Encapsulating Halofuginone Hydrobromide in TPGS Polymeric Micelles Enhances Efficacy Against Triple-Negative Breast Cancer Cells.

BACKGROUND: Halofuginone hydrobromide (HF) is a synthetic analogue of the naturally occurring quinazolinone alkaloid febrifugine, which has potential therapeutic effects against breast cancer, however, its poor water solubility greatly limits its pharmaceutical application. D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of vitamin E, which can self-assemble to form polymeric micelles (PMs) for encapsulating insoluble anti-tumor drugs, thereby effectively enhancing their anti-cancer effects. METHODS: HF-loaded TPGS PMs (HTPMs) were manufactured using a thin-film hydration technique, followed by a series of characterizations, including the hydrodynamic diameter (HD), zeta potential (ZP), stability, drug loading (DL), encapsulation efficiency (EE), and in vitro drug release. The anti-cancer effects and potential mechanism of HTPMs were investigated in the breast cell lines MDA-MB-231 and MCF-7, and normal breast epithelial cell line Eph-ev. The breast cancer-bearing BALB/c nude mouse model was successfully established by subcutaneous injection of MDA-MB-231 cells and used to evaluate the in vivo therapeutic effect and safety of the HTPMs. RESULTS: The optimized HTPMs had an HD of 17.8±0.5 nm and ZP of 14.40±0.1 mV. These PMs exhibited DL of 12.94 ± 0.46% and EE of 90.6 ± 0.85%, along with excellent storage stability, dilution tolerance and sustained drug release in pH-dependent manner within 24 h compared to free HF. Additionally, the HTPMs had stronger inhibitory effects than free HF and paclitaxel against MDA-MB-231 triple-negative breast cancer cells, and little toxicity in normal breast epithelial Eph-ev cells. The HTPMs induced cell cycle arrest and apoptosis of MDA-MB-231 by disrupting the mitochondrial membrane potential and enhancing reactive oxygen species formation. Evaluation of in vivo anti-tumor efficacy demonstrated that HTPMs exerted a stronger tumor inhibition rate (68.17%) than free HF, and exhibited excellent biocompatibility. CONCLUSION: The findings from this study indicate that HTPMs holds great clinical potential for treating triple-negative breast cancer.

Exploring Collagen Parameters in Pure Special Types of Invasive Breast Cancer.

One of the promising tools to evaluate collagen in the extracellular matrix is the second-harmonic generation microscopy (SHG). This approach may shed light on the biological behavior of cancers and their taxonomy, but has not yet been applied to characterize collagen fibers in cases diagnosed as invasive breast carcinoma (BC) of histological special types (IBC-ST). Tissue sections from 99 patients with IBC-ST and 21 of invasive breast carcinoma of no special type (IBC-NST) were submitted to evaluation of collagen parameters by SHG. Tissue microarray was performed to evaluate immunohistochemical-based molecular subtype. In intratumoral areas, fSHG and bSHG (forward-SHG and backward-SHG) collagen parameters achieved their lowest values in mucinous, papillary and medullary carcinomas, whereas the highest values were found in classic invasive lobular and tubular carcinomas. Unsupervised hierarchical cluster analysis and minimal spanning tree using intratumoral collagen parameters allowed the identification of three main groups of breast cancer: group A (classic invasive lobular and tubular carcinomas); group B (IBC-NST, metaplastic, invasive apocrine and micropapillary carcinomas); and group C (medullary, mucinous and papillary carcinomas). Our findings provide further characterization of the tumor microenvironment of IBC-ST. This understanding may add information to build more consistent tumor categorization and to refine prognostication.

A novel orally available seleno-purine molecule suppresses triple-negative breast cancer cell proliferation and progression to metastasis by inducing cytostatic autophagy.

