Cultured Breast Cancer Cells Research Articles

Breast Cancer Cell Cultures on Electrospun Poly(ε-Caprolactone) as a Potential Tool for Preclinical Studies on Anticancer Treatments.

During anticancer drug development, most compounds selected by in vitro screening are ineffective in in vivo studies and clinical trials due to the unreliability of two-dimensional (2D) in vitro cultures that are unable to mimic the cancer microenvironment. Herein, HCC1954 cell cultures on electrospun polycaprolactone (PCL) were characterized by morphological analysis, cell viability assays, histochemical staining, immunofluorescence, and RT-PCR. Our data showed that electrospun PCL allows the in vitro formation of cultures characterized by mucopolysaccharide production and increased cancer stem cell population. Moreover, PCL-based cultures were less sensitive to doxorubicin and electroporation/bleomycin than those grown on polystyrene plates. Collectively, our data indicate that PCL-based cultures may be promising tools for preclinical studies.

HOXA5 Expression Is Elevated in Breast Cancer and Is Transcriptionally Regulated by Estradiol.

HOXA5 is a homeobox-containing gene associated with the development of the lung, gastrointestinal tract, and vertebrae. Here, we investigate potential roles and the gene regulatory mechanism in HOXA5 in breast cancer cells. Our studies demonstrate that HOXA5 expression is elevated in breast cancer tissues and in estrogen receptor (ER)-positive breast cancer cells. HOXA5 expression is critical for breast cancer cell viability. Biochemical studies show that estradiol (E2) regulates HOXA5 gene expression in cultured breast cancer cells in vitro. HOXA5 expression is also upregulated in vivo in the mammary tissues of ovariectomized female rats. E2-induced HOXA5 expression is coordinated by ERs. Knockdown of either ERα or ERβ downregulated E2-induced HOXA5 expression. Additionally, ER co-regulators, including CBP/p300 (histone acetylases) and MLL-histone methylases (MLL2, MLL3), histone acetylation-, and H3K4 trimethylation levels are enriched at the HOXA5 promoter in present E2. In summary, our studies demonstrate that HOXA5 is overexpressed in breast cancer and is transcriptionally regulated via estradiol in breast cancer cells.

An analytical study of Trastuzumab-dendrimer-fluorine drug delivery system in breast cancer therapy in vitro

Trastuzumab is considered to be a fundamental drug for treatment of breast cancer with Her-2 overexpression (Her-2 positive cells). Trastuzumab is a monoclonal antibody that targets the Her-2 receptor. Trastuzumab treatment used in breast cancer therapy require a visualization to validate their delivery and response. The objective of this study was to investigate Trastuzumab-dendrimer-fluorine drug delivery system by synthesis and characterization of a series of fluorinated dendrimers. Materials and methodsTrastuzumab-dendrimer-fluorine drug delivery system is a covalent attachment of Trastuzumab to fluorinated dendrimers. We design synthesis and evaluate main product by using electrophoresis, HPLC and LC–MS techniques. We prepared three-dimensional breast cancer cell culture in bioreactor device. For the cell culture we used MCF-7 cells with Her-2 overexpression to study Trastuzumab-dendrimer-fluorine drug delivery system efficacy. We evaluate efficacy by Magnetic Resonance Imaging (MRI) relaxation time. ResultsAn analytical analysis showed that synthesis of Trastuzumab-dendrimer-fluorine drug delivery system is possible to obtain with a good yield. The results obtained indicated potential of Trastuzumab-dendrimer-fluorine drug delivery system is more efficient than trastuzumab alone. Chromatographic and electrophoretic separations showed that the synthetized conjugates were a Trastuzumab-dendrimer-fluorine drug delivery systems. The hight synthesis efficiency was found. The presence of molecules with lower masses than trastuzumab can have influence on efficiency. ConclusionsTrastuzumab-dendrimer-fluorine drug delivery system is a new form of Trastuzumab to treat breast cancer cells in vitro. Due to presence of 19F nuclei the system can be monitored by MRI measurements.

