the miR-7/TRAF6/NF-κB signalling cascade.

Multifunctional Liposomes Enable Active Targeting and Twinfilin 1 Silencing to Reverse Paclitaxel Resistance in Brain Metastatic Breast Cancer.

Paclitaxel (PTX) is a first-line chemotherapeutic drug for breast cancer, but PTX resistance often occurs in metastatic breast cancer. In addition, due to the poor targeting of chemotherapeutic drugs and the presence of the blood-brain barrier (BBB), it is hard to effectively treat brain metastatic breast cancer using paclitaxel. Thus, it is urgent to develop an effective drug delivery system for the treatment of brain metastatic breast cancer. The current study found that TWF1 gene, an epithelial-mesenchymal transition-associated gene, was overexpressed in brain metastatic breast cancer (231-BR) cells and was associated with the PTX resistance of 231-BR cells. Knockdown of TWF1 by small interference RNA (siRNA) in 231-BR cells could effectively increase the sensitivity of brain metastatic breast cancer cells to paclitaxel. Then, a liposome-based drug delivery system was developed for PTX delivery across BBB, enhancing PTX sensitivity and brain metastases targeting via BRBP1 peptide modification. The results showed that BRBP1-modified liposomes could effectively cross the BBB, specifically accumulate in brain metastases, and effectively interfere TWF1 gene expression in vitro and in vivo, and thus they enhanced proliferation inhibition, cell cycle arrest, and apoptosis induction, thereby inhibiting the formation and growth of brain metastases. In summary, our results indicated that BRBP1-modified and PTX- and TWF1 siRNA-loaded liposomes have the potential for the treatment of brain metastatic breast cancer, which lays the foundation for the development of a new targeted drug delivery system.

The MNK1/2-eIF4E Axis Supports Immune Suppression and Metastasis in Postpartum Breast Cancer.

Breast cancer diagnosed within 10 years following childbirth is defined as postpartum breast cancer (PPBC) and is highly metastatic. Interactions between immune cells and other stromal cells within the involuting mammary gland are fundamental in facilitating an aggressive tumor phenotype. The MNK1/2-eIF4E axis promotes translation of prometastatic mRNAs in tumor cells, but its role in modulating the function of nontumor cells in the PPBC microenvironment has not been explored. Here, we used a combination of in vivo PPBC models and in vitro assays to study the effects of inactivation of the MNK1/2-eIF4E axis on the protumor function of select cells of the tumor microenvironment. PPBC mice deficient for phospho-eIF4E (eIF4ES209A) were protected against lung metastasis and exhibited differences in the tumor and lung immune microenvironment compared with wild-type mice. Moreover, the expression of fibroblast-derived IL33, an alarmin known to induce invasion, was repressed upon MNK1/2-eIF4E axis inhibition. Imaging mass cytometry on PPBC and non-PPBC patient samples indicated that human PPBC contains phospho-eIF4E high-expressing tumor cells and CD8+ T cells displaying markers of an activated dysfunctional phenotype. Finally, inhibition of MNK1/2 combined with anti-PD-1 therapy blocked lung metastasis of PPBC. These findings implicate the involvement of the MNK1/2-eIF4E axis during PPBC metastasis and suggest a promising immunomodulatory route to enhance the efficacy of immunotherapy by blocking phospho-eIF4E. SIGNIFICANCE: This study investigates the MNK1/2-eIF4E signaling axis in tumor and stromal cells in metastatic breast cancer and reveals that MNK1/2 inhibition suppresses metastasis and sensitizes tumors to anti-PD-1 immunotherapy.

Phenotypic Heterogeneity and Metastasis of Breast Cancer Cells.

