Non-cancerous Breast Cell Line Research Articles

BannMI deciphers potential n-to-1 information transduction in signaling pathways to unravel message of intrinsic apoptosis.

Cell fate decisions, such as apoptosis or proliferation, are communicated via signaling pathways. The pathways are heavily intertwined and often consist of sequential interaction of proteins (kinases). Information integration takes place on the protein level via n-to-1 interactions. A state-of-the-art procedure to quantify information flow (edges) between signaling proteins (nodes) is network inference. However, edge weight calculation typically refers to 1-to-1 interactions only and relies on mean protein phosphorylation levels instead of single cell distributions. Information theoretic measures such as the mutual information (MI) have the potential to overcome these shortcomings but are still rarely used. This work proposes a Bayesian nearest neighbor-based MI estimator (BannMI) to quantify n-to-1 kinase dependency in signaling pathways. BannMI outperforms the state-of-the-art MI estimator on protein-like data in terms of mean squared error and Pearson correlation. Using BannMI, we analyze apoptotic signaling in phosphoproteomic cancerous and noncancerous breast cell line data. Our work provides evidence for cooperative signaling of several kinases in programmed cell death and identifies a potential key role of the mitogen-activated protein kinase p38. Source code and applications are available at: https://github.com/zuiop11/nn_info and can be downloaded via Pip as Python package: nn-info.

Apoptosis-mediated anti-proliferative activity of Calligonum comosum against human breast cancer cells, and molecular docking of its major polyphenolics to Caspase-3.

Due to poor diagnosis breast cancer in women has emerged as the most common cause of death disease in developing countries. Medicinal plants have been used for thousands of years and can be useful in healthcare, especially in developing countries. Ethanol extracts of leaves of fire bush or arta (Calligonum comosum; EECC), exhibited significant anticancer potencies against two breast cancer cell lines, MCF-7 and MDA 231. These in vitro effects of EECC indicated potential anticancer activities that were determined to be specific since minimal toxicity was recorded against MCF-12, a non-cancerous breast cell line used as a reference. EECC also induced cell cycle arrest in MCF-7 and MDA 231 as revealed by the increased proportions of sub-G1 cells. Fluorescence-activated cell sorter analysis (FACS), utilizing double staining by annexin V-FITC/propidium iodide, revealed that the observed cytotoxic effects were mediated via apoptosis and necrosis. FACS measurement of thegreater in fluorescence intensity, linked with oxidation of DCFH to DCF, revealed that apoptosis was attributable to production of free radicals. EECC-mediated apoptosis was further validated by observation of up-regulation in the “executioner” enzyme, caspase 3. The current findings reveal that EECC exhibits significant, selective cytotoxicity to breast cancer cells, that proceeds via the generation of ROS, which culminates in apoptosis. The anti-proliferative effects of EECC weres further verified by use of a structure-based, virtual screening between its major bioactive polyphenolic constituents and the apoptosis executioner marker enzyme, caspase-3. Based on their glide score values against the active site of caspase 3, some phyto-constituents present in EECC, such as DL-alpha-tocopherol and campesterol, exhibited distinctive, drug-like potential with no predicted toxicity to non-target cells. Taken together, the usefulness of natural phenolic and flavonoid compounds contained in Calligonum comosum were suggested to be potent anticancer agents.

Synthesis, characterization, crystal structures, and anticancer activity of some new 2,3-dihydro-1,5-benzoxazepines