ZFHX3 Promotes the Proliferation and Tumor Growth of ER-Positive Breast Cancer Cells Likely by Enhancing Stem-Like Features and MYC and TBX3 Transcription.

Simple SummaryBreast cancer is a common malignancy, but the understanding of its cellular and molecular mechanisms is limited. The ZFHX3 transcription factor regulates mammary epithelial cells’ proliferation and differentiation by interacting with estrogen and progesterone receptors. Both these receptors play crucial roles in breast cancer development, but whether ZFHX3 also impacts breast cancer is unknown. In this study, the authors aim to determine if ZFHX3 promotes breast cancer cells’ proliferation and tumor growth and explore the underlying cellular and molecular mechanisms. Higher ZFHX3 expression is associated with worse patient survival in breast cancer, ZFHX3 promotes the proliferation and tumor growth of breast cancer cells, and several breast cancer stem cell factors appear to be involved in the role of ZFHX3 in breast cancer growth. The findings suggest that ZFHX3 is a novel oncogenic molecule promoting breast cancer development. Such a molecule could provide novel opportunities for the treatment of breast cancer.Breast cancer is a common malignancy, but the understanding of its cellular and molecular mechanisms is limited. ZFHX3, a transcription factor with many homeodomains and zinc fingers, suppresses prostatic carcinogenesis but promotes tumor growth of liver cancer cells. ZFHX3 regulates mammary epithelial cells’ proliferation and differentiation by interacting with estrogen and progesterone receptors, potent breast cancer regulators. However, whether ZFHX3 plays a role in breast carcinogenesis is unknown. Here, we found that ZFHX3 promoted the proliferation and tumor growth of breast cancer cells in culture and nude mice; and higher expression of ZFHX3 in human breast cancer specimens was associated with poorer prognosis. The knockdown of ZFHX3 in ZFHX3-high MCF-7 cells decreased, and ZFHX3 overexpression in ZFHX3-low T-47D cells increased the proportion of breast cancer stem cells (BCSCs) defined by mammosphere formation and the expression of CD44, CD24, and/or aldehyde dehydrogenase 1. Among several transcription factors that have been implicated in BCSCs, MYC and TBX3 were transcriptionally activated by ZFHX3 via promoter binding, as demonstrated by luciferase-reporter and ChIP assays. These findings suggest that ZFHX3 promotes breast cancer cells’ proliferation and tumor growth likely by enhancing BCSC features and upregulating MYC, TBX3, and others.

Effects of FGFR inhibitors TKI258, BGJ398 and AZD4547 on breast cancer cells in 2D, 3D and tissue explant cultures

PurposeFibroblast growth factor receptors (FGFR) and pathways are important players in breast cancer (BC) development. They are commonly altered, and BCs exhibiting FGFR gene amplification are currently being studied for drug development. Here, we aimed to compare the effects of three FGFR inhibitors (FGFRis), i.e., non-selective TKI258 and selective BGJ398 and AZD4547, on different BC-derived cell lines (BCCs) and primary tissues.MethodsThe human BCCs MCF-7 and MDA-MB-231(SA) (wild-type FGFR) and MFM223 (amplified FGFR1 and FGFR2) were analyzed for FGFR expression using qRT-PCR, and the effects of FGFRis on FGFR signaling by Western blotting. The effects of FGFRis on proliferation, viability, migration and invasion of BCCs were assessed in 2D cultures using live-cell imaging, and in 3D cultures using phenotypic analysis of organoids. To study radio-sensitization, FGFRi treatment was combined with irradiation. Patient-derived BC samples were treated with FGFRis in explant cultures and immunostained for Ki67 and cleaved caspase 3.ResultsWe found that all FGFRis tested decreased the growth and viability of BC cells in 2D and 3D cultures. BGJ398 and AZD4547 were found to be potent at low concentrations in FGFR-amplified MFM233 cells, whereas higher concentrations were required in non-amplified MCF7 and MDA-MB-231(SA) cells. TKI258 inhibited the migration and invasion, whereas BGJ398 and AZD4547 only inhibited the invasion of MDA-MB-231(SA) cells. FGFRi treatment of MCF7 and MFM223 cells enhanced the inhibitory effect of radiotherapy, but this effect was not observed in MDA-MB-231(SA) cells. FGFRi-treated primary BC explants with moderate FGFR levels showed a tendency towards decreased proliferation and increased apoptosis.ConclusionsOur results indicate that, besides targeting FGFR-amplified BCs with selective FGFRis, also BCs without FGFR amplification/activation may benefit from FGFRi-treatment. Combination with other treatment modalities, such as radiotherapy, may allow the use of FGFRis at relatively low concentrations and, thereby, contribute to better BC treatment outcomes.

