Breast Epithelial Cell Line Research Articles

Expression of RBMS3 in Breast Cancer Progression.

The aim of the study was to evaluate the localization and intensity of RNA-binding motif single-stranded-interacting protein 3 (RBMS3) expression in clinical material using immunohistochemical (IHC) reactions in cases of ductal breast cancer (in vivo), and to determine the level of RBMS3 expression at both the protein and mRNA levels in breast cancer cell lines (in vitro). Moreover, the data obtained in the in vivo and in vitro studies were correlated with the clinicopathological profiles of the patients. Material for the IHC studies comprised 490 invasive ductal carcinoma (IDC) cases and 26 mastopathy tissues. Western blot and RT-qPCR were performed on four breast cancer cell lines (MCF-7, BT-474, SK-BR-3 and MDA-MB-231) and the HME1-hTERT (Me16C) normal immortalized breast epithelial cell line (control). The Kaplan-Meier plotter tool was employed to analyze the predictive value of overall survival of RBMS3 expression at the mRNA level. Cytoplasmatic RBMS3 IHC expression was observed in breast cancer cells and stromal cells. The statistical analysis revealed a significantly decreased RBMS3 expression in the cancer specimens when compared with the mastopathy tissues (p < 0.001). An increased expression of RBMS3 was corelated with HER2(+) cancer specimens (p < 0.05) and ER(-) cancer specimens (p < 0.05). In addition, a statistically significant higher expression of RBMS3 was observed in cancer stromal cells in comparison to the control and cancer cells (p < 0.0001). The statistical analysis demonstrated a significantly higher expression of RBMS3 mRNA in the SK-BR-3 cell line compared with all other cell lines (p < 0.05). A positive correlation was revealed between the expression of RBMS3, at both the mRNA and protein levels, and longer overall survival. The differences in the expression of RBMS3 in cancer cells (both in vivo and in vitro) and the stroma of breast cancer with regard to the molecular status of the tumor may indicate that RBMS3 could be a potential novel target for the development of personalized methods of treatment. RBMS3 can be an indicator of longer overall survival for potential use in breast cancer diagnostic process.

Determining the roles of membrane tension in breast cancer cell migration

Within the past two years, breast cancer has exceeded lung cancer as the most frequently diagnosed cancer, ranking first for incidence and mortality in most of the world. Cancer reaches its deadliest stage when metastatic spread of cells occurs from tumor sites. Cell metastasis relies, in part, on 1) endocytic membrane trafficking and 2) the formation of adhesive contacts with the extracellular matrix. These processes are regulated by mechanical tension on the cell membrane, which varies spatially within migrating cells. While tension gradients assist in maintaining polarity of the cell during migration, recently it has been shown that these gradients create an asymmetry in endocytic dynamics across the plasma membrane, termed as polarized membrane trafficking. Our preliminary data have shown that changes in membrane tension additionally result in the heterogeneous expression of two distinct adhesive structures during proliferation and nondirectional migration of breast cancer cells. Although membrane tension, endocytosis, adhesion, and cell migration have been extensively studied individually, it is unknown how these processes are collectively regulated by breast cancer cells to metastasize. We thus aim to develop experimental and analytical approaches that will allow us to monitor polarized membrane trafficking, the formation of adhesive structures, and membrane tension in several migrating breast epithelial cell lines. This involves using a microfluidic platform that simultaneously allows the leading and trailing edge of a cell to be separately treated during migration and enables the application of inputs native to the cellular microenvironment. Examination of the various regulatory pathways on the cell membrane that contribute to breast cancer metastasis will significantly impact the development of therapeutic targets in breast cancer drug discovery.

Sequential coupling of phases in unjamming transition of epithelial cells via spatiotemporal analysis on physical phenotypes.

