Non-tumorigenic Breast Cell Line Research Articles

AQP3 and AQP5 Modulation in Response to Prolonged Oxidative Stress in Breast Cancer Cell Lines.

Aquaporins are membrane pores regulating the transport of water, glycerol, and other small molecules across membranes. Among 13 human aquaporins, six have been shown to transport H2O2 and are therefore called peroxiporins. Peroxiporins are implicated in cancer development and progression, partly due to their involvement in H2O2 transport. Oxidative stress is linked to breast cancer development but is also a mechanism of action for conventional chemotherapy. The aim of this study is to investigate the effects of prolonged oxidative stress on Aquaporin 3 (AQP3), Aquaporin 5 (AQP5), and signaling pathways in breast cancer cell lines of different malignancies alongside a non-tumorigenic breast cell line. The prolonged oxidative stress caused responses in viability only in the cancer cell lines, while it affected cell migration in the MCF7 cell line. Changes in the localization of NRF2, a transcription factor involved in oxidative stress response, were observed only in the cancer cell lines, and no effects were recorded on its downstream target proteins. Moreover, the prolonged oxidative stress caused changes in AQP3 and AQP5 expression only in the cancer cell lines, in contrast to their non-malignant counterparts. These results suggest peroxiporins are potential therapeutic targets in cancer treatment. However, further research is needed to elucidate their role in the modulation of therapy response, highlighting the importance of research on this topic.

Performance assessment of dielectrically modulated negative capacitance germanium source vertical tunnel FET biosensor for detection of breast cancer cell lines

The paper presents a dielectrically modulated negative capacitance Germanium source vertical tunnel FET (DM-NC-Ge-vTFET) biosensor for detection of non-tumorigenic breast cell line (MCF-10A) and breast cancer cell lines of Hs578T, MDA-MB-231, MCF-7 and T47D. The proposed biosensor exploits the incorporation of ferroelectric (FE) layer with lateral and vertical band-to-band tunneling for improved detection sensitivity. The detecting strategy is determined by variations in the dielectric constant of various breast malignant cell lines. Sensitivity (Sn) is determined for both neutral and charged (positive/negative) biomolecules fordistinctive breast cell lines. Sn is determined by evaluating the shiftsin drain current. Sensitivity of 1.16 × 105 is attained for MCF-10A whereas Sn of 1.04 × 108 is realised for the T47D breast cell line for a completely filled nanogap. Additionally, the impact of FE layer thickness on the biosensor has been evaluated. It is observed that Sn of the proposed biosensor isenhanced for increase infill factor from partially to completely filled nanogap with a peak SNR value of approximately 61100. The biosensor’s selectivity and linearity have been evaluated. Lastly, the DM-NC-Ge-vTFET biosensor is benchmarked against existing published literatures andyields superior results in terms of its approximate sensitivity.

Short-term exposure to ethanol induces transcriptional changes in nontumorigenic breast cells.

Breast cancer is a leading cause of cancer-related deaths in women. Many genetic and behavioral risk factors can contribute to the initiation and progression of breast cancer, one being alcohol consumption. Numerous epidemiological studies have established a positive correlation between alcohol consumption and breast cancer; however, the molecular basis for this link remains ill defined. Elucidating ethanol-induced changes to global transcriptional programming in breast cells is important to ultimately understand how alcohol and breast cancer are connected mechanistically. We investigated induced transcriptional changes in response to a short cellular exposure to moderate levels of alcohol. We treated the nontumorigenic breast cell line MCF10A and the tumorigenic breast cell lines MDA-MB-231 and MCF7, with ethanol for 6 h, and then captured the changes to ongoing transcription using 4-thiouridine metabolic labeling followed by deep sequencing. Only the MCF10A cell line exhibited statistically significant changes in newly transcribed RNA in response to ethanol treatment. Further experiments revealed that some ethanol-upregulated genes are sensitive to the dose of alcohol treatment, while others are not. Gene Ontology and biochemical pathway analyses revealed that ethanol-upregulated genes in MCF10A cells are enriched in biological functions that could contribute to cancer development.

ATP13A4 Upregulation Drives the Elevated Polyamine Transport System in the Breast Cancer Cell Line MCF7.

