Breast Cancer Cell Line Hs578T Research Articles

The dual role of asporin in breast cancer progression.

Asporin has been reported as a tumor suppressor in breast cancer, while asporin-activated invasion has been described in gastric cancer. According to our in silico search, high asporin expresion associates with significantly better relapse free survival (RFS) in patients with low-grade tumors but RFS is significantly worse in patients with grade 3 tumors. In line with other studies, we have confirmed asporin expression by RNA scope in situ hybridization in cancer associated fibroblasts. We have also found asporin expression in the Hs578T breast cancer cell line which we confirmed by quantitative RT-PCR and western blotting. From multiple testing, we found that asporin can be downregulated by bone morphogenetic protein 4 while upregulation may be facilited by serum-free cultivation or by three dimensional growth in stiff Alvetex scaffold. Downregulation by shRNA inhibited invasion of Hs578T as well as of CAFs and T47D cells. Invasion of asporin-negative MDA-MB-231 and BT549 breast cancer cells through collagen type I was enhanced by recombinant asporin. Besides other investigations, large scale analysis of aspartic acid repeat polymorphism will be needed for clarification of the asporin dual role in progression of breast cancer.

Iroquois homeobox 2 suppresses cellular motility and chemokine expression in breast cancer cells.

BackgroundDisseminated tumor cells (DTCs) can be detected using ultrasensitive immunocytochemical assays and their presence in the bone marrow can predict the subsequent occurrence of overt metastasis formation and metastatic relapse. Using expression profiling on early stage primary breast tumors, low IRX2 expression was previously shown to be associated with the presence of DTCs in the bone marrow, suggesting a possible role of IRX2 in the early steps of metastasis formation. The purpose of this study is to gain insights into the significance of IRX2 protein function in the progression of breast cancer.MethodsTo assess the physiological relevance of IRX2 in breast cancer, we evaluated IRX2 expression in a large breast cancer cohort (n = 1992). Additionally, constitutive IRX2 over expression was established in BT-549 and Hs578T breast cancer cell lines. Subsequently we analyzed whether IRX2 overexpression effects chemokine secretion and cellular motility of these cells.ResultsLow IRX2 mRNA expression was found to correlate with high tumor grade, positive lymph node status, negative hormone receptor status, and basal type of primary breast tumors. Also in cell lines low IRX2 expression was associated with mainly basal breast cancer cell lines. The functional studies show that overexpression of the IRX2 transcription factor in basal cell lines suppressed secretion of the pro-metastatic chemokines and inhibited cellular motility but did not influence cell proliferation.ConclusionOur results imply that the IRX2 transcription factor might represent a novel metastasis associated protein that acts as a negative regulator of cellular motility and as a repressor of chemokine expression. Loss of IRX2 expression could therefore contribute to early hematogenous dissemination of breast cancer by sustaining chemokine secretion and enabling mobilization of tumor cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1907-4) contains supplementary material, which is available to authorized users.

The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which originally was described to be expressed in the epididymis and studied for its potential role in male fertility, is highly up-regulated in a number of carcinomas, including breast cancer. Here, we demonstrate that ADGRG2 is a functional receptor, which in transfected HEK293 cells signals with constitutive activity through the adhesion- and migration-related transcription factors serum response element (SRE) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) presumably via coupling to Gα12/13 and Gαq. However, activation of these two pathways appears to occur through distinct molecular activation mechanisms as auto-proteolytic cleavage is essential for SRE activation but not required for NFκB signaling. The overall activation mechanism for ADGRG2 is clearly distinct from the established ADGR activation mechanism as it requires the large extracellular N-terminal domain for proper intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded that the adhesion GPCR ADGRG2 is critically involved in the adhesion and migration of certain breast cancer cells through mechanisms including a non-canonical NFkB pathway and that ADGRG2 could be a target for treatment of certain types of cancer.

Glabridin inhibits cancer stem cell-like properties of human breast cancer cells: An epigenetic regulation of miR-148a/SMAd2 signaling.