Changes in cellular phenotypes are noticeable traits of various pathophysiological events, including cancer metastasis, morphogenesis, and tissue regeneration. For instance, epithelial cells undergo distinct phenotypic transitions known as jamming and unjamming, which correspond to the cessation and awakening of the cell motility, respectively. When epithelial cells become crowded, the pack of cells is halted and maintains the hexagonal shape resulting in a well-designed barrier function. In contrast, the jammed epithelial cells regain motile functions when the cell is exposed to external cues such as growth factors, physical pressure, and empty spaces. Especially, cell migration, a key component for processing pathophysiological situations, requires the unjamming transition (UJT) as a prerequisite. While many researchers have investigated UJT recently, UJT remains largely unexplored. Here, we attempt to induce the UJT of the normal breast epithelial cell line (MCF 10A) by introducing the empty spaces after the cells are densely jammed. The MCF10A monolayer encounters the spaces, cells near the boundary initiate migration toward the empty region and transmits the migratory behavior to the posterior cells. If the cells are simply assumed to be linked particles, the migration transmission will occur gradually. However, the migration of cells in the rear part is formed at a certain time and location. To clarify the intriguing transition in the cellular motility of follower cells, we spatiotemporally quantify physical characteristics, including traction force, intercellular stress, and morphology of constituent cells. The extensive analysis on correlations between physical variables suggests that the UJT of epithelial cells begins with the accumulation of intercellular stress and completes with the cell division-driven relaxation of stress. In conclusion, this study successively proposes the integrated mechanism of UJT, that inclusively involves cell division, traction force, and cellular morphology.

Identification of biological pathways and processes regulated by NEK5 in breast epithelial cells via an integrated proteomic approach

Specific members of the Nima-Related Kinase (NEK) family have been linked to cancer development and progression, and a role for NEK5, one of the least studied members, in breast cancer has recently been proposed. However, while NEK5 is known to regulate centrosome separation and mitotic spindle assembly, NEK5 signalling mechanisms and function in this malignancy require further characterization. To this end, we established a model system featuring overexpression of NEK5 in the immortalized breast epithelial cell line MCF-10A. MCF-10A cells overexpressing NEK5 exhibited an increase in clonogenicity under monolayer conditions and enhanced acinar size and abnormal morphology in 3D Matrigel culture. Interestingly, they also exhibited a marked reduction in Src activation and downstream signalling. To interrogate NEK5 signalling and function in an unbiased manner, we applied a variety of MS-based proteomic approaches. Determination of the NEK5 interactome by Bio-ID identified a variety of protein classes including the kinesins KIF2C and KIF22, the mitochondrial proteins TFAM, TFB2M and MFN2, RhoH effectors and the negative regulator of Src, CSK. Characterization of proteins and phosphosites modulated upon NEK5 overexpression by global MS-based (phospho)proteomic profiling revealed impact on the cell cycle, DNA synthesis and repair, Rho GTPase signalling, the microtubule cytoskeleton and hemidesmosome assembly. Overall, the study indicates that NEK5 impacts diverse pathways and processes in breast epithelial cells, and likely plays a multifaceted role in breast cancer development and progression.3vPKP8VT_3CNKdhee9few9Video

Interacting with tumor cells weakens the intrinsic clockwise chirality of endothelial cells.

Endothelial cells (ECs) possess a strong intrinsic clockwise (CW, or rightward) chirality under normal conditions. Enervating this chirality of ECs significantly impairs the function of the endothelial barrier. Malignant tumor cells (TCs) undergo metastasis by playing upon the abnormal leakage of blood vessels. However, the impact of TCs on EC chirality is still poorly understood. Using a transwell model, we co-cultured the human umbilical vein endothelial cells or human lung microvascular endothelial cells and breast epithelial tumor cell lines to simulate the TC-EC interaction. Using a micropatterning method, we assessed the EC chirality changes induced by paracrine signaling of and physical contact with TCs. We found that the intrinsic clockwise chirality of ECs was significantly compromised by the TC's physical contact, while the paracrine signaling (i.e., without physical contact) of TCs causes minimal changes. In addition, ECs neighboring TCs tend to possess a left bias, while ECs spaced apart from TCs are more likely to preserve the intrinsic right bias. Finally, we found the chirality change of ECs could result from physical binding between CD44 and E-selectin, which activates protein kinase C alpha (PKCα) and induces pseudopodial movement of EC toward TC. Our findings together suggest the crucial role of EC-TC physical interaction in EC chirality and that weakening the EC chirality could potentially compromise the overall endothelial integrity which increases the probability of metastatic cancer spread.

Ionizing Radiation and Estrogen Affecting Growth Factor Genes in an Experimental Breast Cancer Model.