Although intratumoral genomic heterogeneity can impede cancer research and treatment, less is known about the effects of phenotypic heterogeneities. To investigate the role of cell migration heterogeneities in metastasis, we phenotypically sorted metastatic breast cancer cells into two subpopulations based on migration ability. Although migration is typically considered to be associated with metastasis, when injected orthotopically in vivo, the weakly migratory subpopulation metastasized significantly more than the highly migratory subpopulation. To investigate the mechanism behind this observation, both subpopulations were assessed at each stage of the metastatic cascade, including dissemination from the primary tumor, survival in the circulation, extravasation, and colonization. Although both subpopulations performed each step successfully, weakly migratory cells presented as circulating tumor cell (CTC) clusters in the circulation, suggesting clustering as one potential mechanism behind the increased metastasis of weakly migratory cells. RNA sequencing revealed weakly migratory subpopulations to be more epithelial and highly migratory subpopulations to be more mesenchymal. Depletion of E-cadherin expression from weakly migratory cells abrogated metastasis. Conversely, induction of E-cadherin expression in highly migratory cells increased metastasis. Clinical patient data and blood samples showed that CTC clustering and E-cadherin expression are both associated with worsened patient outcome. This study demonstrates that deconvolving phenotypic heterogeneities can reveal fundamental insights into metastatic progression. More specifically, these results indicate that migratory ability does not necessarily correlate with metastatic potential and that E-cadherin promotes metastasis in phenotypically sorted breast cancer cell subpopulations by enabling CTC clustering. SIGNIFICANCE: This study employs phenotypic cell sorting for migration to reveal a weakly migratory, highly metastatic breast cancer cell subpopulation regulated by E-cadherin, highlighting the dichotomy between cancer cell migration and metastasis.

SPDEF targets EMT pathway to inhibit cell migration and invasion in triple‐negative breast cancer

Background Current treatments against triple-negative breast cancer (TNBC) are ineffective due to the high rate of metastasis and therapy resistance. There is, therefore, a pressing need for novel and efficacious therapies against TNBC. The basal-like TNBC has a poor prognosis and a high metastasis rate compared to the luminal-like breast cancer subtypes. Here, set to determine the role of Sam Pointed Domain Ets Transcription Factor also known as Prostate-Derived Ets Factor (SPDEF/PDEF), in TNBC. Methods Clinical data were extracted from the METABRIC breast cancer project, hosted at the TCGA database. MDA-MB-231 and MDA 231-LM2-4175 cells were stably transfected with PDEF/control pBABE retroviral vectors. Cell migration/invasion assays were done to explore the effects of PDEF expression on the motility of TNBC cells. Proliferation was measured under 2D and 3D culture conditions. qRT-PCR and immunoblot were performed to visualize gene and protein expression. Data were graphed and analyzed using Graph Pad Prism Software 8 using one-way ANOVA and the unpaired two-tailed Student's t-test. All data were evaluated in triplicate against vector control cells. Results Basal gene and protein expression screening revealed that the expression of PDEF is significantly lower in highly metastatic breast cancer cells compared to the non-metastatic ones. Data analysis from clinical breast cancer METABRIC cohort revealed the loss of PDEF is associated with tumor metastasis, tumor grade, and poor patient survival. PDEF is lost in highly metastatic and therapy resistant TNBC cells. Re-expression of PDEF diminished cell migration and invasion in TNBC cells. Decreased migration and invasion in response to PDEF was not a result of decreased cell growth and proliferation or increased cell death but a result of suppression of EMT markers in TNBC cells. Conclusions PDEF expression inhibits cell migration and invasion partially by down-regulating EMT-related protein markers, suggesting a critical role for PDEF in suppressing TNBC cell metastasis.

Dual role played by Ca 2+ in the cytotoxicity of human metastatic breast cancer cells

