Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods.

Assessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

Nuclear morphology in breast lesions: refining its assessment to improve diagnostic concordance.

AIMS: Although evaluation of nuclear morphology is important for the diagnosis and categorisation of breast lesions, the criteria used to assess nuclear atypia rely upon the subjective evaluation of several features that may result in inter- and intraobserver variation. This study aims to refine the definitions of cytonuclear features in various breast lesions. METHODS AND RESULTS: ImageJ was used to assess the nuclear morphological features including nuclear diameter, axis length, perimeter, area, circularity and roundness in 160 breast lesions comprising ductal carcinoma in situ (DCIS), invasive breast carcinoma of no special type (IBC-NST), tubular carcinoma, usual ductal hyperplasia (UDH), columnar cell change (CCC) and flat epithelial atypia (FEA). Reference cells included normal epithelial cells, red blood cells (RBCs) and lymphocytes. Reference cells showed size differences not only between normal epithelial cells and RBCs but also between RBCs in varied-sized blood vessels. Nottingham grade nuclear pleomorphism scores 1 and 3 cut-offs in IBC-NST, compared to normal epithelial cells, were < ×1.2 and > ×1.4 that of mean maximum Feret's diameter and < ×1.6 and > ×2.4 that of mean nuclear area, respectively. Nuclear morphometrics were significantly different in low-grade IBC-NST versus tubular carcinoma, low-grade DCIS versus UDH and CCC versus FEA. No differences in the nuclear features between grade-matched DCIS and IBC-NST were identified. CONCLUSION: This study provides a guide for the assessment of nuclear atypia in breast lesions, refines the comparison with reference cells and highlights the potential diagnostic value of image analysis tools in the era of digital pathology.

Quantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy.

Currently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.

Optimally designed theranostic system based folic acids and chitosan as a promising mucoadhesive delivery system for encapsulating curcumin LbL nano-template against invasiveness of breast cancer.

Curcumin is a potential candidate in cancer therapy due to its ability to inhibit many signalling pathways at the same time of exposure because of its unique content of aromatic ring, B diketone, olefinic linker, and O methoxy phenolic groups. Its applications in biomedical therapy is limited because of its sensitivity, and its rapid degradation. In the current study, curcumin inserted into polyelectrolyte pairs (protamine and dextran) and then was functionalized by folic acid conjugated chitosan used for the first time, as theranostic system. Such this strategy allows to improve its mucoadhesion and penetration that increases their accumulation inside cancer cells. CUR-LbL NPs were then used to investigate drug release inside Human Mammary Carcinoma (MCF-7 cell lines) after their incubations for 3 h, 6 h and 24 h. Flow cytometry indicated that the percentages of apoptosis, necrosis and cell cycle arrest were increased significantly in MCF-7 cell lines treated by CUR-LbL NPs. Furthermore, SEM image showed many debris in the section of MCF-7 treated by CUR-LbL NPs. Here, it can be summarized that curcumin functionalized by multi-layered polyelectrolyte capsules can be used as a model to study the fate of the adsorbed nanocarriers and to investigate the drug release inside cells.

CD34+ Stromal Cells/Telocytes as a Source of Cancer-Associated Fibroblasts (CAFs) in Invasive Lobular Carcinoma of the Breast.

Several origins have been proposed for cancer-associated fibroblasts (CAFs), including resident CD34+ stromal cells/telocytes (CD34+SCs/TCs). The characteristics and arrangement of mammary CD34+SCs/TCs are well known and invasive lobular carcinoma of the breast (ILC) is one of the few malignant epithelial tumours with stromal cells that can express CD34 or αSMA, which could facilitate tracking these cells. Our objective is to assess whether tissue-resident CD34+SCs/TCs participate in the origin of CAFs in ILCs. For this purpose, using conventional and immunohistochemical procedures, we studied stromal cells in ILCs (n:42) and in normal breasts (n:6, also using electron microscopy). The results showed (a) the presence of anti-CD34+ or anti-αSMA+ stromal cells in varying proportion (from very rare in one of the markers to balanced) around nests/strands of neoplastic cells, (b) a similar arrangement and location of stromal cells in ILC to CD34+SCs/TCs in the normal breast, (c) both types of stromal cells coinciding around the same nest of neoplastic cells and (d) the coexpression of CD34 and αSMA in stromal cells in ILC. In conclusion, our findings support the hypothesis that resident CD34+SCs/TCs participate as an important source of CAFs in ILC. Further studies are required in this regard in other tumours.

Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

EGFR Expression in HER2-Driven Breast Cancer Cells.

The epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3, and HER4. Heterodimerization has been proposed as a mechanism of resistance to therapy for HER2 overexpressing breast cancer. Here, a method is presented for the simultaneous detection of individual EGFR and HER2 receptors in the plasma membrane of breast cancer cells via specific labeling with quantum dot nanoparticles (QDs). Correlative fluorescence microscopy and liquid phase electron microscopy were used to analyze the plasma membrane expression levels of both receptors in individual intact cells. Fluorescent single-cell analysis of SKBR3 breast cancer cells dual-labeled for EGFR and HER2 revealed a heterogeneous expression for receptors within both the cell population as well as within individual cells. Subsequent electron microscopy of individual cells allowed the determination of individual receptors label distributions. QD-labeled EGFR was observed with a surface density of (0.5-5) × 101 QDs/µm2, whereas labeled HER2 expression was higher ranging from (2-10) × 102 QDs/µm2. Although most SKBR3 cells expressed low levels of EGFR, an enrichment was observed at large plasma membrane protrusions, and amongst a newly discovered cellular subpopulation termed EGFR-enriched cells.

Photodynamic inactivation of circulating tumor cells: An innovative approach against metastatic cancer.

The spread of a primary malignant tumor is the major reason for most of the cancer-associated deaths. To this day, treatment regimen and available drugs are still insufficient to manage these conditions. In this work, a new therapeutic concept based on photodynamic therapy (PDT) of metastasis-initiating cells is introduced. To address this issue, an experimental model was developed to simulate the movement and photodynamic inactivation of circulating tumor cells (CTCs) in vitro. Using curcumin loaded poly(lactic-co-glycolic acid) nanoparticles, a significant reduction in the cell viability of human breast cancer cells (MDA-MB-231) could be achieved after 30 min laser irradiation (λ = 447 nm, P = 100mW) under flow conditions (5 cm s-1). Confocal laser scanning microscopy images confirmed the immediate accumulation of curcumin on the cell membrane and an increased fluorescence signal after irradiation. PDT caused time-dependent morphological cell alterations (i.e. membrane evaginations and disruption) indicating apoptosis and early necrosis. During the photoactivation of curcumin, a blue shift in the absorption spectra and a decrease in the curcumin content could be determined. This study confirms that the presented experimental model is suitable for in vitro investigations of CTCs under in vivo-like conditions, at the same time encouraging the clinical implementation of PDT as an innovative strategy against metastasis.

Tumor size and focality in breast carcinoma: Analysis of concordance between radiological imaging modalities and pathological examination at a cancer center.

CONTEXT: Accurate assessment of clinical and pathological tumor stage is crucial for patient treatment and prognosis. OBJECTIVE: The aim of this study was to assess the concordance between the tumor size and focality between radiological studies and pathology and to evaluate the impact of discrepancies on staging. DESIGN: Patients who underwent surgery for invasive breast carcinoma from January 1, 2014, to December 31, 2015, were identified. RESULTS: Three imaging modalities (mammogram, ultrasound and MRI) were compared with gross examination and final pathology. 1152 preoperative radiological studies were evaluated for focality and 1019 were evaluated for tumor size. For all 3 radiographic modalities, there was a statistically significant difference between the mean tumor size on radiology and the final pathology report (mammogram, P < .001; ultrasound, P = .004; MRI, P < .001). In 29% of radiology studies, there was a discrepancy in stage. The error rate for determining focality was 28% for mammograms, 27% for ultrasounds, and 29% for MRIs. Tumor size from gross examination correlated with microscopic tumor size in 57% of cases, but gross examination had 88% concordance with the final pathology report in determining focality. CONCLUSION: Our study revealed statistically significant differences in mean tumor size reported across all 3 imaging modalities when compared to the final pathology report. MRI had the highest error rate, with a tendency to overestimate tumor size and number of foci. Among all diagnoses, cases of invasive carcinoma with an extensive intraductal component were most prone to discrepancies with imaging.