Patients with triple-negative breast cancer (TNBC) often have a poor prognosis largely due to lack of effective targeted therapy. Using a library of seleno-purines coupled to a high-throughput biochemical enzymatic assays we identified a potent pharmacological enhancer of autophagy (referred herein as SLLN-15) that selectively activated cytostatic macroautophagy/autophagy in TNBC preclinical models. SLLN-15 induced a dose-dependent anti-proliferative activity in the TNBC cell lines MDA-MB-231 and BT-20 via induction of autophagy and autophagic flux. This induction was associated with a selective inhibition of AKT-MTOR signaling. Conversely, rapamycin, a known autophagy inducer and MTOR inhibitor, was unable to duplicate SLLN-15's effect on TNBC cells. Inhibition of autophagy by siRNA-mediated targeting of the autophagy regulators, BECN1, ATG5 and ATG7 or using 3-methyladenine (3-MA), significantly protected against SLLN-15-induced inhibition of cell viability, further supporting that SLLN-15-induced inhibition of cancer cell proliferation was autophagy-dependent. SLLN-15-induced autophagy in TNBC cells was also associated with decreased AURKA expression, decreased AKT phosphorylation and subsequent blockage of the AKT-MTOR pathway. In vivo, oral SLLN-15 revealed a potent anticancer and anti-metastatic activity in mice bearing TNBC. Altogether, this study describes a novel regulator of mammalian autophagy, with potential utility as an experimental therapeutic for TNBCs. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; AURKA: aurora kinase A; AURKB: aurora kinase B; BECN1: beclin 1; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; ERBB2: erb-b2 receptor tyrosine kinase 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase 1; PI: propidium iodide; SQSTM1/p62: sequestosome 1; TNBC: triple-negative breast cancer.

The effects of celecoxib on the proliferation and ultrastructural changes of MDA-MB-231 breast cancer cells.

This study sought to investigate the effects of celecoxib on the proliferation and morphological changes of triple-negative breast cancer (TNBC) MDA-MB-231 cells. In this study, after MDA-MB-231 cells were treated with a certain concentration of celecoxib, a cell counting kit-8 (CCK-8) proliferation assay was used to detect cell viability. Western blotting was utilized to analyze the expression level of caspase-3, which is an apoptosis-related protein. In addition, the morphological changes in the cells and nuclei were determined with fluorescence and electron microscope. Apoptotic nuclei and obvious cytoplasmic vacuolization were observed with a microscope. Collectively, celecoxib can inhibit the proliferation of MDA-MB-231 cells by increasing caspase-3 expression and causing ultrastructural changes.

Deciphering the molecular mechanism underlying anticancer activity of coumestrol in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) represents the highly aggressive subgroup of breast cancers with poor prognosis due to absence of estrogen receptor (ER). Therefore, alternative targeted therapies are required against ER-negative breast cancers. Coumestrol, a phytoestrogen inhibits cell growth of ER-negative breast cancer MDA-MB-231 cells; the exact mechanism has not yet been reported. Unlike normal cells, cancer cells contain elevated copper which play an integral role in angiogenesis. The current focus of the work was to identify any possible role of copper in coumestrol cytotoxic action against breast cancer MDA-MB-231 cells. Results demonstrated that coumestrol inhibited cell viability, induced ROS generation, DNA damage, G1/S cell cycle arrest, up-regulation of Bax and apoptosis induction via caspase-dependent mitochondrial mediated pathway in MDA-MB-231 cells. Further, addition of copper chelator, neocuproine and ROS scavenger, N-acetyl cysteine were ineffective in abrogating coumestrol-mediated apoptosis. This suggests non-involvement of copper and ROS in coumestrol-induced apoptosis. To account for coumestrol-mediated up-regulation of Bax and apoptosis induction, direct binding potential between coumestrol and Bax/Bcl-2 was studied using in silico molecular docking studies. We propose that coumestrol directly enters cells and combines with Bax/Bcl-2 to alter their structures, thereby causing Bax binding to the outer mitochondrial membrane and Bcl-2 release from the mitochondria to initiate apoptosis. Thus, non-copper targeted ROS independent DNA damage is the central mechanism of coumestrol in ER-negative MDA-MB-231 cells. These findings will be useful in better understanding of anticancer mechanisms of coumestrol and establishing it as a lead molecule for TNBC treatment.