Genes associated with growth factors were previously analyzed in a radiation- and estrogen-induced experimental breast cancer model. Such in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line, MCF-10F, to low doses of high linear energy transfer (LET) α particle radiation (150 keV/μm) and subsequent growth in the presence or absence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation after being irradiated with either a single 60 cGy dose or 60/60 cGy doses of alpha particles. In the present report, the profiling of differentially expressed genes associated with growth factors was analyzed in their relationship with clinical parameters. Thus, the results indicated that Fibroblast growth factor2 gene expression levels were higher in cells transformed by radiation or in the presence of ionizing radiation; whereas the fibroblast growth factor-binding protein 1gene expression was higher in the tumor cell line derived from this model. Such expressions were coincident with higher values in normal than malignant tissues and with estrogen receptor (ER) negative samples for both gene types. The results also showed that transforming growth factor alpha gene expression was higher in the tumor cell line than the tumorigenic A5 and the transformed A3 cell line, whereas the transforming growth factor beta receptor 3 gene expression was higher in A3 and A5 than in Tumor2 cell lines and the untreated controls and the E cell lines. Such gene expression was accompanied by results indicating negative and positive receptors for transforming growth factor alpha and the transforming growth factor beta receptor 3, respectively. Such expressions were low in malignant tissues when compared with benign ones. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expressions were found to be present in all BRCA patients that are BRCA-Basal, BRCA-LumA, and BRCA-LumB, except in BRCA-Her2 patients. The results also indicated that the insulin growth factor receptor gene expression was higher in the tumor cell line Tumor2 than in Alpha3 cells transformed by ionizing radiation only; then, the insulin growth factor receptor was higher in the A5 than E cell line. The insulin growth factor receptor gene expression was higher in breast cancer than in normal tissues in breast cancer patients. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expression levels were in stages 3 and 4 of breast cancer patients. It can be concluded that, by using gene technology and molecular information, it is possible to improve therapy and reduce the side effects of therapeutic radiation use. Knowing the different genes involved in breast cancer will make possible the improvement of clinical chemotherapy.

Wnt-pathway inhibitors with selective activity against triple-negative breast cancer: From thienopyrimidine to quinazoline inhibitors.

The Wnt-pathway has a critical role in development and tissue homeostasis and has attracted increased attention to develop anticancer drugs due to its aberrant activation in many cancers. In this study, we identified a novel small molecule series with a thienopyrimidine scaffold acting as a downstream inhibitor of the β-catenin-dependent Wnt-pathway. This novel chemotype was investigated using Wnt-dependent triple-negative breast cancer (TNBC) cell lines. Structure activity relationship (SAR) exploration led to identification of low micromolar compounds such as 5a, 5d, 5e and a novel series with quinazoline scaffold such as 9d. Further investigation showed translation of activity to inhibit cancer survival of HCC1395 and MDA-MB-468 TNBC cell lines without affecting a non-cancerous breast epithelial cell line MCF10a. This anti-proliferative effect was synergistic to docetaxel treatment. Collectively, we identified novel chemotypes acting as a downstream inhibitor of β-catenin-dependent Wnt-pathway that could expand therapeutic options to manage TNBC.

Effectiveness of hypofractionated and normofractionated radiotherapy in a triple‐negative breast cancer model

Breast cancer (BC) is one of the most diagnosed malignant carcinomas in women with a triple-negative breast cancer (TNBC) phenotype being correlated with poorer prognosis. Fractionated radiotherapy (RT) is a central component of breast cancer management, especially after breast conserving surgery and is increasingly important for TNBC subtype prognosis. In recent years, moderately hypofractionated radiation schedules are established as a standard of care, but many professionals remain skeptical and are concerned about their efficiency and side effects. In the present study, two different triple-negative breast cancer cell lines, a non-malignant breast epithelial cell line and fibroblasts, were irradiated daily under normofractionated and hypofractionated schedules to evaluate the impact of different irradiation regimens on radiation-induced cell-biological effects. During the series of radiotherapy, proliferation, growth rate, double-strand DNA break-repair (DDR), cellular senescence, and cell survival were measured. Investigated normal and cancer cells differed in their responses and receptivity to different irradiation regimens, indicating cell line/cell type specificity of the effect. At the end of both therapy concepts, normal and malignant cells reach almost the same endpoint of cell count and proliferation inhibition, confirming the clinical observations in the follow-up at the cellular level. These result in cell lines closely replicating the irradiation schedules in clinical practice and, to some extent, contributing to the understanding of growth rate or remission of tumors and the development of fibrosis.