Various benzoxazepine derivatives have been synthesized and characterized using IR, NMR, GC–MS, and microanalysis. The single-crystal X-ray structures of 2,2-dimethyl-4-[(E)-2-(4-methylphenyl)ethenyl]-2,3-dihydro-1,5-benzoxazepine (RS01), 4-[(E)-2-(2-chlorophenyl)ethenyl] -2,2-dimethyl-2,3-dihydro-1,5-benzoxazepine (RS05), 2,2,4-trimethyl-2,3-dihydrobenzothiazepine (RS11), and 2,2,4-trimethyl-2,3-dihydrobenzoxazepine (RS12) have been discussed. The compounds have been evaluated for their anticancer properties in breast cancer cells. 4-[(E)-2-(2-Chlorophenyl)ethenyl]-2,2-dimethyl-2,3-dihydro-1,5-benzoxazepine (RS03) and RS12 displayed potent cytotoxicity in both benign (MCF-7) and metastatic (MDA-MB-231) breast cancer cells. These compounds were more selective for the MCF-7 cells with RS03 being the most potent compound (IC50 = 15 µM) of the series. Upon further investigation, it was found that RS03 and RS12 induced cell cycle arrest in the G2/M phase and display limited toxicity against the noncancerous breast cell line, MCF-10A.

Breast Cancer-Derived Microvesicles Are the Source of Functional Metabolic Enzymes as Potential Targets for Cancer Therapy.

Membrane-derived extracellular vesicles, referred to as microvesicles (MVs), have been proposed to participate in several cancer diseases. In this study, MV fractions were isolated by differential ultracentrifugation from a metastatic breast cancer (BC) cell line MDA-MB-231 and a non-cancerous breast cell line MCF10A, then analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 1519 MV proteins were identified from both cell lines. The data obtained were compared to previously analyzed proteins from small extracellular vesicles (sEVs), revealing 1272 proteins present in both MVs and sEVs derived from the MDA-MB-231 cell line. Among the 89 proteins unique to MDA-MB-231 MVs, three enzymes: ornithine aminotransferase (OAT), transaldolase (TALDO1) and bleomycin hydrolase (BLMH) were previously proposed as cancer therapy targets. These proteins were enzymatically validated in cells, sEVs, and MVs derived from both cell lines. The specific activity of OAT and TALDO1 was significantly higher in MDA-MB-231-derived MVs than in MCF10A MVs. BLMH was highly expressed in MDA-MB-231-derived MVs, compared to MCF10A MVs. This study shows that MVs carry functional metabolic enzymes and provides a framework for future studies of their biological role in BC and potential in therapeutic applications.

Chemical Profiling, Antioxidant, Cytotoxic Activities and Molecular Docking Simulation of Carrichtera annua DC. (Cruciferae).

Our investigation intended to analyze the chemical composition and the antioxidant activity of Carrichtera annua and to evaluate the antiproliferative effect of C. annua crude and phenolics extracts by MTT assay on a panel of cancerous and non-cancerous breast and liver cell lines. The total flavonoid and phenolic contents of C. annua were 47.3 ± 17.9 mg RE/g and 83.8 ± 5.3 mg respectively. C. annua extract exhibited remarkable antioxidant capacity (50.92 ± 5.64 mg GAE/g) in comparison with BHT (74.86 ± 3.92 mg GAE/g). Moreover, the extract exhibited promising reduction ability (1.17 mMol Fe+2/g) in comparison to the positive control (ascorbic acid with 2.75 ± 0.91) and it displayed some definite radical scavenging effect on DPPH (IC50 values of 211.9 ± 3.7 µg/mL). Chemical profiling of C. annua extract was achieved by LC-ESI-TOF-MS/MS analysis. Forty-nine hits mainly polyphenols were detected. Flavonoid fraction of C. annua was more active than the crude extract. It demonstrated selective cytotoxicity against the MCF-7 and HepG2 cells (IC50 = 13.04 and 19.3 µg/mL respectively), induced cell cycle arrest at pre-G1 and G2/M-phases and displayed apoptotic effect. Molecular docking studies supported our findings and revealed that kaempferol-3,7-O-bis-α-L-rhamnoside and kaempferol-3-rutinoside were the most active inhibitors of Bcl-2. Therefore, C. annua herb seems to be a promising candidate to further advance anticancer research. In extrapolation, the intake of C. annua phenolics might be adventitious for alleviating breast and liver malignancies and tumoral proliferation in humans.

