Human Breast Cell Lines Research Articles

Upregulated hsa_circ_0000129 expression promotes proliferation and migration of breast cancer cells.

Circular RNAs (circRNAs) are considered potential biomarkers in the pathogenesis and detection of several types of cancer. The present study aimed to investigate the role of hsa_circ_0000129 in the pathogenesis and molecular mechanism underlying breast cancer. A total of 68 pairs of breast cancer and corresponding paracancerous tissue samples, three different breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) and a normal human breast cell line (MCF-10A) were used to investigate the expression of hsa_circ_0000129. The effect of hsa_circ_0000129 on cell proliferation, migration and colony formation was assessed in MCF-7 and MDA-MB-468 cells, along with the expression of enhancer of zeste homolog 2 (EZH2). The results demonstrated that hsa_circ_0000129 expression was significantly higher in breast cancer tissues compared with normal tissues. In addition, high hsa_circ_0000129 expression was significantly associated with lymph node metastasis and a higher tumor-node-metastasis stage. Comparisons between the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) and MCF-10A cells indicated similar results. MCF-7 cells overexpressed with hsa_circ_0000129 significantly increased cell proliferation, migration and colony formation compared with the negative control group, the effects of which were reversed following hsa_circ_0000129 knockdown in MDA-MB-468 cells. Furthermore, EZH2 expression was positively associated with hsa_circ_0000129 expression. Taken together, the results of the present study suggest that hsa_circ_0000129 may represent a promising prognostic biomarker for breast cancer. In addition, the role of hsa_circ_0000129 in breast cancer cell lines indicates a mechanism for tumorigenesis, as well as a potent target for the treatment of malignant progression.

Design, synthesis, biological evaluation and molecular modeling investigation of new N'-(2-Thiouracil-5-oyl) hydrazone derivatives as potential anti-breast cancer and anti-bacterial agents

A series of novel N’-(2-thiouracil-5-oyl)hydrazones were designed and chemically synthesized using an active substructure combination method. The synthesized compounds were structurally characterized on the basis of elemental (%) analyses, IR, MS, 1H NMR, and 13C NMR spectra. All the prepared compounds were evaluated in vitro against MCF-7 human breast cell line using MTT assay. The anticancer results showed that compounds 3j, 4a, 3c, 3b, and 3h exhibit the most prominent effect against breast cancer cell line with IC50 values of 3.40, 3.50, 3.60, 3.70, and 3.80 µg/ml, respectively using doxorubicin as a control drug. Moreover, molecular docking studies were also performed in order to identify the binding mode mechanism of these compounds with prospective target, thymidylate synthase (PDB:1JU6). On the other hand, the antibacterial activities of all prepared compounds were screened in vitro against three bacterial strains, namely, Escherichia coli, and Pseudomonas aeruginosa as Gram negative bacteria and Staphylococcus aureus as a Gram positive bacterium using agar well diffusion method. The antibacterial activity results revealed that most of the compounds under test were inactive however, among all the tested compounds, only 3g and 4a, in a concentration of 50 µg/ml showed a high antibacterial activity against the three used bacterial strained. It is worthy to note that the potency of 3g against Escherichia coli was comparable to that of the reference drug, while compound 2 exhibited significant activity against Pseudomonas aeruginosa only and compound 3j displayed low activity against Staphylococcus aureus. The structure-antibacterial activity relationship analysis can be modulated by the presence of aromatic or heteroaromatic moiety containing more lipophilic character significantly contributed to antibacterial activity. In addition, the drug-likeness properties have predicted for the target compounds.

Design, synthesis and biological evaluation of water soluble and non-aggregated silicon phthalocyanines, naphthalocyanines against A549, SNU-398, SK-MEL128, DU-145, BT-20 and HFC cell lines as potential anticancer agents