Small-molecule RL71-triggered excessive autophagic cell death as a potential therapeutic strategy in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has an aggressive phenotype and a poor prognosis owing to the high propensity for metastatic progression and the absence of specific targeted treatment. Here, we revealed that small-molecule RL71 targeting sarco/endoplasmic reticulum calcium-ATPase 2 (SERCA2) exhibited potent anti-cancer activity on all TNBC cells tested. Apart from apoptosis induction, RL71 triggered excessive autophagic cell death, the main contributor to RL71-induced TNBC cell death. RL71 augmented the release of Ca2+ from the endoplasmic reticulum (ER) into the cytosol by inhibiting SERCA2 activity. The disruption of calcium homeostasis induced ER stress, leading to apoptosis. More importantly, the elevated intracellular calcium signals induced autophagy through the activation of the CaMKK-AMPK-mTOR pathway and mitochondrial damage. In two TNBC xenograft mouse models, RL71 also displayed strong efficacy including the inhibition of tumor growth, the reduction of metastasis, as well as the prolongation of survival time. These findings suggest SERCA2 as a previous unknown target candidate for TNBC treatment and support the idea that autophagy inducers could be useful as new therapeutics in TNBC treatment.

Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics.

The extracellular matrix (ECM) is a complex meshwork of insoluble fibrillar proteins and signaling factors interacting together to provide architectural and instructional cues to the surrounding cells. Alterations in ECM organization or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases, and cancer. We provide here optimized protocols to solubilize ECM proteins from normal or tumor tissues, digest the proteins into peptides, analyze ECM peptides by mass spectrometry, and interpret the mass spectrometric data. In addition, we present here two novel R-script-based web tools allowing rapid annotation and relative quantification of ECM proteins, peptides, and intensity/abundance in mass spectrometric data output files. We illustrate this protocol with ECMs obtained from two pairs of tissues, which differ in ECM content and cellularity: triple-negative breast cancer and adjacent mammary tissue, and omental metastasis from high-grade serous ovarian cancer and normal omentum. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD005554.

Fatty acid synthase regulates the chemosensitivity of breast cancer cells to cisplatin-induced apoptosis.

Fatty acid synthase (FASN) is a key enzyme in fat biosynthesis that is over-expressed in advanced breast cancer stages. Cisplatin (CDDP) is a platinum-based drug used in the treatment of certain types of this disease. Although it was shown that FASN inhibition induced apoptosis by enhancing the cytotoxicity of certain drugs in breast cancer, its role in regulating the chemosensitivity of different types of breast cancer cells to CDDP-induced apoptosis is not established yet. Therefore, two different breast cancer cell lines; triple negative breast cancer (TNBC; MDA-MB-231) and triple positive breast cancer (TPBC; BT-474) cells were used to examine such role. We show that TNBC cells had naturally less fat content than TPBC cells. Subsequently, the fat content increased in both cells when treated with Palmitate rather than Oleate, whereas both fatty acids produced apoptotic ultra-structural effects and attenuated FASN expression. However, Oleate increased FASN expression in TPBC cells. CDDP decreased FASN expression and increased apoptosis in TNBC cells. These effects were further enhanced by combining CDDP with fatty acids. We also illustrate that the inhibition of FASN by either siRNA or exogenous inhibitor decreased CDDP-induced apoptosis in TPBC cells suggesting its role as an apoptotic factor, while an opposite finding was observed in TNBC cells when siRNA and fatty acids were used, suggesting its role as a survival factor. To our knowledge, we are the first to demonstrate a dual role of FASN in CDDP-induced apoptosis in breast cancer cells and how it can modulate their chemosensitivity.

The autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing DNA break repair.