Cannabinoid Combination Induces Cytoplasmic Vacuolation in MCF-7 Breast Cancer Cells.

This study evaluated the synergistic anti-cancer potential of cannabinoid combinations across the MDA-MB-231 and MCF-7 human breast cancer cell lines. Cannabinoids were combined and their synergistic interactions were evaluated using median effect analysis. The most promising cannabinoid combination (C6) consisted of tetrahydrocannabinol, cannabigerol (CBG), cannabinol (CBN), and cannabidiol (CBD), and displayed favorable dose reduction indices and limited cytotoxicity against the non-cancerous breast cell line, MCF-10A. C6 exerted its effects in the MCF-7 cell line by inducing cell cycle arrest in the G2 phase, followed by the induction of apoptosis. Morphological observations indicated the induction of cytoplasmic vacuolation, with further investigation suggesting that the vacuole membrane was derived from the endoplasmic reticulum. In addition, lipid accumulation, increased lysosome size, and significant increases in the endoplasmic reticulum chaperone protein glucose-regulated protein 78 (GRP78) expression were also observed. The selectivity and ability of cannabinoids to halt cancer cell proliferation via pathways resembling apoptosis, autophagy, and paraptosis shows promise for cannabinoid use in standardized breast cancer treatment.

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

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

New Palladium(II) and Platinum(II) Complexes Based on Pyrrole Schiff Bases: Synthesis, Characterization, X-ray Structure, and Anticancer Activity.

New palladium (Pd)II and platinum (Pt)II complexes (C1–C5) from the Schiff base ligands, R-(phenyl)methanamine (L1), R-(pyridin-2-yl)methanamine (L2), and R-(furan-2-yl)methanamine (L3) (R-(E)-N-((1H-pyrrol-2-yl) methylene)) are herein reported. The complexes (C1–C5) were characterized by FTIR, 1H and 13C NMR, UV–vis, and microanalyses. Single-crystal X-ray crystallographic analysis was performed for the two ligands (L1–L2) and a Pt complex. Both L1 and L2 belong to P21/n monoclinic and P-1 triclinic space systems, respectively. The complex C5 belongs to the P21/c monoclinic space group. The investigated molar conductivity of the complexes in DMSO gave the range 4.0–8.8 μS/cm, suggesting neutrality, with log P values ≥ 1.2692 ± 0.004, suggesting lipophilicity. The anticancer activity and mechanism of the complexes were investigated against various human cancerous (Caco-2, HeLa, HepG2, MCF-7, and PC-3) and noncancerous (MCF-12A) cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Apopercentage assays, respectively. C5 demonstrated strong DNA-binding affinity for calf thymus DNA (CT-DNA) with a binding constant of 8.049 × 104 M–1. C3 reduced cell viability of all the six cell lines, which included five cancerous cell lines, by more than 80%. The C5 complex also demonstrated remarkably high selectivity with no cytotoxic activity toward the noncancerous breast cell line but reduced the viability of the five cancerous cell lines, which included one breast cancer cell line, by more than 60%. Further studies are required to evaluate the selective toxicity of these two complexes and to fully understand their mechanism of action.

6,N 2-Diaryl-1,3,5-triazine-2,4-diamines: synthesis, antiproliferative activity and 3D-QSAR modeling.

A library of 126 compounds with a 6,N2-diaryl-1,3,5-triazine-2,4-diamine scaffold was prepared using a one-pot, microwave-assisted method from readily available cyanoguanidine, aromatic aldehydes and arylamines. The three-component condensation of these reagents in the presence of hydrochloric acid was followed by the treatment with a base, which promoted a rearrangement of the dihydrotriazine ring and its dehydrogenative aromatization. The antiproliferative properties of the prepared compounds were evaluated using three breast cancer cell lines. The most promising results were obtained in the growth inhibition of the triple negative MDA-MB231 breast cancer cells. The active compounds were also selective against cancer cells and did not affect growth of the non-cancerous MCF-10A breast cell line. Analyzing the structure–activity relationship within the series, we built a 3D-QSAR model for the further design of more potent anticancer compounds.