Cancer has become an important public problem in worldwide since cancer incidence and mortality are growing rapidly. In this study, water soluble and non-aggregated silicon (IV) phthalocyanines and naphthalocyanines containing (3,5-bis{3-[3-(diethylamino)phenoxy]propoxy}phenyl)methoxy groups have been synthesized and characterized to investigate their anticancer potential. Their DNA binding/nuclease, topoisomerases inhibition were investigated using UV–Vis absorption, thermal denaturation and agarose gel electrophoresis. The in vitro cytotoxic properties of the compounds on human lung (A549), breast (BT-20), liver (SNU-398), prostate (DU-145), melanoma (SK-Mel 128) carcinoma and human fibroblast (HFC) normal cell lines were evaluated by using MTT assay. In order to determine the mechanism of cancer cell growth suppression, cell cycle analysis was carried out using flow cytometer on A549 cell line. The Kb values of SiPc1a and SiNc2a were 6.85 ± (0.35) × 106 and 1.72 ± (0.16) × 104 M−1 and Tm values of ct-DNA were calculated as 82.02 °C and 78.07 °C, respectively in the presence of both compounds. The ΔTm values of SiPc1a and SiNc2a were calculated as 6.45 and 2.50 °C, respectively. The nuclease effects of SiPc1a and SiNc2a with supercoiled plasmid pBR322 DNA demonstrated that both compounds did not trigger any DNA nuclease effects at the lowest concentrations without irradiation whereas both compounds in the presence of activating agent (H2O2) showed significant plasmid DNA nuclease actions under irradiation (22.5 J/cm2). SiPc1a and SiNc2a inhibited to topoisomerase I on increasing concentrations whilst they had lower inhibition action toward topoisomerase II that of topoisomerase I. The in vitro cytotoxicity studies displayed that SiPc1a had the highest cytotoxic effects among the tested compounds against A549, SNU-398, SK-MEL128, DU-145, BT-20 and HFC cell lines with CC50 values ranged from 0.49 to 2.99 µM. Furthermore, SiPc1a inhibited cell proliferation by cell cycle arrest in G0/G1 phase. All of these results suggested that SiPc1a is a promising candidate as an anticancer agent.

Normal breast epithelial MCF-10A cells to evaluate the safety of carbon dots

The human normal breast cell line MCF-10A is being widely used as a model in toxicity studies due to its structural similarity to the normal human mammary epithelium. Over the years, application of carbon dots (C-dots) in biomedicine has been increasing due to their photoluminescence properties, biocompatibility, biosafety and possible applications in bioimaging and as drug carriers. In this work we prepared three different C-dots from the same set of carbon and nitrogen precursors (citric acid and urea, respectively) via three distinct bottom-up synthetic routes and their safety was tested against the normal breast cell line MCF-10A. The characterization results demonstrated a similar size range and composition for all the C-dots. The MCF-10A cells were treated with different concentrations of C-dots for 24, 48 and 72 h to evaluate the cell viability over time. For the 24 h incubation, there were no significant decreases in the viability of the MCF-10A cells. For the 48 h treatment, there was a significant decrease in the viability of the cells treated with calcination-based C-dots, but without significant cellular viability changes for microwave and hydrothermal-based C-dots. For 72 h, cells treated with hydrothermal-based C-dots have the most promising viability profile. Also, compared with paclitaxel, these C-dots have a safety profile very close to that of an antineoplastic in non-tumor cells. Our results suggest that these new C-dots have potential as imaging candidates or biosensing tools as well as drug carriers, and further investigation in animal models is needed for future application in medicine.

Schiff base ligand L synthesis and its evaluation as anticancer and antidepressant agent

ObjectivesThe current study focuses on the synthesis of Schiff base ligand L [1(pyridine-2-ylimino) methyl) naphthalene-2-ol], and its applications as anticancer agent against human lung (H-460) and breast (MCF-7) cell lines, and as antidepressant agent. MethodsThe Schiff base ligand L was synthesized by aldol condensation reaction by reacting commercially available 2-hydroxy-1-napthaldehyde with 2-amino-pyridine. The synthesized ligand was characterized by different spectroscopic techniques and evaluated for its anticancer potential against H-460 and MCF-7 cell lines. The antidepressant activity was evaluated by means of elevated plus maze model using Diazepam as reference drug. ResultsA dose dependent inhibitory response was recorded against both the cell line used. For highest tested dose (500 µg/ml), 89.20% of anticancer activity with IC50 = 23.99 µg/ml against H-460 was observed while an IC50 of 31.68 µg/ml against MCF-7 indicated the potential of this compound to be used as anticancer agent. A dose dependent response was also recorded for the synthesized ligand when tested as antidepressant in elevated plus maze model. ConclusionThe synthesized Schiff base ligand L exhibited good anticancer and antidepressant activities that could be used as alternative drug to treat the related health complications. However, further toxicological and clinical studies are needed to confirm the safe use of this compound.