Introduction Cancer cells exhibit enhanced potential to metastasize via increased acto-myosin cross bridges formation which requires increased Ca2+ influx via cell surface voltage-gated Ca2+ channels (VGCC) leading to activation of myosin light chain kinase (MLCK). The MLCK then phosphorylates myosin and forms acto-myosin cross-bridges. The role of MLCK, and calcium channel in cancer cell growth has not been extensively tested. Objective In this study, we tested the individual effects of blocking VGCC and MLCK on MCF-7 cell (human metastatic breast cancer cell) survival. We also treated the cells with high KCl to induce increased Ca2+ influx and test if the effects of KCl will be opposite to that of VGCC blocker. Methods The experimental design of this study is primarily focused on the administration of specific inhibitors to MCF-7 cells with an interest in the dose-dependent response of the cells to varying concentrations of the treatments as well as a thorough comparison of the cell morphology, viability, and proliferation to the effects of the 1% BME control. The three drugs that have been investigated so far are Nifedipine, a voltage-gated calcium blocker, Nifedipine in the presence of KCl [in order to artificially depolarize the cell membrane to open the VGCC], and ML-7, a myosin light chain kinase inhibitor. The cell viability was then assessed through an MTT colorimetric assay at 562 nm in order to visually and quantitatively assess the results. Additionally, transfection studies are planned to be carried out to substantiate pharmacological findings of inhibitors. Results & Conclusion Nifedipine dose-dependently inhibited MCF-7 cell viability. Following were the % absorbance values with increasing doses of nifedipine. All values are relative to control without nifediine considered as 100%; [0.625 um Nif – 43.05%, 1.25 um Nif – 42.19%, 2.5 um Nif – 40.15%, 5 um Nif -39.53%, 10 um Nif – 38.19%, 20 um Nif – 25.1%, 40 um Nif – 22.96%, 1% BME –39.26%]. The 1% BME (bitter melon extract) is the positive control in our studies as our lab have found BME to be toxic to MCF-7 cells. ML-7 also dose-dependently inhibited MCF-7 cell viability. Following were the % absorbance values with increasing doses of ML-7. All values are relative to control without ML-7 considered as 100%; (0.5 um ML7- 70.01%, 1 um ML7 – 65.74%, 2 um ML7 – 57.71%, 4 um ML7 – 49.36%, 8 um ML7 – 48.28%, 16 um ML7 – 34.13%, 1% BME – 36.17%]. When we stimulated cells with increasing doses of KCl, increasing toxicity was seen as reflected by the decreasing absorbance values. We tested the effects of KCl in the presence or absence of nifedipine. Following were the % absorbance values with increasing doses of KCl and/or nifedipine. All values are relative to control (neither KCl nor nifedipine) considered as 100%; [40 mm KCl – 29.55%, 20 mm KCl – 44.024%, 10 mm KCl –56.51%, 40 um Nif – 22.81%, 40 mm KCl + 40 um Nif – 13.69%, 20 mm KCl + 40 um Nif –15.87%, 10 mm KCl + 40 um Nif – 30.91%, EtOH – 86.85%, 1% BME – 35.27%.]. Thus, either inhibiting Ca2+ influx with nifedipine or opening VGCC with high KCl both exerted cytotoxic effects on MCF-7 cells. This suggests that Ca2+ is critical for the viability of these cells but at the same time too much Ca2+ is toxic to the cells.

Hematopoietic protein RELT stains prominently in breast cancer and binds the cytoskeletal protein Filamin A.

Abstract Receptor Expressed in Lymphoid Tissues (RELT) is a human TNFR superfamily member expressed most prominently in cells and tissues of the hematopoietic system. Mouse knockout studies indicate that RELT functions in part by negatively regulating T-cell activation. RELT has two homologous binding partners, RELL1 and RELL2, and collectively, these proteins are described as RELT family members. This study sought to identify novel protein interactions with RELT family members and to compare the expression of RELT in normal and diseased tissues. A yeast two-hybrid screen utilizing RELL1 as bait identified the cytoskeletal protein Filamin A (FlnA); physical interaction between FlnA and RELT family members was confirmed by in vitro co-immunoprecipitation. A truncated mutant of FlnA disrupts the ability of RELT family members to activate the p38 pathway. Previous reports indicate that autoantibodies against RELT are associated with breast cancer and that RELL2 prevents migration and invasion of the metastatic breast cancer cell line MDA-MB-231 (231), while multiple studies suggest a link between FlnA and breast cancer. We therefore sought to examine the association of RELT family members with breast cancer. Western blotting revealed significant expression of endogenous RELT family members in the breast cancer cell lines 231 and MCF-7. Immunohistochemistry (IHC) revealed a higher intensity of RELT staining in breast cancer tissues versus normal mammary tissue. Additionally, overexpression of RELT family members enhanced apoptosis in 231 cells. Collectively, these results further our understanding of signal transduction pathways associated with RELT family members and provide evidence that RELT is upregulated in breast cancer.

QAP21 reduces stemness and mobility of metastatic breast cancer cells involving D1DR activation

QAP21 reduces stemness and mobility of metastatic breast cancer cells involving D1DR activation

Quartz Crystal Microbalance (QCM) Based Biosensor Functionalized by HER2/neu Antibody for Breast Cancer Cell Detection