Expression of MTERF3 gene in breast carcinoma and the relationship with clinicopathological characteristics.

BackgroundMitochondrial transcription termination factor 3 (MTERF3) is a negative regulator of mitochondrial transcription. It is a modular factor involves in mitochondrial ribosome biogenesis and protein synthesis. However, the association between MTERF3 and breast cancers remains largely unknown. The aim of this study was to investigate the expression of MTERF3 in breast carcinoma and to analyze its clinicopathological significance, and to examine the potential prognostic value of MTERF3 in breast cancer.MethodsThe protein expression levels of MTERF3 in MCF7 (Luminal A), BT-474 (Luminal B), SKBR3 (HER2 overexpression), MDA-MB-468 (Basal like) and MCF10A cell lines were detected by Western blotting. Immunohistochemistry (IHC), Western blotting, and semiquantitative RT-PCR were performed to analyze the protein and mRNA expression levels of MTERF3 in 58 breast cancer tissues and 58 noncancerous breast tissues. The MTERF3 expression data and clinical information from breast cancer patients were downloaded from the TCGA dataset by using the R3.6.1 software. Then the relationship between the expression level of MTERF3 and clinicopathological characteristics and the prognostic value was analyzed. A Cox regression model was performed for the multivariate analysis of the factors that affected the prognosis of breast cancer. The association between the expression levels of MTERF3 and other mitochondrial regulatory genes was analyzed with GEPIA.ResultsMTERF3 is upregulated in breast cancer cell lines compared to noncancerous breast cell line. The IHC results showed that the MTERF3 protein was located in the cytoplasm, and the rate of positive expression in breast cancer tissue was significantly upregulated compared with the adjacent normal tissue. The mRNA and protein expression levels of MTERF3 in breast cancer tissues were significantly higher than that in breast tissues. Moreover, the expression of MTERF3 was significantly correlated with ER status, PR status, breast cancer molecular typing, cancer type, histological diagnosis and primary site (P<0.05). Further analysis showed MTERF3 expression was not related to prognosis. Multivariate Cox regression analysis showed that age, metastasis status and tumor type were independent prognostic factors for breast cancer patients. The expression levels of MTERF3 were positively correlated with the TFAM, TFB1M, TFB2M, MTERF1, TEFM and MFN1 genes but negatively correlated with the MTERF4 and PINK1 genes. In addition, the expression levels of MTERF3 were not correlated with the MTERF2 gene.ConclusionsMTERF3 was significantly upregulated in breast cancer cells and tissues compared with noncancerous cells and tissues. Moreover, the expression level of MTERF3 was correlated with ER status, PR status, breast cancer molecular typing, cancer type, histological diagnosis and primary site. These findings suggested that the upregulation of MTERF3 may be used as a diagnostic and therapeutic target in breast carcinoma.

Nalbuphine suppresses breast cancer stem-like properties and epithelial-mesenchymal transition via the AKT-NF\u03baB signaling pathway

BackgroundCancer pain is a debilitating disorder of human breast cancer and a primary determinant of the poor quality of life, and relieving pain is fundamental strategy in the cancer treatment. However, opioid analgesics, like morphine and fentanyl, which are widely used in cancer pain treatment have been reported to enhance stem-like traits and epithelial-mesenchymal transition (EMT) of breast cancer cells. As such, it is vital to make the best choice of analgesic for breast cancer management.MethodsMTT assays and colony formation assays were performed to examine tumor cell proliferation upon nalbuphine treatment. RT-PCR, western blot, flow cytometry, sphere formation, immunohistochemistry, transwell assays, wound healing assays and mouse xenograft were used to assess the biological effects of nalbuphine treatment.ResultsNalbuphine inhibited breast cancer cell growth and tumorigenesis, with little effect on noncancerous breast cell lines. Nalbuphine suppressed cancer stem-like traits and EMT in both breast cancer cells and mouse xenograft tumor tissues. Additionally, activation of AKT reversed the nalbuphine-induced inhibition of cancer stem-like properties, tumorigenesis and EMT.ConclusionsOur results demonstrate a new role of nalbuphine in inhibiting cancer stem-like properties and EMT in addition to relieving pain, which suggests that nalbuphine may be effective in breast cancer treatment.