Organotin(IV) complexes derived from 1,4-naphthalenedicarboxylic acid: synthesis, structure, in vitro cytostatic activity

Seven organotin(IV) complexes, [(R3Sn)2L]n (R = Me 1, R = n-Bu 2), [(Ph3Sn)2L] (3), [(R2Sn)2L(μ3-O)]n (R = Me 4, R = n-Bu 5), [R2SnL(1,10-phen)]n (R = Me 6), [(R2SnCl)2L(1,10-phen)2] (R = n-Bu 7) derived from 1,4-naphthalenedicarboxylic acid (H2L) have been synthesized and characterized by elemental analysis, FT-IR, PXRD, NMR and X-ray crystallography. The single crystal diffraction reveals that complexes 1 and 2 represent 2D network structures, which both contain tetranuclear 26-membered macrocycles. Complexes 3 and 7 display dinuclear tin monomers, which can form 1D infinite chain by C-H···O and C-H···π interactions. Meanwhile, complexes 4 and 5 display 2D network containing tetraorganodistannoxane unit, while complex 6 adopts a 1D infinite chain structure, which further constructs 2D supramolecular architecture through C-H···O intermolecular interactions. What's more, in vitro cytostatic activity of the complexes 1-3 against cervical carcinoma cell lines (HeLa), hepatocellular carcinoma cell lines (HepG-2) and human normal breast cell lines (HBL-100) have been investigated, and the results show that organotin derivatives with n-butyl and phenyl group (2 and 3) exhibit significantly higher anticancer activity than complex 1 with methyl group. Meanwhile, organotin(IV) complexes (1-3) display lower cytotoxicity than corresponding organotin(IV) precursors [trimethyltin chloride, bis(tri-n-butyltin) oxide and triphenyltin chloride] in HBL-100 cells. Furthermore, cytostatic assessments of complex 3 against HepG-2 cells reveal that the effect of cytostatic and apoptotic may through ROS-mediated pathway.

Ionic liquid mediated synthesis and in vitro mechanistic exploration of polycyclic cage‐like heterocyclic hybrid

AbstractA three‐component, [3 + 2]‐cycloaddition/annulation domino protocol is described for the synthesis in excellent yield of a polycyclic cage‐like heterocyclic hybrid (PCHH) that comprises various advantaged structural units viz., α,β‐unsaturated ketone moiety, 4‐pyridinone and pyrroloisoquinoline in a cage‐like framework. The antitumor activity of PCHH on human breast (MCF7), colon (HCT116), cervical (JURKAT) and lung (NCI‐H460) malignant cell lines inhibited the propagation of all cell lines. This hybrid molecule displayed increased broad‐spectrum anticancer activity with higher doses of PCHH. Furthermore, the compound induced 45.21% of early apoptosis and 46.32% of late apoptosis in the Jurkat cancer cell line. Cell cycle analysis showed that this cage‐like compound caused cell cycle arrest of Jurkat cells at the S phase and sub G0/G1 phase. Additionally, it led to increased DNA fragmentation and mitochondrial membrane permeabilization through activation of caspase‐3 enzyme. Present investigation demonstrates the specific cytotoxic activity of the cage‐like compound and the induction of apoptosis through the intrinsic pathway of Jurkat cells.

Effect of Presence of Aliphatic Glycine in the Anti-cancer Platinum Complex Structure on Human Serum Albumin Binding