In breast cancer, the cancer stem cells (CSCs) are thought to be the main cause of metastasis and recurrence. Targeting of CSCs or cancer cells with stem cell-like properties has become a new approach for the treatment of breast cancer. Glabridin (GLA), a phytochemical from the root of Glycyrrhiza glabra, exhibited effective antitumor properties in various human cancer cells. However, the roles of GLA in the regulation of CSC-like properties and the underlying molecular mechanisms remain unclear. Here, we reported that GLA attenuated the CSC-like properties through microRNA-148a (miR-148a)/transforming growth factor beta (TGFβ)-SMAD2 signal pathway in vitro and in vivo. In MDA-MB-231 and Hs-578T breast cancer cell lines, GLA enhanced the expression of miR-148a through DNA demethylation. By targeting of the SMAD2-3'-UTR, miR-148a blocked the expression/activation of SMAD2, and in turn, restored the epithelial characteristics, adhesive abilities, and CSC-like properties. Furthermore, in mouse xenograft models, we also confirmed that GLA attenuated the tumor growth, mesenchymal characteristics, and CSCs-like properties via demethylation-activated miR-148a. Our findings suggested a potential treatment strategy to reduce the CSCs-like properties, and therefore enhance the effectiveness of breast cancer therapy.

Synthesis and anti-cancer screening of novel heterocyclic-(2H)-1,2,3-triazoles as potential anti-cancer agents.

trans-Cyanocombretastatin A-4 (trans-CA-4) analogues have been structurally modified to afford their more stable CA-4-(2H)-1,2,3-triazole analogues. Fifteen novel, stable 4-heteroaryl-5-aryl-(2H)-1,2,3-triazole CA-4 analogues (8a-i, 9 and 11a-e) were evaluated for anti-cancer activity against a panel of 60 human cancer cell lines. These analogues displayed potent cytotoxic activity against both hematological and solid tumor cell lines with GI50 values in the low nanomolar range. The most potent compound, 8a, was a benzothiophen-2-yl analogue that incorporated a 3,4,5-trimethoxyphenyl moiety connected to the (2H)-1,2,3-triazole ring system. Compound 8a exhibited GI50 values of <10 nM against 80% of the cancer cell lines in the panel. Three triazole analogues, 8a, 8b and 8g, showed particularly potent growth inhibition against the triple negative Hs578T breast cancer cell line with GI50 values of 10.3 nM, 66.5 nM and 20.3 nM, respectively. Molecular docking studies suggest that these compounds bind to the same hydrophobic pocket at the interface of α- and β-tubulin that is occupied by colchicine and cis-CA-4, and are stabilized by Van der Waals' interactions with surrounding amino acid residues. Compound 8a was found to inhibit tubulin polymerization in vitro with an IC50 value of 1.7 µM. The potent cytotoxicity of these novel compounds and their inhibition of tubulin dynamics make these triazole analogues promising candidates for development as anti-cancer drugs.

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&amp;lt;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&amp;lt;0.0001). Matrigel invasion was reduced in the Hs578t cell line only (p&amp;lt;0.002). however, cell attachment was decreased in both cell lines. (p &amp;lt;0.0001 for Hs578t, and &amp;lt; 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.

Analysis of cell surface antigens by Surface Plasmon Resonance imaging

Surface Plasmon Resonance (SPR) is most commonly used to measure bio-molecular interactions. SPR is used significantly less frequent for measuring whole cell interactions. Here we introduce a method to measure whole cells label free using the specific binding of cell surface antigens expressed on the surface of cancer cells and specific ligands deposited on sensor chips using an IBIS MX96 SPR imager (SPRi). As a model system, cells from the breast cancer cell line HS578T, SKBR3 and MCF7 were used. SPRi responses to Epithelial Cell Adhesion Molecule (EpCAM) antibody and other ligands coated on the sensor chips were measured. SPR curves show a response attributable to the sedimentation of the cells and a specific binding response on top of the initial response, the magnitude of which is dependent on the ligand density and the cell type used. Comparison of SPRi with flow cytometry showed similar EpCAM expression on MCF7, SKBR3 and HS578T cells.

Deleted in breast cancer 1 (DBC1) deficiency results in apoptosis of breast cancer cells through impaired responses to UV-induced DNA damage

DBC1 (deleted in breast cancer 1) participates in the regulation of cell survival and death in response to various stimuli. In particular, DBC1 promotes cell death upon DNA damage through inhibition of SIRT1 deacetylase. However, the SIRT1-independent functions of DBC1 in the regulation of DNA damage response are less well understood. Therefore, we analyzed the DNA damage response in Hs578T breast cancer cell line in which the DBC1–SIRT1 interaction is barely detectable. DBC1–siRNA transfected cells showed a failure in the DNA damage checkpoint and the accumulation of genomic damage following UV irradiation. In addition, DBC1-deficient cells exhibited less JNK activation. Finally, the interruptions of signaling in DBC1-depleted cells contributed to cell death in response to UV irradiation. Overall, these data suggest that DBC1 is essential for a fully efficient and effective response to UV irradiation. Therefore, DBC1 plays a critical role in maintaining genomic stability and cellular integrity following UV-induced genotoxic stress.

