MCF-10A Human Mammary Epithelial Cells Research Articles

Abstract A084: DEK overexpression in the murine mammary gland promotes epithelial hyperplasia

Abstract Introduction: Breast cancer (BC) remains a leading cause of cancer-associated mortality for women in the USA, which makes it critically important to identify and study molecular drivers of pathogenesis. We have previously identified the chromatin modifying DEK protein to have oncogenic properties. DEK is a histone H3 chaperone that is over-expressed in over 60% of breast cancers, particularly triple negative breast cancers, and is associated with poor survival. To date, all studies regarding the role of DEK in BC have manipulated DEK expression in cultured immortalized MCF10A human mammary epithelial cells and BC cell lines. However, the oncogenic functions of DEK in vivo and in normal mammary epithelium have remained unexplored. Methods: We utilized a tetracycline-off regulated, DEK overexpressing (DEK-OE) transgenic mouse model that targets mammary epithelium through the mouse mammary tumor virus (MMTV) promoter. We subsequently performed bulk RNA-Seq transcriptomic analyses, western blotting, and both histological and morphological assessments of control vs. DEK-OE mammary gland tissue to identify the oncogenic effects of DEK over-expression in vivo. Results: Bulk RNA sequencing and gene ontology analyses identified cell cycle regulation as a significantly upregulated biological process in DEK-OE versus control glands. Subsequent studies confirmed upregulation of proteins that promote the G1-S cell cycle transition including cyclins A and D and CDKs 2, 4, and 6 as well as increased numbers of Ki67+ cells. We also observed down-regulation of genes that inhibit entry the G1-S transition, such as p21 and p27. This pro-proliferative molecular status correlated with the development of mammary gland hyperplasia. Importantly, in silico analyses demonstrated gene correlations between DEK and cell cycle regulators that were also evident in human breast cancers. One potential mechanism for this transcriptional deregulation is that we identified increased histone H3K27me3 epigenetic modifications in DEK-OE cells, in both mouse and human models, which may occur through EZH2 activity. Conclusions: Combined, our work indicates that DEK is not a tumor initiator, but its oncogenic activities are likely due to its ability to promote proliferation of mammary epithelial cells. Furthermore, we report a novel function for DEK in the epigenetic regulation of histone H3 K27 trimethylation, which is mediated by the well-known EZH2 oncogene in triple negative breast cancers. Citation Format: Megan Johnstone, Ashley Leck, Taylor Lange, Lisa Privette Vinnedge. DEK overexpression in the murine mammary gland promotes epithelial hyperplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A084.

Synergistic Suppression of NF1 Malignant Peripheral Nerve Sheath Tumor Cell Growth in Culture and Orthotopic Xenografts by Combinational Treatment with Statin and Prodrug Farnesyltransferase Inhibitor PAMAM G4 Dendrimers.

Neurofibromatosis type 1 (NF1) is a disorder in which RAS is constitutively activated due to the loss of the Ras-GTPase-activating activity of neurofibromin. RAS must be prenylated (i.e., farnesylated or geranylgeranylated) to traffic and function properly. Previous studies showed that the anti-growth properties of farnesyl monophosphate prodrug farnesyltransferase inhibitors (FTIs) on human NF1 malignant peripheral nerve sheath tumor (MPNST) cells are potentiated by co-treatment with lovastatin. Unfortunately, such prodrug FTIs have poor aqueous solubility. In this study, we synthesized a series of prodrug FTI polyamidoamine generation 4 (PAMAM G4) dendrimers that compete with farnesyl pyrophosphate for farnesyltransferase (Ftase) and assessed their effects on human NF1 MPNST S462TY cells. The prodrug 3-tert-butylfarnesyl monophosphate FTI-dendrimer (i.e., IG 2) exhibited improved aqueous solubility. Concentrations of IG 2 and lovastatin (as low as 0.1 μM) having little to no effect when used singularly synergistically suppressed cell proliferation, colony formation, and induced N-RAS, RAP1A, and RAB5A deprenylation when used in combination. Combinational treatment had no additive or synergistic effects on the proliferation/viability of immortalized normal rat Schwann cells, primary rat hepatocytes, or normal human mammary epithelial MCF10A cells. Combinational, but not singular, in vivo treatment markedly suppressed the growth of S462TY xenografts established in the sciatic nerves of immune-deficient mice. Hence, prodrug farnesyl monophosphate FTIs can be rendered water-soluble by conjugation to PAMAM G4 dendrimers and exhibit potent anti-tumor activity when combined with clinically achievable statin concentrations.