Polyamine homeostasis is disturbed in several human diseases, including cancer, which is hallmarked by increased intracellular polyamine levels and an upregulated polyamine transport system (PTS). Thus far, the polyamine transporters contributing to the elevated levels of polyamines in cancer cells have not yet been described, despite the fact that polyamine transport inhibitors are considered for cancer therapy. Here, we tested whether the upregulation of candidate polyamine transporters of the P5B transport ATPase family is responsible for the increased PTS in the well-studied breast cancer cell line MCF7 compared to the non-tumorigenic epithelial breast cell line MCF10A. We found that MCF7 cells presented elevated expression of a previously uncharacterized P5B-ATPase, ATP13A4, which was responsible for the elevated polyamine uptake activity. Furthermore, MCF7 cells were more sensitive to polyamine cytotoxicity, as demonstrated by cell viability, cell death and clonogenic assays. Importantly, the overexpression of ATP13A4 WT in MCF10A cells induced a MCF7 polyamine phenotype, with significantly higher uptake of BODIPY-labeled polyamines and increased sensitivity to polyamine toxicity. In conclusion, we established ATP13A4 as a new polyamine transporter in the human PTS and showed that ATP13A4 may play a major role in the increased polyamine uptake of breast cancer cells. ATP13A4 therefore emerges as a candidate therapeutic target for anticancer drugs that block the PTS.

3D-printing-assisted synthesis of paclitaxel-loaded niosomes functionalized by cross-linked gelatin/alginate composite: Large-scale synthesis and in-vitro anti-cancer evaluation

Breast cancer is one of the most lethal cancers, especially in women. Despite many efforts, side effects of anti-cancer drugs and metastasis are still the main challenges in breast cancer treatment. Recently, advanced technologies such as 3D-printing and nanotechnology have created new horizons in cancer treatment. In this work, we report an advanced drug delivery system based on 3D-printed gelatin-alginate scaffolds containing paclitaxel-loaded niosomes (Nio-PTX@GT-AL). The morphology, drug release, degradation, cellular uptake, flow cytometry, cell cytotoxicity, migration, gene expression, and caspase activity of scaffolds, and control samples (Nio-PTX, and Free-PTX) were investigated. Results demonstrated that synthesized niosomes had spherical-like, in the range of 60–80 nm with desirable cellular uptake. Nio-PTX@GT-AL and Nio-PTX had a sustained drug release and were biodegradable. Cytotoxicity studies revealed that the designed Nio-PTX@GT-AL scaffold had <5 % cytotoxicity against non-tumorigenic breast cell line (MCF-10A) but showed 80 % cytotoxicity against breast cancer cells (MCF-7), which was considerably more than the anti-cancer effects of control samples. In migration evaluation (scratch-assay), approximately 70 % reduction of covered surface area was observed. The anticancer effect of the designed nanocarrier could be attributed to gene expression regulation, where a significant increase in the expression and activity of genes promoting apoptosis (CASP-3, CASP-8, and CASP-9) and inhibiting metastasis (Bax, and p53) and a remarkable decrease in metastasis-enhancing genes (Bcl2, MMP-2, and MMP-9) were observed. Also, flow cytometry results declared that Nio-PTX@GT-AL reduced necrosis and increased apoptosis considerably. The results of this study prove that employing 3D-printing and niosomal formulation is an effective approach in designing nanocarriers for efficient drug delivery applications.

Synthesis and characterization of novel Schiff base-silicon (IV) phthalocyanine complex for photodynamic therapy of breast cancer cell lines

BackgroundPhotodynamic therapy is an alternative anticancer treatment approach that promises high therapeutic efficacy. In this study, it is aimed to investigate the PDT-mediated anticancer effects of newly synthesized silicon phthalocyanine (SiPc) molecules on MDA-MB-231, MCF-7 breast cancer cell lines, and non-tumorigenic MCF-10A breast cell line. MethodsNovel bromo substituted Schiff base (3a), its nitro homolog (3b), and their silicon complexes (SiPc-5a and SiPc-5b) were synthesized. Their proposed structures were confirmed by FT-IR, NMR, UV–vis and MS instrumental techniques. MDA-MB-231, MCF-7 and MCF-10A cells were illuminated at a light wavelength of 680 nm for 10 min, giving a total irradiation dose of 10 j/cm2. MTT assay was used to determine the cytotoxic effects of SiPc-5a and SiPc-5b. Apoptotic cell death was analyzed using flow cytometry. Changes in the mitochondrial membrane potential were determined by TMRE staining. Intracellular ROS generation was observed microscopically using H2DCFDA dye. Colony formation assay and in vitro scratch assay were performed to analyze the clonogenic activity and cell motility. Transwell migration and matrigel invasion analyzes were conducted to observe changes in the migration and invasion status of the cells. ResultsThe combination of SiPc-5a and SiPc-5b with PDT exhibited cytotoxic effects on cancer cells and triggered cell death. SiPc-5a/PDT and SiPc-5b/PDT decreased mitochondrial membrane potential and increased intracellular ROS production. Statistically significant changes were detected in cancer cells' colony-forming ability and motility. SiPc-5a/PDT and SiPc-5b/PDT reduced cancer cells' migration and invasion capacities. ConclusionThe present study identifies PDT-mediated antiproliferative, apoptotic, and anti-migratory characteristics of novel SiPc molecules. The outcomes of this study emphasize the anticancer properties of these molecules and suggest that they may be evaluated as drug-candidate molecules for therapeutic purposes.

