Epithelial Breast Cancer Cells Research Articles

Application of self-organizing maps to AFM-based viscoelastic characterization of breast cancer cell mechanics

Cell mechanical properties have been proposed as label free markers for diagnostic purposes in diseases such as cancer. Cancer cells show altered mechanical phenotypes compared to their healthy counterparts. Atomic Force Microscopy (AFM) is a widely utilized tool to study cell mechanics. These measurements often need skilful users, physical modelling of mechanical properties and expertise in data interpretation. Together with the need to perform many measurements for statistical significance and to probe wide enough areas in tissue structures, the application of machine learning and artificial neural network techniques to automatically classify AFM datasets has received interest recently. We propose the use of self-organizing maps (SOMs) as unsupervised artificial neural network applied to mechanical measurements performed via AFM on epithelial breast cancer cells treated with different substances that affect estrogen receptor signalling. We show changes in mechanical properties due to treatments, as estrogen softened the cells, while resveratrol led to an increase in cell stiffness and viscosity. These data were then used as input for SOMs. Our approach was able to distinguish between estrogen treated, control and resveratrol treated cells in an unsupervised manner. In addition, the maps enabled investigation of the relationship of the input variables.

Functionalized Fluorescent Nanostructures Generated from Self-Assembly of a Cationic Tripeptide Direct Cell-Selective Chemotherapeutic Drug Delivery.

Nanodrug delivery systems (NDDs) capable of conveying chemotherapeutics directly into malignant cells without harming healthy ones are of significant interest in the field of cancer therapy. However, the development of nanostructures with the requisite biocompatibility, inherent optical properties, cellular penetration ability, encapsulation capability, and target selectivity has remained elusive. In an effort to develop cell-selective NDDs, we have synthesized a cationic tripeptide Boc-Arg-Trp-Phe-OMe (PA1), which self-assembles into well-ordered spheres in 100% aqueous medium. The inherent fluorescence properties of the peptide PA1 were shifted from the ultraviolet to the visible region by the self-assembly. These fluorescent nanostructures are proteolytically stable, photostable, and biocompatible, with characteristic blue fluorescence signals that permit us to monitor their intracellular entry in real time. We also demonstrate that these tripeptide spherical structures (TPSS) have the capacity to entrap the chemotherapeutic drug doxorubicin (Dox), shuttle the encapsulated drug within cancerous cells, and initiate the DNA damage signaling cascade, which culminates in apoptosis. Next, we functionalized the TPSS with an epithelial-cell-specific epithelial cell adhesion molecule aptamer. Aptamer-conjugated PA1 (PA1-Apt) facilitated efficient Dox delivery into the breast cancer epithelial cell line MCF7, resulting in cell death. However, cells of the human cardiomyocyte cell line AC16 were resistant to the cell killing actions of PA1-Apt. Together, these data demonstrate that not only can the self-assembly of cationic tripeptides like PA1 be exploited for efficient drug encapsulation and delivery but their unique chemistry also allows for functional modifications, which can improve the selectivity of these versatile NDDs.

Investigation of breast cancer metabolic plasticity in the tumor microenvironment by single-cell organelle phenotyping.

Metabolic plasticity, i.e., the capability of cells to modify their metabolic state, is a hallmark of cancer and an important factor in the formation of metastases as well as a major contributor to chemoresistance and tumor recurrence. Furthermore, the tumor microenvironment, namely the collection of environmental properties (pH, nutrient availability, fatty acids) and cell types that surround the main tumor mass, is regarded to as the main driver of the metabolic state of tumor cells. Unfortunately, no technique can non-invasively assess the metabolic state of single cells in living samples, limiting our understanding of the heterogeneity and dynamics of metabolic state transitions. Here, we apply a technique, named ESPRESSO (Environmental Sensors Profiling Relayed by Subcellular Structures and Organelles) that combines organelle-specific environment-sensitive probes (ESPs) with hyperspectral imaging and quantitative bioimage analysis. We use a mixture of lipid droplet-, mitochondria- and lysosome-specific ESPs to quantify of morphological (e.g., number, size, organization) and functional (e.g., membrane potential, pH, polarity) characteristics to identify the metabolic state in single, living cells. To understand the differences in metabolic plasticity of non-tumorigenic breast epithelial (MCF10a) and triple negative breast cancer (MDA-MB-231) cells, we determined their metabolic state with ESPRESSO under a variety of stress and environmental conditions, identifying the characteristic metabolic signature of the cell lines under those conditions. We further investigated the response to diverse chemotherapy drugs (doxorubicin, paclitaxel, curcumin) in an effort to understand their effect on cancer and healthy cells, highlighting the environmental conditions that would favor the resistance to a specific drug treatment. Taken together, we provide a framework to identify the metabolic response of cancer and non-tumorigenic cells under different drugs, stressors and environmental conditions, providing in-depth characterization of their metabolic response at the single cell level.