Crosstalk between aryl hydrocarbon receptor (AhR) and BCL-2 pathways suggests the use of AhR antagonist to maintain normal differentiation state of mammary epithelial cells during BCL-2 inhibition therapy

IntroductionActivating the aryl hydrocarbon receptor upon exposure to environmental pollutants promotes development of breast cancer stem cell (CSCs). BCL-2 family proteins protect cancer cells from the apoptotic effects of chemotherapeutic drugs. However, the crosstalk between AhR and the BCL-2 family in CSC development remains uninvestigated. ObjectivesThis study explored the interaction mechanisms between AhR and BCL-2 in CSC development and chemoresistance. MethodsA quantitative proteomic analysis study was performed as a tool for comparative expression analysis of breast cancer cells treated by AhR agonist. The basal and inducible levels of BCL-2, AhR, and CYP1A1 in vitro breast cancer and epithelial cell lines and in vivo mice animal models were determined by RT-PCR, Western blot analysis, immunofluorescence, flow cytometry, silencing of the target, and immunohistochemistry. In addition, an in silico toxicity study was conducted using DEREK software. ResultsActivation of the AhR/CYP1A1 pathway in mice increased EpCAMHigh/CD49fLow CD61+ luminal progenitor-like cells in early tumor formation but not in advanced tumors. In parallel, a chemoproteomic study on breast cancer MCF-7 cells revealed that the BCL-2 protein expression was the most upregulated upon AhR activation. The crosstalk between the AhR and BCL-2 pathways in vitro and in vivo modulated the CSCs features and chemoresistance. Interestingly, inhibition of BCL-2 in mice by venetoclax (VCX) increased EpCAMHigh/CD49fLow CD61+ luminal progenitor-like cells, causing inhibition of epithelial lineage markers, disruption of mammary gland branching and induced the epithelial-mesenchymal transition in mammary epithelial cells (MECs). The combined treatment of VCX and AhR antagonists in mice corrected the abnormal differentiation in MECs and protected mammary gland branching and cell identity. ConclusionsThis is the first study to report crosstalk between AhR and BCL-2 in breast CSCs and provides the rationale for using a combined treatment of BCL-2 inhibitor and AhR antagonist for more effective cancer prevention and treatment.

Synthesis, conformation and cytotoxic activity of short hybrid peptides containing conformationally constrained 1-(aminomethyl)cyclohexanecarboxylic acid and gabapentin

The present work describes the synthesis,conformation and cytotoxic activities of short β/γ hybrid peptides, Boc-β2,2-Ac6c-Gpn-NHMe, BG1; Boc-(β2,2-Ac6c-Gpn)2-OMe, BG2; Boc-(β2,2-Ac6c-Gpn)3-OMe, BG3; H-β2,2-Ac6c-Gpn-NHMe, BG4; H-(β2,2-Ac6c-Gpn)2-OMe, BG5; H-(β2,2-Ac6c-Gpn)3-OMe, BG6, Boc-β2,2-Ac6c-Gpn-OMe, BG7 and H-β2,2-Ac6c-Gpn-OMe, BG8. Mixed C6/C7 conformations were observed for β/γ hybrid peptides. Further, BG1-BG8 were screened against MCF-7 (Breast cancer), A549 (Lung Cancer), PC-3 (Prostate cancer), HCT-116 (Colon cancer), and MDA-MB-231 (Breast cancer) cell lines. Among all, BG6 exhibited potent cytotoxicity against all cancer cell lines with IC50 ranging from 1.6 μM to 6.3 μM with relatively low cytotoxicity against normal epithelial breast cell line fR-2 and human embryonic kidney cell line HEK-293. Minimal hemolytic activity was observed for BG6 against human erythrocytes. Peptide BG6 displayed anti-migratory and anti-invasive potentials showing strong interactions with intrinsic apoptotic markers Bcl-2, Bax, and cleaved-PARP, as well as the induction of the mitochondria maladjustment mediated apoptosis.

Cell Adhesion Molecules Affected by Ionizing Radiation and Estrogen in an Experimental Breast Cancer Model.

Cancer develops in a multi-step process where environmental carcinogenic exposure is a primary etiological component, and where cell–cell communication governs the biological activities of tissues. Identifying the molecular genes that regulate this process is essential to targeting metastatic breast cancer. Ionizing radiation can modify and damage DNA, RNA, and cell membrane components such as lipids and proteins by direct ionization. Comparing differential gene expression can help to determine the effect of radiation and estrogens on cell adhesion. An in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line MCF-10F to low doses of high linear energy transfer α particle radiation and subsequent growth in the presence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation that showed gradual phenotypic changes including altered morphology, increase in cell proliferation relative to the control, anchorage-independent growth, and invasive capability before becoming tumorigenic in nude mice. This model was used to determine genes associated with cell adhesion and communication such as E-cadherin, the desmocollin 3, the gap junction protein alpha 1, the Integrin alpha 6, the Integrin beta 6, the Keratin 14, Keratin 16, Keratin 17, Keratin 6B, and the laminin beta 3. Results indicated that most genes had greater expression in the tumorigenic cell line Tumor2 derived from the athymic animal than the Alpha3, a non-tumorigenic cell line exposed only to radiation, indicating that altered expression levels of adhesion molecules depended on estrogen. There is a significant need for experimental model systems that facilitate the study of cell plasticity to assess the importance of estrogens in modulating the biology of cancer cells.