Local Disease-Free Survival Rate (LSR) Application to Personalize Radiation Therapy Treatments in Breast Cancer Models.

Cancer heterogeneity represents the main issue for defining an effective treatment in clinical practice, and the scientific community is progressively moving towards the development of more personalized therapeutic regimens. Radiotherapy (RT) remains a fundamental therapeutic treatment used for many neoplastic diseases, including breast cancer (BC), where high variability at the clinical and molecular level is known. The aim of this work is to apply the generalized linear quadratic (LQ) model to customize the radiant treatment plan for BC, by extracting some characteristic parameters of intrinsic radiosensitivity that are not generic, but may be exclusive for each cell type. We tested the validity of the generalized LQ model and analyzed the local disease-free survival rate (LSR) for breast RT treatment by using four BC cell cultures (both primary and immortalized), irradiated with clinical X-ray beams. BC cells were chosen on the basis of their receptor profiles, in order to simulate a differential response to RT between triple negative breast and luminal adenocarcinomas. The MCF10A breast epithelial cell line was utilized as a healthy control. We show that an RT plan setup based only on α and β values could be limiting and misleading. Indeed, two other parameters, the doubling time and the clonogens number, are important to finely predict the tumor response to treatment. Our findings could be tested at a preclinical level to confirm their application as a variant of the classical LQ model, to create a more personalized approach for RT planning.

A Translational Study of a Silicon Phthalocyanine Substituted with a Histone Deacetylase Inhibitor for Photodynamic Therapy.

In this study, we synthesized and characterized a silicon phthalocyanine substituted with 3-hydroxypyridin-2-thione (SiPc-HDACi), designed to be a chemophotodynamic therapy agent acting as a histone deacetylase inhibitor, and we determined its photophysical, photochemical, and photobiological properties. Next, we evaluated its anticancer efficacy on MCF-7, double positive and MDA-MB-231, triple negative breast cancer cell lines, as well as on a healthy human endothelial cell line (HUVEC). Our results indicate that SiPc-HDACi can target nucleoli of cells, effectively inducing apoptosis while promoting cell cycle arrest thanks to its high singlet oxygen yield and its histone deacetylase downregulating properties, suggesting a powerful anticancer effect on breast cancer in vitro. Our further studies will be conducted with primary breast cancer cell culture to give a better insight into the anticancer mechanism of the compound.

The cell-surface anchored serine protease TMPRSS13 promotes breast cancer progression and resistance to chemotherapy.

Breast cancer progression is accompanied by increased expression of extracellular and cell-surface proteases capable of degrading the extracellular matrix as well as cleaving and activating downstream targets. The type II transmembrane serine proteases (TTSPs) are a family of cell-surface proteases that play critical roles in numerous types of cancers. Therefore, the aim of this study was to identify novel and uncharacterized TTSPs with differential expression in breast cancer and to determine their potential roles in progression. Systematic in silico data analysis followed by immunohistochemical validation identified increased expression of the TTSP family member, TMPRSS13 (transmembrane protease, serine 13), in invasive ductal carcinoma patient tissue samples compared to normal breast tissue. To test whether loss of TMPRSS13 impacts tumor progression, TMPRSS13 was genetically ablated in the oncogene-induced transgenic MMTV-PymT tumor model. TMPRSS13 deficiency resulted in a significant decrease in overall tumor burden and growth rate, as well as a delayed formation of detectable mammary tumors, thus suggesting a causal relationship between TMPRSS13 expression and the progression of breast cancer. Complementary studies using human breast cancer cell culture models revealed that siRNA-mediated silencing of TMPRSS13 expression decreases proliferation, induces apoptosis, and attenuates invasion. Importantly, targeting TMPRSS13 expression renders aggressive triple-negative breast cancer cell lines highly responsive to chemotherapy. At the molecular level, knockdown of TMPRSS13 in breast cancer cells led to increased protein levels of the tumor-suppressive protease prostasin. TMPRSS13/prostasin co-immunoprecipitation and prostasin zymogen activation experiments identified prostasin as a potential novel target for TMPRSS13. Regulation of prostasin levels may be a mechanism that contributes to the pro-oncogenic properties of TMPRSS13 in breast cancer. TMPRSS13 represents a novel candidate for targeted therapy in combination with standard of care chemotherapy agents in patients with hormone receptor-negative breast cancer or in patients with tumors refractory to endocrine therapy.