Cancer-specific cytotoxicity of Ringer’s acetate solution irradiated by cold atmospheric pressure plasma

Cold atmospheric pressure plasmas are promising medical tools that can assist in cancer treatment. While the medical pathology mechanism is substantially understood, knowledge of the contribution of reactive species formed in plasma and the mode of activation of biochemical pathways is insufficient. Herein, we present a concept involving antitumoral plasma-activated organics, which is envisaged to increase cytotoxicity levels against cancer cells. Ringer′s acetate solution was irradiated by low-temperature plasma at atmospheric pressure and possible reaction pathways of the compound generation are presented. The chemical compounds formed by plasma treatment and their effects on non-tumorigenic breast epithelial cells (MCF-10A) and breast cancer cells (MCF-7) were investigated. The cell viability results have shown that plasma-derived compounds have both, stimulatory and inhibitory effects on cell viability, depending on the concentration of the generated compounds in the irradiated liquids. Previous studies have shown that oxidative stresses involving reactive oxygen and nitrogen species (RONS) can be used to kill cancer cells. Hence, while RONS offers promising first-step killing effects, cell viability results have shown that plasma-derived compounds, such as acetic anhydride and ethyl acetate, have the potential to play important roles in plasma-based cancer therapy.

Apoptotic Induction in Human Cancer Cell Lines by Antimicrobial Compounds from Antarctic Streptomyces fildesensis (INACH3013)

The Antarctic Streptomyces fildesensis has been recognized for its production of antimicrobial compounds with interesting biological activities against foodborne bacteria and multi-resistant strains, but not for its potential antiproliferative activity and mechanisms involved. Two bioactive ethyl acetate extract (EAE) fractions were purified via thin-layer chromatography and High-Performance Liquid Chromatography (HPLC), showing that orange-colored compounds displayed antimicrobial activity against pathogenic bacteria even after shock thermal treatment. The UV–VIS features of the active compounds, the TLC assay with actinomycin-D pure standard, Fourier transform infrared (FTIR) spectra and the ANTISMASH analysis support the presence of actinomycin-like compounds. We demonstrated that S. fildesensis displays antiproliferative activity against human tumor cell lines, including human breast cancer (MCF-7), prostate cancer (PC-3), colon cancer (HT-29) and non-tumoral colon epithelial cells (CoN). The half-maximal effective concentrations (EC50) ranged from 3.98 µg/mL to 0.1 µg/mL. Our results reveal that actinomycin-like compounds of S. fildesensis induced apoptosis mediated by caspase activation, decreasing the mitochondrial membrane potential and altering the cell morphology in all tumoral and non-tumoral cell lines analyzed. These findings confirm the potential of the psychrotolerant Antarctic S. fildesensis species as a promising source for obtaining potential novel anticancer compounds.

Abstract A013: Loss of MLL3 epigenetic regulator drives metastasis by promoting a hybrid epithelial-mesenchymal transition state

