Hs578T Breast Cancer Research Articles

Xanthone-1,2,4-triazine and Acridone-1,2,4-triazine Conjugates: Synthesis and Anticancer Activity.

A total of 21 novel xanthone and acridone derivatives were synthesized using the reactions of 1,2,4-triazine derivatives with 1-hydroxy-3-methoxy-10-methylacridone, 1,3-dimethoxy-, and 1,3-dihydroxanthone, followed by optional dihydrotiazine ring aromatization. The synthesized compounds were evaluated for their anticancer activity against colorectal cancer HCT116, glioblastoma A-172, breast cancer Hs578T, and human embryonic kidney HEK-293 tumor cell lines. Five compounds (7a, 7e, 9e, 14a, and 14b) displayed good in vitro antiproliferative activities against these cancer cell lines. Compounds 7a and 7e demonstrated low toxicity for normal human embryonic kidney (HEK-293) cells, which determines the possibility of further development of these compounds as anticancer agents. Annexin V assay demonstrated that compound 7e activates apoptotic mechanisms and inhibits proliferation in glioblastoma cells.

Controlled Release of Doxorubicin for Targeted Chemo-Photothermal Therapy in Breast Cancer HS578T Cells Using Albumin Modified Hybrid Nanocarriers.

Hybrid nanomaterials have attracted research interest owing to their intriguing properties, which may offer new diagnostic options with triggering features, able to realize a new kind of tunable nanotherapeutics. Hybrid silica/melanin nanoparticles (NPs) containing silver seeds (Me-laSil_Ag-HSA NPs) disclosed relevant photoacoustic contrast for molecular imaging. In this study we explored therapeutic function in the same nanoplatform. For this purpose, MelaSil_Ag-HSA were loaded with doxorubicin (DOX) (MelaSil_Ag-HSA@DOX) and tested to assess the efficiency of drug delivery combined with concurrent photothermal treatment. The excellent photothermal properties allowed enhanced cytotoxic activity at significantly lower doses than neat chemotherapeutic treatment. The results revealed that MelaSil_Ag-HSA@DOX is a promising platform for an integrated photothermal (PT) chemotherapy approach, reducing the efficacy concentration of the DOX and, thus, potentially limiting the several adverse side effects of the drug in in vivo treatments.

Longdaysin inhibits Wnt/β-catenin signaling and exhibits antitumor activity against breast cancer.

BackgroundCK1 is involved in regulating Wnt/β-catenin signaling and represents a promising target for the treatment of breast cancer. A purine derivative longdaysin has recently been identified as a novel modulator of cellular circadian rhythms through targeting the protein kinases CK1δ, CK1α, and ERK2. However, the antitumor activity of longdaysin and its underlying mechanisms remain unclear.MethodsThe inhibitory effect of longdaysin on Wnt/β-catenin signaling was investigated using the SuperTOPFlash reporter system. The levels of phosphorylated LRP6, total LRP6, DVL2, active β-catenin, and total β-catenin were examined by Western blot. The expression of Wnt target genes was determined using real-time PCR. The ability of colony formation of breast cancer cells was measured by colony formation assay. The effects of longdaysin on cancer cell migration and invasion were assessed using transwell assays. The effect of longdaysin on cancer stem cells was tested by sphere formation assay. The in vivo antitumor effect of longdaysin was evaluated using MDA-MB-231 breast cancer xenografts.ResultsLongdaysin suppressed Wnt/β-catenin signaling through inhibition of CK1δ and CK1ε in HEK293T cells. In breast cancer Hs578T and MDA-MB-231 cells, micromolar concentrations of longdaysin attenuated the phosphorylation of LRP6 and DVL2 and reduced the expression of active β-catenin and total β-catenin, leading to the downregulation of Wnt target genes Axin2, DKK1, LEF1, and Survivin. Furthermore, longdaysin inhibited the colony formation, migration, invasion, and sphere formation of breast cancer cells. In MDA-MB-231 breast cancer xenografts, treatment with longdaysin suppressed tumor growth in association with inhibition of Wnt/β-catenin signaling.ConclusionLongdaysin is a novel inhibitor of the Wnt/β-catenin signaling pathway. It exerts antitumor effect through blocking CK1δ/ε-dependent Wnt signaling.