Nanoparticle-based immunotherapy of breast cancer using recombinant Helicobacter pylori proteins

Breast cancer is the most common cancer in women worldwide and is associated with substantial medical and economic burden. We report the development of a hybrid immunotherapeutic system based on recombinant Nap protein from Helicobacter pylori (HP-Nap) for the treatment of breast tumors. Chitosan nanoparticles with pseudo-spherical morphology and positive zeta potential were synthesized as carriers for HP-Nap. In vitro study was performed on mouse breast cancer cell line (4T1) and human breast cancer cell lines (MCF7). In vivo study was done on 4T1 tomural mice. TUNEL assay and real time PCR test were performed on tumor mice receiving the nanoparticle treatment. The nanoparticle-protein complex induced apoptosis in vitro in cultured breast cancer cells. In-vivo studies on inbred, female BALB/c mice confirmed the shrinkage of tumor mass after administration of the nanoparticle complex containing HP-Nap. The TUNEL assay further confirmed apoptosis in extracted mouse breast cancer cells. A decrease in the expression of VEGF and MMP9 genes was observed in 4T1 cells as shown by real time PCR. Our data suggesting that the therapeutic nanocomplex may have led to decreased tumor growth in mice through changing the production rate of cytokines and increasing tumoricidal activities of the immune system.

Abstract 1782: N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target

Abstract N-myristoyltransferases (NMTs) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. This activity regulates numerous membrane-bound signal transduction pathways important in cancer biology, and the pan-NMT inhibitor PCLX-001 is in clinical development as a cancer therapy. The physiologic distribution and relative contributions of the two human NMTs, NMT1 and NMT2, remain poorly understood as previous studies used polyclonal antibodies with potential cross-reactivity. We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to the human isotypes of NMT1 and NMT2. These mAbs were used to perform an immunohistochemical (IHC) analysis of the abundance and distribution of NMT1 and NMT2 in normal human breast epithelial samples and a large (n=703) cohort of primary breast adenocarcinomas from the BCIRG001 study. While NMT1 protein was readily identifiable in most normal and transformed breast epithelial tissue, NMT1 abundance was associated with higher overall histologic grade, higher Ki67, and lower hormone expression. While NMT2 protein was readily detected in normal breast epithelial tissue, NMT2 protein was undetectable in the majority of malignant breast cancers, but detectable NMT2 protein correlated with significantly poorer overall survival outcomes (hazard ratio for death 1.36; p < 0.029) and significantly worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. NMT status was unrelated to HER2 status. NMT1 and NMT2 protein abundances were positively correlated with each other. Treatment of cultured breast cancer cells with the pan-NMT inhibitor PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts induced significant dose-dependent tumor growth inhibition in vivo. These results support the further evaluation of NMT immunohistochemistry for selection of patients for NMT inhibitor therapy, and clinical trials of NMT inhibition in breast cancer patients. Citation Format: John R. Mackey, Justine Lai, Utkarsh Chauhan, Wei-Feng Dong, Darryl Glubrect, Sunita Ghosh, Gilbert Bigras, Raymond Lai, Luc G. Berthiaume. N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1782.