Abstract Phenotypic plasticity associated with the hybrid epithelial-mesenchymal transition (EMT) state is crucial to metastatic seeding and outgrowth. However, the mechanisms controlling the induction of hybrid EMT remain poorly defined. We showed that deletion of the epigenetic regulator MLL3, a tumor suppressor frequently altered in human cancer, promoted the acquisition of the hybrid EMT state in both epithelial and mesenchymal breast cancer cells by facilitating EMT and mesenchymal-epithelial transition, respectively, distinct from other known EMT regulators mediating unidirectional changes. Consequently, MLL3 deletion greatly increased metastasis by enhancing metastatic colonization. MLL3 loss led to increased IFNg signaling, which is required for the induction of hybrid EMT cells and the enhanced metastatic capacity. Mechanically, MLL3 loss enhanced IFNg signaling by increasing H3K27ac intensity in the enhancers of a subset of IFNg response genes in both epithelial and mesenchymal tumor cells. We further showed that MLL3-deficient cells were sensitive to BET inhibition, which effectively suppressed the growth of MLL3-mutant primary tumors and metastases. These results uncovered a key driver of the hybrid EMT state in breast cancer that could be targeted therapeutically. Citation Format: Jihong Cui, Chi Zhang, Ji-Eun Lee, Boris Bartholdy, Dapeng Yang, Yu Liu, Piril Erler, Phillip Galbo, Dayle Q. Hodge, Danwei Huangfu, Deyou Zheng, Kai Ge, Wenjun Guo. Loss of MLL3 epigenetic regulator drives metastasis by promoting a hybrid epithelial-mesenchymal transition state [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A013.

Synthesis of Iron oxide Nanoparticle using Propolis from Northern Cyprus and Evaluation of its Antibacterial, Anticancer Potential on MDA-MB 231 cells

Research in nanotechnology has progressed in the last decades, and it is purposeful to explore the applications of biocompatible nanoparticles in general and their anticancer attribute specifically. This study is aimed at examining the cytotoxic effects of biocompatible Northern Cyprus propolis extract of iron oxide nanoparticles (PE-Fe2O3-NPs) on MDA-MB-231 epithelial human breast cancer cell line, in vitro. Propolis extract were used to obtain PE-Fe2O3-NPs and further characterized using several methods like ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, scanning electronic microscopy and X-ray diffraction analysis. The PE-Fe2O3-NPs has a distinctive Surface Plasmon Resonance band at 350 nm. The prepared PE-Fe2O3-NPs had a size of 108 nm by diameter with a zeta potential of +33.9 mV which indicates a good stability of the nanoparticles’ while the size of Fe2O3-NPs was 89.40 nm by diameter. Antibacterial result showed that Fe2O3-NPs has highest Minimum Inhibitory Concentration and Minimum Bactericidal Concentration as compared to PE-Fe2O3-NPs with different concentration of propolis extract as regards the tested microorganisms. This indicates that PE-Fe2O3-NPs possesses a more effective threat to eliminating pathogenic bacteria at lesser dosages, be it Gram-ve or Gram+ve. PE-Fe2O3-NPs effectively halted the spread of MDA-MB-231 cancer cell lines and thus, prove to be commendable for anticancer biomedical uses.

Cytotoxic Activity of Leaves Essential oils of Aeollanthus heliotropioides and Ocimum urticaefolium (Lamiaceae) from Cameroon on the Breast Cancer (MCF-7) and Epithelial (ARPE-19) Cells

Background: The aim of this work was to study the cytotoxicity of leaves essential oils and their identified constituents of Aeollanthus heliotropioides and Ocimum urticaefolium (Lamiaceae) from Cameroon. Materials and Methods: Leaves essential oils were obtained by hydrodistillation and were analysed by gas chromatography (GC) and GC/mass spectrometry (MS). The cytotoxic activity was evaluated in Human breast cancer (MCF-7) and normal epithelial (ARPE-19) cells line by MTT assay. Results: The essential oils of Aeollanthus heliotropioides and Ocimum urticaefolium leaves were found to most constitute by sesquiterpene components with 52.8% and 67.3% respectively than monoterpenes. The major constituents of the essential oil of Aeollanthus heliotropioides are linalool (43.4%), (E)-(Z)-farnesene (27.7%), (δ- Cadinol (13.0%), germacrene D (3.7%), α-caryophyllene (3.1%). Eugenol (26%), (Z)--ocimene(5.3%), α- caryophyllene (5%), germacrene D (4.4%), δ-muurolène (17%) , spathulenol (14.2%), δ-Cadinol (11.3%), (E)-Z-farnesol (5.4%) and 6E)-2Z-farnesol (3.6%) are the main major components of Ocimum urticaefolium essential oil. The cytoxicity of Aeollanthus heliotropioides and Ocimum urticaefolium oils indicate that the oils have a good cytotoxicity against breast cancer cells (MCF-7) growth with IC50 values of 0. 620μL/mL and 0.422 μL/mL. The highest selectivity was obtained with the essential oil Aeollanthus heliotropioides (1.72) which was more cytotoxic against MCF-7 than against normal cell line (ARPE-19) with IC50 values of 0.620 μL/mL and 1.07 μL/mL, respectively. Conclusion: This study suggests for the first time the ability of Aeollanthus heliotropioides and Ocimum urticaefolium essential oils to inhibit human tumor cell growth. They could be considered as a potent source of alternate lead compounds against breast cancer.