A 50 Hz magnetic field influences the viability of breast cancer cells 96 h after exposure

BackgroundThe exposure of breast cancer to extremely low frequency magnetic fields (ELF-MFs) results in various biological responses. Some studies have suggested a possible cancer-enhancing effect, while others showed a possible therapeutic role. This study investigated the effects of in vitro exposure to 50 Hz ELF-MF for up to 24 h on the viability and cellular response of MDA-MB-231 and MCF-7 breast cancer cell lines and MCF-10A breast cell line.Methods and resultsThe breast cell lines were exposed to 50 Hz ELF-MF at flux densities of 0.1 mT and 1.0 mT and were examined 96 h after the beginning of ELF-MF exposure. The duration of 50 Hz ELF-MF exposure influenced the cell viability and proliferation of both the tumor and nontumorigenic breast cell lines. In particular, short-term exposure (4–8 h, 0.1 mT and 1.0 mT) led to an increase in viability in breast cancer cells, while long and high exposure (24 h, 1.0 mT) led to a decrease in viability and proliferation in all cell lines. Cancer and normal breast cells exhibited different responses to ELF-MF. Mitochondrial membrane potential and reactive oxygen species (ROS) production were altered after ELF-MF exposure, suggesting that the mitochondria are a probable target of ELF-MF in breast cells.ConclusionsThe viability of breast cells in vitro is influenced by ELF-MF exposure at magnetic flux densities compatible with the limits for the general population and for workplace exposures. The effects are apparent after 96 h and are related to the ELF-MF exposure time.

Comparison of the Toxicological Effects of Pesticides in Non-Tumorigenic MCF-12A and Tumorigenic MCF-7 Human Breast Cells.

Humans are exposed to residues of organophosphate and neonicotinoid pesticides, commonly used in agriculture. Children are particularly vulnerable and, among possible adverse outcomes, the increased incidence of premature mammary gland development (thelarche) has raised concern. We evaluated the toxicological effects of chlorpyrifos (CPF), imidacloprid (IMI) and glyphosate (GLY) at exposure concentrations occurring in children on the tumorigenic MCF-7 and non-tumorigenic MCF-12A breast cell lines, as representative of the target organ model, assessing cytotoxicity, apoptosis, necrosis, intracellular reactive oxygen species (ROS) and ATP levels, 17β-estradiol secretion and gene expression of nuclear receptors involved in mammary gland development. The pesticides decreased cell vitality in MCF-7 and cell proliferation in MCF-12A cells. ATP levels were decreased in MCF-7 cells by pesticides and apoptosis was increased in MCF-12A cells only by GLY (2.3 nM). ROS production was decreased by pesticides in both cell lines, except IMI (1.6 nM) in MCF-7 cells. Endocrine disrupting activity was highlighted by induction of 17β-estradiol secretion and modulation of the gene expression of estrogen alpha and beta, progesterone, androgen, and aryl hydrocarbon receptors in both cell lines. The use of MCF-7 and MCF-12A cells highlighted dissimilar modes of action of each pesticide at low human relevant concentrations.

In vitro studies of Annona muricata L. extract-loaded electrospun scaffolds for localized treatment of breast cancer.

This paper presents in vitro studies of the sustained release of Annona muricata leaf extracts (AME) from hybrid electrospun fibers for breast cancer treatment. Electrospun hybrid scaffolds were fabricated from crude AME extracts, poly(lactic-co-glycolic acid)/gelatin (PLGA/Ge) and pluronic F127. The physicochemical properties of the AME extract and scaffolds were studied. The antiproliferative effects of the scaffolds were also assessed on breast cancer (MCF-7 and MDA-MB-231) and non-tumorigenic breast (MCF10A) cell lines. Scanning electron microscope micrographs revealed a random network of micro- and submicron fibers. In vitro drug release profiles, governed by quasi-Fickian diffusion at pH7.4 and non-Fickian super case II at pH6.7, showed initial burst AME release from the PLGA/Ge-AME and PLGA/Ge-F127/AME fibers at pH7.4, and burst release from PLGA/Ge-F127/AME (not observed from PLGA/Ge-AME) at pH6.7. Then, a slower, sustained release of the remaining AME from the fibers, attributed to the onset of degradation of the PLGA/Ge backbone, was observed for the next 72 hr. The cumulative release of AME was 89.33 ± 0.73% (PLGA/Ge-AME) and 51.17 ± 7.96% (PLGA/Ge-F127/AME) at pH7.4, and 9.27 ± 2.3% and 73.5 ± 4.5%, respectively, at pH6.7. Pluronic F127 addition increased the drug loading capacity and prolonged the sustained AME release from the fibers. The released AME significantly inhibited the in vitro growth of the breast cancer cells more than the non-tumorigenic cells, due to the induction of apoptosis, providing evidence for using pluronic F127-containing electrospun fibers for sustained and localized AME delivery to breast cancer cells.

