Positive Human Breast Cancer Cell Research Articles

Phloretin Inhibits the Proliferation of Breast Cancer Cells Through the Down-regulation of Estrogen Receptor α.

Phloretin is a natural flavonoid compound found in some plants, such as apples and pears, as well as in the bark of apple trees. Phloretin has been shown to have inhibitory effects on glucose transporters in cells and can potentially inhibit the growth of cancer cells. However, the mechanism by which phloretin regulates the expression of estrogen receptor alpha (ERα), a key transcription factor in breast cancer, is still unclear. This study investigated how phloretin affects the growth of ERα positive human breast cancer cells. The growth of breast cancer cell lines, including MCF7 and T47D, was examined using cell proliferation and colony formation assays. Western blotting and semi-quantitative RT-PCR were used to examine protein and mRNA levels, respectively. Localization of cellular proteins was analyzed using subcellular fractionation. Transient transfection and reported gene assays were used to elucidate the impact of phloretin on cell proliferation and ERα transactivation. Phloretin decreased ERα expression at the mRNA and protein levels in MCF7 and T47D cells. It also inhibited the binding of ERα to the estrogen response element present in the promoter of target genes. Moreover, treatment with phloretin inhibited the expression of cyclin D1 and breast cancer marker gene pS2, which are known ERα target genes. Consequently, it inhibited the growth of ERα-positive human breast cancer cells. Furthermore, inhibition of breast cancer growth by phloretin was found to be mediated through both the ERα and ERK1/ERK2 pathways. Phloretin, a dihydrochalcone extracted from natural sources, exhibits the ability to regulate ERα function and suppress breast cancer cell proliferation.

Synthesis and In Vitro Comparison of DOTA, NODAGA and 15-5 Macrocycles as Chelators for the 64Cu-Labelling of Immunoconjugates

The development of 64Cu-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators (BFCs) that contain functional groups allowing both convenient bioconjugation and stable copper complexes to limit in vivo bioreduction, transmetallation and/or transchelation. The excellent in vivo kinetic inertness of the pentaazamacrocyclic [64Cu]Cu-15-5 complex prompted us to investigate its potential for the 64Cu-labelling of monoclonal antibodies (mAbs), compared with the well-known NODAGA and DOTA chelators. To this end, three NODAGA, DOTA and 15-5-derived BFCs, containing a pendant azadibenzocyclooctyne moiety, were synthesised and a robust methodology was determined to form covalent bonds between them and azide-functionalised trastuzumab, an anti-HER2 mAb, using strain-promoted azide-alkyne cycloaddition. Unlike the DOTA derivative, the NODAGA- and 15-5-mAb conjugates were radiolabelled with 64Cu, obtaining excellent radiochemical yields, under mild conditions. Although all the radioimmunoconjugates showed excellent stability in PBS or mouse serum, [64Cu]Cu-15-5- and [64Cu]Cu-NODAGA-trastuzumab presented higher resistance to transchelation when challenged by EDTA. Finally, the immunoreactive fraction of the radioimmunoconjugates (88-94%) was determined in HER-2 positive BT474 human breast cancer cells, confirming that the bioconjugation and radiolabelling processes implemented had no significant impact on antigen recognition.

Advantages and Disadvantages of Two <i>In Vitro</i> Assays in Evaluating Aromatase Activity: “A Cell-Based and a Cell-Free Assay”

Aromatase is an enzyme that catalyzes the conversion of androgens to estrogens. While inhibition of aromatase is a useful approach for treating breast cancer, it may also have toxicological consequences due to its endocrine disrupting/modulating effect. In this study, sensitivity and performance of two in vitro assays -a cell free and a cell-based- for evaluating aromatase activity were investigated by testing known aromatase inhibitors and partial validation of the methods was performed. Advantages and disadvantages of these methods are also discussed. Aromatase activity was evaluated via two in vitro models; direct measurement with a cell-free assay using a fluorescent substrate and recombinant human enzyme and indirect evaluation with a cell-based assay where cell proliferation was determined in estrogen receptor positive human breast cancer cells (MCF-7 BUS) in the absence of estrogen and the presence of testosterone. In the cell-free direct measurement assay, reference compounds ketoconazole and aminoglutethimide have been shown to inhibit the aromatase enzyme with half-maximal inhibitory concentration (IC50) values concordant with literature. In cell-based indirect measurement assay, only ketoconazole dose-dependently inhibited cell proliferation with 3.47 x 10-7 M IC50. Inter-assay and intra-assay reproducibility of both methods was found to be within acceptable deviation levels. Both methods can be successfully applied. However, to evaluate the potential aromatase activity of the novel compounds in vitro, it seems better to perform both the cell-based and the cell-free assays that allows low-moderate biotransformation and eliminate cytotoxicity potential, respectively.