Stabilization of Zataria essential oil with pectin-based nanoemulsion for enhanced cytotoxicity in monolayer and spheroid drug-resistant breast cancer cell cultures and deciphering its binding mode with gDNA

Efficacy of chemotherapy is limited by the resistance of cancer cells. Phytochemicals especially Essential Oils (EOs) provide an alternative mode of cancer therapy. However, EOs utilization is restricted because of low bioavailability, and high degradation. Nanoemulsification is a method developed to overcome these obstacles. Accordingly, Citrus-Pectin nanoemulsion of Zataria Essential Oil (CP/ZEONE) was prepared to evaluate the anticancer activity and the mechanisms responsible for the caused cytotoxicity. Physical properties and FTIR spectra of CP/ZEONE were characterized. CP/ZEONE progressively improves the suppression of viability of drug-resistant MCF-7, MDA-MB-231 breast cancer cells, and spheroids. It triggers apoptosis by increasing Reactive Oxygen Species (ROS), mitochondrial membrane potential (MMP) loss, DNA damage, G2 and S-phase arrest in MDA-MB-231 cells and spheroids respectively. Additionally, spectroscopy techniques revealed the interaction of CP/ZEONE with DNA via the formation of a groove binding/partial intercalative complex. Thus, ZEO-loaded CP Nano-particles can be further explored as a promising antiproliferative and therapeutic candidate against cancer.

Epigenetic Evaluation of N-(2-hydroxyphenyl)-2-Propylpentanamide, a Valproic Acid Aryl Derivative with Activity Against HeLa Cells.

Valproic acid (VPA) is an HDAC inhibitor (HDACI) with an anticancer activity, but is hepatotoxic. N-(2-hydroxyphenyl)-2-propylpentanamide (o-OH-VPA) is a VPA aryl derivative designed in silico as a selective inhibitor of HDAC8 with biological properties against HeLa, rhabdomyosarcoma and breast cancer cell cultures. We studied the epigenetic mechanism of o-OH-VPA as an HDACI and evaluated whether it was toxic to normal cells. HeLa cells and primary human fibroblasts were used for this study as carcinogenic and normal cells, respectively. Cell survival was evaluated by MTT assay, whereas viability and doubling time were determined by the Trypan-blue method. HDAC activity was tested using the colorimetric HDAC activity assay. The expression of p21 was analyzed by PCR and HDAC8 expression was also evaluated by real-time PCR. Cell cycle and caspase-3 activity were analyzed by flow cytometry and caspase-3 colorimetric assay, respectively. o-OH-VPA (IC50 = 0.1 mM) was fifty-eight times more effective than VPA (IC50 = 5.8 mM) to reduce HeLa cell survival. Furthermore, o-OH-VPA increased the doubling time of HeLa cells by 33% with respect to the control. o-OH-VPA acted as HDACI in HeLa cells without affecting the HDAC8 expression, arresting the cell cycle of HeLa cells in the G0/G1 phase due to the increase in p21 expression with the inhibition of caspase-3 activity without exhibiting toxicity toward normal cells. Our results revealed that o-OH-VPA is an HDACI with a selective effect against HeLa cells but without the known toxicity exerted by most pan-HDACIs on normal cells.

Dedifferentiation of MCF-7 Breast Cancer Continuous Cell Line, Development of Breast Cancer Stem Cells (BCSCs) Enriched Culture and Biomarker Analysis