The Impact of Insulin on Low-dose Metronomic Vinorelbine and Mafosfamide in Breast Cancer Cells.

Breast cancer (BC) may be affected by diabetes and anti-diabetic medication, as well as its therapeutic agents. Low-dose metronomic chemotherapy (LDMC) is an available treatment option in BC. We investigated the impact of insulin on low-dose metronomic vinorelbine and mafosfamide in BC cell lines. Human BC cell lines T-47D, MCF-7, MDA-MB-231, BT-549 and non-tumorigenic breast cell line MCF-10A were exposed to 0.01 μg/ml and 10 μg/ml insulin in combination with low-dose metronomic vinorelbine or mafosfamide. The cell viability was determined after 24-72 hours using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Insulin, especially at a concentration of 10 μg/ml, seemed to increase viability of vinorelbine-treated hormone receptor-positive BC cells, whereas low-dose mafosfamide treatment tended to be potentiated by insulin in triple-negative cells. Our findings suggest that insulin may influence the cytotoxic activity of LDMC depending on insulin concentration, type of cytotoxic drug used and BC cell line.

Abstract PS18-30: Loss of NF1 leads to rho GTPase activation and sensitivity to multiple agents in breast cancer

Abstract Background: The Ras signaling pathway is a key oncogenic growth signaling pathway. The neurofibromatosis gene (NF1) is a tumor suppressor gene and negative regulator of Ras. Approximately 20% of breast cancers experience loss of active NF1 protein, leading to unabated Ras activity. Germline inactivation of NF1 leads to neurofibromatosis, which can be treated with MEK inhibitors. The functional consequences of NF1 loss have not been thoroughly explored in breast cancer. Therefore, we created a cellular model for the loss of NF1 in breast cells in order to identify novel therapies that would target NF1 null breast cells. Methods: We used CRISPR CAS9 to knock out (KO) NF1 through targeted disruption of both NF1 alleles in the human non-tumorigenic breast cell line MCF-10A. Immunoblotting confirmed loss of the protein and this cell line was used to identify changes in cellular signaling that resulted from the loss of NF1. We then used a rational approach to select drugs that may target the cellular changes in NF1 null cells. Finally, the candidate drugs were tested in a tumor xenograft model in mice. Results: Loss of NF1 endowed cells with a more transformed phenotype, including EGF growth independence and anchorage-independent growth. We observed increased activation of MAPK and activated Rho GTPase. The MEK inhibitors trametinib and PD0325901 (IC50=0.25 nM and 0.16 nM, respectively) inhibited growth of NF1-null, but not parent cells. We explored inhibitors of other proteins in the MAPK signaling pathway, including the Raf inhibitors sorafenib and vemurafenib and the dual MAP3K1/MAP2K4 inhibitor LY2228820. However, these compounds did not selectively inhibit growth of NF1-null cells, suggesting that Raf and MAP3K1/MAP2K4 are not vulnerable targets in NF1-null cells. Vincristine and zoledronic acid can indirectly affect Rho GTPase function. NF1-null cells were more sensitive to both as single agents (IC50=1.1 nM and 6.9 μM, respectively) than parental cells. We also tested multiple combinations of these drugs and observed benefits from several combinations that exceeded single agent use. Similar sensitivity was not observed to docetaxel or ixabepilone. We then tested the drugs as single agents and in combination against mouse tumor xenograft models with the human breast cancer cell lines Hs578T (NF1 null) or MCF-7 (wild type NF1). Treatment with single agent vincristine or zoledronic acid for 19 days resulted in statistically-significant decreases of 37.3% and 38.9%, respectively (p&amp;lt;0.05) in Hs578T tumor xenografts. The combination treatment resulted in an enhanced reduction in tumor size of 48.8% with a higher level of significance (p&amp;lt;0.01). Treatment of mice with MCF-7 bearing tumors with single agent vincristine resulted in a statistically significant decrease of 35.2% (p&amp;lt;0.05); however, this effect was not observed using the combination treatment, as the change in growth compared to saline treatment was not statistically significant. Conclusions: Our results suggest that loss of NF1 results in a transformed phenotype. Our results provide the first evidence that vincristine in combination with zoledronic acid may be efficacious in treating breast cancers. Additionally, we provide evidence that other drugs and drug combinations may be more effective at treating breast cancers with NF1 loss than MEK inhibitors, warranting further exploration. Citation Format: Melody A. Cobleigh, Matthew Najor, Satnam Brar, Sanja Turturro, Liam Portt, Timothy Yung, Abde M Abukhdeir. Loss of NF1 leads to rho GTPase activation and sensitivity to multiple agents in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-30.