Docosahexaenoic acid (DHA) inhibits pro-angiogenic effects of breast cancer cells via down-regulating cellular and exosomal expression of angiogenic genes and microRNAs.

AIMS: Docosahexaenoic acid (DHA) as an omega 3 free fatty acid has been reported to exert anti-angiogenesis effects. However, our current understanding regarding the precise mechanisms of such effects is still limited. Exosomes secreted by cancer cells may act as angiogenesis promoters. The aim of the study was to determine altered expression levels of HIF-1α, TGF-ß, VEGFR, Snail1, Snail2 and SOX2 and their regulating microRNAs in MDA-MB-231 and BT-474 cell lines after treatment with DHA in both normoxic and hypoxic conditions. MAIN METHODS: Human breast cancer cell lines including MDA-MB-231 and BT-474 were treated for 24 h with 100 uM DHA under normoxic and hypoxic conditions. Exosomes were isolated from untreated and treated cells and characterized by transmission electron microscopy (TEM) and western blotting. RNAs from cells and isolated exosomes were extracted and cDNAs were synthesized. Expression levels of miRNAs and their pro-angiogenic target genes were analyzed using quantitative real-time PCR (qRT-PCR). KEY FINDINGS: We showed significant decrease in the expression of pro-angiogenic genes including HIF1-α, TGF-ß, SOX2, Snail1, Snail2 and VEGFR in cells and also their secreted exosomes after treatment with DHA in normoxic and hypoxic conditions. Also the expression levels of tumor suppressor miRs including miR-101, miR-199, miR-342 were increased and the expression levels of oncomiRs including mir-382 and miR-21 were decreased after treatment with DHA in cells and exosomes. SIGNIFICANCE: DHA can alter the expression of pro-angiogenic genes and microRNA contents in breast cancer cells and their derived-exosomes in favor of the inhibition of angiogenesis. Our data demonstrated new insight into DHA's anti-cancer action to target not only breast cancer cells but also their derived exosomes to suppress tumor angiogenesis.

A novel 4-aminoquinazoline derivative, DHW-208, suppresses the growth of human breast cancer cells by targeting the PI3K/AKT/mTOR pathway.

Breast cancer is one of the most frequent cancers among women worldwide. However, there is still no effective therapeutic strategy for advanced breast cancer that has metastasized. Aberrant activation of the PI3K/AKT/mTOR pathway is an essential step for the growth of human breast cancers. In our previous study, we designed and synthesized DHW-208 (2,4-difluoro-N-(5-(4-((1-(2-hydroxyethyl)-1H-pyrazol-4-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)benzenesulfonamide) as a novel pan-PI3K inhibitor. This study aimed to assess the therapeutic efficacy of DHW-208 in breast cancer and investigate its underlying mechanism. We found that DHW-208 inhibited the growth, proliferation, migration, and invasion of breast cancer cells. Moreover, DHW-208 induced breast cancer cell apoptosis via the mitochondrial pathway and induced G0/G1 cell-cycle arrest. In vitro results show that DHW-208 is a dual inhibitor of PI3K and mTOR, and suppress the growth of human breast cancer cells by targeting the PI3K/AKT/mTOR pathway. Consistent with the in vitro results, in vivo studies demonstrated that DHW-208 elicits an antitumor effect by inhibiting the PI3K/AKT/mTOR-signaling pathway with a high degree of safety in breast cancer. Above all, we report for the first time that DHW-208 suppressed the growth of human breast cancer cells by inhibiting the PI3K/AKT/mTOR-signaling pathway both in vivo and in vitro. Our study may provide evidence for the use of DHW-208 as an effective, novel therapeutic candidate for the treatment of human breast cancers in clinical trials.

Diagnostic performance of peripheral leukocyte telomere G-tail length for detecting breast cancer.