Abstract 4879: Inhibition of amino acid transporters as a novel mechanism of action of the repurposed anti-cancer agent tranexamic acid

Abstract Tranexamic Acid (TA) is an anti-fibrinolytic agent that inhibits Plasminogen activation and is used to control bleeding. Past studies have suggested that the ability of TA to block Plasminogen activation may be useful for ablating the growth or invasion of cancer. Based on the similarity of TA to the amino acid Lysine we hypothesize that TA might exhibit novel mechanisms of action independent of blocking Plasminogen activation. Analysis of a series of signaling pathways indicated that TA inhibits phosphorylation of S6K1 and STAT3 on sites required for their activation and reduces expression of the MYC oncogene in a concentration-dependent manner. Interestingly, MYC transformed MCF10A human mammary epithelial cells exhibited enhanced sensitivity to TA-mediated reduction of cell viability, suggesting that TA may exhibit selectivity for MYC overexpressing breast cancers. Treatment of breast tumor bearing mice with TA strongly blocked tumor growth and was associated with extensive cancer cell death compared with control tumors. Analysis of extracts from TA or vehicle treated tumors showed that TA blocked S6K1 phosphorylation in vivo in a statistically significant manner. Since S6K1 phosphorylation is regulated by amino acid levels, we hypothesized that due to the structural similarity between TA and Lysine and Arginine, TA may block Lysine or Arginine uptake by blocking cationic amino acid transporters (CATs). Molecular docking simulations utilizing the structure of the bacterial CAT predicted that TA binds to CAT with affinity similar to that of Lysine. Amino acid uptake experiments performed with radiolabeled Arginine and Lysine showed that TA blocked both Arginine and Lysine absorption by cancer cells in a concentration-dependent manner. Together, these results indicate a novel mechanism by which TA inhibits uptake of Arginine and Lysine, triggering loss of S6K1 activity in parallel with tumor growth inhibition. Initial studies suggest the feasibility of designing TA analogs with significantly improved potency for amino acid transporters, and with corresponding increases in selective toxicity to breast cancer cells. Based on these novel mechanisms of TA action, we expect TA to exhibit anti-cancer activity against a broad range of human malignancies. Citation Format: Brian K. Law, Mary E. Law, Amanda F. Ghilardi, Elham Yaaghubi, Brad J. Davis, Zaafir M. Dulloo, Mengxiong Wang, Olga A. Guryanova, Coy D. Heldermon, Stephan C. Jahn, Ronald K. Castellano. Inhibition of amino acid transporters as a novel mechanism of action of the repurposed anti-cancer agent tranexamic acid. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4879.

Epigallocatechin-3-gallate Delivered in Nanoparticles Increases Cytotoxicity in Three Breast Carcinoma Cell Lines.

The anticancer activity of epigallocatechin-3-gallate (EGCG), orally administrated, is limited by poor bioavailability, absorption, and unpredictable distribution in human tissues. EGCG charged nanoparticles may represent an opportunity to overcome these limitations. We assayed two different kinds of lipid nanoparticles (LNPs and LNPs functionalized with folic acid) charged with EGCG on three breast carcinoma cell lines (MCF-7, MDA-MB-231, and MCF-7TAM) and the human normal MCF10A mammary epithelial cells. Both LNPs loaded with EGCG, at low concentrations, induced a significant cytotoxicity in the three breast carcinoma cells but not in MCF10A cells. In view of a future application, both LNPs and LNPs-FA were found to be very suitable for in vitro studies and useful to improve EGCG administration in vivo. Since they are produced by inexpensive procedures using bioavailable, biocompatible, and biodegradable molecules, they represent an applicable tool for a more rationale use of EGCG as an anti-cancer agent.