Abstract 829: SAS1B protein: Determining whether ASTL or SAS1B has a role in tumor progression

Abstract The matrix metalloproteinase Sperm Acrosomal SLLP1 Binding (SAS1B) is an ideal molecular and cellular candidate for both female contraception and for cancer therapies. SAS1B translation is restricted among adult tissues to the ovary and oocytes, and SAS1B first appears in oocytes of follicles at the primary-secondary transition and also in tumors of varied types and stages. Research conducted using SAS1B antibodies suggested that the protein is localized at the cell membrane of cancer cells and can get internalized in a classical endocytosis assay and can be killed using an immunotoxin linked onto antibodies. The current study aims at deciphering whether the ASTL gene or the SAS1B protein is important for tumor progression, invasion and metastasis through cell biology and molecular biology tools and ensuring tumor cell-killing properties via an ADC-mediated killing using biochemistry and molecular biology tools. PCR, immunohistochemistry, Immunofluorescence, and Western blots revealed that a noncancerous breast cell line, MCF10A5E, is SAS1B null and can be used to study cancer-like properties once stably transfected with the SAS1B construct. Previous work in the lab indicated that an SAS1B-ADC strategy is a viable immunotherapy that could benefit patients who have failed to respond to chemotherapy, if indeed SAS1B plays a role in cancer development and progression. The outcomes of an SAS1B-ADC in a breast cancer model will be presented. Citation Format: Zunair S. Khokhar, Eusebio S. Pires, Alexander J. Kim, Ryan D'Souza. SAS1B protein: Determining whether ASTL or SAS1B has a role in tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 829.

Effects of local anesthetics on breast cancer cell viability and migration

BackgroundBreast cancer accounts for nearly a quarter of all cancers in women worldwide, and more than 90% of women diagnosed with breast cancer undergo mastectomy or breast-conserving surgery. Retrospective clinical studies have suggested that use of regional anesthesia leads to improved patient outcomes. Laboratory studies have reported that breast cancer cells are inhibited by some local anesthetics at millimolar concentration. Here, we present a comprehensive analysis of the effects of six common local anesthetics on two human breast cancer cell lines. We used concentrations ranging from those corresponding to plasma levels during regional block by local anesthetic (plasma concentration) to those corresponding to direct infiltration of local anesthetic.MethodsHuman breast cancer cell lines, MDA-MB-231 and MCF7, were incubated with each of six local anesthetics (lidocaine, mepivacaine, ropivacaine, bupivacaine, levobupivacaine, and chloroprocaine) (10 μM ~ 10 mM) for 6 to 72 h. Assays for cell viability, cytotoxicity, migration, and cell cycle were performed.ResultsHigh concentrations (> 1 mM) of local anesthetics applied to either MDA-MB-231 or MCF7 cells for 48 h significantly inhibited cell viability and induced cytotoxicity. At plasma concentrations (~ 10 μM) for 72 h, none of the local anesthetics affected cell viability or migration in either cell line. However, at 10 × plasma concentrations, 72-h exposure to bupivacaine, levobupivacaine or chloroprocaine inhibited the viability of MDA-MB-231 cells by > 40% (p < 0.001). Levobupivacaine also inhibited the viability of MCF7 cells by 50% (p < 0.001). None of the local anesthetics affected the viability of a non-cancerous breast cell line, MCF10A. MDA-MB-231 cell migration was inhibited by 10 × plasma concentrations of levobupivacaine, ropivacaine or chloroprocaine and MCF7 cell migration was inhibited by mepivacaine and levobupivacaine (p < 0.05). Cell cycle analysis showed that the local anesthetics arrest MDA-MB-231 cells in the S phase at both 1 × and 10 × plasma concentrations.ConclusionsLocal anesthetics at high concentrations significantly inhibited breast cancer cell survival. At 10 × plasma concentrations, the effect of local anesthetics on cancer cell viability and migration depended on the exposure time, specific local anesthetic, specific measurement endpoint and specific cell line.