The heterogeneity and metastatic features of cancer cells lead to a great number of casualties in the world. Additionally, its diagnosis as well as its treatment is highly expensive. Therefore, development of simple but effective diagnostic systems which detect the molecular markers of cancer is of great importance. The molecular changes on cancer cell membranes serve as targets, such as HER2/neu receptor which is detected on the surface of highly metastatic breast cancer cells. We have aimed to develop a specific and simple quartz crystal microbalance (QCM)-based system to identify HER2/neu expressing breast cancer cells via a receptor-specific monoclonal antibody. First, the QCM chip was coated with polymeric nanoparticles composed of hydroxyethylmethacrylate (HEMA) and ethylene glycol dimethacrylate (EDMA). The nanoparticle coated QCM chip was then functionalized by binding of HER2/neu antibody. The breast cancer cells with/without HER2/neu receptor expression, namely, SKBR3, MDA-MB 231 and also mouse fibroblasts were passed over the chip at a rate of 10–500 cells/mL and the mass changes (Δm) on cell/cm2 unit surface of sensor were detected in real-time. The detection limit of the system was 10 cells/mL. Thus, this QCM-based HER2/neu receptor antibody functionalized system might be used effectively in the detection of HER2/neu expressing SKBR3 breast cancer cells.

Widespread Alternative Splicing Changes in Metastatic Breast Cancer Cells

Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types.

The Influence of Cold Atmospheric Pressure Plasma-Treated Media on the Cell Viability, Motility, and Induction of Apoptosis in Human Non-Metastatic (MCF7) and Metastatic (MDA-MB-231) Breast Cancer Cell Lines.

Breast cancer remains the most common type of cancer, occurring in middle-aged women, and often leads to patients’ death. In this work, we applied a cold atmospheric pressure plasma (CAPP)-based reaction-discharge system, one that is unique in its class, for the production of CAPP-activated media (DMEM and Opti-MEM); it is intended for further uses in breast cancer treatment. To reach this aim, different volumes of DMEM or Opti-MEM were treated by CAPP. Prepared media were exposed to the CAPP treatment at seven different time intervals and examined in respect of their impact on cell viability and motility, and the induction of the apoptosis in human non-metastatic (MCF7) and metastatic (MDA-MB-231) breast cancer cell lines. As a control, the influence of CAPP-activated media on the viability and motility, and the type of the cell death of the non-cancerous human normal MCF10A cell line, was estimated. Additionally, qualitative and quantitative analyses of the reactive oxygen and nitrogen species (RONS), generated during the CAPP operation in contact with analyzed media, were performed. Based on the conducted research, it was found that 180 s (media activation time by CAPP) should be considered as the minimal toxic dose, which significantly decreases the cell viability and the migration of MDA-MB-231 cells, and also disturbs life processes of MCF7 cells. Finally, CAPP-activated media led to the apoptosis of analyzed cell lines, especially of the metastatic MDA-MB-231 cell line. Therefore, the application of the CAPP system may be potentially applied as a therapeutic strategy for the management of highly metastatic human breast cancer.

Quantitative Approach for Protein Analysis in Small Cell Ensembles by an Integrated Microfluidic Chip with MALDI Mass Spectrometry.

Increasing evidence has demonstrated that cells are individually heterogeneous. Advancing the technologies for single-cell analysis will improve our ability to characterize cells, study cell biology, design and screen drugs, and aid cancer diagnosis and treatment. Most current single-cell protein analysis approaches are based on fluorescent antibody-binding technology. However, this technology is limited by high background and cross-talk of multiple tags introduced by fluorescent labels. Stable isotope labels used in mass cytometry can overcome the spectral overlap of fluorophores. Nevertheless, the specificity of each antibody and heavy-metal-tagged antibody combination must be carefully validated to ensure detection of the intended target. Thus, novel single-cell protein analysis methods without using labels are urgently needed. Moreover, the labeling approach targets already known motifs, hampering the discovery of new biomarkers relevant to single-cell population variation. Here, we report a combined microfluidic and matrix-assisted laser desorption and ionization (MALDI) mass spectrometric approach for the analysis of protein biomarkers suitable for small cell ensembles. All necessary steps for cell analysis including cell lysis, protein capture, and digestion as well as MALDI matrix deposition are integrated on a microfluidic chip prior to the downstream MALDI-time-of-flight (TOF) detection. For proof of principle, this combined method is used to assess the amount of Bcl-2, an apoptosis regulator, in metastatic breast cancer cells (MCF-7) by using an isotope-labeled peptide as an internal standard. The proposed approach will eventually provide a new means for proteome studies in small cell ensembles with the potential for single-cell analysis and improve our ability in disease diagnosis, drug discovery, and personalized therapy.