C/EBP\u03b2 isoform-specific regulation of migration and invasion in triple-negative breast cancer cells

The transcription factor C/EBPβ is a master regulator of mammary gland development and tissue remodelling during lactation. The CEBPB-mRNA is translated into three distinct protein isoforms named C/EBPβ-LAP1, -LAP2 and -LIP that are functionally different. The smaller isoform LIP lacks the N-terminal transactivation domains and is considered to act as an inhibitor of the transactivating LAP1/2 isoforms by competitive binding for the same DNA recognition sequences. Aberrantly high expression of LIP is associated with mammary epithelial proliferation and is found in grade III, estrogen receptor (ER) and progesterone (PR) receptor-negative human breast cancer. Here, we show that reverting the high LIP/LAP ratios in triple-negative breast cancer (TNBC) cell lines into low LIP/LAP ratios by overexpression of LAP reduces migration and matrix invasion of these TNBC cells. In addition, in untransformed MCF10A human mammary epithelial cells overexpression of LIP stimulates migration. Knockout of CEBPB in TNBC cells where LIP expression prevails, resulted in strongly reduced migration that was accompanied by a downregulation of genes involved in cell migration, extracellular matrix production and cytoskeletal remodelling, many of which are epithelial to mesenchymal transition (EMT) marker genes. Together, this study suggests that the LIP/LAP ratio is involved in regulating breast cancer cell migration and invasion. This study together with studies from others shows that understanding the functions the C/EBPβ-isoforms in breast cancer development may reveal new avenues of treatment.

Replacement of hydrochloride in metformin hydrochloride with caprylic acid to investigate its effects on MCF-7 and MDA-MB-231 breast cancer cell lines

AimsMetformin hydrochloride is a highly hydrophilic molecule with low permeability. In the present study, to develop an effective drug to treat the metastatic breast cancer, metformin caprylic acid was synthesized using metformin hydrochloride as a permeable agent. Main methodsThe cytotoxic effects of various concentrations of metformin caprylic acid and metformin hydrochloride (0 to 20 mM) on MCF-7 and MDA-MB-231 breast cancer cells and MCF-10A human mammary epithelial cell line were assessed by the MTT assay. Furthermore, Annexin V, PI staining, and cell flow cytometry assays were performed to evaluate the apoptotic effects. The invasion and migration ability of these cells were evaluated following treatment with equal concentration (3 mM) of the two compounds. Key findingsThe treatment of tested cell lines with an equal concentration of two chemicals decreased cell viability in a time and dose-dependent manner, where in all cases, metformin caprylic acid caused significantly more apoptosis and invasion inhibition than that of metformin hydrochloride (*p < 0.05). Chemical structures of both compounds were confirmed by FTIR and 1H NMR, 13C NMR. Both chemicals inhibited the migration of MCF-7 and MCF-10A cells, but had no effect on MDA-MB-231 migration. All data are expressed as mean ± SD (n = 3). SignificanceIt seems that in an equal concentration, the similarity of the hydrophobic tail of caprylic acid to the cell membrane improves its entrance, while decreasing the drug excretion.

Cold atmospheric plasma for preventing infection of viruses that use ACE2 for entry.

Rational: The mutating SARS-CoV-2 potentially impairs the efficacy of current vaccines or antibody-based treatments. Broad-spectrum and rapid anti-virus methods feasible for regular epidemic prevention against COVID-19 or alike are urgently called for.Methods: Using SARS-CoV-2 virus and bioengineered pseudoviruses carrying ACE2-binding spike protein domains, we examined the efficacy of cold atmospheric plasma (CAP) on virus entry prevention.Results: We found that CAP could effectively inhibit the entry of virus into cells. Direct CAP or CAP-activated medium (PAM) triggered rapid internalization and nuclear translocation of the virus receptor, ACE2, which began to return after 5 hours and was fully recovered by 12 hours. This was seen in vitro with both VERO-E6 cells and human mammary epithelial MCF10A cells, and in vivo. Hydroxyl radical (·OH) and species derived from its interactions with other species were found to be the most effective CAP components for triggering ACE2 nucleus translocation. The ERα/STAT3(Tyr705) and EGFR(Tyr1068/1086)/STAT3(Tyr705) axes were found to interact and collectively mediate the effects on ACE2 localization and expression.Conclusions: Our data support the use of PAM in helping control SARS-CoV-2 if developed into products for nose/mouth spray; an approach extendable to other viruses utilizing ACE2 for host entry.