Rose Bengal Decorated NaYF4:Tb Nanoparticles for Low Dose X-ray-Induced Photodynamic Therapy in Cancer Cells.

The use of scintillating nanoparticles (ScNPs) in X-ray-induced photodynamic therapy (X-PDT) is a technique for deep tissue-localized tumor therapy with few side effects. ScNPs transfer X-ray-induced energy to photosensitizers, which generate massive amounts of reactive oxygen species (ROS) and kill cancer cells. Here we fabricated rose bengal (RB)-installed, Tb3+-rich NaYF4 nanocrystals (NaYF4:Tb@RB), in which optically inert Y3+ enables highly efficient energy transfer via high amounts of Tb3+ doping. NaYF4:Tb was prepared via solvothermal synthesis to have an average size of 7.6 nm, followed by coating with poly(maleic anhydride-alt-1-octedecene)-poly(ethylene glycol) with a molecular weight of 2000 (C18PMH-PEG2k). Further, RB was covalently conjugated to carboxyl groups generated from PMH on NaYF4:Tb using an ethylenediamine linker. NaYF4:Tb@RB exhibited a hydrodynamic diameter of ∼75 nm with a ζ-potential of -12 mV. NaYF4:Tb@RB efficiently generated ROS in cultured luciferase-expressing murine epithelial breast cancer (4T1-luc) cells under low dose X-ray irradiation (0.5 Gy). The ROS generation amounts of NaYF4:Tb@RB were 1.5-2-fold higher than those of NaGdF4:Tb@RB, in which host nanocrystals were prepared with optically active Gd3+. Flow cytometric and confocal microscopic analyses showed higher intracellular ROS production of NaYF4:Tb@RB, compared to NaYF4:Tb and RB, resulting in higher X-ray-induced DNA damage in cultured 4T1-luc cells. Ultimately, NaYF4:Tb@RB elicited significant cytotoxicity after X-ray irradiation (0.5 Gy), while inducing marginal cytotoxicity without X-ray irradiation. Altogether, this research proposes a promising ScNP design for efficient X-PDT agents that make the better use of incident X-ray energy while causing the fewest side effects.

The Effect of Acetylsalicylic Acid (Asa) on the Mechanical Properties of Breast Cancer Epithelial Cells.

In most communities, the risk of developing breast cancer is increasing. By affecting the cyclooxygenase 1 and 2 (COX-1 and COX-2) enzymes and actin filaments, acetylsalicylic acid (Aspirin) has been shown to reduce the risk of breast cancer and prevent cell migration in both laboratory and clinical studies. The purpose of this study is to determine the mechanical properties of normal and cancerous breast tissue cells, as well as the short-term effect of aspirin on cancer cells. To this end, the mechanical properties and deformation of three cell types were investigated: healthy MCF-10 breast cells, MCF-7 breast cancer cells, and MCF-7 breast cancer cells treated with a 5 μM aspirin solution. Atomic Force Microscopy (AFM) was used to determine the mechanical properties of the cells. Cell deformation was analyzed in all groups, and Young's modulus was calculated using the Hertz model. According to the obtained data, cancer cells deformed at a rate half that of healthy cells. Nonetheless, when aspirin was used, cancer cells deformed similarly to healthy cells. Additionally, healthy cells' Young's modulus was calculated to be approximately three times that of cancer cells, which was placed closer to that of healthy cells by adding aspirin to Young's modulus. Cell strength appears to have increased due to aspirin's intervention on actin filaments and cytoskeletons, and the mechanical properties of breast cancer cells have become more similar to those of normal cells. The likelihood of cell migration and metastasis decreases as cell strength increases.