In this work, a new water-soluble Pt(II) complex was synthesized with aliphatic glycine ligand with a formula of cis-[Pt(NH3)2(isopentylgly)]NO3, as an anti-cancer drug, and characterized. To determine the binding constant of the human serum albumin (HSA, the most abundant carrier proteins in the human circulatory system) to this complex and the binding site of the complex on HSA, the melting point of HSA and the kinetics of this interaction were investigated to introduce an anti-breast cancer drug with fewer side effects. HSA interaction with the complex was studied via a spectroscopic method at 27 and 37 °C and physiological situation (I = 10 mM, pH = 7.4) and molecular docking. The toxicity value of this complex was obtained against the human cancer breast cell line of MCF-7. The thermodynamic parameters of enthalpy and entropy were also achieved in the empirical procedure. Due to the spontaneity of the interaction, Gibbs free energy variation was obtained negative. The binding constant of this complex to HSA was 3.9 × 105 (M−1). Empirical results showed that the quenching mechanism was static. Hill coefficients, Hill constant, complex aggregation number around protein, number of binding sites, and protein melting temperature with complex were obtained. The kinetics of this interaction was also investigated, which showed that this interaction follows a second-order kinetic. The molecular docking data indicated that the position of the interaction of complex on the protein was the site I in the sub-second IIA. Also, the hydrogen bonding and the hydrophobic interaction as the dominant binding forces were seen in complex–HSA formation. This interaction with positive cooperativity was recognized via a superior hydrogen bond. The reasonable binding constant was also obtained, which could ultimately be a good option as an anti–breast cancer drug.

R-sulforaphane modulates the expression profile of AhR, ER\u03b1, Nrf2, NQO1, and GSTP in human breast cell lines

Our previous study showed remarkable differences in the effect of R-sulforaphane (R-SFN) on the expression of CYPs 19, 1A1, 1A2, and 1B1 in ER(+) MCF7, ER( −) MDA-MB-231, and non-tumorigenic immortalized MCF10A (8). This study aimed to evaluate the effect of R-SFN on phase II enzymes induction and expression of AhR, Nrf2, and ERα in the same breast cell lines. The results showed increased expression of GSTP as a result of treatment with R-SFN in breast cancer cells. An increased NQO1 transcript and protein levels were found in all breast cells, with the most significant increase in MCF7 cells. Similarly, the enhancement of Nrf2 expression was noticed in all tested cells. AhR gene transcript and protein were decreased in MCF7 cells. In MDA-MB-231, increased AhR mRNA was not confirmed at the protein level. No differences were found in the expression of ERα. Overall, the results of the present study extended our earlier suggestions on the possible interference of R-SFN with estrogens homeostasis in breast cancer cells differing in ERα status, as well as in non-tumorigenic immortalized breast epithelial cells. While some of R-SFN effects might be beneficial and useful in breast cancer prevention, the others, particularly GSTP induction, may lead to adverse effects.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Recognition of healthy and cancerous breast cells: Sensing the differences by dielectric spectroscopy.

The response of human cells to applied electrical signals depends on the cellular health status, because it is influenced by the composition and structure of the main cellular components. Therefore, electrical impedance-based techniques can be considered as sensitive tools to investigate healthy or disease state at cellular level. The goal of this study is to show that different types of in vitro cellular lines, related to different health status, can be differentiated using impedance spectra analysis. Three different types of human breast cell line, corresponding to healthy, cancerous, and metastatic adenocarcinoma cells, were measured by means of electrical impedance spectroscopy. By modeling the investigated cells with proper resistive and capacitive circuital elements, the magnitude of the cell electrical components and spectra of real and imaginary part of dielectric permittivity were obtained. The latter were subsequently examined with a commonly adopted mathematical model, in order to estimate the values of specific dielectric parameters for the three different cellular lines. The relative variation of cellular capacitance with respect to that of the culture medium, estimated at 100Hz, has a larger value for the two types of cancerous cells with respect to the noncancerous type. Furthermore, the ratio between the real and imaginary part of the dielectric permittivity function has larger values for metastatic cells with respect to the normal and nonmetastatic ones. Therefore, the mentioned relative capacitance allows to discriminate between normal and cancerous cells, whereas the results obtained for the dielectric function can discriminate between metastatic and nonmetastatic cells. This study can be considered as an exploratory investigation of evaluating in vitro the health status of humans cells using selected electrical impedance parameters as potential markers. The obtained results highlight that a standard cultureware system, provided with interdigitated electrodes and appropriate impedance parameters, that is, cellular capacitance and the ratio between the imaginary and real part of cellular dielectric function, can be used to discriminate between healthy and cancerous breast cell lines, as well as different malignancy degrees.