The proteomic analysis of breast cell line exosomes reveals disease patterns and potential biomarkers

Cancer cells release small extracellular vesicles, exosomes, that have been shown to contribute to various aspects of cancer development and progression. Differential analysis of exosomal proteomes from cancerous and non-tumorigenic breast cell lines can provide valuable information related to breast cancer progression and metastasis. Moreover, such a comparison can be explored to find potentially new protein biomarkers for early disease detection. In this study, exosomal proteomes of MDA-MB-231, a metastatic breast cancer cell line, and MCF-10A, a non-cancerous epithelial breast cell line, were identified by nano-liquid chromatography coupled to tandem mass spectrometry. We also tested three exosomes isolation methods (ExoQuick, Ultracentrifugation (UC), and Ultrafiltration–Ultracentrifugation) and detergents (n-dodecyl β-d-maltoside, Triton X-100, and Digitonin) for solubilization of exosomal proteins and enhanced detection by mass spectrometry. A total of 1,107 exosomal proteins were identified in both cell lines, 726 of which were unique to the MDA-MB-231 breast cancer cell line. Among them, 87 proteins were predicted to be relevant to breast cancer and 16 proteins to cancer metastasis. Three exosomal membrane/surface proteins, glucose transporter 1 (GLUT-1), glypican 1 (GPC-1), and disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), were identified as potential breast cancer biomarkers and validated with Western blotting and high-resolution flow cytometry. We demonstrated that exosomes are a rich source of breast cancer-related proteins and surface biomarkers that may be used for disease diagnosis and prognosis.

STAT6 expression and trastuzumab resistance in HER2+ breast cancer.

e13006 Background: Approximately 20% of breast cancers are HER2-positive. Trastuzumab has improved patient outcomes significantly for these cancers. However, acquired resistance occurs in almost all patients with metastatic breast cancer. Therefore, identifying molecular changes that are associated with trastuzumab resistance is worthwhile. STAT6 is a transcription factor that regulates a variety of genes involved in cell cycle regulation, growth inhibition, and apoptosis. STAT6 expression is lost in approximately 3% of breast cancers, but little work has been done in the context of trastuzumab resistance in breast cancer. Methods: In isogenic cell line pairs, we observed that trastuzumab-resistant cells expressed significantly lower levels of STAT6 compared to trastuzumab-sensitive cells. Therefore, in order to study the consequences of STAT6 loss in HER2+ breast cancer, we knocked out both alleles of the STAT6 gene using somatic cell gene targeting. Results: We observed that loss of STAT6 resulted in resistance to trastuzumab treatment in HER2-over-expressing cells. Additionally, loss of STAT6 resulted in anchorage-independent growth and changed expression of several genes involved in epithelial to mesenchymal transition. Functional studies revealed that STAT6 loss caused a non-tumorigenic human breast cell line to form tumors in mice. Conclusions: Loss of STAT6 in breast cells results in enhanced growth properties and resistance to trastuzumab. This study suggests that STAT6 may play a role in the pathophysiology of HER2+ human breast cancer. Disclaimer: The work herein was completed while AMA was a faculty member at Rush University. AMA is currently an employee with the U.S. Food and Drug Administration. The views and data in this publication do not reflect the opinions of the U.S. Government or The U.S. Food and Drug Administration.