Frankincense oil-loaded nanoemulsion formulation of paclitaxel and erucin: A synergistic combination for ameliorating drug resistance in breast cancer: In vitro and in vivo study.

Nanoformulation-based combinational drug delivery systems are well known to overcome drug resistance in cancer management. Among them, nanoemulsions are well-known and thermodynamically stable drug delivery systems suitable for carrying hydrophobic drugs and phytoconstituents to tackle drug-resistant cancers. In the present study, we have investigated the effect of paclitaxel in combination with erucin (natural isothiocyanate isolated from the seeds of Eruca sativa) loaded in the frankincense oil-based nanoemulsion formulation. The choice of frankincense oil for the current study was based on reported research investigations stating its magnificient therapeutic potential against breast cancer. Optimized nanoemulsion of paclitaxel (PTX) and erucin (ER) combination (EPNE) provided sustained release and exhibited enhanced cytotoxicity towards human epithelial breast cancer cells (T-47D) as compared to individual ER and PTX. EPNE was further assessed for its antitumor activity in the 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer mice model. EPNE significantly decreased the levels of hepatic and renal parameters along with oxidative stress in breast cancer mice. Furthermore, EPNE also showed decreased levels of inflammatory cytokines TNF-α, IL-6. Histopathological examinations revealed restoration of the tumorous breast to normal tissues in EPNE-treated breast cancer mice. Therefore, EPNE can act as a viable lead and therapeutic option for drug-resistant breast cancer.

Recombinant Oncolytic Adenovirus Combined with Cyclophosphamide Induces Synergy in the Treatment of Breast Cancer in vitro and in vivo.

PurposeOncolytic virus therapy has gradually become an integral approach in cancer treatment. We explored the therapeutic effects of the combination of a dual cancer-selective anti-tumor recombinant adenovirus (Ad-Apoptin-hTERTp-E1a) and cyclophosphamide on breast cancer cells.MethodsThe inhibition of MCF-7 and MDA-MB-231 breast cancer cells by Ad-Apoptin-hTERTp-E1a (Ad-VT), cyclophosphamide, and Ad-VT + Cyclophosphamide was investigated using the CCK-8 assay. The combination index (CI) was calculated using CalcuSyn software to determine the best combination based on the inhibition rates of the different treatment combinations. The CCK-8 assay and crystal violet staining were used to detect the cytotoxicity of the combined Ad-VT and cyclophosphamide in breast cancer cells and breast epithelial cells. Subsequently, Hoechst staining, annexin V flow cytometry, and JC-1 staining were used to analyze the inhibitory pathway of Ad-VT plus cyclophosphamide on breast cancer cells. Cell migration and invasion of breast cancer cells were assessed using the cell-scratch and Transwell assays. The anti-tumor effects of different treatment groups in a tumor-bearing nude mouse model also were analyzed.ResultsThe treatment combination of Ad-VT (40 MOI) and cyclophosphamide (400 µM) significantly inhibited MCF-7 and MDA-MB-231 cells and reduced the toxicity of cyclophosphamide in normal cells. Ad-VT primarily induced breast cancer cell apoptosis through the endogenous apoptotic pathway. Apoptosis was significantly increased after treatment with Ad-VT plus cyclophosphamide. The combination significantly inhibited the migration and invasion of MCF-7 and MDA-MB-231 cells. The in vivo experiments demonstrated that exposure to Ad-VT plus cyclophosphamide significantly inhibited tumor growth and extended the survival time of the nude mice.ConclusionAd-VT plus cyclophosphamide reduced toxicity and exhibited increased efficacy in treating breast cancer cells.

A Promising Method for the Determination of Cell Viability: The Membrane Potential Cell Viability Assay.