The anti-proliferative effect of β-carotene against a triple-negative breast cancer cell line is cancer cell-specific and JNK-dependent

BackgroundBreast cancer is a major cause of cancer-related mortality in women worldwide. Triple-negative breast cancer presents an aggressive behavior and a poor response to therapeutic. Cancer progression is associated with reprogramming of metabolic pathways for glutamine, glucose and folic acid. MethodsIn this study, we characterized the antitumoral effect (effects on cell proliferation, culture growth, viability, migration, oxidative stress levels, cell cycle and apoptosis) of carotenoids on a triple-negative human breast cancer cell line (MDA-MB-231 cell line) and investigated interference with nutrient cellular uptake as a contributing mechanism. ResultsOf the four tested carotenoids (β-carotene, crocin, fucoxanthin, astaxanthin), β-carotene presented the most interesting antitumoral effect, by reducing cell proliferation, migration, viability and culture growth, inducing apoptosis and by interfering with cell cycle (S phase arrest). β-carotene significantly increased 3H-deoxy-D-glucose uptake but did not affect neither 3H-glutamine nor 3H-folic acid uptake. Also, it did not interfere with oxidative stress levels. The anti-proliferative effect of β-carotene involves the JNK intracellular pathway, and this carotenoid was able to enhance the anti-proliferative effect of doxorubicin. Importantly, β-carotene did not affect cell viability, proliferation, cell cycle and migration rates of MCF-12A cells, a non-tumoral human breast epithelial cell line. Conclusionβ-carotene presents potential as co-adjuvant to doxorubicin for triple-negative breast cancer treatment.

Biosynthesis of copper oxide nanoparticles mediated Annona muricata as cytotoxic and apoptosis inducer factor in breast cancer cell lines

This study investigated for the first time a simple bio-synthesis approach for the synthesis of copper oxide nanoparticles (CuO NPs) using Annona muricata L (A. muricata) plant extract to test their anti-cancer effects. The presence of CuONPs was confirmed by UV–visible spectroscopy, Scanning electron microscope (SEM), and Transmission electron microscope (TEM). The antiproliferative properties of the synthesized nanoparticles were evaluated against (AMJ-13), (MCF-7) breast cancer cell lines, and the human breast epithelial cell line (HBL-100) as healthy cells. This study indicates that CuONPs reduced cell proliferation for AMJ-13 and MCF-7. HBL-100 cells were not significantly inhibited for several concentration levels or test periods. The outcomes suggest that the prepared copper oxide nanoparticles acted against the growth of specific cell lines observed in breast cancer. It was observed that cancer cells had minor colony creation after 24 h sustained CuONPs exposure using (IC50) concentration for AMJ-13 was (17.04 µg mL−1). While for MCF-7 cells was (18.92 µg mL−1). It indicates the uptake of CuONPs by cancer cells, triggering apoptosis. Moreover, treatment with CuONPs enhanced Lactate dehydrogenase (LDH) production, probably caused by cell membrane damage, creating leaks comprising cellular substances like lactate dehydrogenase. Hence, research results suggested that the synthesized CuONPs precipitated anti-proliferative effects by triggering cell death through apoptosis.

First Report on Comparative Essential Oil Profile of Stem and Leaves of Blepharispermum hirtum Oliver and Their Antidiabetic and Anticancer Effects.