(E)-2-Styrylanthracene-9,10-dione: A new type of fluorescent probe core and its application in specific mitochondria imaging

Herein, a new type of fluorescent probe core, (E)-2-styrylanthracene-9,10-dione (EK01), was developed which displayed strong fluorescence quantum yield (Φ = 0.867 in DMF; Φ = 0.561 in acetone; Φ = 0.616 in CH2Cl2; Φ = 0.265 in DMSO), good photostability, large stokes shift (90 nm–120 nm) and molar extinction coefficients (0.5875 × 104–0.7609 × 104 mol−1 L cm−1). During cell assays and co-localization experiments, EK01 showed excellent cell membrane permeability and low cytotoxicity against MCF-10A (human mammary epithelial cell line) and HT-29 (human colorectal adenocarcinoma cell line). Particularly, we surprisingly discovered that EK01 could selectively aggregate in mitochondria and specific stain it in a green emissive fluorescent form, which means that EK01 could be a real-time specifically monitor of mitochondria in living cells with a high signal-to-noise ratio. Hence a new mitochondria imaging method was established which is incubating EK01 with living cells for 1 h at a final concentration of 6–12 μM, then visualizing under a confocal microscope at 395 nm. It is worth noting that the fluorescence efficiency of EK01 is not outstanding in organisms, it has much stronger fluorescence efficiency in other organic solvent systems (such as DMF, acetone and CH2Cl2). Therefore, as a new type of fluorescent core that is easy to synthesis and graft, we believe that (E)-2-styrylanthracene-9,10-diones have the potential to develop a variety of fluorescence platforms applying in different fields.

Further lead optimization on Bax activators: Design, synthesis and pharmacological evaluation of 2-fluoro-fluorene derivatives for the treatment of breast cancer.

To further pursue potent Bax activators with better safety profiles for the treatment of breast cancer, structural optimization was conducted based on lead compound CYD-4-61 through several strategies, including scaffold hopping on the 2-nitro-fluorene ring, replacement of the nitro group with bioisosteres to avoid potential toxicity, and further optimization on the upper pyridine by exploring diverse alkylamine linkers as a tail or replacing the pyridine with bioisosteric heterocycles. F-containing compound 22d (GL0388) exhibited a good balance between the activity and toxicity, displaying submicromolar activities against a variety of cancer cell lines with 5.8-10.7-fold selectivity of decreased activity to MCF-10A human mammary epithelial cell line. Compound 22d dose-dependently blocked colony formation of breast cancer cells and prevented the migration and invasion of MDA-MB-231cells. Mechanism of action studies indicate that 22d activated Bax, rendering its insertion into mitochondrial membrane, thereby leading to cytochrome c release from the mitochondria into the cytoplasm, subsequently inducing release of apoptotic biomarkers. Further invivo efficacy studies of 22d in human breast cancer xenografts arisen from MDA-MB-231cells demonstrated that this drug candidate significantly suppressed tumor growth, indicating the therapeutic promise of this class of compounds for the treatment of breast cancer as well as the potential for developing F-radiolabeled imaging ligands as anticancer chemical probes.

Searching for Small Molecules as Antibacterials: Non-Cytotoxic Diarylureas Analogues of Triclocarban.

Triclocarban (TCC), a broad-spectrum lipophilic antimicrobial agent, is a diarylurea derivative that has been used for more than 60 years as a major ingredient of toys, clothing, food packaging materials, food industry floors, medical supplies and especially of personal care products, such as soaps, toothpaste and shampoo. In September 2016, the U.S. FDA banned nineteen antimicrobial ingredients, including TCC, in over-the-counter consumer antiseptic wash products, due to their toxicity. Withdrawal of TCC has prompted efforts to search for new antimicrobial compounds. In this paper, we present the synthesis and biological evaluation, as antibiotic and non-cytotoxic agents, of a series of diarylureas, analogues of TCC. These compounds are characterized by an intriguingly simple chemistry and can be easily synthesized. Among the synthesized compounds, 1ab and 1bc emerge as the most interesting compounds as they show the same activity of TCC (MIC = 16 µg/mL) against Staphylococcus aureus, and a higher activity than TCC against Enterococcus faecalis (MIC = 32 µg/mL versus MIC = 64 µg/mL). Moreover, 1ab and 1bc show no cytotoxicity towards the human mammary epithelial cells MCF-10A and embryonic kidney epithelial cells Hek-293, in opposition to TCC, which exhibits a marked cytotoxicity on the same cell lines and shows a good antitumor activity on a panel of cell lines tested.