TNF-α gene knockout in triple negative breast cancer cell line induces apoptosis.

Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine involved in the promotion and progression of cancer, including triple negative breast cancer cells. Thus, there is significant interest in understanding the molecular signaling pathways that connect TNF-α with the survival of tumor cells. In our experiments, we used as an in vitro model for triple negative breast cancer the cell line Hs578T. The purpose of this study is to determine the gene expression profiling of apoptotic signaling networks after blocking TNF-α formation by using specially designed siRNA molecules to target TNF-α messenger RNA. Knockdown of TNF-α gene was associated with cell proliferation inhibition and apoptosis, as observed by monitoring the cell index using the xCELLigence RTCA System and flow cytometry. PCR array technology was used to examine the transcript levels of 84 genes involved in apoptosis. 15 genes were found to be relevant after comparing the treated group with the untreated one of which 3 were down-regulated and 12 up-regulated. The down-regulated genes are all involved in cell survival, whereas the up-regulated ones are involved in and interact with pro-apoptotic pathways. The results described here indicate that the direct target of TNF-α in the Hs578T breast cancer cell line increases the level of certain pro-apoptotic factors that modulate different cellular networks that direct the cells towards death.

Dysadherin expression promotes the motility and survival of human breast cancer cells by AKT activation

High dysadherin expression has been recognized as a biological predictor of metastasis and poor prognosis for many different cancer types; however, the molecular mechanisms of how dysadherin affects cancer progression are still poorly understood. In this study, we examined whether AKT signaling could link dysadherin expression with downstream events that promote the metastatic potential of human breast cancer cells. Immunohistochemical analysis of breast cancer tissues showed that dysadherin expression was highly associated with elevated expression of phospho-AKT. The introduction of dysadherin cDNA into BT-474, MCF-7 and T-47D breast cancer cell lines enhanced their levels of AKT phosphorylation, while knockdown of dysadherin in MDA-MB-231 and Hs578T breast cancer cell lines suppressed AKT phosphorylation. Treatment with the AKT inhibitor triciribine suppressed dysadherin-mediated pro-metastatic effects, including epithelial-mesenchymal transition, cell motility and drug resistance. These findings suggest that dysadherin might contribute to breast cancer progression through AKT activation.

Profiling and comparative analysis of glycoproteins in Hs578BST and Hs578T and investigation of prolyl 4-Hydroxylase alpha polypeptide II expression and influence in breast cancer cells

To identify potential cancer related glycoproteins in breast cancer cells, we enriched N-linked glycoproteins by lentil lectin from the human breast cancer cell line Hs578T and the normal breast cell line Hs578BST for proteomic comparison. Glycoproteins were separated and compared by two-dimensional electrophoresis. Twenty-four glycoproteins were identified that expressed remarkably differently, among which nine were involved in the progress of collagen synthesis. Prolyl 4-hydroxylase alpha polypeptide II (P4HA2) expression and influence in breast cancer was further investigated. Immunohistochemistry revealed that P4HA2 was upregulated in breast tumor cells compared with its adjacent normal tissues. Moreover, overexpression and RNA interference of P4HA2 showed that P4HA2 expression suppressed cell proliferation and migration in Hs578T in vitro.

Abstract 486A: Biological characterization of recombinant maspin in breast cancer cells