Determining the viability of cells is fraught with many uncertainties. It is often difficult to determine whether a cell is still alive, approaching the point of no return, or dead. Today, there are many methods for determining cell viability. Most rely on an indirect determination of cell death (metabolism, molecular transport, and leakage, to name a few). In contrast, we have developed a promising novel method for a “direct” determination of cell viability. The potential method assesses cell membrane integrity (which is essential for all viable cells) by measuring the electrical potential of the cell membrane. To test the assay, we chose two different cell types, blood macrophages (TLT) and breast cancer epithelial cells (MCF 7). We exposed them to seven different toxic scenarios (arsenic (V), UV light, hydrogen peroxide, nutrient starvation, Tetrabromobisphenol A, fatty acids, and 5-fluorouracil) to induce different cell death pathways. Under controlled test conditions, the assay showed good accuracy when comparing the toxicity assessment with well-established methods. Moreover, the method showed compatibility with live cell imaging. Although we know that further studies are needed to confirm the performance of the assay in other situations, the results obtained are promising for their wider application in the future.

Epigenetic and transcriptome responsiveness to ER modulation by tissue selective estrogen complexes in breast epithelial and breast cancer cells.

Selective estrogen receptor modulators (SERMs), including the SERM/SERD bazedoxifene (BZA), are used to treat postmenopausal osteoporosis and may reduce breast cancer (BCa) risk. One of the most persistent unresolved questions regarding menopausal hormone therapy is compromised control of proliferation and phenotype because of short- or long-term administration of mixed-function estrogen receptor (ER) ligands. To gain insight into epigenetic effectors of the transcriptomes of hormone and BZA-treated BCa cells, we evaluated a panel of histone modifications. The impact of short-term hormone treatment and BZA on gene expression and genome-wide epigenetic profiles was examined in ERαneg mammary epithelial cells (MCF10A) and ERα+ luminal breast cancer cells (MCF7). We tested individual components and combinations of 17β-estradiol (E2), estrogen compounds (EC10) and BZA. RNA-seq for gene expression and ChIP-seq for active (H3K4me3, H3K4ac, H3K27ac) and repressive (H3K27me3) histone modifications were performed. Our results show that the combination of BZA with E2 or EC10 reduces estrogen-mediated patterns of histone modifications and gene expression in MCF-7ERα+ cells. In contrast, BZA has minimal effects on these parameters in MCF10A mammary epithelial cells. BZA-induced changes in histone modifications in MCF7 cells are characterized by altered H3K4ac patterns, with changes at distal enhancers of ERα-target genes and at promoters of non-ERα bound proliferation-related genes. Notably, the ERα target gene GREB1 is the most sensitive to BZA treatment. Our findings provide direct mechanistic-based evidence that BZA induces epigenetic changes in E2 and EC10 mediated control of ERα regulatory programs to target distinctive proliferation gene pathways that restrain the potential for breast cancer development.

Investigation of the Mechanism of hsa_circ_000 1429 Adsorbed miR-205 to Regulate KDM4A and Promote Breast Cancer Metastasis.

This study investigates the mechanism of hsa_circ_0001429 adsorbing miR-205 and regulating the expression of KDM4A to promote breast cancer metastasis and its mechanism. Mammary epithelial cells MCF-10A and human breast cancer cell lines BT474, SKBr-3, ZR-75-30, and MCF7 are cultured, and the mRNA expressions of hsa_circ_000 1429, miR-205, and KDM4A are detected by qRT-PCR; hsa_circ_000 1429 binds to miR-205, and miR-205 targets KDM4A. RIP verifies that hsa_circ_000 1429 binds to AGO2; RNA pull down results prove that hsa_circ_000 1429 binds to miR-205; MTT detects cell proliferation; transwell assay detects cell migration and invasion ability; flow cytometry detects cell apoptosis rate. The expressions of KDM4A, migration, and invasion-related factors, N-cadherin and MMP-9 protein, are detected by blot. hsa_circ_000 1429 may upregulate the KDM4A gene by adsorbing miR-205. Therefore, it will promote the proliferation, migration, and invasion of breast cancer cells and inhibit their apoptosis.