Oxocrebanine: A Novel Dual Topoisomerase inhibitor, Suppressed the Proliferation of Breast Cancer Cells MCF-7 by Inducing DNA Damage and Mitotic Arrest

BackgroundDNA topoisomerase (Topo) inhibition plays key role in breast cancer treatment. Stephania hainanensis H. S. Lo et Y. Tsoong (S. hainanensis), a Li nationality plant that has abundant aporphine alkaloids, can inhibit Topo. PurposeTo identify a dual Topo inhibitor, a deep and systematic study of active aporphine alkaloids in S. hainanensis and their mechanisms of inhibiting breast cancer proliferation and Topo activity are essential. Study designThis study aimed to assess the anti-breast cancer and Topo inhibitory activities of oxocrebanine and explore the underlying mechanisms. MethodsThe growth inhibitory activities of 12 compounds in S. hainanensis were screened by MTT assay in MCF-7, SGC-7901, HepG-2 cells, and compared with the effects on human normal mammary epithelial MCF-10A cells as non cancer control cells. The Topo inhibitory activity was assessed by DNA relaxation and unwinding assays, kDNA decatenation assay and western blot. Cell cycle and autophagy analyses were carried out with flow cytometry and staining. Acridine orange staining and α-tubulin morphology were observed by fluorescence microscopy. Western blot was used to examine microtubule assembly dynamics and the expression levels of key proteins associated with DNA damage, autophagy and mitotic arrest. ResultsOxocrebanine was the anti-breast cancer active alkaloid in S. hainanensis. It exhibited the best inhibitory effect on MCF-7 cells with an IC50 of 16.66 μmol/l, and had only weak effect on the proliferation of MCF-10A cells. Oxocrebanine inhibited Topo I and II α in a cell-free system and in MCF-7 cells. The DNA unwinding assay suggested that oxocrebanine intercalated with DNA as a catalytic inhibitor. Oxocrebanine regulated the levels of Topo I and IIα and DNA damage-related proteins. Oxocrebanine led to the mitotic arrest, and these effects occurred through both p53-dependent and p53-independent pathways. Oxocrebanine induced autophagy, abnormal α-tubulin morphology and stimulated enhanced microtubule dynamics. ConclusionOxocrebanine was the anti-breast cancer active aporphine alkaloid in S. hainanensis. Oxocrebanine was a Topo I/IIα dual inhibitor, catalytic inhibitor and DNA intercalator. Oxocrebanine caused DNA damage, autophagy, and mitotic arrest in MCF-7 cells. Oxocrebanine also disrupted tubulin polymerization. Accordingly, oxocrebanine held a great potential for development as a novel dual Topo inhibitor for effective breast cancer treatment.

Induction of Tumor-Selective Cytotoxicity by Myrtus communis L. Extract and its Bioactive Metabolite 1, 8-cineole

Breast cancer is the most common type of cancer in women. Despite current treatments, new treatments should be investigated. The antioxidant and apoptotic activities of plant metabolites have attracted increasing interest in the molecular pharmacology. Here, we investigated the anti-proliferative and apoptotic potential of Myrtus communis L. extracts, used in traditional medicine. To monitor cancer-selective activities, the extract and its most prominent components were assayed in parallel with human MCF7 breast cancer and normal MCF10A mammary epithelial cells. Indeed, we observed a potential therapeutic window of concentrations with significantly higher toxicity for the tumor cells. GC-MS confirmed the presence of α-pinene, 1,8-cineole, linalool as major components. Analyzing these pure terpenoids revealed exclusive toxicity of 1,8-cineole for MCF7 cells, whereas no toxicity was seen for MCF10A cells. In contrast, both α-pinene and linalool did not exhibit tumor selectivity but were toxic for the tumor and normal cells. These data suggest that the anti-proliferative activity of the oil may be mediated by 1,8-cineole with the other components counteracting this potential therapeutic activity, which apperantely acted preferentially on epithelial cells. To gain mechanistic insight, we tested by RT-qPCR whether the essential oil affected apoptosis mediators. Indeed, BCL2 expression was dramatically down-regulated in MCF7 while it was up regulated in MCF10A. Minor differences were observed for BAX, while APAF1 expression was up-regulated in MCF7 and down-regulated in MCF10A. Our data suggest that in M. communis extracts, 1,8-cineole mediates tumor-selective apoptosis by deregulating BCL2 and APAF1.