Abstract Mammary serine protease inhibitor (maspin) is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily and is involved in mammary gland development. Loss of maspin expression is commonly observed during breast cancer progression while re-expression of maspin confers tumor suppressive capabilities. Cellular localization of maspin within the cytosol, nucleus, and association in complexes at the plasma membrane confer different properties in cancer cells adding to the complexity of maspin function. The recombinant form of maspin (rMas) when added to growth media of maspin-null cancer cells recapitulates many of the effects of exogenous maspin re-expression including inhibition of invasion through 3D matrices. These effects highlight the potential of rMas for therapeutic development as a novel adjuvant therapy. The aim of this study was to characterize the uptake, processing and distribution of wild-type and mutated forms of rMas in cancer cells. Using the MDA-MB-231, BT549 and Hs578T breast cancer cell lines we demonstrate that rMas is directly internalized by cancer cells and degraded rapidly. Time-lapse confocal microscopy using Alexa Fluor 594 labeled rMas and nuclear/cytoplasmic extraction of rMas following cellular uptake demonstrate that rMas is predominantly localized and processed in the cytosol. Wild-type rMas inhibited cancer cell invasion through collagen based matrices and this effect was independent of nuclear localization. Interestingly, site-directed mutagenesis of rMas to induce reported polymorphisms (Ile to Val 66, Pro to Ser 176, Leu to Val 187, Ile to Val 319) abrogated the ability of rMas to inhibit invasion. These preliminary experiments contribute to the characterization of rMas following internalization by cancer cells and aid in the therapeutic development of rMas as a potential therapy. Additionally, mutational data from this study support growing evidence that mutations in the maspin gene alter functionality in vitro, and may correlate to clinical outcomes in patients in which maspin is expressed in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 486A. doi:1538-7445.AM2012-486A

TGFβ1 Inhibition Increases the Radiosensitivity of Breast Cancer CellsIn Vitroand Promotes Tumor Control by RadiationIn Vivo

To determine whether inhibition of TGFβ signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. TGFβ-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFβ type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFβ signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFβ neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. Human breast cancer cell lines pretreated with TGFβ small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFβ growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFβ type I receptor kinase inhibitor. Moreover, TGFβ neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. These results show that TGFβ inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy.

Melanoma-associated antigen family protein-D4: clinical significance and functional relevance in breast cancer

Melanoma-associated antigen (MAGE) family genes are broadly expressed during development and are involved in the regulation of cell survival, cell cycle progression and apoptosis. MAGE family proteins are generally described as tumour-specific antigens and as representing ideal targets for cancer immunotherapy. In the current study, we identified melanoma-associated antigen protein-D4 (MAGE-D4), a recently characterised MAGE family member, as a new prognostic biomarker and potential therapeutic target for breast cancer. Specifically, in a whole genome microarray analysis of 103 cases of invasive breast tumours, MAGE-D4 expression was observed in 43.8% of tumours, while undetectable in normal breast tissue. Multivariate and univariate analyses also indicated MAGE-D4 expression to be associated with tumour grade, spread to lymph nodes and shortened times to relapse (P = 0.0369) and death (P = 0.0133) from time of cancer diagnosis; suggesting a role for MAGE-D4 in tumour progression. To further investigate the involvement of MAGE-D4 in breast cancer cell biology, the phenotypic effects of this gene were characterised in vitro. We observed a marked upregulation of MAGE-D4 expression - at both mRNA and protein levels - in the breast cancer cell line Hs578T compared with the syngenic Hs578Bst breast cell line. Interestingly, RNA interference-mediated knockdown of MAGE-D4 expression in Hs578T cells significantly reduced cell migration and invasion and correlated with inhibition of STAT3 and NF-κB p65 subunit phosphorylation, thus affecting two common signalling pathways involved in regulating cancer progression. Moreover, monolayer cell growth rate was not affected by MAGE-D4 gene knockdown, while growth in soft agar was significant compromised. Our results indicate that MAGE-D4 contributes to the tumorigenesis of breast cancer cells by regulating migration, invasion and anchorage-independent growth, and therefore may represent a novel target for the detection and treatment of breast cancer.

Abstract 5550: Autophagic potentiation of radiosensitization by vitamin D3 and the analog, EB1089