Abstract 2425: Mechano-response via ATP alters calcium signaling in breast epithelial cells with oncogenic KRas mutation

Abstract The majority of breast cancer patient deaths occur when tumor cells migrate away from the primary tumors and disseminate metastatically. Cancer cells at the invasive front mimic the behaviors of non-tumorigenic epithelial cells at wound edges but show less coordinated migration comparatively. While most wound healing studies use a timeframe of 24-48 hours, our previous studies have shown that ATP released from wounded cells initiates a calcium (Ca2+) signal within seconds that spreads to neighboring cells through activation of the P2Y2 surface receptor. RNAseq data of MCF10A mammary epithelial cells with stepwise mutations of PTEN knockout and KRas activation alone or in combination, demonstrate that P2Y2 is downregulated specifically by active KRas. Fluorescence microscopy was used to measure Ca2+ signaling and mitochondrial membrane potential in parental and mutant MCF10A cells. Change in total fluorescence was measured and compared to baseline using Fluo-4, a Ca2+ fluorescent indicator, and TMRM (tetramethylrhodamine, methyl ester), a fluorescent mitochondrial membrane potential dye on Nikon analysis software. We show that KRas activation disrupts ATP stimulation and Ca2+ signaling. This change was further quantified using a FlexStation III plate reader with Fluo-4 to read specific fluorescent changes in Ca2+ after ATP addition. ATP-stimulated Ca2+ was also disrupted in other breast tumor cell lines harboring Ras activating mutations, MDA-MB-231 and MDA-MB-436, both showing a similar decrease in P2Y2 expression. These data show that ATP stimulation can be used to further understand the differences in Ca2+ signaling and mechanical-response between normal breast epithelial cells and cancer cells. Clarifying these molecular mechanisms could reveal targets for new cancer treatments, especially since 90% of human tumors are epithelial carcinomas. Citation Format: Makenzy Mull, Stephen Pratt, Rachel Lee, Abanoub Gad, Katarina Chang, David Annis, Keyata Thompson, Michele Vitolo, Liron Boyman, Stuart Martin. Mechano-response via ATP alters calcium signaling in breast epithelial cells with oncogenic KRas mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2425.

GRWD1 affects the proliferation, apoptosis, invasion and migration of triple negative breast cancer through the Notch signaling pathway.

Breast cancer is a highly heterogeneous tumor, among which triple negative breast cancer (TNBC) is the most invasive and prone to recurrence and metastasis. The present study aimed to investigate the regulatory mechanisms of glutamate-rich WD-repeat-containing protein 1 (GRWD1) in TNBC cells. The expression of GRWD1 in the normal human breast epithelial cells and human breast cancer cells was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The transfection effects of small interfering RNA (siRNA)-GRWD1 and overexpression (Ov)-Notch1 were also confirmed by RT-qPCR and western blotting. The proliferation, apoptosis, invasion and migration of transfected cells were in turn analyzed by Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine, Matrigel and wound healing assays. The expression of proteins related to proliferation, apoptosis, metastasis, epithelial-mesenchymal transition and the Notch signaling pathway was detected by western blotting. As a result, GRWD1 expression was upregulated in breast cancer cells and was revealed to be highest in MDA-MB-231 and HCC1937 cells. GRWD1 knockdown suppressed TNBC cell proliferation, invasion and migration and promoted TNBC cell apoptosis. Furthermore, the expression of Notch1 and Notch4 was inhibited by GRWD1 knockdown. The expression of downstream genes of the Notch signaling pathway Hes1, Hes5, Hey1, Hey2, p21, c-Myc, cyclin D1, human epidermal growth factor 2 receptor and NF-κB were all suppressed after siRNA-GRWD1 transfection. However, Notch1 overexpression reversed the effect of GRWD1 knockdown on biological behaviors of TNBC cells. In conclusion, GRWD1 knockdown could suppress the proliferation, invasion and migration and promoted apoptosis of TNBC cells through inhibiting the Notch signaling pathway.

Formulation and Characterization of Raloxifene Nanostructured Lipid Carriers for Permeability and Uptake Enhancement Applications.