Cytotoxicity, Regulation of Apoptotic and Anti-apoptotic Gene Expression by IL-27 in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines

Breast cancer is one of the commonest cancers among women. Conventional therapies cause adverse side effects in patients. Cytokine immunotherapy such as interleukin-27 (IL-27) has been sought as an alternative cancer treatment in recent years. IL-27 has been shown to improve anticancer immunity and anti-angiogenesis in cancers, however, its effect on apoptotic and anti-apoptotic gene expression especially in breast cancers is yet to be explored. Cytotoxicity of IL-27 in non-cancerous (184b5) and cancerous breast cell lines was first determined for 24-72 h in this study. The results indicated that IL-27 treatment did not retard 184b5 cell growth, however, did inhibit MCF-7 (48 h) and MDAMB-231 (72 h) cell growth with IC50 at 442 and 457 ng/ml, respectively. Apoptotic (TRAIL, FADD, FAS, caspase-3 and caspase-8) and anti-apoptotic (BCL-2, AKT, and COX-2) genes were then amplified from untreated (control) and treated breast cancer cells and studied. TRAIL, caspase-3, caspase-8 gene expression was significantly (p < 0.05) upregulated in treated MCF-7 (442 ng/ml) and MDA-MB-231 (457 ng/ml) cells. Expression of FADD and FAS genes was not detected in both control and treated MCF-7 and MDA-MB-231 cells. COX-2 gene was also not expressed by MCF-7 cells, but reduced significantly (p < 0.05) in treated MDA-MB-231 cells. In MDA-MB-231 cells, IL-27 treatment seemed to slightly enhance the expression of AKT and BCL-2 genes which, on the other hand, was downregulated in treated MCF-7 cells. Conclusively, IL-27 is able to inhibit breast cancer cell growth and regulate apoptotic and anti-apoptotic gene expression in breast cancer cells. DOI : http://dx.doi.org./10.17576/JSKM-2018-03

Differential Response of Two Human Breast Cancer Cell Lines to the Phenolic Extract from Flaxseed Oil.

Many studies have evidenced that the phenolic components from flaxseed (FS) oil have potential health benefits. The effect of the phenolic extract from FS oil has been evaluated on two human breast cancer cell lines, MCF7 and MDA-MB231, and on the human non-cancerous breast cell line, MCF10A, by SRB assay, cellular death, cell cycle, cell signaling, lipid peroxidation and expression of some key genes. We have evidenced that the extract shows anti-proliferative activity on MCF7 cells by inducing cellular apoptosis, increase of the percentage of cells in G0/G1 phase and of lipid peroxidation, activation of the H2AX signaling pathway, and upregulation of a six gene signature. On the other hand, on the MDA-MB2131 cells we verified only an anti-proliferative activity, a weak lipid peroxidation, the activation of the PI3K signaling pathway and an up-regulation of four genes. Overall these data suggest that the extract has both cytotoxic and pro-oxidant effects only on MCF7 cells, and can act as a metabolic probe, inducing differences in the gene expression. For this purpose, we have performed an interactomic analysis, highlighting the existing associations. From this approach, we show that the phenotypic difference between the two cell lines can be explained through their differential response to the phenolic extract.

A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells.