Abstract 2393: APOBEC3 enzymes induce chromosomal translocations in solid cancers

Abstract Introduction: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases provides a defense for the host genome against exogenous viruses and endogenous retro-elements, but their enzymatic activity may cause host genomic instability via a conversion of cytosine to uracil during RNA and DNA editing. While it is well established that activation-induced cytidine deaminase (AID; a B-cell specific APOBEC family enzyme) induces genomic instability by initiating mutations and oncogenic chromosomal translocations involved in the development and progression of lymphoma, it is unclear whether APOBEC3 enzymes could play a similar role in non-hematopoietic cells. Recently, APOBEC-signature mutation (C-to-T transition) has been identified in almost half of all human cancers, including breast and lung cancers. However, enzymes that trigger chromosomal translocation in solid cancers are largely underdetermined. In this study, we investigated the role of APOBEC3 proteins in chromosomal translocation formation and validated APOBEC3-induced chromosomal translocations in solid cancers. Methods: We generated genome-wide chromosomal translocation maps of human APOBEC3 enzymes in both mouse and human cells by adapting the high-throughput, genome-wide translocation sequencing (HTGTS) technique. We exploited immortalized mouse fibroblasts carrying a 25x I-SceI meganuclease cassette targeted in the c-myc locus and also human MCF10A (breast), Beas-2B (lung), and PC-9 (non-small cell lung cancer) cells by generating DNA DSBs in either the c-myc locus or the EML4 locus by the CRISPR/Cas9 system. Results: All human APOBEC3 enzymes have cytidine deaminase activity on their preferred target motifs with different potency. By overexpressing APOBEC3 proteins in AID−/− B cell, we found that APOBEC3 enzymes, in contrast to AID, did not induce IgG1 class switch recombination (CSR) indicating a different mechanism of action between AID and other APOBEC3 enzymes. By HTGTS, we demonstrated that APOBEC3s and AID induced significantly higher number of translocations in novel hotspots compared to control fibroblasts. Strikingly, bioinformatics analysis of the junctions revealed that APOBEC3 proteins induce chromosomal translocations with similar frequency, but only partially overlap with AID. In addition, by analyzing individual genes or translocation targets, we observed similar but distinct effects on translocation frequency for each member of the APOBEC3 family. Interestingly, AID preferentially targets genomic regions associated with high transcriptional activity, but APOBEC3Cs do not. The analysis of HTGTS from human breast and lung cell lines revealed the similar pattern of translocations found in mouse fibroblasts. Remarkably, APOBEC3 off-target hotspots are distinct and unique in each cell type. Conclusions: We showed that human APOBEC3 enzymes enhance chromosomal translocations in non-hematopoietic cells with similar potency to AID, suggesting that APOBEC3 enzymes could be initiating genomic instability in solid tumors where AID is not expressed. Thus, APOBEC3 expression has to be tightly controlled in non-hematopoietic cells to limit its genotoxic effects. Citation Format: Taek-Chin Cheong, Qi Wang, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2393.

Abstract 1927: Evaluating molecular determinants of elesclomol sensitivity in breast cancer cells: An implication for metastatic properties