The current research was designed to explore the Blepharispermum hirtum Oliver (Asteraceae) stem and leaves essential oil (EO) composition extracted through hydro-distillation using gas chromatography-mass spectrometry (GC-MS) analysis for the first time. The EOs of the stem and leaves of B. hirtum were comparatively studied for the in vitro antidiabetic and anticancer potential using in vitro α-glucosidase and an MTT inhibition assay, respectively. In both of the tested samples, the same number of fifty-eight compounds were identified and contributed 93.88% and 89.07% of the total oil composition in the EOs of the stem and leaves of B. hirtum correspondingly. However, camphene was observed as a major compound (23.63%) in the stem EO, followed by β-selinene (5.33%) and β-elemene (4.66%) and laevo-β-pinene (4.38%). While in the EO of the leaves, the dominant compound was found to be 24-norursa-3,12-diene (9.08%), followed by β-eudesmol (7.81%), β-selinene (7.26%), thunbergol (5.84%), and caryophyllene oxide (5.62%). Significant antidiabetic potential was observed with an IC50 of 2.10 ± 0.57 µg/mL by the stem compared to the EO of the leaves of B. hirtum, having an IC50 of 4.30 ± 1.56 µg/mL when equated with acarbose (IC50 = 377.71 ± 1.34 µg/mL). Furthermore, the EOs offered considerable cytotoxic capabilities for MDA-MB-231. However, the EO of the leaves presented an IC50 = 88.4 ± 0.5 μg/mL compared to the EO of the stem of B. hirtum against the triple-negative breast cancer (MDA-MB-231) cell lines with an IC50 = 123.6 ± 0.8 μg/mL. However, the EOs were also treated with the human breast epithelial (MCF-10A) cell line, and from the results, it has been concluded that these oils did not produce much harm to the normal cell lines. Hence, the present research proved that the EOs of B. hirtum might be used to cure diabetes mellitus and human breast cancer. Moreover, further studies are considered to be necessary to isolate the responsible bioactive constituents to devise drugs for the observed activities.

Atractylenolide I Inhibits Triple-Negative Breast Cancer Cell Proliferation and Promotes Apoptosis via Blocking the Janus Kinase 2/Signal Transducer and Activator of Transcription 3 Signaling Pathway

Bioactive agent Atractylenolide I (AT-1) has been shown to possess therapeutic value for treating various malignancies. The purpose of the current study is to clarify the potential effect of AT-1 on the development of triple-negative breast cancer (TNBC) and to investigate relevant signaling pathways involved in its mechanism. MTT assay was used to assess the effect of different concentrations of AT-1 on cell survival rate in MCF-10A normal breast epithelial cell line and MDA-MB-231 TNBC cell line. Exogenous IL-6 and WP1066 respectively acted as the agonist and the inhibitor of JAK2/STAT3. Determination of MDA-MB-231 cell viability, proliferation and apoptosis employed MTT, colony formation assay and TUNEL. Western blotting was conducted to measure the expression of proliferation- and apoptosis-related proteins. The viability of MCF-10A cells was unaffected by AT-1, whereas in MDA-MB-231 cells the proliferation level was decreased and the apoptosis level was increased after AT-1 treatment. IL-6 partially restored the expression of AT-1-blocked JAK2/STAT3, and WP1066 inhibited JAK2/STAT3 expression in combination with IL-6 and AT-1. Furthermore, compared with the AT-1 group, co-incubation of IL-6 and AT-1 partially restored the proliferative capacity and reduced the apoptosis of MDA-MB-231 cells, while WP1066 reversed these effects in combination with IL-6 and AT-1. AT-1 suppressed proliferation and promoted apoptosis in TNBC cells likely through inhibiting the activation of JAK2/STAT3 signaling pathway. The findings from this study may provide guidance for future studies on AT-1 and theoretical basis of AT-1 pharmacological activities.

Cytotoxic Activity of Carrageenan on Malignant MCF-7 Breast Cancer and the non-Malignant SVCT Breast Epithelial Cell Lines

Objective: Recent studies have shown that sulfated polysaccharides obtained from marine algae have many biological and physiological activities. Kappa-carrageenan, a sulfated polysaccharide obtained from red marine algae, is known to have anti-proliferative effects in various cancer cell lines. In this study, the cytotoxic effect of kappa-carrageenan on two different cell lines, namely the malignant MCF-7 breast cancer cell line and the non-malignant SVCT breast epithelial cell lines, was investigated in an in vitro model. Materials &amp;amp; Methods: Three different concentrations of carrageenan were prepared for each cell in its own medium, with an initial dose of 1000 µg/ml (Dilution I: 1000 µg/ml; Dilution II: 250 µg/ml; Dilution III: 62.5 µg/ml). Cells were incubated with carrageenan at the doses set for each experimental group. Cells incubated in a carrageenan-free medium comprised the control group. Results: To measure cell viability, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) analysis was performed. Cell morphology was investigated by the acridine orange (AO)/propidium iodide (PI) fluorescent staining method. The present results indicated that carrageenan caused cytotoxic effects on both malignant and non-malignant cell lines. Conclusion: Considering that the different phenotypic features of the subtypes of the MCF-7 cell line may affect cell viability and cell proliferation, cell selection should be performed very carefully in cytotoxicity studies. We suggest that using the MCF-7 cell line for cytotoxicity experiments needs to contemplate this important phenomenon for further experimental setups.