The telomere G-tail (G-tail) plays an essential role in maintaining chromosome stability. In this study, we assessed the leukocyte G-tail length of breast cancer (BC) patients and cancer-free individuals and evaluated the association between the G-tail length and the presence of BC. A significant shortening of the median G-tail length was observed in BC patients compared with cancer-free individuals and was found in the early phase of BC. Our study indicated that the leukocyte G-tail length might be a potential biomarker for BC detection.

Evaluation of lumican effects on morphology of invading breast cancer cells, expression of integrins and downstream signaling.

The small leucine-rich proteoglycan lumican regulates estrogen receptors (ERs)-associated functional properties of breast cancer cells, expression of matrix macromolecules, and epithelial-to-mesenchymal transition. However, it is not known whether the ER-dependent lumican effects on breast cancer cells are related to the expression of integrins and their intracellular signaling pathways. Here, we analyzed the effects of lumican in three breast cancer cell lines: the highly metastatic ERß-positive MDA-MB-231, cells with the respective ERß-suppressed (shERßMDA-MB-231), and lowly invasive ERα-positive MCF-7/c breast cancer cells. Scanning electron microscopy, confocal microscopy, real-time PCR, western blot, and cell adhesion assays were performed. Lumican effects on breast cancer cell morphology were also investigated in 3-dimensional collagen cultures. Lumican treatment induced cell-cell contacts and cell grouping and inhibited microvesicles and microvilli formation. The expression of the cell surface adhesion receptor CD44, its isoform and variants, hyaluronan (HA), and HA synthases was also investigated. Lumican inhibited the expression of CD44 and HA synthases, and its effect on cell adhesion revealed a major role of α1, α2, α3, αVß3, and αVß5 integrins in MDA-MB-231 cells, but not in MCF-7/c cells. Lumican upregulated the expression of α2 and ß1 integrin subunits both in MDA-MB-231 and in shERßMDA-MB-231 as compared to MCF-7/c cells. Downstream signaling pathways for integrins, such as FAK, ERK 1/2 MAPK 42/44, and Akt, were found to be downregulated by lumican. Our data shed light to the molecular mechanisms responsible for the anticancer activity of lumican in invasive breast cancer.

Structural Studies of Epithelial Mesenchymal Transition Breast Tissues.

At the supramolecular level, the proliferation of invasive ductal carcinoma through breast tissue is beyond the range of standard histopathology identification. Using synchrotron small angle x-ray scattering (SAXS) techniques, determining nanometer scale structural changes in breast tissue has been demonstrated to allow discrimination between different tissue types. From a total of 22 patients undergoing symptomatic investigations, different category breast tissue samples were obtained in use of surgically removed tissue, including non-lesional, benign and malignant tumour. Structural components of the tissues were examined at momentum transfer values between q = 0.2 nm-1 and 1.5 nm-1. From the SAXS patterns, axial d-spacing and diffuse scattering intensity were observed to provide the greatest discrimination between the various tissue types, specifically in regard to the epithelial mesenchymal transition (EMT) structural component in malignant tissue. In non-lesional tissue the axial period of collagen is within the range 63.6-63.7 nm (formalin fixed paraffin embedded (FFPE) dewaxed) and 63.4 (formalin fixed), being 0.9 nm smaller than in EMT cancer-invaded regions. The overall intensity of scattering from cancerous regions is a degree of magnitude greater in cancer-invaded regions. Present work has found that the d-spacing of the EMT positive breast cancer tissue (FFPE (dewaxed)) is within the range 64.5-64.7 nm corresponding to the 9th and 10th order peaks. Of particular note in regard to formalin fixation of samples is that no alteration is observed to occur in the relative differences in collagen d-spacing between non-lesional and malignant tissues. This is a matter of great importance given that preserved-sample and also retrospective study of samples is greatly facilitated by formalin fixation. Present results indicate that as aids in tissue diagnosis SAXS is capable of distinguishing areas of invasion by disease as well as delivering further information at the supramolecular level.

GANT61 and curcumin-loaded PLGA nanoparticles for GLI1 and PI3K/Akt-mediated inhibition in breast adenocarcinoma.