Pomegranate Extract Alters Breast Cancer Stem Cell Properties in Association with Inhibition of Epithelial-to-Mesenchymal Transition

ABSTRACTCancer stem cells (CSCs) have become an important target population in cancer therapy and prevention due to their ability to self-renew, initiate tumors, and resist therapy. We examined whether pomegranate extract (PE) alters characteristics of breast CSCs. Ability to grow as mammospheres is a hallmark of breast CSCs. PE inhibited mammosphere formation in two different cell lines, neoplastic mammary epithelial HMLER and breast cancer Hs578T. In addition, mammosphere-derived cells from PE treatment groups showed reduced mammosphere formation for at least two serial passages. These data indicate that PE inhibits CSC's ability to self-renew. In addition, incubation of mammospheres with PE reversed them into adherent cultures, indicating promotion of CSC differentiation. Epithelial-to-mesenchymal transition (EMT) is a key program in generating CSCs and maintaining their characteristics. Thus, we examined the effect of PE on EMT. PE reduced cell migration, a major feature of the EMT phenotype. In addition, PE downregulated genes involved in EMT, including the EMT-inducing transcription factor Twist family basic helix-loop-helix transcription factor 1 (TWIST1). This suggests that PE suppresses CSC characteristics in part due to inhibition of EMT. The ability of PE to suppress CSCs can be exploited in the prevention of breast cancer.

Overexpression of miR-509 Increases Apoptosis and Inhibits Invasion via Suppression of Tumor Necrosis Factor-α in Triple-Negative Breast Cancer Hs578T Cells.

Triple-negative breast cancer (TNBC) is associated with high recurrence rates of metastasis and death. miR-509 has been reported to be a tumor suppressor in many cancers, but its effect in TNBC has not yet been identified. In this article, we explored the effects of miR-509 on the malignant phenotype of TNBC cells, including proliferation, apoptosis, migration, and invasion. We transiently transfected TNBC cells, Hs578T, with miR-509 mimic. Upon transfection, the expression of miR-509 was upregulated about 50-fold compared with cells transfected with scramble mimic. Overexpression of miR-509 inhibited cell proliferation, induced cell apoptosis, and suppressed cell invasion of Hs578T cells. Moreover, tumor necrosis factor-α (TNF-α) was involved in miR-509-mediated suppressive effects of TNBC cells, as being treated with TNF-α could partially abolish the suppressive effects of miR-509. Collectively, these data suggest that miR-509 could reverse the malignant phenotype of TNBC cells, probably by suppressing TNF-α.

Bioactive metabolites from Chaetomium aureum: Structure elucidation and inhibition of the Hsp90 machine chaperoning activity

Chemical investigation of the EtOAc extract of the fungus Chaetomium aureum, an endophyte of the Moroccan medicinal plant Thymelaea lythroides, afforded one new resorcinol derivative named chaetorcinol, together with five known metabolites. The structures of the isolated compounds were determined on the basis of one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry as well as by comparison with the literature. All compounds were tested for their activity towards the Hsp90 chaperoning machine in vitro using the progesterone receptor (PR) and rabbit reticulocyte lysate (RRL). Among the isolated compounds, only sclerotiorin efficiently inhibited the Hsp90 machine chaperoning activity. However, sclerotiorin showed no cytotoxic effect on breast cancer Hs578T, MDA-MB-231 and prostate cancer LNCaP cell lines. Interestingly, deacetylation of sclerotiorin increased its cytotoxicity toward the tested cell lines over a period of 48h.

Abstract P5-04-16: Extracellular matrix 1 (ECM1) knockdown alters cellular characteristics, CD44 expression and other metastasis associated genes in breast cancer cells

Abstract Introduction: ECM1 is known to be over-expressed in multiple tumor cell lines and primary tumors, including breast cancers. ECM1 overexpression is also an independent predictor of poor prognosis in primary breast carcinomas. While similar associations have also been reported in other tumors, the mechanisms by which ECM1 affects prognosis have not been elucidated. Methods: ECM1 was silenced in the Hs578t and MDA-MB231 breast cancer cell lines using siRNA. The cells were evaluated for morphological changes, migration (wound-healing assay, TScratch program), matrigel invasion and cell attachment. Real Time RT-PCR using a 94-geneTumor Metastasis Array (TaqMan), was performed to assess the effects of ECM1 downregulation on in Hs578t cells. Alterations in RNA expression were validated at the protein level using western blots. Differences between groups were evaluated using the student's t-test and p<0.05 was considered statistically significant. Results: ECM1 silenced cells demonstrated altered morphology with a more elongated appearance to the cells, when compared those transfected with non-targeting siRNA. Reduced cell migration was noted for both silenced Hs578t and MB231 cell lines (both p<0.0001). Matrigel invasion was reduced in the Hs578t cell line only (p<0.002). however, cell attachment was decreased in both cell lines. (p <0.0001 for Hs578t, and < 0.01 for MB231). ECM1 downregulation affected the expression of several metastasis associated genes including CD44, Metalloproteinase-2, fibronectin at the RNA level. CD44 expression was significantly reduced in ECM1-silenced cells by western blot. Conclusion: ECM1 appears to be involved in maintenance of tumor cell shape, migration, invasion and attachment properties in the cell lines tested, and also affects expression of CD44 protein, a known tumor prognostic factor. ECM1 may be a key molecule affecting tumor progression and metastasis, which is a complex process involving these components. Our findings suggest mechanisms by which ECM1 overexpression may affect tumor prognosis. Further work is needed to evaluate if altering ECM1 expression may be a novel therapeutic option for these tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-16.