An In Vitro Approach for Investigating the Safety of Lipotransfer after Breast-Conserving Therapy.

The application of lipotransfer after breast-conserving therapy (BCT) and irradiation in breast cancer patients is an already widespread procedure for reconstructing volume deficits of the diseased breast. Nevertheless, the safety of lipotransfer has still not been clarified yet due to contradictory data. The goal of this in vitro study was to further elucidate the potential effects of lipotransfer on the irradiated remaining breast tissue. The mammary epithelial cell line MCF-10A was co-cultured with the fibroblast cell line MRC-5 and irradiated with 2 and 5 Gy. Afterwards, cells were treated with conditioned medium (CM) from adipose-derived stem cells (ADSC), and the effects on the cellular functions of MCF-10A cells and on gene expression at the mRNA level in MCF-10A and MRC-5 cells were analyzed. Treatment with ADSC CM stimulated transmigration and invasion and decreased the surviving fraction of MCF-10A cells. Further, the expression of cytokines, extracellular, and mesenchymal markers was enhanced in mammary epithelial cells. Only an effect of ADSC CM on irradiated fibroblasts could be observed. The present data suggest epithelial–mesenchymal transition-like changes in the epithelial mammary breast cell line. Thus, the benefits of lipotransfer after BCT should be critically weighed against its possible risks for the affected patients.

Heteroleptic silver(I), nickel(II), and copper(II) complexes of N4‐substituted thiosemicarbazones and ciprofloxacin: Theoretical, in vitro anti‐proliferative, and in silico molecular modeling and pharmacokinetics studies

In this study, a series of new heteroleptic silver(I), nickel(II), and copper(II) complexes of the type [M(L1–3)(Cfx)n] (1–9), where L1–3 = N4‐substituted thiosemicarbazones (TSCs), Cfx = ciprofloxacin, M = Ag(I), Ni(II), or Cu(II), and n = 1 or 2, have been synthesized and characterized by spectral methods. The spectral data of the complexes suggest bidentate behavior of the TSC ligand, which coordinate with metal ion through thione sulphur and hydrazine nitrogen atoms, and neutral bidentate behavior of the ciprofloxacin drug that coordinates with metal ion through pyridone oxygen and one of the carboxylate oxygen atoms. The structural properties of the silver(I) complexes (1–3) were studied by density functional theory calculations, which indicate the distorted tetrahedral geometry with lower energy gap for complex 3. All the heteroleptic complexes were screened for their in vitro anti‐proliferative activity against four cancerous (lung [A549], estrogen negative [MDA‐MB‐231], estrogen positive [MCF‐7] and Henrietta's cancer cells [HeLa]), and one non‐cancerous (non‐tumorigenic human breast epithelial cell line [MCF‐10a]) cell lines by MTT reduction assay. The obtained results revealed the lower activity of silver(I) complexes and higher activity of copper(II) complexes against the tested cancer cell lines. The complexes 6 and 9 induce apoptosis in MDA‐MB‐231 cells, which was evidenced by acridine orange/ethidium bromide staining method. The molecular docking studies revealed hydrogen bonding and hydrophobic interactions between the complexes and the epidermal growth factor receptor. The in silico analysis of the complexes for Lipinski's “rule of five” and absorption, distribution, metabolism, and excretion properties predicted the drug‐likeness of the synthesized complexes.