Current conventional mono and combination therapeutic strategies often fail to target breast cancer tissue effectively due to tumor heterogeneity comprising cancer stem cells (CSCs) and bulk tumor cells. This is further associated with drug toxicity and resistivity in the long run. A nanomedicine platform incorporating combination anti-cancer treatment might overcome these challenges and generate synergistic anti-cancer effects and also reduce drug toxicity. GANT61 and curcumin were co-delivered via polymeric nanoparticles (NPs) for the first time to elicit enhanced anti-tumor activity against heterogeneous breast cancer cell line MCF-7. We adopted the single-emulsion-solvent evaporation method for the preparation of the therapeutic NPs. The GANT61-curcumin PLGA NPs were characterized for their size, shape and chemical properties, and anti-cancer cell studies were undertaken for the plausible explanation of our hypothesis. The synthesized GANT61-curcumin PLGA NPs had a spherical, smooth surface morphology, and an average size of 347.4 d. nm. The NPs induced cytotoxic effects in breast cancer cells at a mid-minimal dosage followed by cell death via autophagy and apoptosis, reduction in their target protein expression along with compromising the self-renewal property of CSCs as revealed by their in vitro cell studies. The dual-drug NPs thus provide a novel perspective on aiding existing anti-cancer nanomedicine therapies to target a heterogeneous tumor mass effectively.

Core needle biopsy of benign, borderline and in-situ problematic lesions of the breast: Diagnosis, differential diagnosis and immunohistochemistry.

Core needle biopsy (CNB) is the most common sampling technique for the histologic evaluation of breast abnormalities. Diagnosing benign proliferative, borderline and some in-situ lesions in CNB is challenging and subject to a significant degree of interobserver variability. In addition, due to the inherent limitations of CNB, "upgrading" to a more significant pathology at excision is an important consideration for some lesions. Pathologists carry a major responsibility in patient diagnosis, risk stratification and management. Familiarity with the histologic features and the clinical significance of these common and problematic lesions encountered in CNB is necessary for adequate treatment and patient follow-up. This review will focus on benign, atypical and in-situ epithelial proliferations, papillary lesions, radial sclerosing lesions, adenosis and cellular fibroepithelial lesions. Highlights of histologic features, useful strategies for accurate diagnosis, basic immunohistochemistry and management will be presented.

Proteomic profiling of extracellular vesicles allows for human breast cancer subtyping.

Extracellular vesicles (EVs) are a potential source of disease-associated biomarkers for diagnosis. In breast cancer, comprehensive analyses of EVs could yield robust and reliable subtype-specific biomarkers that are still critically needed to improve diagnostic routines and clinical outcome. Here, we show that proteome profiles of EVs secreted by different breast cancer cell lines are highly indicative of their respective molecular subtypes, even more so than the proteome changes within the cancer cells. Moreover, we detected molecular evidence for subtype-specific biological processes and molecular pathways, hyperphosphorylated receptors and kinases in connection with the disease, and compiled a set of protein signatures that closely reflect the associated clinical pathophysiology. These unique features revealed in our work, replicated in clinical material, collectively demonstrate the potential of secreted EVs to differentiate between breast cancer subtypes and show the prospect of their use as non-invasive liquid biopsies for diagnosis and management of breast cancer patients.

Withaferin-A Inhibits Growth of Drug-Resistant Breast Carcinoma by Inducing Apoptosis and Autophagy, Endogenous Reactive Oxygen Species (ROS) Production, and Inhibition of Cell Migration and Nuclear Factor kappa B (Nf-κB)/Mammalian Target of Rapamycin (m-TOR) Signalling Pathway.