BACKGROUND: Cancer stem cells (CSCs) eradication might serve as a robust approach for cancer eradication. MCF-7 as breast cancer continuous cell line is known to contain breast CSCs (BCSCs) for its capability to maintain its original tumor population. CSCs enriched culture is a fundamental tool for CSCs targeted therapy development. Effective and unsophisticated CSCs dedifferentiation protocol for producing CSCs enriched culture is needed.METHODS: MCF-7 cells were cultured initially in Dulbecco's Modified Eagle Medium (DMEM) low glucose medium then changed to DMEM:F12. Serum starvation was performed during each medium refreshment gradually with fetal bovine serum (FBS) concentration of 10%, 5%, 2.5% until reaching 1% FBS concentration. Stable MCF-7 culture was then adapted to serum free culture system, containing DMEM:F12, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and B27 supplement as dedifferentiation protocol for 18 days. Cluster of differentiation (CD)44 and CD24 double staining immunocytochemistry was performed to evaluate cell stemness.RESULTS: The population of cells expressing BCSCs markers (CD44+/CD24low) in non-adherent single cells subpopulation was significantly increased after the dedifferentiation procedure (70.39%) compared to control groups (0.71%) (p<0.05). In contrast, the expression of BCSCs marker in adherent single cells subpopulation and for both adherent and non-adherent mammosphere the BCSCs markers showed a stable expression.CONCLUSION: BCSCs enrichment of breast cancer cell cultures from MCF-7 breast cancer cell line can be performed. Breast cancer cell plasticity is observed during the dedifferentiation protocol. Development of dedifferentiation inducing protocols can serve as an important foundation for breast cancer therapy development through BCSCs elimination.KEYWORDS: breast neoplasms, cell line, dedifferentiation, immunohistochemistry, neoplastic stem cells

Matrix Stiffness Regulates Chemosensitivity, Stemness Characteristics, and Autophagy in Breast Cancer Cells.

The biomechanical environment of natural or synthetic extracellular matrices (ECMs) is identified to play a considerable role in embryonic development in stem cell fate and also in cancer development and fibrotic diseases. However, rare evidence shows the impact of biomechanical signals such as ECM stiffness on cancer cell stemness and autophagy, which makes huge contributions to cancer and many developmental and physiological processes. Furthermore, the influence and mechanism of ECM stiffness on autophagy in cancer cells remains unclear. Herein, we employed fibronectin-coated polyacrylamide hydrogels as the substrates for culturing breast cancer cells. We found that a soft environment was beneficial for the maintenance of cancer stem cell (CSC) population in breast cancer cells, which likely led to aggravated chemoresistance. Conversely, nutritional deprivation-induced autophagy was elevated along with increasing matrix stiffness. In addition, we found that though the central regulator of mechanotransduction, the yes-associated protein, YAP, was beneficial for autophagy activation, unexpectedly, it was not the main cause of rigid substrate promoting autophagy. In contrast, the YAP was crucial for a compliant environment for maintaining breast cancer stem cells and promoting chemotherapeutic resistance. We also found that the Rho-ROCK-ERK signal pathway and actin cytoskeleton were essential for the regulation of autophagy by matrix stiffness. Taken together, our study showed the important influence of ECM stiffness on stemness and autophagy in breast cancer cells and revealed the possible signal pathway involved in the mechanotransduction in autophagy activation, which provides significant implications for the study of cancer progression and design of hydrogels for tissue engineering in clinical therapy.

Epithelial Mesenchymal Transition and Progression of Breast Cancer Promoted by Diabetes Mellitus in Mice Are Associated with Increased Expression of Glycolytic and Proteolytic Enzymes.

The development of breast cancer (BC) is influenced by age, overweight, obesity, metabolic syndrome, and diabetes mellitus (DM), which are associated with hyperglycemia, glucose intolerance, insulin resistance, and oxidative stress. High glucose concentration increases a metastatic phenotype in cultured breast cancer cells, promoting cell proliferation, reactive species production (ROS), epithelial mesenchymal transition (EMT), and expression of proteolytic enzymes. Our aim was to determine whether diabetes mellitus favor BC progression in mice and its association with changes in the content of ROS and glycolytic and proteolytic enzymes. Diabetes was induced in 7-week-old Balb/c mice, under 6-h fasting with a unique i. p. dose of streptozotocin 120mg/kg. Furthermore, 4T1 breast cancer cells were injected beneath the nipple to induce tumors. G6PD, GAPDH, ENO1, uPA, uPAR, PAI-1, β-catenin, Snail, vimentin, and E-cadherin were measured by western blot and MPP-9 and MMP-2 by gel zymography. TBARS were measured as markers of the lipid peroxidation. Lower survival and increased tumor growth, together with marked EMT, were found in diabetic in comparison with nondiabetic mice. The effects of diabetes were associated with enhanced lipid peroxidation and higher levels of glycolytic (G6PD, GAPDH, and ENO1) and proteolytic (uPA, MMP-9) enzymes. Possibly, hyperglycemia and ROS led to faster progression of breast cancer in diabetic mice, fomenting EMT and the expression of glycolytic and proteolytic enzymes. These enzymes participate in the supply of energy and precursors for macromolecular biosynthesis and extracellular matrix degradation during breast cancer progression.