Beyond hemostasis: a snake venom serine protease with potassium channel blocking and potential antitumor activities

Snake venom serine proteases (SVSPs) are complex and multifunctional enzymes, acting primarily on hemostasis. In this work, we report the hitherto unknown inhibitory effect of a SVSP, named collinein-1, isolated from the venom of Crotalus durissus collilineatus, on a cancer-relevant voltage-gated potassium channel (hEAG1). Among 12 voltage-gated ion channels tested, collinein-1 selectively inhibited hEAG1 currents, with a mechanism independent of its enzymatic activity. Corroboratively, we demonstrated that collinein-1 reduced the viability of human breast cancer cell line MCF7 (high expression of hEAG1), but does not affect the liver carcinoma and the non-tumorigenic epithelial breast cell lines (HepG2 and MCF10A, respectively), which present low expression of hEAG1. In order to obtain both functional and structural validation of this unexpected discovery, where an unusually large ligand acts as an inhibitor of an ion channel, a recombinant and catalytically inactive mutant of collinein-1 (His43Arg) was produced and found to preserve its capability to inhibit hEAG1. A molecular docking model was proposed in which Arg79 of the SVSP 99-loop interacts directly with the potassium selectivity filter of the hEAG1 channel.

AhR ligands reactivate LINE-1 retrotransposon in triple-negative breast cancer cells MDA-MB-231 and non-tumorigenic mammary epithelial cells NMuMG

Breast cancer is the most common cancer type in females worldwide. Environmental exposure to pesticides affecting hormonal homeostasis does not necessarily induce DNA mutations but may influence gene expression by disturbances in epigenetic regulation. Expression of long interspersed nuclear element-1 (LINE-1) has been associated with tumorigenesis in several cancers. In nearly all somatic cells, LINE-1 is silenced by DNA methylation in the 5́′UTR and reactivated during disease initiation and/or progression. Strong ligands of aryl hydrocarbon receptor (AhR) activate LINE-1 through the transforming growth factor-β1 (TGF-β1)/Smad pathway. Hexachlorobenzene (HCB) and chlorpyrifos (CPF), both weak AhR ligands, promote cell proliferation and migration in breast cancer cells, as well as tumor growth in rat models. In this context, our aim was to examine the effect of these pesticides on LINE-1 expression and ORF1p localization in the triple-negative breast cancer cell line MDA-MB-231 and the non-tumorigenic epithelial breast cell line NMuMG, and to evaluate the role of TGF-β1 and AhR pathways. Results show that 0.5 μM CPF and 0.005 μM HCB increased LINE-1 mRNA expression through Smad and AhR signaling in MDA-MB-231. In addition, the methylation of the first sites in 5́′UTR of LINE-1 was reduced by pesticide exposure, although the farther sites remained unaffected. Pesticides modulated ORF1p localization in MDA-MB-231: 0.005 μM HCB and 50 μM CPF increased nuclear translocation, while both induced cytoplasmic retention at 0.5 and 5 μM. Moreover, both stimulated double-strand breaks, enhancing H2AX phosphorylation, coincidentally with ORF1p nuclear localization. In NMuMG similar results were observed, since they heighten LINE-1 mRNA levels. CPF effect was through AhR and TGF-β1 signaling, whereas HCB action depends only of AhR. In addition, both pesticides increase ORF1p expression and nuclear localization. Our results provide experimental evidence that HCB and CPF exposure modify LINE-1 methylation levels and induce LINE-1 reactivation, suggesting that epigenetic mechanisms could contribute to pesticide-induced breast cancer progression.

Lipid-Functionalized Graphene Loaded with hMnSOD for Selective Inhibition of Cancer Cells.

Combination therapies utilize multiple mechanisms to target cancer cells to minimize cancer cell survival. Graphene provides an ideal platform for combination therapy due to its photothermal properties and high loading capacity for cancer-fighting molecules. Lipid functionalization of graphene extends its potential as a therapeutic platform by improving its biocompatibility and functionality. Previous studies involving graphene demonstrated its usage as a therapeutic vehicle; however, the effect of bare and engineered graphene structures on oxidative stress has not been comprehensively investigated. Because oxidative stress has been linked to cancer progression, it is vital to examine the generation of reactive oxygen species (ROS) in response to therapeutic platforms. This study functionalizes reduced graphene oxide (rGO) with lipids and the antioxidant enzyme human manganese superoxide dismutase (hMnSOD) and presents a detailed characterization of cellular responses to bare and functionalized rGO nanostructures in tumorigenic and nontumorigenic breast cell lines. Each cell type displayed distinct responses depending on whether they were normal, nonmetastatic, or metastatic cells. Bare rGO significantly reduced cell growth and substantially increased ROS production in all cell lines and instigated necrosis in metastatic breast cancer cells. Cell proliferation decreased in cancerous breast cells upon introduction of lipid-rGO, which correlated with peroxidation of lipids coating the rGO. In contrast, lipid-rGO nanostructures had minimal impact on proliferation and lipid peroxidation for normal breast cells. Lipid-rGO nanostructures with bound hMnSOD inhibited the proliferation of metastatic cancer cells while preventing necrosis and avoiding the negative side effects on normal cells associated with chemotherapeutic agents. Together, the results confirm the importance of functionalizing rGO for therapeutic applications and present an additional modality for the usage of graphene to selectively target cancer cells.