According to the World Health Organization (WHO), breast cancer is the most common cancer affecting women worldwide. In the USA ~12.3% of all women are expected to be diagnosed with various types of breast cancer, exhibiting varying degrees of therapeutic response rates. Therefore, the identification of novel anti-breast cancer drugs is of paramount importance. The 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore was incorporated into a number of cytotoxins. Three of the resulting dienones, 2a, 2b and 2c, were tested for their anti-neoplastic potencies in a variety of human breast cancer-derived cell lines, including the triple negative MDA-MB-231 cell line and its metastatic variant, using a live-cell bio-imaging method. Special emphasis was put on dienone 2c, since its anti-cancer activity and its mode of inflicting cell death have so far not been reported. We found that all three dienones exhibited potent cytotoxicities towards the breast cancer-derived cell lines tested, whereas significantly lower toxicities were observed towards the non-cancerous human breast cell line MCF-10A. The dienones 2b and 2c exhibited the greatest selective cytotoxicity at submicromolar concentration levels. We found that these two dienones induced phosphatidylserine externalization in MDA-MB-231 cells in a concentration-dependent manner, suggesting that their cytotoxic effect might be mediated by apoptosis. This possibility was confirmed by our observation that the dienone 2c can induce mitochondrial depolarization, caspase-3 activation, cell cycle disruption and DNA fragmentation in MDA-MB-231 cells. Our findings indicate that dienone 2c uses the mitochondrial/intrinsic pathway to inflict apoptosis in triple negative MDA-MB-231 breast cancer-derived cells. This observation warrants further assessment of dienone 2c as a potential anti-breast cancer drug.

Modulation of expression of heat shock proteins and apoptosis by Flueggea leucopyrus (Willd) decoction in three breast cancer phenotypes.

BackgroundDuring the past few years, there has been an increasing interest among the Traditional and Folk medical practitioners of Sri Lanka in the use of a decoction prepared from Flueggea leucopyrus (Willd.) for treating various cancers including breast cancer. In the present study, the cytotoxicity of this decoction and its effects on Heat Shock Protein (HSP) expression and apoptosis were compared in three breast cancer phenotypes, to scientifically evaluate if a decoction prepared from F. leucopyrus (Willd.) is useful for the treatment of breast cancer.MethodsCytotoxic potential of the F. leucopyrus decoction was determined by evaluating its effects in MCF-7, MDA-MB-231 and SKBR-3 breast cancer cell lines, and MCF-10A (non-cancerous) breast cell line, by use of the Sulphorhodamine (SRB) assay. The effect of the decoction on HSP gene expression in the above cells was evaluated by (a) Real time reverse transcription PCR (RT-PCR) and (b) Immunofluorescence analysis of HSP protein expression. Effects of the decoction on apoptosis were evaluated by (a) fluorescent microscopic examination of apoptosis related morphological changes and (b) DNA fragmentation (c) Caspase 3/7 assay.ResultsF. leucopyrus decoction can mediate significant cytotoxic effects in all three breast cancer cells phenotypes (IC50 values: 27.89, 99.43, 121.43 μg/mL at 24 h post incubation periods, for MCF-7, MDA-MB-231, SKBR-3 respectively) with little effect in the non-cancerous breast cell line MCF-10A (IC50: 570.4 μg/mL). Significant (*P <0.05) inhibitions of HSP 90 and HSP 70 expression were mediated by the decoction in MCF-7 and MDA-MB-231, with little effect in the SKBR-3 cells. Clear apoptotic morphological changes on Acridine orange/Ethidium bromide staining and DNA fragmentation were observed in all three breast cancer cell lines. Caspase 3/7 were significantly (*P <0.05) activated only in MDA-MB-231 and SKBR-3 cells indicating caspase dependent apoptosis in these cells and caspase independent apoptosis in MCF-7 cells.ConclusionsModulation of HSP 90 and HSP 70 expressions is a possible mechanism by which the decoction of F. leucopyrus mediates cytotoxic effects MCF-7 and MDA-MB-231 cells. This effect appears to correlate with enhanced apoptosis in these cells. In SKBR-3 cells, mechanisms other than HSP inhibition may be utilized to a greater extent by the decoction to mediate the observed cytotoxic effects. Overall findings suggest that the decoction has the potential to be exploited further for effective treatment of breast cancer.