Photobleaching Imprinting Enhanced Background Rejection in Line-Scanning Temporal Focusing Microscopy.

Compared with two-photon point-scanning microscopy, two-photon temporal focusing microscopy (2pTFM) provides a parallel high-speed imaging strategy with optical sectioning capability. Owing to out-of-focus fluorescence induced by scattering, 2pTFM suffers deteriorated signal-to-background ratio (SBR) for deep imaging in turbid tissue, Here, we utilized the photobleaching property of fluorophore to eliminate out-of-focus fluorescence. According to different decay rates in different focal depth, we extract the in-focus signals out of backgrounds through time-lapse images. We analyzed the theoretical foundations of photobleaching imprinting of the line-scanning temporal focusing microscopy, simulated implementation for background rejection, and demonstrated the contrast enhancement in MCF-10A human mammary epithelial cells and cleared Thy1-YFP mouse brains. More than 50% of total background light rejection was achieved, providing higher SBR images of the MCF-10A samples and mouse brains. The photobleaching imprinting method can be easily adapted to other fluorescence dyes or proteins, which may have application in studies involving relatively large and nontransparent organisms.

An Electrophilic Deguelin Analogue Inhibits STAT3 Signaling in H-Ras-Transformed Human Mammary Epithelial Cells: The Cysteine 259 Residue as a Potential Target.

Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signals that are often constitutively activated in many cancerous or transformed cells and some stromal cells in the tumor microenvironment. Persistent STAT3 activation in malignant cells stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation. STAT3 undergoes activation through phosphorylation on tyrosine 705, which facilitates its dimerization. Dimeric STAT3 translocates to the nucleus, where it regulates the transcription of genes involved in cell proliferation, survival, etc. In the present study, a synthetic deguelin analogue SH48, discovered by virtual screening, inhibited the phosphorylation, nuclear translocation, and transcriptional activity of STAT3 in H-ras transformed human mammary epithelial MCF-10A cells (MCF10A-ras). We speculated that SH48 bearing an α,β-unsaturated carbonyl group could interact with a thiol residue of STAT3, thereby inactivating this transcription factor. Non-electrophilic analogues of SH48 failed to inhibit STAT3 activation, lending support to the above supposition. By utilizing a biotinylated SH48, we were able to demonstrate the complex formation between SH48 and STAT3. SH48 treatment to MCF10A-ras cells induced autophagy, which was verified by staining with a fluorescent acidotropic probe, LysoTracker Red, as well as upregulating the expression of LC3II and p62. In conclusion, the electrophilic analogue of deguelin interacts with STAT3 and inhibits its activation in MCF10A-ras cells, which may account for its induction of autophagic death.

Design, synthesis and biological evaluation of novel heptamethine cyanine dye-erlotinib conjugates as antitumor agents

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) has been proved as a target for the treatment of non-small cell lung cancer (NSCLC) with specific gene mutations. However, EGFR-TK inhibitors (EGFR-TKIs) need to enter cancer cells and then competitively interact with the active site of tyrosine kinase receptors to suppress the downstream signaling pathway to inhibit tumor proliferation. In this study, in order to improve the tumor cell targeting ability of EGFR-TKI, EGFR-TKI erlotinib was conjugated with the cancer cell-targeting heptamethine cyanine dyes to form seventeen novel erlotinib-dye conjugates. The efficiency of tumor targeting properties of conjugates against cancer cell growth and EGFR-TK inhibition was evaluated in vitro. The result revealed that most erlotinib-dye conjugates exhibited stronger inhibitory effect on A549, H460, H1299 and MDA-MB-231 cell lines than the parent drug erlotinib. Meanwhile, representative compounds exhibited weak cytotoxicity on human normal mammary epithelial MCF-10A cells. Moreover, the conjugate CE17 also showed ~14-fold higher EGFR-TK inhibition activity (IC50 = 0.124 μM) than erlotinib (IC50 = 5.182 μM) in A549 cell line. Finally, molecular docking analysis verified that the erlotinib moiety of compound CE17 could form hydrogen bond with Met-769 and occupy active cavity of EGFR-TK. Therefore, we believed the integration strategy between heptamethine cyanine dyes and EGFR-TKI will contribute to enhancing the therapeutic effect of EGFR-TKI for NSCLC treatment.