Abstract Pre-clinical and clinical studies employing the experimental therapeutic elesclomol have produced contradictory findings. To better understand the mechanisms of response, we sought to identify molecular determinants that may predict elesclomol sensitivity. After stratifying human breast cell lines (n=8) into elesclomol-sensitive (n=4) and elesclomol-insensitive (n=4) groups, we first evaluated published predictors of elesclomol sensitivity, including lactate dehydrogenase (LDH) expression and various genomic aberrations. Though low levels of LDH have been reported to associate with elesclomol response in clinical samples, our data showed no statistically significant difference in LDHA (p=0.5) or LDHB (p=0.3) expression between elesclomol-sensitive and insensitive groups. Mining a human pan-cancer cell line dataset using the Genomics of Drug Sensitivity Database (GDSC) revealed an association between SMARC4A mutation and elesclomol sensitivity (MWW p=0.01) and between FLT3 chromosomal gain and elesclomol resistance (MWW p=0.00005), but the same investigation in a breast cancer cell line dataset produced no statistically significant genomic aberrations in association with elesclomol sensitivity. Indeed, none of our elesclomol-sensitive breast cell lines carried a SMARC4A mutation according to the Cancer Cell Line Encyclopedia, nor did they differentially express FLT3 relative to the elesclomol-insensitive lines as determined by expression profile data (p=1). Therefore, LDH expression and GDSC-defined genomic aberrations may not effectively predict elesclomol sensitivity in breast cancer. We then utilized transcriptomic data for each of our elesclomol-sensitive and insensitive cell lines to identify differentially-expressed genes (DEGs) and their functional annotations. A total of 456 DEGs (LFC>2, p<0.05) associated with elesclomol sensitivity based on an assessment of expression profiles within a single dataset using NCBI GEO2R. Interestingly, the DEGs tended to be enriched for molecules involved in cell adhesion (n=29, adj. p=0.02) according to an analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID). To better understand the relevance of these data, we considered that a loss of cell adhesion precedes invasion/metastasis, and discovered that our elesclomol-sensitive cell lines harbored a high invasive potential (n=2/2), whereas our insensitive lines harbored a low invasive potential (n=4/4). Therefore, an expression profile that lends to aberrant adhesion or high invasion potential may be used to predict sensitivity to elesclomol. Our findings not only introduce a relationship between an important hallmark of cancer, activating invasion and metastasis, and elesclomol sensitivity, but also rationalize future studies that explore elesclomol for the prevention of breast cancer metastasis. Citation Format: Allison Dyevoich, Koran Harris, Lance Miller, Carl D. Langefeld, Elizabeth Alli. Evaluating molecular determinants of elesclomol sensitivity in breast cancer cells: An implication for metastatic properties [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1927.

Comparative studies of two vegetal extracts from Stokesia laevis and Geranium pratense: polyphenol profile, cytotoxic effect and antiproliferative activity

AbstractIn this study, two ethanolic extracts, fromStokesia aster(Slae26) andGeranium pratense(Gpre36) respectively, were evaluated in order to assess the cytotoxic activity and potential antiproliferative activity upon the nontumorigenic human epithelial cell line derived from the mammary gland (MCF-12A) and the human breast tumor cell line (BT-20). The selection of the plant species was done on the basis of their chemical composition, specifically combinations of luteolin derivatives with caffeic and gallic acid derivatives. Therefore, theS. laevisethanolic extract proved its capacity to inhibit the viability of both normal and tumor breast cell lines (i.e., up to 90% cell viability inhibition, IC50= 42 µg/mL). On the contrary, theG. pratenseethanolic extract proved weak stimulatory effects on the viability of the two human breast cell lines studied. The obtained results were discussed in the contexts of computational studies and drug-likeness bioactivity of seven common luteolin derivatives: luteolin, luteolin-7-O-glucoside/cynaroside, luteolin-5-O-glucoside/galuteolin, luteolin-6-C-glucoside/isoorientin, luteolin-8-C-glucoside/orientin, luteolin-3′,4′-di-O-glucoside and luteolin-7,3′-di-O-glucoside. Computational studies have revealed that the hydrophilic behavior of luteolin derivatives (log Pvalues) does not follow other tested parameters (e.g., polar surface area values), possibly explaining different efficacy concerning the biological propertiesin vitro. These predictions could be a starting point for studies on the biochemical mechanism by which luteolin derivatives induce biological effects.

Abstract A47: In silico organoids: A model for deconvolution of microenvironmental and drug effects on tumor growth

Abstract In vivo tumor microenvironments are heterogeneous in their architecture, cellular contents, metabolic landscape, and extracellular matrix (ECM) composition. Moreover, these microenvironments can undergo temporal and spatial changes. This may alter tumor responses to anticancer therapies. To examine the complex and dynamic changes within tumors and in tumor microenvironments, we developed a computational single cell-based model of the three-dimensional multicellular organotypic cultures: Organoid3D. This model was calibrated to experimental data and reproduced growth dynamics and morphologies of four human breast cell lines: a nontumorigenic epithelial MCF-10A, a mildly tumorigenic MCF-10AT1, and metastatic MCF-10CA-1a and MCF-10CA-1d lines grown in various microenvironmental conditions. By explicitly including the fibril structure of the ECM in the model, we examined how ECM properties promote or suppress the growth of in silico tumor organoids. The quantitative integration of experimental data for organoids exposed to different concentrations of doxorubicin allowed us to derive hypotheses on relative importance of microenvironmental factors and chemotherapeutic treatments on organoid growth. Thus, the in silico organoid model interrogated with experimental data provides a tool for fast and broad hypotheses testing and presents an opportunity to explore experimental conditions beyond what is physically feasible in laboratory experiments. Citation Format: Katarzyna A. Rejniak, Sharan Poonja, Jessica Kingsley, Shreya Mathur, Ibrahim Chamseddine, Aleksandra Karolak, Dmitry A. Markov, Lisa J. McCawley. In silico organoids: A model for deconvolution of microenvironmental and drug effects on tumor growth [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A47.