Raloxifene (RLX), a biopharmaceutical classification system (BCS) class II drug, is a selective estrogen receptor modulator (SERM) having an estrogenic effect on the bone and an antiestrogenic effect on the endometrium and breast. Low solubility, high permeability, high metabolism, and low bioavailability are the characteristics of raloxifene. Although 60% is absorbed orally, raloxifene shows extremely poor bioavailability (2%) owing to its low solubility and extensive (>90%) intestinal/hepatic first-pass metabolism. Hence, it becomes important to increase the solubility of raloxifene to enhance its bioavailability. In this study, raloxifene nanostructured lipid carriers (RNLCs) were prepared using the melt dispersion ultrasonication method. The prepared RNLCs were characterized, and the in vitro studies were carried out in the human epithelial breast cancer cell line (MCF-7). The RNLCs had a size of 114.8 ± 0.98 nm and a zeta potential of +9.21 ± 0.58 mV. Transmission electron microscopy (TEM) images showed particle size ranging from 65 to 120 nm. With an entrapment efficiency of 75.04% ± 2.75%, the RNLCs showed sustained release over 7 days compared with the raloxifene drug solution. The prepared RNLCs were successfully taken up by the MCF-7 cells in a time-dependent manner, and the RNLCs showed increased cell cytotoxicity compared with the raloxifene drug. Using the parallel artificial membrane permeability assay (PAMPA), the permeability rate for raloxifene solution was calculated to be 8 × 10-6 cm/s, and for the RNLCs, it was calculated to be 17.8 × 10-6 cm/s. Hence, from the permeability rate calculated, we could conclude that raloxifene, when formulated as nanostructured lipid carriers, showed increased permeability. Overall, the prepared RNLCs were found to be superior to the raloxifene drug as such.

Antiproliferative and anticancer activities of Neurada procumbens L. against epithelial carcinoma and breast cancer cell lines

Neurada procumbens L. commonly called as sand button, is used as a medicinal herb by Bedouin in the Arabia Peninsula for heart and respiratory functions. This little known plant has not been investigated in detail for its biological activities. Hence, we have made an attempt to investigate the plant for its anticancer activities. Methanol and chloroform extracts of N.procumbens were evaluated for antiproliferative and anticancer activities in HEp-2 and MCF-7 cancer cell lines. Humanforeskin epithelial cells showed an IC50 value of 34 μg and 149 μg for chloroform and methanol extracts, respectively. Thechloroform extract of N. procumbens @3 μg showed 50% reduction of HEp-2 cell population in 24 h, while 5.5 μg showedsimilar effects in MCF-7 cells. The results of the methanol extracts were little varied, where 49 μg showed slow inhibitionof HEp-2 cells over a period of 72 h. In another similar observation, 76 μg of methanol extract inhibited 50% MCF-7 cellproliferation. The concentrations which could inhibit complete turnover of the experimental cancer cell lines at 24 h were>5 μg and 6 μg of chloroform extract and > 60 μg and 80 μg of methanol extract for HEp-2 and MCF-7, respectively. Themicro therapeutic index of methanol extract in HEp-2 and MCF-7 were 3 and 1.9, while chloroform extract was 11.3 and6.1. This is an initial observation of N. procumbens that shows antiproliferative activities against cancer cell lines.

Learning the rules of collective cell migration using deep attention networks.

Collective, coordinated cellular motions underpin key processes in all multicellular organisms, yet it has been difficult to simultaneously express the 'rules' behind these motions in clear, interpretable forms that effectively capture high-dimensional cell-cell interaction dynamics in a manner that is intuitive to the researcher. Here we apply deep attention networks to analyze several canonical living tissues systems and present the underlying collective migration rules for each tissue type using only cell migration trajectory data. We use these networks to learn the behaviors of key tissue types with distinct collective behaviors-epithelial, endothelial, and metastatic breast cancer cells-and show how the results complement traditional biophysical approaches. In particular, we present attention maps indicating the relative influence of neighboring cells to the learned turning decisions of a 'focal cell'-the primary cell of interest in a collective setting. Colloquially, we refer to this learned relative influence as 'attention', as it serves as a proxy for the physical parameters modifying the focal cell's future motion as a function of each neighbor cell. These attention networks reveal distinct patterns of influence and attention unique to each model tissue. Endothelial cells exhibit tightly focused attention on their immediate forward-most neighbors, while cells in more expansile epithelial tissues are more broadly influenced by neighbors in a relatively large forward sector. Attention maps of ensembles of more mesenchymal, metastatic cells reveal completely symmetric attention patterns, indicating the lack of any particular coordination or direction of interest. Moreover, we show how attention networks are capable of detecting and learning how these rules change based on biophysical context, such as location within the tissue and cellular crowding. That these results require only cellular trajectories and no modeling assumptions highlights the potential of attention networks for providing further biological insights into complex cellular systems.