Triple negative breast cancer (TNBC), characterized by an abundance of treatment-resistant breast cancer stem cells (CSCs), has a poorer prognosis than other types of breast cancers. Despite its aggressiveness, no effective targeted therapy exists for TNBC. Here, we demonstrate that CQ effectively targets CSCs via autophagy inhibition, mitochondrial structural damage, and impairment of double-stranded DNA break repair. Electron microscopy demonstrates CQ-induced mitochondrial cristae damage, which leads to mitochondrial membrane depolarization with a significant reduction in the activity of cytochrome c oxidase and accumulation of superoxide and double-stranded DNA breaks. CQ effectively diminishes the TNBC cells' ability to metastasize in vitro and in a TNBC xenograft model. When administered in combination with carboplatin, CQ effectively inhibits carboplatin-induced autophagy. This combination treatment significantly diminishes the expression of DNA repair proteins in CSC subpopulations, resulting in tumor growth reduction in carboplatin-resistant BRCA1 wild-type TNBC orthotopic xenografts. As TNBC's high treatment failure rate has been attributed to enrichment of CSCs, CQ, an autophagy inhibitor with anti-CSC effects, may be an effective adjunct to current TNBC chemotherapy regimens with carboplatin.

A paclitaxel-loaded recombinant polypeptide nanoparticle outperforms Abraxane in multiple murine cancer models.

Packaging clinically relevant hydrophobic drugs into a self-assembled nanoparticle can improve their aqueous solubility, plasma half-life, tumour-specific uptake and therapeutic potential. To this end, here we conjugated paclitaxel (PTX) to recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into ∼60 nm near-monodisperse nanoparticles that increased the systemic exposure of PTX by sevenfold compared with free drug and twofold compared with the Food and Drug Administration-approved taxane nanoformulation (Abraxane). The tumour uptake of the CP-PTX nanoparticle was fivefold greater than free drug and twofold greater than Abraxane. In a murine cancer model of human triple-negative breast cancer and prostate cancer, CP-PTX induced near-complete tumour regression after a single dose in both tumour models, whereas at the same dose, no mice treated with Abraxane survived for >80 days (breast) and 60 days (prostate), respectively. These results show that a molecularly engineered nanoparticle with precisely engineered design features outperforms Abraxane, the current gold standard for PTX delivery.

Mammographic and sonographic features of triple-negative invasive carcinoma of no special type.

The aim of this study was to compare the mammography, ultrasound (US) and histologic features of triple-negative (TN) invasive carcinoma of no special type (NST) to non-TN invasive carcinoma of NST. The second aim was to assess whether the distinct imaging characteristics of TN breast cancer would persist after controlling for the histologic features. A total of 344 invasive carcinomas of NST in 337 patients from January 2007 to February 2008 were included in this study. Two radiologists retrospectively reviewed the mammography and US findings using the American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) lexicon and our institution's criteria. On mammography, TN invasive carcinoma of NST most commonly presented as a mass with round shape and non-spiculated margin. On US, it was more likely to have internal hypoechogenicity, an abrupt boundary and posterior acoustic enhancement. TNBC lacked major suspicious imaging findings such as an irregular shape, spiculated margin and calcification.

Nuclear localization of cancer stem cell marker CD133 in triple-negative breast cancer: a case report.

AIM AND BACKGROUND: It has been recently demonstrated that the detection of stem cell niches in triple-negative (TN) breast cancer may provide good prognostic clues for this tumor. METHODS AND STUDY DESIGN: We investigated the subcellular expression and localization of the cancer stem cell marker CD133 in a TN breast cancer biopsy from a 42-year-old Caucasian woman with a histological diagnosis of high-grade invasive ductal breast carcinoma by immunohistochemistry, flow cytometry and quantitative real-time PCR (qRT-PCR). RESULTS: We describe for the first time in a TN breast cancer the nuclear mislocalization of CD133, which normally shows membrane localization and more sporadically cytoplasmic localization. We also found this aberrant expression with qRT-PCR analysis but not flow cytometry. CONCLUSIONS: Nuclear localization of CD133 may be an indicator of poor prognosis in TN breast cancer, as it is known that surface molecules, when moving into the nucleus, can act as transcriptional regulators by interfering with molecular pathways directly connected to the proliferation and differentiation of tumor cells.