Abstract Introduction: While chemotherapy and radiation treatment may be initially effective at suppressing breast cancer growth, disease recurrence and tumor metastasis are major problems in the clinic. A number of strategies have been developed in an attempt to enhance the response to radiation and prevent tumor cell recovery by interfering with cytoprotective signaling. Our current studies are designed to investigate the nature of cell death when 1-25 Vitamin D3 (1,25D3) is utilized to enhance sensitivity to fractionated radiation (FIR) in the MCF-7, BT474, and Hs578t breast cancer cell lines. Results: Previous studies in our laboratory indicate that a 72 hour exposure of MCF-7 cells to 100nM of the Vitamin D3 analog, EB1089, prior to 5×2Gy FIR resulted in an 80% reduction in viable cell number over 96 hours post-irradiation. EB1089 also sensitized MCF-7 cells to FIR in terms of clonogenic survival. Although apoptosis, mitotic catastrophe and senescence are all evident under these experimental conditions, our studies strongly suggest that autophagy plays a central role in the radiosensitization effects of EB1089 as well as attenuation of the proliferative recovery that is observed with irradiation alone. To further define the role of autophagy in radiation sensitization by EB 1089, cells were exposed to the autophagy inhibitor, chloroquine, prior to EB1089 and irradiation. Chloroquine had no effect on sensitivity to radiation alone and enhanced sensitivity to tamoxifen, consistent with the cytoprotective effects of autophagy in the case of this estrogen antagonist. Inhibition by chloroquine of autophagy induced by EB1089 + FIR did not interfere with the apparent radiosensitization by EB1089, as the cells appeared to shift to an apoptotic mode of cell death. Studies are in progress utilizing genetic knockdown of the expression of autophagy related genes such as ATG5 and Beclin-1 to confirm that interference with autophagy drives the cells towards alternative modes of cell death. Additional studies in progress are designed to evaluate the effects of 1,25D3 treatment on radiation sensitivity in breast tumor cells that are resistant to irradiation such as Hs578t breast cancer cells, in addition focusing on breast tumor cells that over express Her-2/neu (MCF7/HER2, isogenic with the parental MCF-7 cell line, as well as p53 mutant BT474 cells with endogenous over expression of Her-2). Conclusions: Vitamin D irreversibly commits MCF7 breast tumor cells towards cell death upon subsequent exposure to irradiation. Inhibition of autophagy drives the cell towards alternative modes of cell death, primarily apoptosis (but possibly mitotic catastrophe). EB1089 and 1,25D3 may prove to also promote sensitivity to radiation (and chemotherapy) in breast tumor cells that are intrinsically resistant to treatment through the overexpression of cytoprotective signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5550.

Toll‐like receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors

Toll-like receptor 9 (TLR9) activates the innate immune response when exposed to non-methylated CpG-DNA. TLR9 was recently shown to be expressed by cancer cells which have been previously characterized by global hypomethylation. We set out to examine the expression and molecular activity of TLR9 in breast and ovarian cancer cells. Firstly, we confirmed higher levels of hypomethylated DNA in the serum of patients with metastatic breast cancer (n = 18) versus age-matched tumor-free women (n = 18). In breast cancer cell lines and tissues, TLR9 mRNA expression was associated with estrogen-receptor (ER) status (n = 124, P = 0.005). Expression also correlated with increasing tumor grade in both breast (P = 0.03) and ovarian cancer specimens (n = 138, P = 0.04). Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) breast cancer tissues revealed higher TLR9 protein expression in hormone-receptor (HR)-negative specimens (n = 116, P < 0.001). Using an in vitro scratch assay, we observed that cell lines transfected to overexpress TLR9 demonstrated increased cellular migration when stimulated with CpG-DNA. When assessing the molecular activity of TLR9 in breast cancer, we found a strong positive correlation of nuclear factor-kappa B (NF-kappaB) activity with TLR9 mRNA expression (correlation coefficient r = 0.7, P < 0.001). Finally, immunofluorescence analysis of BT-20 and Hs578T breast cancer cell lines showed partial colocalizations of CpG-DNA with TLR9, which diminished when the cells were exposed to methylated CpG-DNA (mCpG-DNA) or control GpC-DNA. In summary we demonstrate that TLR9 expression is associated with poor differentiation in breast and ovarian cancer specimens, and that TLR9 overexpression and stimulation with hypomethylated DNA augments the migratory capacity of cancer cell lines.

Mining of serum glycoproteins by an indirect approach using cell line secretome.

Glycosylation is the most important and abundant post-translational modification in serum proteome. Several specific types of glycan epitopes have been shown to be associated with various types of disease. Direct analysis of serum glycoproteins is challenging due to its wide dynamic range. Alternatively, glycoproteins can be discovered in the secretome of model cell lines and then confirmed in blood. However, there has been little experimental evidence showing cell line secretome as a tractable target for the study of serum glycoproteins. We used a hydrazine-based glycocapture method to selectively enrich glycoproteins from the secretome of the breast cancer cell line Hs578T. A total of 132 glycoproteins were identified by nanoLC-MS/MS analysis. Among the identified proteins, we selected 13 proteins that had one or more N-glycosylation motifs in the matched peptides, which were included in the Secreted Protein Database but not yet in the Plasma Proteome Database (PPD), and whose antibodies were commercially available. Nine out of the 13 selected proteins were detected from human blood plasma by western analysis. Furthermore, eight proteins were also detected from the plasma by targeted LC-MS/MS, which had never been previously identified by data-dependent LC-MS/MS. Our results provide novel proteins that should be enrolled in PPD and suggest that analysis of cell line secretome with subfractionation is an efficient strategy for discovering disease-relevant serum proteins.