Zebrafish embryo extracts enhance 5-FU anti-cancer effects upon breast cancer cells.

The inhibition of the metastatic capability of cancer cells is a pivotal aim of current anticancer strategies. We investigated herein the anti-migrating and anti-invasive properties of Zebrafish embryo extracts (SL) - an integrative formula comprising morphogenetic factors extracted from zebrafish embryos - alone or in association with 5-Fluoro-Uracil (5-FU), when added to metastatic breast cancer cells (MDA-MB-231) and in normal epithelial breast cells (MCF10A) committed toward an inflammatory phenotype upon TGF-β1 stimulation. Invasiveness, migrating capability, cytoskeleton architecture and related molecular factors involved in the epithelial-mesenchymal transition were studied after treatment with 5-FU, with and without SL. Remarkably, in both circumstances, embryo extracts amplify the migratory inhibition triggered by the anticancer drug 5-Fu. The fact that such an effect is noticed in normal as well as in cancerous cells suggests that the critical target of embryo extracts is specifically represented by the migrating/invasive phenotype. However, while 5-FU was unable in antagonizing the invasiveness of cancerous cells, the association with SL can significantly impair the invasive capability of tumor cells. These findings are noticeably associated with the reversion of the EMT phenotype in SL-treated cells, as documented by the contemporary downregulation of TCTP and some EMT-related molecular effectors, like α-SMA and Vimentin. Embryo fish extracts significantly counteract the migrating and invasive phenotype of cancerous and inflammatory breast cells treated with the chemotherapeutic drug 5-FU. The availability of a compound able to amplify 5-Fu activity while significantly hampering the invasive phenotype of breast cancer should provide invaluable benefits, namely if we consider that this compound is substantially deprived of side-effects.

The relationship between expression of VIMENTIN and CD146 genes in breast cancer

Abstract Objectives CD146 is an adhesive molecule that was originally reported on malignant melanoma cells as a protein crucial for cell adhesion. It is now known that high expression of the CD146 protein is not only characteristic of melanoma, but it occurs on a number of cancers, contributing to worse prognosis and increased aggressiveness. Independent in vitro studies in breast cancer have shown that CD146 protein alone can induce a change in epithelial to mesenchymal transcriptional profile, which is the basis of the tumor aggressiveness and metastasis. Methods In the following work, the correlation coefficients were analyzed between the genes of the mesenchymal profile and the CD146 gene in 10 independent transcriptomic data of breast cancer patients. Results The analysis confirmed the relationship between CD146 expression and mesenchymal profile genes, pointing VIMENTIN as the gene which expression is most strongly correlated with the CD146, suggesting that both genes, CD146 and VIM may be directly controlled by the same mechanism or regulate one another. Conclusions The analysis points a potential route for research on the CD146 gene expression, which may lead to understanding of its regulation in breast cancer, contributing to the development of new therapeutic strategies targeting highly metastatic breast cancer cells.

N-cadherin in osteolineage cells modulates stromal support of tumor growth

Tumor growth and metastases are dependent on interactions between cancer cells and the local environment. Expression of the cell–cell adhesion molecule N-cadherin (Ncad) is associated with highly aggressive cancers, and its expression by osteogenic cells has been proposed to provide a molecular “dock” for disseminated tumor cells to establish in pre-metastatic niches within the bone. To test this biologic model, we conditionally deleted the Ncad gene (Cdh2) in osteolineage cells using Osx-cre (cKO). Contrary to expectations, the metastatic breast cancer cell line PyMT-BO1 was able to form tumors in bone and to induce osteolysis in cKO as well as in control mice. Despite absence of Ncad, bone marrow stromal cells isolated from cKO mice were able to engage in direct cell–cell interactions with tumor cells expressing either N- or E-cadherin. However, subcutaneous PyMT-BO1 and B16F10 tumors grew larger in cKO relative to control littermates. Cell tracking experiments using the Ai9 reporter revealed the presence of Osx+ and Ncad+ cells in the stroma of extra-skeletal tumors and in a small population of lung cells. Gene expression analysis by RNAseq of Osx+ cells isolated from extra-skeletal tumors revealed alterations of pro-tumorigenic signaling pathways in cKO cells relative to control Osx+ cells. Thus, Ncad in Osx+ cells is not necessary for the establishment of bone metastases, but in extra-skeletal tumors it regulates pro-tumorigenic support by the microenvironment.