Abstract 3123: The Notch pathway inhibits TGF-β signaling in breast cancer through HEYL-mediated crosstalk.

Abstract Acquired resistance to Transforming growth factor-β (TGF-β) is a key step in the early stages of tumorigenesis. Mutations of TGF-β signaling components, often found in other cancers, are rare in breast cancer, and little is known about the development of this resistance in breast cancer. On the other hand, activation of Notch pathway is known to play a substantial role in promoting breast cancer development. We hypothesized that crosstalk between these two pathways occurs through HEYL, a basic helix-loop-helix (bHLH) transcription factor, and a known direct target of Notch. We found that HEYL is overexpressed in 40% of breast cancers. Expression of Notch increased HEYL expression while knockdown of RBP-J, a critical mediator of Notch, reduced HEYL expression in HS578T and MDAMB-231 breast cancer cells. Taking into account the contradictory biological effects of Notch and TGF-β signaling, we sought to examine whether HEYL inhibits the TGF-β pathway. TGF-β treatment or Smad3 overexpression significantly increased the luciferase activity of the TGF-β responsive reporter vector, p3TP-Luc, and the P15 (CDKN2B) gene promoter, but this transactivation was strongly inhibited by HEYL. Bimolecular fluorescence complementation assays confirmed the interaction between Smad 3 and HEYL; immunoprecipitation assays with deletion constructs showed that the Basic domain of HEYL interacts with the MH2 domain of Smad3, and that their interaction is necessary for HEYL to inhibit TGF-β signaling. In addition, using a tet-off inducible breast cancer cell model, HEYL was shown to transcriptionally up-regulate many genes that drive metastasis and tumor angiogenesis. Supporting this concept, HEYL/HER2 double transgenic mice showed nearly 50% increase in lung metastasis (20% vs 46%) compared to HER2/neu mice. Similarly, lung colonization occurred in 100% of mice injected with MDAMB231 while HEYL-shRNAs reduced incidence to 50%. Therefore, HEYL promotes breast cancer growth and metastasis in Smad-dependent and Smad-independent mechanisms. Citation Format: Han Liangfeng, Adam Diehl, Nguyen K. Nguyen, Preethi Korangath, Teo Weiwen, Zhe Zhang, Scott Kominsky, Pedram Argani, Goran Landberg, Saraswati V. Sukumar. The Notch pathway inhibits TGF-β signaling in breast cancer through HEYL-mediated crosstalk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3123. doi:10.1158/1538-7445.AM2013-3123

Identification and characterization of a novel gene, c1orf109, encoding a CK2 substrate that is involved in cancer cell proliferation

BackgroundIn the present study we identified a novel gene, Homo Sapiens Chromosome 1 ORF109 (c1orf109, GenBank ID: NM_017850.1), which encodes a substrate of CK2. We analyzed the regulation mode of the gene, the expression pattern and subcellular localization of the predicted protein in the cell, and its role involving in cell proliferation and cell cycle control.MethodsDual-luciferase reporter assay, chromatin immunoprecipitation and EMSA were used to analysis the basal transcriptional requirements of the predicted promoter regions. C1ORF109 expression was assessed by western blot analysis. The subcellular localization of C1ORF109 was detected by immunofluorescence and immune colloidal gold technique. Cell proliferation was evaluated using MTT assay and colony-forming assay.ResultsWe found that two cis-acting elements within the crucial region of the c1orf109 promoter, one TATA box and one CAAT box, are required for maximal transcription of the c1orf109 gene. The 5′ flanking region of the c1orf109 gene could bind specific transcription factors and Sp1 may be one of them. Employing western blot analysis, we detected upregulated expression of c1orf109 in multiple cancer cell lines. The protein C1ORF109 was mainly located in the nucleus and cytoplasm. Moreover, we also found that C1ORF109 was a phosphoprotein in vivo and could be phosphorylated by the protein kinase CK2 in vitro. Exogenous expression of C1ORF109 in breast cancer Hs578T cells induced an increase in colony number and cell proliferation. A concomitant rise in levels of PCNA (proliferating cell nuclear antigen) and cyclinD1 expression was observed. Meanwhile, knockdown of c1orf109 by siRNA in breast cancer MDA-MB-231 cells confirmed the role of c1orf109 in proliferation.ConclusionsTaken together, our findings suggest that C1ORF109 may be the downstream target of protein kinase CK2 and involved in the regulation of cancer cell proliferation.