Abstract 13: Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro

Abstract Despite the well-documented benefits of circulating tumor DNA (ctDNA) based liquid biopsy, including screening, prognostication, and treatment monitoring, there remain several concerns with its application. Chief among these concerns are imperfect sensitivity and specificity, particularly pre-diagnosis and after tumor resection, when ctDNA burden is low. Cell death, either through apoptosis or necrosis, has been the most widely accepted mechanism of DNA release. Recent studies have also suggested active release through extracellular vesicles as a paradigm for DNA release. However, this pathway remains highly debated in terms of its contribution, mechanism, and even credibility. To address the deficiencies in ctDNA detection in the clinic and the lack of basic mechanistic knowledge of cf/ctDNA release, we performed an in vitro CRISPR screen on MCF10A cells to identify regulators of DNA release. This normal breast epithelial cell line was chosen because MCF10A cells release DNA into cell culture media with a similar fragmentation pattern to that observed in human blood. After lentiviral infection with the genome-wide Brunello CRISPR-Cas9 knockout library, the population of knockouts identified in the genomic DNA of cells attached to the plates was compared to the population of knockouts found in the DNA released into the cell-depleted culture media. Genes with skewed representation were considered potential regulators of DNA release. Our analysis identified several genes in the TRAIL apoptotic pathway including FADD, as well as several genes encoding RNA-binding proteins including Sam68. Knockout of Sam68 and FADD in MCF10As, MDA-MB-468 breast cancer cells, and NCI-H841 lung cancer cells lead to decreases in DNA release. Previous studies have hypothesized that the fragment size of released DNA is dependent on release mechanism. However, the decreases observed in our knockout cell lines were not limited to a specific fragment size of DNA. Application of the small molecule apoptosis inhibitor Z-VAD-FMK also recapitulated the DNA release phenotype seen in our knockout cell lines. From these results, we conclude that apoptosis is the major source of DNA release from cancer cells, and that fragment size is not the indicator of release mechanism it has been presumed to be. Mechanistic studies of DNA release such as this could lead to the ability to modify the amount of ctDNA in circulation, which would significantly improve accurate detection in the clinic. Citation Format: Brad A. Davidson, Adam X. Miranda, Sarah Croessmann, Ben Ho Park. Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro [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 13.

Abstract 3024: Lipid treatment induces a neural-like phenotype in non-transformed breast epithelial cells

Abstract Introduction. Genes involved in lipid metabolism have been shown to be overexpressed in the contralateral unaffected breast of women with estrogen receptor negative (ER-) breast cancer (BC) (PMID: 28263391). Exposure of non-transformed MCF10A breast epithelial cells to lipids results in altered chromatin structure, epigenomic reprogramming and changes in gene expression. Within the upregulated genes are numerous gene sets related to nervous system development, neurons and synapses. Neural genes are more highly expressed in Triple Negative Breast Cancers (TNBC) compared to non-TNBC, and several studies have shown a connection between neural stemness and tumorigenicity. We hypothesized that stem-like cells have a survival advantage when exposed to lipids and that lipid-induced molecular changes underlying malignant transformation are connected with a neural-like phenotype. Methods. To validate the association of lipids and neural genes, a second non-transformed, estrogen and progesterone receptor negative breast epithelial cell line, MCF12A, was exposed to vehicle or octanoic acid (OA) for 24 hours. Gene expression was assayed by RNA-seq and OA responsive genes were identified. Gene set enrichment analysis was utilized to identify gene sets affected by OA. The 954 neural genes identified to be upregulated in TNBC (PMID: 32080331) were compared to OA responsive genes in both cell lines. The Aldefluor assay was used to identify stem-like (ALDH+) cells in vehicle and OA treated MCF10A cells. To determine if lipid-exposed cells adopt a neural-like phenotype, MCF10A cells were grown on Poly-D-Lysine/Laminin (PDL/LM) coated plates. Results. 5,684 and 5,305 genes were up- and down-regulated (FDR &amp;lt; 0.01), respectively, in MCF12A OA treated cells. Numerous gene sets were significantly associated with OA exposure, including many neural-related pathways. More than 50% of the gene sets identified following OA-exposure were common to both cell lines. 418 and 303 neural genes upregulated in TNBC were significantly (FDR &amp;lt; 0.01) upregulated after OA treatment in MCF10A and MCF12A, respectively. Cells growing on PDL/LM plates assumed the normal MCF10A phenotype in presence of vehicle while OA-treated cells adopted a neuronal-like phenotype. Upon OA treatment the percentage of ALDH+ cells increased by a minimum of 10%. Conclusions. OA treatment was associated with the enrichment of ALDH+ cells, expression of neuronal-related genes and the development of neural-like phenotype. The upregulation neural genes upregulated in TNBC upon OA treatment may in part explain the association of lipid exposure and ER- BC. The upregulation of neural-related genes, and the development of neural-like phenotype suggests that lipid exposure results in cell state instability or plasticity, leading to reprogramming/selecting cells with a multi-potential embryonic or stem-like state that has been associated with tumor progression. Citation Format: Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, Susan Clare. Lipid treatment induces a neural-like phenotype in non-transformed breast epithelial cells [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 3024.