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.

SAT-130 The Intermediate Prolactin Receptor Is a Breast Cancer Oncogene

Epidemiological, cellular, and genetic analyses indicate the hormone prolactin (PRL) and its cognate receptor in humans (hPRLr) are significantly involved in breast cancer pathogenesis. Recent evidence demonstrated that a truncated mouse PRLr (mPRLrT) is oncogenic when expressed with canonical long mPRLr (mPRLrL). mPRLrT shares significant sequence homology with a naturally-occurring and widely-expressed hPRLr isoform, the intermediate hPRLr (hPRLrI). As determined by tissue microarray (TMA), hPRLrI is expressed in >85% of breast cancer, with expression increasing as a function of both tumor grade and Ki67 status. To confirm the oncogenic potential of hPRLrI, isoform-specific hPRLrI knock-down (KD) was performed in breast cancer cell line MCF7. hPRLrI KD resulted in a significant decrease in proliferation, migration, and anchorage-independent growth. Given the homology between mPRLrT and hPRLrI, we hypothesized hPRLrI may similarly induce transformation, when expressed alongside wild-type long hPRLr (hPRLrL). hPRLrL/I co-expression in the immortalized but not transformed human breast cell line MCF10A resulted in a significant increase in proliferation, migration, and anchorage-independent growth. These results were not observed following overexpression of either isoform alone, demonstrating that hPRLrL/I co-expression is necessary to induce transformation of normal mammary epithelia. To test our hypothesis in vivo, we established MCF10A xenografts using female NSG mice. Following intraductal injection, we observed rapid tumor growth in the hPRLrL/I cohort, significantly over that of expressing either isoform alone. To determine mechanisms of transformation, we examined both differential protein stability and altered signaling events. In analyzing receptor degradation, a cycloheximide assay revealed hPRLrL stability is increased when heterodimerized with hPRLrI. hPRLrL turnover is impaired in breast cancer, indicating this phenomenon may be involved in the observed hPRLrI-mediated transformation. Regarding differential signaling, we examined the Jak2/Stat5a pathway. Jak2 is a promiscuous kinase whose oncogenic actions are well-characterized, while Stat5a is a transcription factor whose activities are critical in attenuating the oncogenicity of Jak2. Following PRL stimulation, it was observed that hPRLrL/I co-expression induced approximately two-fold greater Jak2-Y1007/1008 phosphorylation (pJak2) compared to that induced by hPRLrL expression alone. Further, it was observed that hPRLrL/I co-expression induced ten-fold less Stat5a-Y694 phosphorylation (pY-Stat5a) than hPRLrL expression alone. These data indicate unchecked pJak2 activity may also be a contributing mechanism in the observed transformation. Overall, these results demonstrate that hPRLrI, alongside hPRLrL, is sufficient for transformation of normal breast tissue.

Agonist Effects of Propranolol on Non-Tumor Human Breast Cells.