Chemical constituents and in vitro anticancer activity of Typhonium flagelliforme (Araceae)

Aim of the study Typhonium flagelliforme is an indigenous plant of Malaysia and is used by the local communities to treat cancer. This study aims to identify the chemical constituents of Typhonium flagelliforme particularly those which have antiproliferative properties towards human cancer cell lines. Materials and methods Purification of the chemical constituents by various chromatographic procedures was guided by the antiproliferative activity. Identification of the chemical constituents was carried out by various spectroscopic techniques including high resolution MS and NMR. The antiproliferative activity was assayed using MTT on NCI-H23 (lung cancer) and HS578T (breast cancer) cell lines. Microscopic observation and DeadEnd™ colourimetric TUNEL assay was used to identify the apoptotic mode of cell death. Results and conclusion Four pheophorbide related compounds, namely pheophorbide-a, pheophorbide-a′, pyropheophorbide-a and methyl pyropheophorbide-a were identified in the most active fraction, D/F19. These constituents exhibited antiproliferative activity against cancer cells and the activity increased following photoactivation. However, the greater antiproliferative activity exhibited by D/F19 itself compared to the pheophorbides and its other subfractions suggests some form of synergistic action between the constituents. The inhibitory effect of D/F19 and the pheophorbides was apoptotic in the absence of light. Other chemical constituents that have been identified in this study include hexadecanoic acid, oleic acid, linoleic acid, linolenic acid, campesterol, stigmasterol and β-sitosterol. Most of the chemical constituents identified in this plant have not been reported previously.

Common Polymorphism in the Phosphatase PHLPP2 Results in Reduced Regulation of Akt and Protein Kinase C

PHLPP2 (PH domain leucine-rich repeat protein phosphatase 2) terminates Akt and protein kinase C (PKC) activity by specifically dephosphorylating these kinases at a key regulatory site, the hydrophobic motif (Ser-473 in Akt1). Here we identify a polymorphism that results in an amino acid change from a Leu to Ser at codon 1016 in the phosphatase domain of PHLPP2, which reduces phosphatase activity toward Akt both in vitro and in cells, in turn resulting in reduced apoptosis. Depletion of endogenous PHLPP2 variants in breast cancer cells revealed the Ser-1016 variant is less functional toward both Akt and PKC. In pair-matched high grade breast cancer samples we observed retention of only the Ser allele from heterozygous patients (identical results were observed in a pair-matched normal and tumor cell line). Thus, we have identified a functional polymorphism that impairs the activity of PHLPP2 and correlates with elevated Akt phosphorylation and increased PKC levels.

Inhibition of Human Breast Cancer Cell Growth and Enzymatic Activity by a Fermented Nutraceutical

Human breast cancer cell lines MCF-7 (ER-positive) and Hs578T (ER-negative) were cultured and one lot incubated for 48 h with 5-50 mug/ml of a fermented phytocompound (MK: Manda-Koso, Innoshima, Japan). In vitro, it appeared a dose-dependent decrease of cell viability (5-57%) in MK group in both cell lines (P < 0.001, plateau: 30 microg/ml), decreased beta-galactosidase activity, enhanced apoptosis, and inversely increased Bax/Bcl2 ratio (P < 0.01) with an upregulation of p53 (P < 0.05). In the in vivo model, Balb-c mice were inoculated with tumor cells and the treatment group was fed with 20 mg of MK. Tumor weight in MK-fed group was time-course reduced by 22% to 51% at 2 and 4 weeks, respectively (P < 0.05) with increased survival (P < 0.05). Tumour tissue of MK-fed mice showed a downregulated Bcl-2 with increased Bax/Bcl-2 ratio, reduced PCNA, and activated caspase 3. Although more studies are ongoing to foster the clinical applicability of MK integrated within a rational chemopreventive and therapeutic strategy, a p53-mediated mechanism is likely to play a relevant role, besides its reported antioxidant capacity, NK cell activity enhancement, cancer-cytostatic activity properties.