BACKGROUND The main purpose of this study was to assess in vitro and in vivo the anticancer effect of withaferin-A in human breast carcinoma cells (MDA-MB-231), and to assess its effects on autophagy, cell apoptosis, ROS production, cell migration and invasion, and Nf-kappaB/m-TOR signalling pathway. MATERIAL AND METHODS Proliferation of MDA-MB-231 cells at various doses of the drug was studied by CCK8 cell viability assay. Effects on cell apoptosis were studied by fluorescence microscopy in combination with flow cytometry and Western blot analysis. Effects on autophagy were evaluated by transmission electron microscopy and Western blot. Effects on cellular migration were examined in vitro by wound healing assay. RESULTS The results indicated that withaferin-A led to significant reduction of MDA-MB-231 cell viability. The anticancer action of withaferin-A was shown to be due to the stimulation of autophagy, which was accompanied by enhancement of LC3 expression. Withaferin-A prompted mitochondrial apoptosis, which was also associated with increased level of Bax and decreased Bcl-2 in MDA-MB-231 cells. It was also observed that withaferin-A has decreases cellular migration and invasion of the tested human breast cancer cells. The effects of withaferin-A were also investigated in vivo, and it was found that this molecule could inhibit the growth of tumor xenografts in tested mice. Withaferin-A led to suppression of the Nf-kappaB/m-TOR signalling pathway. CONCLUSIONS In brief, the withaferin-A molecule has great potential as an anticancer agent against drug-resistant breast cancer, and as such needs to be further studied in detail.

Folate-coated, long-circulating and pH-sensitive liposomes enhance doxorubicin antitumor effect in a breast cancer animal model.

Long circulating pH-sensitive liposomes have been shown to effectively deliver doxorubicin (DOX) to tumors and reduce its toxic effects. Folic acid receptors are upregulated in a wide variety of solid, epithelial tumors, including breast cancer. In order to improve liposomal endocytosis and antitumor activity, folic acid has been added to nanoparticles surfaces to exploit overexpression of folate receptors in tumor cells. The purpose of this study was to evaluate the antitumor activity in vitro and in vivo of long circulating pH-sensitive folate-coated DOX-loaded liposomes (SpHL-DOX-Fol) in a 4T1 breast cancer model system in vitro and in vivo. Biodistribution studies were performed and in vivo electrocardiographic parameters were evaluated. A higher tumor uptake for radiolabeled SpHL-Fol (99mTc-SpHL-Fol) 4 h after intravenous administration was observed in comparision with non-folate-coated liposomes (99mTc-SpHL). Antitumor activity showed that SpHL-DOX-Fol treatment led to a 68% growth arrest and drastically reduce pulmonary metastasis foci. Additionally, eletrocardiographic parameters analysis revealed no dispersion in the QT and QTc interval was observed in liposomal treated mice. In summary, this novel multifunctional nanoplatform deomonstrated higher tumor uptake and antitumor activity. SpHL-DOX-Fol represents a drug delivery platform to improve DOX tumor delivery and reduce dose-limiting toxicity.

Goniothalamin Induces Necroptosis and Anoikis in Human Invasive Breast Cancer MDA-MB-231 Cells.

Goniothalamin (GTN) is toxic to several types of cancer cells in vitro. However, its effects on non-apoptotic cell death induction of human cancer cells have been poorly documented. Here, an investigation of the anti-cancer activity of GTN and the molecular signaling pathways of non-apoptotic cell death in the invasive human breast cancer MDA-MB-231 cell line were undertaken. Apoptotic cell death was suppressed by using a pan-caspase inhibitor (Benzyloxycarbonyl-Val-Ala-Asp-[O-methyl]-fluoromethylketone), z-VAD-fmk) as a model to study whether GTN induced caspase-independent cell death. In the anoikis study, MDA-MB-231 cells were cultured on poly-(2-hydroxyethyl methacrylate)- or poly-HEMA- coated plates to mimic anoikis-resistance growth and determine whether GTN induced cell death and the mechanisms involved. GTN and z-VAD-fmk induced human breast cancer MDA-MB-231 cells to undergo necroptosis via endoplasmic reticulum (ER) and oxidative stresses, with increased expressions of necroptotic genes such as rip1, rip3, and mlkl. GTN induced MDA-MB-231 cells to undergo anoikis via reversed epithelial-mesenchymal transition (EMT) protein expressions, inhibited the EGFR/FAK/Src survival signaling pathway, and decreased matrix metalloproteinase secretion.