Failure of pesticides to alter migration of cancerous and non-cancerous breast cell lines in vitro

Organochlorine pesticides are routinely used in agricultural processes across the United States.

Protein kinase D1 regulates ERα-positive breast cancer cell growth response to 17β-estradiol and contributes to poor prognosis in patients.

About 70% of human breast cancers express and are dependent for growth on estrogen receptor α (ERα), and therefore are sensitive to antiestrogen therapies. However, progression to an advanced, more aggressive phenotype is associated with acquisition of resistance to antiestrogens and/or invasive potential. In this study, we highlight the role of the serine/threonine-protein kinase D1 (PKD1) in ERα-positive breast cancers. Growth of ERα-positive MCF-7 and MDA-MB-415 human breast cancer cells was assayed in adherent or anchorage-independent conditions in cells overexpressing or depleted for PKD1. PKD1 induces cell growth through both an ERα-dependent manner, by increasing ERα expression and cell sensitivity to 17β-estradiol, and an ERα-independent manner, by reducing cell dependence to estrogens and conferring partial resistance to antiestrogen ICI 182,780. PKD1 knockdown in MDA-MB-415 cells strongly reduced estrogen-dependent and independent invasion. Quantification of PKD1 mRNA levels in 38 cancerous and non-cancerous breast cell lines and in 152 ERα-positive breast tumours from patients treated with adjuvant tamoxifen showed an association between PKD1 and ERα expression in 76.3% (29/38) of the breast cell lines tested and a strong correlation between PKD1 expression and invasiveness (P < 0.0001). In tamoxifen-treated patients, tumours with high PKD1 mRNA levels (n = 77, 50.66%) were significantly associated with less metastasis-free survival than tumours with low PKD1 mRNA expression (n = 75, 49.34%; P = 0.031). Moreover, PKD1 mRNA levels are strongly positively associated with EGFR and vimentin levels (P < 0.0000001). Thus, our study defines PKD1 as a novel attractive prognostic factor and a potential therapeutic target in breast cancer.

Abstract 1472: The differential expression of miRNAs in breast cancer cell lines

Abstract Breast cancer is a heterogenous disease that is the second leading cause of death amongst women in the United States. Triple negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by its loss of estrogen, progesterone and HER2 receptor expression which limits its targets for effective drug therapies. Micro Ribonucleic Acids (miRNAs) are a class of small, endogenous non-coding ribonucleic acids that seem to play an essential role in gene regulation. Changes in the patterns of miRNA expression profiles are serving as potential biomarkers for tumor diagnosis, prognosis of disease-specific outcomes, and the prediction of therapeutic responses in cancer. We analyzed miRNA array (Agilent) expression profiles in triple negative breast cancer cell lines (HCC70, HCC1806, MDA-MB-157) and non-cancerous (AG11132) African American women breast cell lines. We revealed that the TNBC cells exhibited a different miRNA expression pattern when compared to the normal breast cancer cell line. We analyzed the hybridization data files using standard Microsoft Excel techniques and verified our findings using PARTEK Genomics Suite. We combined the data sets from the TNBC cell lines and compared the miRNA array expression profiles to the normal control cell line AG11132. We revealed that there were significant dysregulation of miRNAs including those of the let-7 family, miR-16, miR-31, and miR-25b-1, miR34a, and miR-95 in the triple negative cells as compared to the normal cells. These miRNAs have been described in previous scientific studies to be associated with tumorigenesis in breast cancer. These studies are initial observations that we will follow up with molecular techniques such as real-time PCR and siRNA to validate the results in order to establish more direct correlations to miRNAs and their roles in triple negative breast cancer. Citation Format: Checo J. Rorie, Brianna L. Arrington, Sherette S. Godfrey, Armeshia S. McCoy, Kashenya M. Gurley. The differential expression of miRNAs in breast cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1472. doi:10.1158/1538-7445.AM2014-1472