Abstract P3-11-12: Epigenetic modulation to deepen and prolong the response to PARP inhibitors

Abstract Background: BRCA1 and BRCA2 are key mediators of DNA damage repair response including the homologous recombination repair (HRR) pathway. Mutations in these genes predispose women to various cancers including breast, ovarian and pancreatic cancers. A new class of drugs, the poly (ADP-ribose) polymerase inhibitors (PARPi), were designed to leverage impaired DNA repair in BRCA-mutated tumors and are FDA approved for various cancers (breast, ovarian). Efforts to combine PARPi with cytotoxic agents show increased efficacy but overlapping toxicities hamper their tolerability. Further, the short duration of response to PARPi monotherapy in diseases like breast cancer has inspired the search for PARPi combinations with other agents including epigenetic modifiers. To overcome these hurdles, we explored synergistic interactions between PARPi and DNA methyltransferases inhibitors (DNMTi) such as decitabine, a cytidine analog. When incorporated in newly synthesized DNA strands during S phase, these agents will trigger a covalent protein-DNA complex formation causing cell cycle arrest and cell death. Material &amp; Methods: BRCA wild-type (WT) and BRCA-mutated breast cancer cell lines were used for in vitro analyses: SUM149PT and HCC-3153 (BRCA1-mutated), MDA-MB-231 and BT-549 (BRCA-WT), UCRP231A (a CRISPR/Cas9 engineered BRCA2-mutated from parental MDA-MB-231), and MCF10A (a BRCA-WT non-tumorigenic breast cell line). The combined effect of talazoparib (PARPi) and decitabine (DNMTi) was analyzed using Combenefit® software to determine their synergistic interaction. Cell cycle analyses, dead/alive cells ratio analyses, as well as pH2AX levels were evaluated. In vivo analyses included xenograft models with SUM149PT as well as the parental BRCA-WT MDA-MB-231 and BRCA2-mutated MDA-MB-231 (UCRP231A) cells in immunodeficient NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ mice. Results: Our preclinical data in BRCA-deficient breast cancer cell lines, demonstrated a synergistic inhibition of cell growth and enhancement of cell death at concentrations of talazoparib and decitabine where each agent individually had minimal efficacy. In cells with intact HRR pathways, the drug combination showed an enhanced inhibition of cell growth but was not synergistic in inducing cell death compared to BRCA deficient cell lines. This difference was further manifested by different cell cycle profiles: non-BRCA mutated cells arrested in S-phase while BRCA-mutated cells progressed through S-phase without arrest and entered cell death from G2/M. In vivo mouse data showed a significant synergistic interaction between the two drugs without enhanced toxicity. Conclusion: The ability to induce a synergistic interaction between talazoparib and decitabine at low drug concentrations present a promising therapeutic strategy for patients with HRR-deficient tumors. Ongoing experiments are being conducted to generate additional engineered BRCA- and other HRR-deficient cell lines (ATM, CHEK2 and others) through CRISPR/Cas9 DNA editing system and further delineate the differential mechanism of interaction between the HRR-deficient and HRR-intact cell lines in preparation for clinical testing. Citation Format: Romain Pacaud, Mallika Dhawan, Scott Thomas, Elysia Roche, Jose Garcia, Nela Pawlowska, Pamela Munster. Epigenetic modulation to deepen and prolong the response to PARP inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-12.

Synthesis, Molecular Docking and Cytotoxic Activity Evaluation of Organometallic Thiolated Gold(I) Complexes.

The complex [(PhCH2NC)AuCl], 1, was prepared by the reaction of [(Me2S)AuCl], A, with an equimolar amount of benzyl isocyanide (PhCH2NC) ligand. Through a salt metathesis reaction, the chloride ligand in 1 was replaced by potassium benzothiazole-2-thiolate (Kbt) and potassium benzoimidazole-2-thiolate (Kbi) to afford complexes (PhCH2NC)Au(κ1-S-bt)], 2a and (PhCH2NC)Au(κ1-S-bi)], 2b, respectively, which were characterized by NMR spectroscopy. The cytotoxic activities of 2a and 2b were evaluated against three human cancer cell lines, including A549 (lung), SKOV3 (ovary), and MCF-7 (breast). Our results indicated that 2a exhibited comparable cytotoxicity on investigated cell lines with cisplatin. It showed a good anti-proliferative activity with IC50 of 19.46, 11.76 and 13.27 μM against A549, SKOV3 and MCF-7 cell lines, respectively. The effects of these complexes on the proliferation of the non-tumorigenic epithelial breast cell line (MCF-10A) showed their good selectivity between the tumorigenic and non-tumorigenic cell lines. Molecular docking simulation studies were also conducted to determine the specific binding site and binding mode of the synthesized gold complexes to DNA and thioredoxinreductase (TrxR) as their proposed targets.