Chemo-Photothermal Combination Therapy of HER-2 Overexpressing Breast Cancer Cells with Dual-Ordered Mesoporous Carbon@Silica Nanocomposite.

In cancer treatment, the complexity of tumors seriously affects the therapeutic potential of the treatment. Treatments with combination therapy result in more potent effects than monotherapy or their theoretical combination in cancer treatment. Photothermal therapy (PTT) includes applying phototherapeutic agents that cause local hyperthermia responsible for the thermal ablation of tumor cells after applying near-infrared light and is often applied with other combination therapies. In this study, the chemo-PTT potential of synthesized drug-loaded and targeted GEM/TRA-MC@Si nanocomposite on Her2 positive breast cancer cell line (SK-BR-3) and human triple-negative breast cancer cell line (MDA-MB-231) was investigated using NIR application asin vitro. First, the cell viability (IC50) value of the GEM/TRA-MC@Si nanocomposite was determined as 25µg/µL. Then, chemo-PTT was performed, and the viability of the cells was evaluated. In addition, the live/dead cell rate was established by staining with the Calcein-AM and EthD-1, and apoptosis tests were completed. When the surface temperature of Her2 positive SK-BR-3 cells exceeded 47°C during PTT with an irradiation time of > 100s, it caused cell death. In this study, it was demonstrated that in vitro PTT (1W/cm2, 180s) was applied using GEM/TRA-MC@Si nanocomposite (25µg/mL) on her2 + SK-BR-3 cell line, which contributed to the reduction of cell viability. In addition, this study demonstrates that chemo-PTT with targeted GEM/TRA-MC@Si nanocomposite induced SK-BR-3 cell viability and can initiate cell death through the apoptosis pathway under optimized irradiation conditions. Herewith chemo-PTT combination therapy of targeted GEM-TRA/MC@Si nanocomposite was found to be effective on SK-BR-3 cells as in vitro.

The aromatase inhibitor letrozole restores FADS2 function in ER+ MCF7 human breast cancer cells

PurposePlasticity in fatty acid metabolism is increasingly recognized as a major feature influencing cancer progression and efficacy of treatments. Estrogen receptor positive MCF7 human breast cancer cells have long been known to have no FADS2-mediated Δ6-desaturase activity. Our objective was to examine the effect of estrogen and the “antiestrogen” aromatase inhibitor letrozole, on Δ5- and Δ6-desaturase synthesized fatty acids in vitro. MethodsEicosa-11,14-dienoic acid (20:2n-6), a known substrate for both FADS1 and FADS2, was used as a sentinel of relative FADS2 and FADS1 activity. MCF7 cells and four additional estrogen responsive wild type cell lines (HepG2, SK-N-SH, Y79 and Caco2) were studied. FAME were quantified by GC-FID and structures identified by GCCACI-MS/MS. ResultsIn all five cell lines, estrogen caused a dose dependent decrease in sciadonic acid (5,11,14–20:3, ScA) via apparent inhibition of FADS1 activity, and had no effect on FADS2 catalyzed synthesis of dihomo-gamma linolenic acid (8,11,14–20:3; DGLA). In MCF7 cells, letrozole caused a dose dependent increase in FADS2-catalyzed DGLA synthesis, which plateaued in SK-N-SH cells. ConclusionLetrozole restores Δ6-desaturase mediated synthesis of the anti-inflammatory PGE1-precursor DGLA in vitro and is the first endocrine-active agent to have opposing effects on FADS1 and FADS2 catalyzed activities.