Reconstruction of angle-resolved backscattering through a multimode fiber for cell nuclei and particle size determination

We demonstrate reconstruction of angle-resolved optical backscattering after transmission through a multimode fiber. Angle-resolved backscattering is an important tool for particle sizing, and has been developed as a diagnostic modality for detecting epithelial precancer. In this work, we fully characterized the transfer function of a multimode fiber using a plane-wave illumination basis across two dimensions. Once characterized, angle-resolved scattering information which has been scrambled by multimodal propagation can be easily and accurately reconstructed. Our technique was validated using a Mie theory-based inverse light scattering analysis (ILSA) algorithm on polystyrene microsphere phantoms of known sizes. To demonstrate the clinical potential of this approach, nuclear morphology was determined from the reconstructed angular backscattering from MCF-10A human mammary epithelial cell samples and validated against quantitative image analysis (QIA) of fluorescence microscopy images.

Abstract P1-03-11: In vivo rapid discovery of tumor suppressors in breast cancer

Abstract In breast cancer, genome sequencing of patient have identified hundreds of genetic mutations associated with cancer, but which of these mutations drive tumor formation and which are simply bystanders is largely unknown. This is especially true for mutations which individually occur in a small percentage of patients, but when considered together these less common mutations make a significant proportion of the mutational landscape of breast cancer. To functionally test these genes, we performed a CRISPR screen in vivo to identify which of these genes function as tumor suppressors in a mouse model of breast cancer. To do this, we created a library of sgRNAs targeting 215 putative tumor suppressor genes that are found to be mutated in human patients but had not been previously validated as tumor suppressors. We delivered this library via lentivirus to the adult mammary glands of tumor susceptible mice so each lentiviral infected cell has a knockout of one of the genes in the library as well as an oncogenic PIK3CA mutation. Importantly, each infected cell is surrounded by unmodified normal cells, mimicking the initiation of human breast tumors. As expected, mice injected with the putative tumor suppressor library formed tumor significantly faster and reached endpoint sooner than control library injected mice. Tumors from tumor suppressor library injected mice were deep sequenced which revealed known tumor suppressors such as APC and NF1, as well as an enrichment in multiple epigenetic regulators previously unidentified as tumor suppressors in breast cancer. Further validation using independent individual sgRNAs targeting each of the 4 most commonly targeted epigenetic regulators confirmed their role as tumor suppressors. To test the role of these genes in a human context, we used CRISPR to knockout these epigenetic regulators in normal human mammary epithelial MCF10A cells carrying the same PIK3CA mutation. MCF10A lines with epigenetic regulator knockout formed abnormal and protrusive spheres when seeded in matrigel and were capable of forming tumors when engrafted into immunocompromised mice unlike the parent MCF10A line, indicative of transformation into cancerous cells. To identify the mechanism of action of these epigenetic regulators as tumor suppressors, RNA-seq and ATAC-seq were performed on tumors with knockout of epigenetic genes. When compared to control tumors without epigenetic knockout, pathway analysis revealed a role for these epigenetic regulators in EMT, differentiation and cell metabolism. Analysis of ATAC-seq data is currently underway to identify enhancers associated with these transcriptional changes. Additionally, primary cell lines were isolated from knockout tumors and used to perform a drug screen to find drug sensitivities specific to these regulators. This screen identified several compound classes, including CBP/p300 inhibitors which are more effective at killing epigenetic knockout tumor cell lines than control tumor cell lines or normal mouse mammary epithelial (NMUMG) cells. Overall, these data are the first to identify several epigenetic regulators as tumor suppressors in breast cancer and to test them in a parallel and syngeneic background to establish their mechanism and drug susceptibility. By studying these genes, we seek to not only find treatments that target tumors in which these genes are mutated but also to understand the greater role of epigenetic regulators in tumor suppression. Citation Format: Ellen R Langille, Sampath Loganathan, Daniel Schramek. In vivo rapid discovery of tumor suppressors in breast cancer [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 P1-03-11.