The effect of bovine milk lactoferrin on human breast cancer cell lines

The evidence that biologically active food components are key environmental factors affecting the incidence of many chronic diseases is overwhelming. However, the full extent of such components in our diet is unknown, as is our understanding of their mechanisms of action. Beyond the interaction of these food components with the gut and intestinal immune functions, whey proteins such as lactoferrin are being tested as anticancer agents. Lactoferrin is an iron-binding protein that has been reported to inhibit several types of cancer. In the present work, the effects of bovine milk lactoferrin on human breast cancer HS578T and T47D cells were studied. The cells were either untreated or treated with lactoferrin concentrations ranging from 0.125 to 125 μM. Lactoferrin decreased the cell viability of HS578T and T47D by 47 and 54%, respectively, and increased apoptosis about 2-fold for both cell lines. Proliferation rates decreased by 40.3 and 63.9% for HS578T and T47D, respectively. For the T47D line, cell migration decreased in the presence of the protein. Although the mechanisms of action are not fully known, the results gathered in this work suggest that lactoferrin interferes with some of the most important steps involved in cancer development.

The Impact of Estrogen Receptor-beta on the Actions of Endoxifen in Breast Cancer Cells.

Abstract Background: We have previously demonstrated that endoxifen is the most potent tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing ERα (Wu, Hawse et al. Cancer Res 2009;69:1722). These studies support the clinical findings which demonstrate that women with genetically impaired CYP2D6 metabolism have significantly reduced endoxifen levels and a higher risk of breast cancer recurrence. More recently, data has emerged from several laboratories suggesting that expression of ERβ in breast tumors leads to increased benefits from tamoxifen therapy. However, the role of ERβ in mediating endoxifen action has yet to be explored.Methods: Using U2OS osteosarcoma cells, as well as Hs578T and MCF7 breast cancer cells, stably expressing ERβ, we examined the effects of endoxifen on ERβ protein levels by western blotting, ERβ transcriptional activity by real-time RT-PCR, and on ERβ inhibition of breast cancer cell growth through the use of proliferation assays.Results: We have discovered that endoxifen stabilizes ERβ protein, unlike its targeted degradation of ERα, and induces ERα/ERβ heterodimerization in a concentration dependent manner. We have also found that endoxifen is a more potent inhibitor of estrogen induced gene expression in MCF7 cells stably expressing ERβ. Concentrations of endoxifen that mimic patients with impaired CYP2D6 activity (20-40 nM) do not significantly alter E2-induced cell proliferation in MCF7 cells expressing only ERα. However, these concentrations of endoxifen significantly reduce the estrogen induced proliferation rate of MCF7 cells expressing ERβ.Conclusions: Our data demonstrate that endoxifen stabilizes ERβ protein, induces ER heterodimerization and more potently inhibits the actions of estrogen in breast cancer cells expressing ERβ. The presence of ERβ sensitizes cells to the anti-estrogenic effects of endoxifen and growth inhibition occurs at substantially lower endoxifen concentrations (20-40 nM) than in cells that express only ERα (100 nm). Our data suggest that tamoxifen therapy may be beneficial even in patients with reduced CYP2D6 metabolism when ERβ is expressed. These studies also suggest that the increased effectiveness of tamoxifen therapy in patients whose tumors express ERβ may be due to the effects of endoxifen. Finally, these findings underscore the need to further elucidate the role of ERβ in the biology and treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 405.

Identification of Anaplastic Lymphoma Kinase as a Receptor for the Growth Factor Pleiotrophin

Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.

Induction of Superoxide Dismutase and Cytotoxicity by Manganese In Human Breast Cancer Cells

MnCl2induced manganese-containing superoxide dismutase (MnSOD) expression (mRNA, immunoreactive protein, and enzyme activity) in human breast cancer Hs578T cells. The induction of MnSOD immunoreactive protein in Hs578T cells was inhibited by tiron (a metal chelator and superoxide scavenger), pyruvate (a hydrogen peroxide scavenger), or 2-deoxy-d-glucose (DG, an inhibitor of glycolysis and the hexose monophosphate shunt), but not by 5,5-dimethyl-1-pyrroline-1-oxide (a superoxide scavenger),N-acetyl cysteine (a scavenger for reactive oxygen species and precursor of glutathione), diphenylene iodonium (an inhibitor of flavoproteins such as NADPH oxidase and nitric oxide synthase), or SOD (a superoxide scavenger). Northern blotting demonstrated that tiron or DG affected at the mRNA level, while pyruvate affected Mn-induced MnSOD expression at both the mRNA and protein levels. These results demonstrate that Mn can induce MnSOD expression in cultured human breast cancer cells. Mn also induced apoptosis and necrosis in these cells. Since inhibitors of Mn-induced MnSOD induction did not affect cell viability, MnSOD induction is probably not the cause of the Mn-induced cell killing.