Novel decorated nanostructured lipid carrier for simultaneous active targeting of three anti-cancer agents

Cancer-targeted co-delivery of therapeutic agents has been recognized as an effective strategy for increasing efficacy and reducing side effects of therapeutic agents. In this study, we used methotrexate (MTX) alone as a targeting moiety and chemotherapeutic agent and in combination with docetaxel (DTX) and doxorubicin (DOX) as chemotherapeutic agents to stop cancer cell proliferation with the aid of newly designed nanostructured lipid carriers (NLCs). The physicochemical properties of our designed nanocomplexes were evaluated by DLS, FT-IR spectroscopy, SEM, and TEM. Moreover, the targeting efficiency of the designed and synthesized nanoplatforms was evaluated on the folate receptor (FR) positive human breast cancer cell line (MCF-7) and FR negative human alveolar basal epithelial cells (A549). The NLCs/DTX/DOX/CS and NLCs/DTX/DOX/CS-MTX complexes significantly increased the cell cytotoxicity and the cell apoptosis rate. However, the complexes significantly reduced the capability of colony formation and cell migration. Our results revealed that NLCs/DTX/DOX/CS-MTX had synergistic cytotoxicity, reactive oxygen spaces, autophagy, and the apoptosis induction ability with an enhanced cellular internalization rate in FR-positive cancer cells, thorough MTX recognition capability. We conclude that the NLCs/DTX/DOX/CS-MTX complex is a new promising paradigm for breast cancer-targeted co-delivery.

Estrogenic activity of ethyl gallate and its potential use in hormone replacement therapy

We aimed to compare the estrogenic activities of compounds isolated from Moutan Cortex Radicis (MRC, Paeonia suffruticosa Andrews) and identify their potential use in hormone replacement therapy. We quantified seven marker components (gallic acid, oxypaeoniflorin, paeoniflorin, ethyl gallate, benzoic acid, benzoylpaeoniflorin, and paeonol) in MRC using a high-performance liquid chromatography simultaneous analysis assay. To investigate the estrogenic activity of MRC and the seven marker components, an E-screen assay was conducted using the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line. Among them, ethyl gallate caused cell proliferation in a concentration-dependent manner at concentrations above 25 µM and was clearly suppressed by combination treatment with the ER antagonist ICI 182,780. Therefore, ethyl gallate may be a compound of MRC that can increase the estrogenic effect in ER-positive MCF-7 cells.

HDAC3-ERα Selectively Regulates TNF-α-Induced Apoptotic Cell Death in MCF-7 Human Breast Cancer Cells via the p53 Signaling Pathway.

Tumor necrosis factor-α (TNF-α) plays a significant role in inflammation and cancer-related apoptosis. We identified a TNF-α-mediated epigenetic mechanism of apoptotic cell death regulation in estrogen receptor-α (ERα)-positive human breast cancer cells. To assess the apoptotic effect of TNF-α, annexin V/ propidium iodide (PI) double staining, cell viability assays, and Western blotting were performed. To elucidate this mechanism, histone deacetylase (HDAC) activity assay and immunoprecipitation (IP) were conducted; the mechanism was subsequently confirmed through chromatin IP (ChIP) assays. Finally, we assessed HDAC3–ERα-mediated apoptotic cell death after TNF-α treatment in ERα-positive human breast cancer (MCF-7) cells via the transcriptional activation of p53 target genes using luciferase assay and quantitative reverse transcription PCR. The TNF-α-induced selective apoptosis in MCF-7 cells was negatively regulated by the HDAC3–ERα complex in a caspase-7-dependent manner. HDAC3 possessed a p53-binding element, thus suppressing the transcriptional activity of its target genes. In contrast, MCF-7 cell treatment with TNF-α led to dissociation of the HDAC3–ERα complex and substitution of the occupancy on the promoter by the p53–p300 complex, thus accelerating p53 target gene expression. In this process, p53 stabilization was accompanied by its acetylation. This study showed that p53-mediated apoptosis in ERα-positive human breast cancer cells was negatively regulated by HDAC3–ERα in a caspase-7-dependent manner. Therefore, these proteins have potential application in therapeutic strategies.

Bisphenolic compounds alter gene expression in MCF-7 cells through interaction with estrogen receptor α