PHLIP(Var7)-P1AP suppresses tumor cell proliferation in MDA-MB-231 triple-negative breast cancer by targeting protease activated receptor 1

PurposeProtease-activated receptor 1 (PAR1) is a signaling protein ubiquitously present on the surface of tumor cells, and its homologous protein fragment, PAR1-activating peptide (P1AP), can inhibit protein signal transduction of PAR1/G in tumor cells. pH (Low) insertion peptide (pHLIP) can target the acidic tumor microenvironment (TME) and can be used as an excellent carrier to deliver P1AP to tumor cells for therapeutic purposes.MethodsPAR1 expression on the surface of MDA-MB-231 cells and human MCF10A mammary epithelial cells was observed. The binding between fluorescent-labeled pHLIP(Var7)-P1AP and MDA-MB-231 cells under different pH values was analyzed. The effect of pHLIP(Var7)-P1AP on the proliferation of MDA-MB-231 cells was analyzed under the conditions of pH 7.4 and 6.0.ResultsPAR1 was highly expressed on the surface of MDA-MB-231 cells. In an acidic environment (pH 6.0 and 5.0), fluorescent-labeled pHLIP(Var7)-P1AP and MDA-MB-231 cells had a high binding ability, and the binding ability increased with the decrease in pH. In an acidic environment (pH 6.0), pHLIP(Var7)-P1AP significantly inhibited MDA-MB-231 cell proliferation. With 0.5 μg, 1 μg, 2 μg, 4 μg, and 8 μg of pHLIP(Var7)-P1AP, the cell proliferation inhibition rates were 3.39%, 5.27%, 14.29%, 22.14%, and 35.69%, respectively.ConclusionPAR1 was highly expressed on the surface of MDA-MB-231 cells. pHLIP(Var7)-P1AP can effectively target MDA-MB-231 cells in an acidic environment and inhibit the growth of MDA-MB-231 cells by inhibiting the signal transduction of PAR1/G protein.

Caveolin-1 was involved in the reduced adiosensitivity to X-ray in human mammary epithelial MCF10A cells

Caveolin-1 was involved in the reduced adiosensitivity to X-ray in human mammary epithelial MCF10A cells

Characterization and Cytotoxic Activity of &amp;lt;i&amp;gt;Dalbergia latifolia&amp;lt;/i&amp;gt; Wood Extract

<i>Dalbergia latifolia</i> commonly known as the Indian rosewood, commonly found in association with Teak trees population in and around Western Ghat areas of Tamil Nadu and Kerala in India. From the heartwood of roots and trunk many phytochemical contents were investigated. A wide spectrum of flavanoids namely, flavones, isoflavones, neoflavones and chalcones have been obtained. Specifically, one of the flavonoids compound chalcone compound-Isoliquiritigenin (ILTG, 2',4',4'-trihydroxychalcone) shows various pharmacological properties, including cytotoxic activities. This study revealed the cytotoxic effect of Isoliquiritigenin, efficiently controls the invasive capacity of breast cancer cells MCF10A human mammary epithelial cell line <i>through</i> uplifting G1-phase cell cycle, even arrest at low concentration, also accelerate the extracellular signal regulated kinase signaling pathway to topup the proteins combined with apoptosis and arrest cancer. The cytotoxicity <i>invitro</i> of isoliquitrigenin against MCF10A ATCC cells was investigated by MTT assay. Apoptosis was evaluated by microscopy and flow cytometry. Isoliquiritigenin exhibited the most potent anticancer activity against MCF 10A ATCC with an IC 50 of 10.00 µg/ml. The methanolic extract expressed cytotoxic effect with an IC50 value of 20 µg/ml. The compounds were also tested for their toxicity on normal human cell lines-LO2 and were found to be nontoxic. Administering the wood extract approaches may help reduce side effects in patients under conventional chemotherapy. These potential molecule may exerts their co-active effects through various pathways and helpful for the development of novel drugs; also conservation of these important germplasm is mandatory.