The β-blocker propranolol (PROP) has been proposed as a repurposed treatment for breast cancer. The similarity of action between β-agonists and antagonists found on breast cells encouraged us to compare PROP and isoproterenol (ISO, agonist) signaling pathways on a human breast cell line. Cell proliferation was measured by cell counting and DNA-synthesis. Cell adhesion was measured counting the cells that remained adhered to the plastic after different treatments. Changes in actin cytoskeleton were observed by fluorescence staining and Western Blot. ISO and PROP caused a diminution of cell proliferation and an increase of cell adhesion, reverted by the pure β-antagonist ICI-118551. ISO and PROP induced a reorganization of actin cytoskeleton increasing F-actin, p-COFILIN and p-LIMK. While ISO elicited a marked enhancement of cAMP concentrations and an increase of vasodilator-stimulated phosphoprotein (VASP) and cAMP response element-binding protein (CREB) phosphorylation, PROP did not. Clathrin-mediated endocytosis inhibition or β-arrestin1 dominant-negative mutant abrogated PROP-induced cell adhesion and COFILIN phosphorylation. The fact that PROP has been proposed as an adjuvant drug for breast cancer makes it necessary to determine the specific action of PROP in breast models. These results provide an explanation for the discrepancies observed between experimental results and clinical evidence.

Cantharidin-loaded functional mesoporous titanium peroxide nanoparticles for non-small cell lung cancer targeted chemotherapy combined with high effective photodynamic therapy.

BackgroundAlthough photodynamic therapy (PDT) has emerged as a potential alternative to conventional chemotherapy, the low reactive oxygen species (ROS) yield of the photosensitizer such as TiO2 nanoparticles has limited its application. In addition, it is difficult to achieve effective tumor treatment with a single tumor therapy.MethodsWe used TiOx nanocomposite (YSA‐PEG‐TiOX) instead of TiO2 as a photosensitizer to solve the problem of insufficient ROS generation in PDT. Benefiting from the desired mesoporous structure of TiOx, Cantharidin (CTD), one of the active components of mylabris, is loaded into TiOx for targeted combination of chemotherapy and PDT. The cellular uptake in human non‐small cell lung carcinoma cell line (A549) and human normal breast cell line (MCF 10A) was evaluated by confocal microscopy. in vitro cytotoxicity was evaluated using Cell Counting Kit‐8 assay. The ROS was detected via a chemical probe DCFH‐DA and the photodynamic treatment effect of YSA‐PEG‐TiOx was further evaluated by a living‐dead staining. The cell apoptosis was detected by the flow cytometry.ResultsOur findings showed that the modification of YSA peptide improved the cytotoxicity of YSA‐PEG‐TiOX/CTD to EphA2 overexpressing A549 non‐small cell lung cancer (NSCLC) than non‐YSA modified counterparts. In addition, TiOx generated adequate ROS under X‐ray irradiation to further kill cancer cells. Flow analysis results also proved the superiority of this combined treatment.ConclusionsYSA‐PEG‐TiOX nanoparticles could significantly increase ROS production under X‐ray exposure and provide a new drug delivery nanocarrier for CTD in combination with PDT to achieve effective NSCLC treatment.

Assessment of cellular response to drug/nanoparticles conjugates treatment through FTIR imaging and PLS regression study

This work explores the potential of Fourier transform infrared (FTIR) spectroscopic imaging at the single-cell level combined with partial least squares (PLS) regression to investigate the cell’s response to drug/gold nanoparticles (AuNPs) conjugates. In a broader context, this study provides not only the effective differentiation between non-tumorigenic, mildly malignant and malignant cells, but above all proposes a novel approach to define the sensitivity of cancer cells to drugs. AuNPs can be widely and effectively used in biosensing and bioimaging applications. Three human breast cell lines, i.e. MCF10A (non-tumorigenic), MCF7 (metastatic, pleural effusion, mildly malignant) and MDA-MB-231 (metastatic, pleural effusion, malignant) have been exposed to different concentration of AuNPs (15 mg/L and 10 mg/L) and AuNPs/α-methyl-DL-tryptophan conjugates conjugates (10 mg/L NPs @ 3.75 × 10−6 M and 10 mg/L NPs @ 3.75 × 10−5 M). It is implied that the class values predicted by the PLS regression model indicate a very good separation between non-tumorigenic, mildly malignant and malignant cells. Furthermore, the FTIR-PLS approach shows that bands responsible for the segregation between the control cells and the cells treated with drug/AuNPs conjugate result from the changes in RNA/DNA, lipids and amide I and II vibrations. Drug/AuNPs conjugate affects the nucleic acids in all of the studied cell types. The performed analysis suggests that MCF-7 cell line is particularly sensitive to the drug treatment, which result in the drug interacting with nucleic acids and DNA conformational changes.