Quinoline‑ and Benzoselenazole-Derived Unsymmetrical Squaraine Cyanine Dyes: Design, Synthesis, Photophysicochemical Features and Light-Triggerable Antiproliferative Effects against Breast Cancer Cell Lines.

Photodynamic therapy is an innovative treatment approach broadly directed towards oncological diseases. Its applicability and efficiency are closely related to the interaction of three main components, namely a photosensitizer, light and molecular triplet oxygen, which should drive cell death. Recently, several studies have demonstrated that squaraine cyanine dyes have a set of photophysical and photochemical properties that have made of these compounds’ potential photosensitizers for this therapeutic modality. In the present research work, we describe the synthesis and characterization of four quinoline- and benzoselenazole-derived unsymmetrical squaraine cyanine dyes. Except for the precursor of aminosquaraine dyes, i.e., O-methylated derivative, all dyes were evaluated for their behavior and absorption capacity in different organic and aqueous solvents, their ability to form singlet oxygen, their light-stability, and in vitro phototherapeutic effects against two human breast cancer cell cultures (BT-474 and MCF-7). Regardless of the nature of the used solvents, the synthesized dyes showed intense absorption in the red and near-infrared spectral regions, despite the formation of aggregates in aqueous media. Dyes showed high light-stability against light exposure. Despite the low ability to produce singlet oxygen, aminosquaraine dyes demonstrated worthy in vitro phototherapeutic activity.

Targeting breast cancer stem-like cells using chloroquine encapsulated by a triphenylphosphonium-functionalized hyperbranched polymer.

Cancer stem cells (CSCs) have garnered increasing attention over the past decade, as they are believed to play a crucial role in tumor progression and drug resistance. Accumulating evidence provides insight into the function of autophagy in maintenance and survival of CSCs. Here, we studied the impact of a mitochondriotropic triphenylphosphonium-functionalized dendrimeric nanocarrier on cultured breast cancer cell lines, grown either as adherent cells or as mammospheres that mimic a stem-like phenotype. The nanocarrier manifested a substantial cytotoxicity both alone as well as after encapsulation of chloroquine, a well-known autophagy inhibitor. The cytotoxic effects of the nanocarrier could be ascribed to interference with mitochondrial function. Importantly, mammospheres were selectively sensitive to encapsulated chloroquine and this depends on the expression of the gene encoding ATM kinase. Ataxia-telangiectasia mutated (ATM) kinase is an enzyme that functions as an essential signaling mediator that enables growth of cancer stem cells through the regulation of autophagy. We noted that this ATM-dependent sensitivity of mammospheres to encapsulated chloroquine was independent of the status of the tumor suppressor gene p53. Our study suggests that breast cancer stem cells, as they are modeled by mammospheres, are sensitive to encapsulated chloroquine, depending on the expression of the ATM kinase, which is thereby characterized as a potential biomarker for sensitivity to this type of treatment.

Tracking the invasion of breast cancer cells in paper-based 3D cultures by OCT motility analysis.

3D paper-based cultures (PBCs) are easy-to-use and provide a biologically representative microenvironment. By stacking a sheet of cell-laden paper below sheets containing cell-free hydrogel, we form an assay capable of segmenting cells by the distance they invaded from the original cell-seeded layer. These invasion assays are limited to end-point analyses with fluorescence-based readouts due to the highly scattering nature of the paper scaffolds. Here we demonstrate that optical coherence tomography (OCT) can distinguish living cells from the surrounding extracellular matrix (ECM) or paper fibers based upon their intracellular motility amplitude (M). M is computed from fluctuation statistics of the sample, rejects shot noise, and is invariant to OCT signal attenuation. Using OCT motility analysis, we tracked the invasion of breast cancer cells over a 3-day period in 4-layer PBCs (160-300 µm thick) in situ. The cell population distributions determined with OCT are highly correlated with those obtained by fluorescence imaging, with an intraclass correlation coefficient (ICC) of 0.903. The ability of OCT motility analysis to visualize live cells and quantify cell distributions in PBC assays in situ and longitudinally provides a novel means for understanding how chemical gradients within the tumor microenvironment affect cellular invasion.