Plasticizers released from microplastic are increasingly viewed with concern. While adverse health effects induced by bisphenol A and its analogues on marine animals are well documented in the literature, the endocrine potential of bisphenolic compounds on human health remains elusive. We applied next generation sequencing (NGS) with the estrogen receptor α (ERα) positive human breast cancer cell line MCF-7 treated with 17-β-estradiol (E2), bisphenol A (BPA), bisphenol B (BPB), bisphenol Z (BPZ) and tetramethyl bisphenol A (4MeBPA). We used molecular docking, microscale thermophoresis, ERα activation assay, and cell cycle experiments on MCF-7 and ERα overexpressing HEK293 cells to verify the impact of the compounds on ERα. 14 genes were found upregulated (ADORA1, DDIT4, CELSR2, FOSL2, JUN, HSPA13, IER3, IGF1R, PGR, RUNX2, SLC7A11, SLC7A2, SLC7A5, STC2) and 3 genes were downregulated (BCAS3, PHF19, PRKCD) in almost all samples. These genes are associated with cell growth, invasion, migration, apoptosis and cancer development. We further confirmed the binding, activation and proliferative effect of BPA, BPB, BPZ, and 4MeBPA on ERα. We provide evidence for the endocrine potential of bisphenolic compounds and give insights into their molecular effects in MCF-7 cells.

Curcumin‐Induced Apoptosis Pathways in Positive and Negative Human Epidermal Growth Factor Receptor Breast Cancer Cells

AimCurcumin, have several advantages and potential health benefits, such as low cytotoxicity and minimal development of resistance, as compared to chemically‐based therapies, and have anti‐cancerous efficacies, including induction of apoptosis, in cancer cells of various tissue origins. The aim of this study was toelucidate the mechanisms of curcumin‐induced apoptosis in human epidermal growth factor receptor positive (BT474) and negative (MCF‐7) breast cancer cell lines.MethodsCytotoxic activity of curcumin was measured by the sulphorhodamine B assay. We studied the induction of apoptosis by flow cytometry and detection of an apoptosis‐associated PARP cleavage product via Western blotting. Westernblotting was also used to measure Cytochrome C translocation, which determines mitochondrial involvement and caspase‐7 activation. The Caspase cascade was measured by activity levels of caspase‐3, caspase‐8, and caspase‐9 using chromogenic substrates.ResultsCurcumin CC50 (concentration that kills 50% of the cells) was 25.92 ±5.13μM for MCF‐7, and 49.96 ± 5.98 μM for BT474 cells. Flow cytometry results indicated a 13.36 ±5.96% expression increase and a 6.87 ± 1.42% expression increase in early stage apoptosis (Annexin V positively stained cells) in MCF‐7 and BT474 cells, respectively. Late stage apoptosis (dual stained Annexin V/Propidium Iodide (PI)cells) in curcumin exposed MCF‐7 cells was increased by 3.45 ± 0.94% expression, but no increase was seen in BT474 cells. PARP cleavage product (89kD) was detected at similar levels in both curcumin exposed MCF‐7 and BT474 cells and in a dose dependent manner. Curcumin‐exposedMCF‐7 cells, but not BT474 cells, showed substantial release of Cytochrome C from the mitochondria into the cytoplasm. Curcumin significantly upregulatedcaspase‐8 activity without modulating the activity of caspase‐9 in both BT474 and MCF‐7 cells. However, Caspase‐3 activity was upregulated only in BT474 cells. MCF‐7 cells reportedly have a gene deletion for Caspase‐3 and therefore do not express it. Caspase‐7 was then tested in MCF‐7 cells, but no upregulation was measured.ConclusionCytotoxicity of Curcumin is low for both MCF‐7 and BT474 cells and concentrations are easily achievable. Apoptosis induction by Curcumin exposure was detected by both PARP cleavage and flow cytometry. Flow cytometry showed that curcumin induced predominantly early stage apoptosis (Annexin V stained) in both BT474 and MCF‐7 cells, with a late‐stage apoptosis increase seen only in MCF‐7. Caspase studies determined an extrinsic pathway (Caspase‐8 and Caspase‐3 positive, Caspase‐9 negative) in BT474 cells. MCF‐7 cells were also Caspase‐8 positive, Caspase‐9 negative, but Caspase‐3 was not detected. In the absence of Caspase‐3, Caspase‐7 might serve as an effector caspase. However, Curcumin exposure did not upregulate its expression in MCF‐7 cells. While curcumin activated the mitochondria‐independent extrinsic pathway in BT474 cells (no translocation of Cytochrome C into the cytoplasm), a mitochondria‐dependent (translocation of Cytochrome C into the cytoplasm), extrinsic with possible intrinsic pathway involvement was activated in MCF‐7 cells. Further studies are needed to elucidate the altered apoptotic pathway utilized in MCF‐7 cells.