TrkA overexpression in non-tumorigenic human breast cell lines confers oncogenic and metastatic properties.

TrkA overexpression occurs in over 20% of breast cancers, including triple-negative breast cancers (TNBC), and has recently been recognized as a potential driver of carcinogenesis. Recent clinical trials of pan-Trk inhibitors have demonstrated targeted activity against tumors harboring NTRK fusions, a relatively rare alteration across human cancers. Despite this success, current clinical trials have not investigated TrkA overexpression as an additional therapeutic target for pan-Trk inhibitors. Here, we evaluate the cancerous phenotypes of TrkA overexpression relative to NTRK1 fusions in human cells and assess response to pharmacologic Trk inhibition. To evaluate the clinical utility of TrkA overexpression, a panel of TrkA overexpressing cells were developed via stable transfection of an NTRK1 vector into the non-tumorigenic breast cell lines, MCF10A and hTERT-IMEC. A panel of positive controls was generated via stable transfection with a CD74-NTRK1 fusion vector into MCF10A cells. Cells were assessed via various in vitro and in vivo analyses to determine the transformative potential and targetability of TrkA overexpression. TrkA overexpressing cells demonstrated transformative phenotypes similar to Trk fusions, indicating increased oncogenic potential. TrkA overexpressing cells demonstrated growth factor-independent proliferation, increased PI3Kinase and MAPKinase pathway activation, anchorage-independent growth, and increased migratory capacity. These phenotypes were abrogated by the addition of the pan-Trk inhibitor, larotrectinib. In vivo analysis demonstrated increased tumorgenicity and metastatic potential of TrkA overexpressing breast cancer cells. Herein, we demonstrate TrkA overexpressing cells show increased tumorgenicity and are sensitive to pan-Trk inhibitors. These data suggest that TrkA overexpression may be an additional target for pan-Trk inhibitors and provide a targeted therapy for breast cancer patients.

Cytotoxic activity and volatile components of peel oil of Citrus volkameriana

Citrus volkameriana Pasq. (Family: Rutacea) is a very common edible and medicinal plant growing in several places in the world including Egypt. We previously studied the toxicity of components of the leaves; however, the peel oil has not been fully analyzed. In the current study, peel oil of C. volkameriana was prepared by hydro-distillation method, and was analyzed for its components using gas chromatography and gas chromatography–mass spectrometry. The recovered oil was formulated as oil in water emulsion using a wet gum technique with gum acacia as an emulsifier. Two compounds were selected from the identified oil components, emulsified using the same technique, and tested for their cytotoxicity along with the emulsified oil on five human tumor cell lines, as well as on one human non-tumorigenic epithelial breast cell line. Our results showed that limonene (68.5%) was found to be the major oil constituent, followed by γ-terpinene (11.3%). Other minor oil components include thymol methyl ether (4%), 4-terpineol (2.9%), cymol (2.4%) and α-pinene (2.1%). The oil showed a very strong cytotoxic effect on the five human tumor cells tested with the highest effect on human hepatoma HepG2 cells (IC50 = 0.038 μL/mL), followed by the effect on human laryngeal carcinoma HEp2 cells (IC50 = 0.045 μL/mL), human breast adenocarcinoma MCF7 cells (IC50 = 0.075 μL/mL), human lung carcinoma A549 cells (IC50 = 0.093 μL/mL), and human cervical cancer Hela cells (IC50 = 0.13 μL/mL). In contrast, the oil showed a lower cytotoxic effect on the non-tumorigenic breast MCF10A cells (IC50 = 0.106 μL/mL). D-limonene possessed similar cytotoxic results as the oil, suggesting a vital role in the cytotoxic effect obtained with the oil. Furthermore, α-pinene showed a strong cytotoxic effect on MCF7 cells (IC50 = 0.072 μL/mL) with a weaker effect on the MCF10A cells (IC50 = 0.185 μL/mL). In conclusion, Citrus volkameriana peel oil and its components have the potential to act as promising cytotoxic agents.