SAT-124 Optimization of Experimental Conditions for Mimicking Hypoxia in Cultured Breast Cancer Cells by Using Cobalt(II) Chloride (CoCl2)

Hypoxia is a common phenomenon in solid tumor development caused by a decrease in either oxygen concentration or oxygen pressure as a result of rapid tumor cell growth. Hypoxia is characterized by stabilization of the alpha subunit of the hypoxia-inducible factor (HIF-1α) and its nuclear translocation and heterodimerization with HIF-1β. Activation of this signaling pathway involves multiple downstream effectors including carbonic anhydrase 9 (CA9, s. CAIX). A reliable method to mimic hypoxia utilizes cobalt(II) chloride (CoCl2), which directly induces the expression of HIF-1α. The aim of this study was to optimize the experimental conditions for CoCl2 treatment of breast cancer cells in vitro using three human breast cancer cell lines (MDA-MB-231, T-47D, and MCF-7 cells). We performed time- and concentration-response experiments, using various concentrations of CoCl2 (50, 100, 200, and 300 μM) for 24 and 48 hours, and measured the expression of HIF-1α and CA9 by qRT-PCR and Western blot analyses. Results demonstrated that CoCl2 downregulated HIF-1α mRNA levels but upregulated CA9 mRNA levels in a concentration- and time-dependent manner. Concomitantly, CoCl2 treatment resulted in a significant induction of HIF-1α protein levels. We further investigated the effect of the CoCl2 concentrations listed above on cell apoptosis using an in situ apoptosis detection kit. The results demonstrated that concentrations of CoCl2 up to 100 μM had no significant effect on cell apoptosis.

IGFBP-1 Expression Promotes Tamoxifen Resistance in Breast Cancer Cells via Erk Pathway Activation.

Insulin-like growth factor (IGF) system plays a significant role in many cellular processes, including proliferation, and survival. In estrogen receptor positive breast cancer, the level of circulating IGF-1 is positively associated with the incidence and at least 50% of cases have elevated IGF-1R signaling. Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα-positive breast cancer. Unfortunately, tamoxifen resistance is a frequent occurrence in patients receiving treatment and the molecular mechanisms that underlie tamoxifen resistance not adequately defined. It has recently been reported that the inhibition of IGF-1R activation and the proliferation of breast cancer cells upon tamoxifen treatment is mediated by the accumulation of extracellular insulin-like growth factor binding protein 1 (IGFBP-1). Elevated IGFBP-1 expression was observed in tamoxifen-resistant (TamR) MCF-7 and T-47D cells lines suggesting that the tamoxifen-resistant state is associated with IGFBP-1 accumulation. MCF-7 and T-47D breast cancer cells stably transfected with and IGFBP-1 expression vector were generated (MCF7-BP1 and T47D-BP1) to determine the impact of breast cancer cell culture in the presence of increased IGFBP-1 expression. In these cells, the expression of IGF-1R was significantly reduced compared to controls and was similar to our observations in tamoxifen-resistant MCF-7 and T-47D cells. Also similar to TamR breast cancer cells, MCF7-BP1 and T47D-BP1 were resistant to tamoxifen treatment, had elevated epidermal growth factor receptor (EGFR) expression, increased phospho-EGFR (pEGFR), and phospho-Erk (pErk). Furthermore, tamoxifen sensitivity was restored in the MCF7-BP1 and T47D-BP1 upon inhibition of Erk phosphorylation. Lastly, the transient knockdown of IGFBP-1 in MCF7-BP1 and T47D-BP1 inhibited pErk accumulation and increased tamoxifen sensitivity. Taken together, these data support the conclusion that IGFBP-1 is a key component of the development of tamoxifen resistance in breast cancer cells.