The significance of Runx2 mediating alcohol-induced Brf1 expression and RNA Pol III gene transcription

Runx2 (Runt-related transcription factor 2) is a key transcription factor which is associated with osteoblast differentiation and expressed in ER+ (estrogen receptor positive) human breast cancer cell lines. Runx2 also participates in mammary gland development. Deregulation of RNA Pol III genes (polymerase III-dependent genes) is tightly linked to tumor development, while Brf1 (TFIIB-related factor 1) specifically regulates these gene transcription. However, nothing is known about the effect of Runx2 on Brf1 expression and Pol III gene transcription. Expression of Runx2, Brf1 and Pol III genes from the samples of human breast cancer and cell culture model were determined by the assays of RT-qPCR, immunoblot, luciferase reporter activity, immunohistochemistry, chromatin immunoprecipitation and Immunofluorescence. High expression of Runx2 is observed in the cases of breast cancer. The patients of high Runx2 expression at early stages display longer survival period, whereas the cases of high Runx2 at advanced stages reveal faster recurrence. The identification of signaling pathway indicates that JNK1 and c-Jun mediate Runx2 transcription. Repression of Runx2 reduces Brf1 expression and Pol III gene transcription. Further analysis indicates that Runx2 is colocalized with Brf1 in nucleus of breast cancer tissue. Both Runx2 and Brf1 synergistically modulate Pol III gene transcription. These studies indicate that Brf1 overexpression is able to be used as an early diagnosis biomarker of breast cancer, while high Runx2 expression indicates long survival period and faster recurrence. Runx2 mediates the deregulation of Brf1 and Pol III genes and its abnormal expression predicts the worse prognosis of breast cancer.

Evaluation of Newcastle disease virus mediated dendritic cell activation and cross-priming tumor-specific immune responses ex vivo.

We have developed an oncolytic Newcastle disease virus (NDV) that has potent in vitro and in vivo anti-tumor activities and attenuated pathogenicity in chickens. In this ex vivo study using the same recombinant NDV backbone with GFP transgene (NDV-GFP, designated as rNDV), we found that rNDV induces maturation of monocyte-derived immature dendritic cells (iDCs) by both direct and indirect mechanisms, which promote development of antigen-specific T cell responses. Addition of rNDV directly to iDCs culture induced DC maturation, as demonstrated by the increased expression of costimulatory and antigen-presenting molecules as well as the production of type I interferons (IFNs). rNDV infection of the HER-2 positive human breast cancer cell line (SKBR3) resulted in apoptotic cell death, release of proinflammatory cytokines, and danger-associated molecular pattern molecules (DAMPs) including high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70). Addition of rNDV-infected SKBR3 cells to iDC culture resulted in greatly enhanced upregulation of the maturation markers and release of type I IFNs by DCs than rNDV-infected DCs only. When co-cultured with autologous T cells, DCs pre-treated with rNDV-infected SKBR3 cells cross-primed T cells in an antigen-specific manner. Altogether, our data strongly support the potential of oncolytic NDV as efficient therapeutic agent for cancer treatment.

Lupiwighteone induces caspase-dependent and -independent apoptosis on human breast cancer cells via inhibiting PI3K/Akt/mTOR pathway

Breast cancer is one of the most common causes of mortality in women. Lupiwighteone has anticancer effects in prostate cancer cells and neuroblastoma cells. However, the molecular and cellular mechanisms of lupiwighteone effects on human breast cancer cells are not as well known. In the present study, we investigated the effects of lupiwighteone on the proliferation and apoptosis of two different human cancer cells; MCF-7, an estrogen receptor (ER)-positive human breast cancer cell, and MDA-MB-231, a triple negative human breast cancer cell. Lupiwighteone treatment decreased the viability of MCF-7 and MDA-MB-231 cells. Lupiwighteone treatment resulted in apoptotic cell death in breast cancer cells, which was characterized by DNA fragmentation, accumulation of apoptotic cells, and nuclear condensation. We also showed that treatment with lupiwighteone induced caspase-dependent apoptosis (up-regulation of caspase-3, -7, -8, -9, PARP, and Bax or down-regulation of Bid, Bcl-2), induction of caspase-independent apoptosis (up-regulation of AIF and Endo G on cytosol), and inhibition of the PI3K/Akt/mTOR signaling pathway (down-regulation of PI3K, p-Akt, and p-mTOR) in both MCF-7 and MDA-MB-231 cells. These results suggest that lupiwighteone induces caspase-dependent and -independent apoptosis in both breast cancer cell lines via inhibiting PI3K/Akt/mTOR pathway.

Effect of CCT137690 on long non-coding RNA expression profiles in MCF-7 and MDA-MB-231 cell lines.

Long non-coding RNAs (lncRNAs) are involved in a range of biological processes, such as cellular differentiation, migration, apoptosis, invasion, proliferation, and transcriptional regulation. The aberrant expression of lncRNAs plays a significant role in several cancer types. Aurora kinases are increasingly expressed in various malignancies; accordingly, the inhibition of these enzymes may represent a novel approach for the treatment of various cancers. CCT137690, an Aurora kinase inhibitor, displays an anti-proliferative activity in human cancer cell lines. The aim of the present study was to investigate the anti-proliferative and cytotoxic effects of CCT137690 on estrogen receptor (ER)-positive human breast cancer cell line (MCF-7) and ER-negative human breast cancer cell line (MDA-MB-231). In addition, this study was targeted toward determining the changes induced in lncRNA expression levels following the initiation of Aurora kinase inhibitor treatment. The cytotoxic effects of CCT137690 were determined by means of the xCELLigence system. Furthermore, the anti-proliferative role of CCT137690 in breast cancer was investigated by checking the changes in lncRNA expression profiles using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The half-maximal inhibitory concentrations (IC50) of CCT137690 were determined as 4.5 µM (MCF-7) and 7.27 µM (MDA-MB-231). Several oncogenic lncRNAs (e.g., PRINS, HOXA1AS, and NCRMS) were downregulated in both ER-negative and ER-positive cell lines. On the other hand, tumor suppressor lncRNAs (e.g., DGCR5 and IGF2AS) were upregulated in the ER-positive cell line. After CCT137690 treatment, HOXA11AS and PCAT-14 lncRNAs were downregulated in the ER-positive cell lines. In addition, MER11C, SCA8, BC200, HOTAIR, PCAT-1, UCA1, SOX2OT, and HULC lncRNAs were downregulated in the ER-negative cell lines. The results of the present study indicated that Aurora kinase inhibitor CCT137690 could be a potential anti-cancer agent for breast cancer treatment.

GRB7 dependent proliferation of basal-like, HER-2 positive human breast cancer cell lines is mediated in part by HER-1 signaling.

GRB7 gene encodes a multi-domain signal transduction molecule and is part of the core of the HER-2 amplicon. GRB7 is commonly co-amplified and overexpressed with HER-2 in human breast cancer. This study addresses the role of GRB7 in HER-2 positive human breast cancers resistant to HER-2 targeted therapy. HCC1954, 21MT1, and JIMT1 are basal like HER-2 positive breast cancer cell lines based on expression profiling. These three cell lines are resistant to trastuzumab and lapatinib treatment. Knockdown of GRB7 protein expression with siRNA transfection as well as lentiviral vector mediated shRNA over-expression decreased the growth of HCC1954, 21MT1, and JIMT1 cells in vitro and the growth of tumor xenografts these cells formed in animal models. When assayed by ki-67 staining and TUNEL assay, the mechanism of reduced tumor xenograft growth appeared to be distinct. Reduced proliferation and increased apoptosis were seen in 21MT1 cells, while reduced proliferation was seen in HCC1954 cells and increased apoptosis in JIMT1 cells. Phospho-proteome profiling found HER-1 tyrosine phosphorylation was reduced with GRB7 knock down in JIMT1 cells. Immuno-blotting and immuno-precipitation experiments found HER-1 phosphorylation was reduced with GRB7 knock down in all three cell lines. HER-1 knock down via siRNA transient transfection as well as blocking HER-1 function with panitumumab decreased proliferation of all three cell lines in vitro. Our study finds that GRB7 has an essential growth promoting function which is mediated in part by HER-1 activation. The potential of HER-1 targeting in therapy resistant HER-2 positive breast cancer merits further study.

Developing Tamoxifen-Based Chemical Probes for Use with a Dual-Modality Fluorescence and Optical Coherence Tomography Imaging Needle

Fluorescent small molecules based on the chemotherapeutic tamoxifen have been synthesised for use with an imaging needle capable of acquiring simultaneous fluorescence and optical coherence tomography (OCT) images. The chemical probes are based on the active metabolite of the drug, 4-hydroxytamoxifen that is coupled with a diamine linker to commercially available Alexa Fluor or 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes. The tamoxifen derivatives were then added to cultures of live oestrogen receptor positive MCF-7 human breast cancer cells and imaged using the miniaturised fibre-optic device enclosed within a 23-gauge needle (outer diameter 640 μm). The OCT images showed the micro-architecture of the cell culture, while the fluorescence identified oestrogen receptor positive cells. Both dyes were found to have suitable excitation and emission properties and are good candidates to further develop as probes for fluorescence-guided surgery.

Caesalpinia pulcherrima sarrests cell cycle and triggers reactive oxygen species-induced mitochondrial-mediated apoptosis and necroptosis via modulating estrogen and estrogen receptors

Background: Caesalpinia pulcherrima belonging to the family Fabaceae is used in India as a traditional medicine for a variety of ailments. Globally, traditional medicines are presently being used for the treatment of cancer. Objective: The present study was aimed at investigating the chemomodulatory potential of C. pulcherrima flowers in breast cancer and explaining its possible mechanism. Materials and Methods: The cytotoxic potential of ethyl acetate fraction of C. pulcherrima (EAFCP) flower was tested in MCF-12A (normal breast), MCF-7 (estrogen receptor [ER] positive), and MDA-MB-453 (human epidermal growth factor receptor 2 positive) human breast cancer cells by sulforhodamine B assay. Chemomodulatory potential was evaluated in vivo against N-methyl-N-nitrosourea (MNU)-induced mammary carcinoma in female Sprague Dawley® rats. The mechanism for anticancer potential was screened by in vitro studies involving Annexin V-FITC assay (apoptosis), cell cycle patterns, intracellular reactive oxygen species, and mitochondrial membrane potential measurement (FACS based) followed by docking study on estrogen receptor-alpha (ER-α). Results: The fractions showed perceptible cell growth inhibition potency (IC50

PO-234 Transwell co-culture with human breast adipocytes alters the proteome expression profiles of MCF-7 and MDA-MB-231 human breast cancer cells

IntroductionBreast tumours grow within adipose tissue, and stromal adipocytes are likely the first cell type encountered by breast cancer cells as they metastasize. Adipocytes at the invasive edge of human breast tumours exhibit a modified phenotype, and consequently these adipocytes have been named Cancer Associated Adipocytes (CAA). Previous in vitro research has shown that co-culture with CAA promotes breast cancer cell resistance to radiation and some therapeutic agents, as well as breast cancer cell invasiveness both in vitro and in vivo. The aim of the current research was to identify cellular proteins that are differentially regulated in breast cancer cells co-cultured with CAA.Material and methodsThis study collected human breast adipose tissue samples, from which pre-adipocytes were isolated, differentiated into mature adipocytes, and co-cultured with hormone receptor positive (MCF-7) and negative (MDA-MB-231) human breast cancer cells for 3 days in a transwell co-culture system. The proteomes of co-cultured and control breast cancer cells were compared quantitatively using iTRAQ labelling and LC-coupled LTQ-Orbitrap tandem mass spectrometry. Validation of iTRAQ results was performed by Western blotting.Results and discussionsThis study identified and quantified a total of 1126 and 1218 proteins expressed in MCF-7 and MDA-MB-231 cells, respectively. Of these, 85 in MCF-7 and 63 in MDA-MB-231 had a fold change >1.5 between co-culture and control breast cancer cell samples. Overall, MCF-7 cells had more proteins downregulated (n=53) than upregulated (n=32) after co-culture, whereas, more proteins were upregulated (n=51) than downregulated (n=12) in MDA-MB-231 cells. Enrichment analysis revealed an upregulation of proteins involved in metabolic pathways, namely TCA cycle proteins in MCF-7 cells and glycolysis proteins in MDA-MB-231 cells. The metabolic enzyme PGK1 was upregulated in response to co-culture with CAA in both cell lines, and is associated with poorer overall survival and resistance to the chemotherapy agent paclitaxel in breast cancer. Western blotting results validated mass spectrometry ratios for three candidate proteins.ConclusionOverall, this study describes for the first time the local effect CAA have on the expression of proteins in breast cancer cells in vitro, and provides a comprehensive platform for further research investigating local interactions between breast cancer cells and CAA.

Synergistic antitumor activity by combining trastuzumab with retinoic acid in HER2 positive human breast cancer cells.

Breast cancer can be classified into molecular subtypes. Tumors overexpressing HER2 protein are more aggressive and metastatic; hence, patients have a poor prognosis. Anti-HER2 strategies, such as the monoclonal antibody Trastuzumab (Tz), have therefore been developed. Despite this progress, not all patients respond to the treatment. Retinoic acid (RA) has been proposed as an adjuvant treatment of breast carcinoma because of its ability to inhibit cell growth. We evaluated the effect of Tz in combination with RA on the viability, adhesion, migration, invasion and expression of migration-related proteins in SKBR3 and BT-474 human breast cancer cells. MTT, pharmacological interaction analysis, immunofluorescence, adhesion/migration/invasion and Western blot assays were performed. The coadministration of both drugs synergistically decreased cell survival. Tz+RA significantly decreased adhesion/migration/invasion in both cell types. Tz+RA strongly reduced FAK and HER2 expression and induced nuclear FAK translocation. In addition, a granular distribution of HER2 receptor was observed after the combined treatment. In conclusion, the coadministration of both drugs in patients with this type of cancer could contribute to the improvement of their prognosis and reduce the adverse effects of therapy because the applied Tz doses would be lower due to the adjuvant effect of RA.

Abstract P2-08-03: An essential role of GRB7 in promoting the growth of therapy resistant HER-2 positive human breast cancer cells in culture and animal models

Abstract Background- GRB7 gene encodes a multi-domain signal transduction molecule and is part of the core of the HER-2 amplicon. GRB7 is commonly co-amplified and over-expressed with HER-2 in human breast cancer. Earlier studies found a functional role of GRB7 in breast cancer. The role of GRB7 in HER-2 positive human breast cancer resistant to HER-2 targeted therapy remains unexplored however. Materials and Methods- HCC-1954, 21MT1 and JimT1 are human HER-2 positive breast cancer cell lines that are resistant to trastuzumab and lapatinib treatment. Transient knock down of GRB7 protein expression was achieved with siRNA transfection and stable knock down with lentiviral vector mediated shRNA over-expression. Cell lines transfected with non-targeting siRNA or shRNA serve as negative controls. Knock down of GRB7 protein expression is verified by Western blotting. The growth of human breast cancer cell lines after GRB7 knock down in vitro is measured with the CellTiter Glo assay as well as the Incucyte live cell imaging. Activation status of specific signaling pathways was examined with phospho-specific antibody by immune-blotting and immune-precipitation. To assess the growth promoting function of GRB7 in human breast cancer cell lines in vivo, polyclonal HCC-1954, 21MT1 and JimT1 cells, with GRB7 knock down or their corresponding negative control, were orthotopically injected into the mammary fat pads of female immune-deficient NSG mice. The growth rates of these tumors, measured serially with caliper, and final tumor weights were compared between GRB7 knock down and the negative control. The proliferation rate and apoptosis of these tumors were studied with ki-67 staining and Tunel assay.The effects of GRB7 knock down on signaling were investigated with a proteome profiler receptor tyrosine kinase kit (R&D). The role of signaling molecules differentially activated in the growth of breast cancer cells by GRB7 knock down was examined utilizing siRNA mediated knock down, and antibody and small molecule inhibitors. Results- GRB7 knock down decreased the growth of HCC-1954, 21MT1 and JimT1 cells in vitro and the growth of tumor xenograft these cells formed in animal models. When assayed by ki67 staining and Tunel assay, the mechanism of reduced tumor xenograft growth appeared to be distinct. Reduced proliferation and increased apoptosis were seen in 21MT1 cells, while reduced proliferation was seen in HCC-1954 cells and increased apoptosis in JimT1 cells. Protein profiling found that tyrosine phosphorylation of candidate signaling molecules was reduced with GRB7 knock down in JimT1 cells. Immuno-blotting and immuno-precipitation experiments were performed to evaluate these effects in other cell lines. The effect of targeting these molecules in breast cancer cell growth by siRNA and inhibitors is being examined. Discussion- GRB7 has essential growth promoting function in therapy resistant HER-2 positive human breast cancer cells. GRB7 knock down has pleiotropic effects on signaling in various cellular contexts. The potential of targeting GRB7 signaling in treating therapy resistant HER-2 positive breast cancer merits further study. Citation Format: Luoh S-W, Wagoner W, Lai X, Hu Z, Chin K, Ramsey B. An essential role of GRB7 in promoting the growth of therapy resistant HER-2 positive human breast cancer cells in culture and animal models [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-08-03.