Fluorescent fructose derivatives for imaging breast cancer cells

Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells.     

Immunocytochemical localization in normal and transformed human cells in tissue culture using a monoclonal antibody to the src protein of the Harvey strain of murine sarcoma virus

Using a rat monoclonal antibody directed against the p21 src protein of the Harvey strain of Murine Sarcoma Virus (MSV), we have examined the reactivity of human cells in tissue culture using immunofluorescence and electron microscopy. Qualitative results indicated that untransformed mouse and human fibroblastic cells have undetectable amounts of p21; these levels were greatly increased after transformation with Harvey MSV. A group of human tumor cell lines adapted to tissue culture were examined and almost all of the epithelial tumor lines showed significant localization with this antibody. Notable exceptions were two melanoma cell lines which were negative for p21 by immunofluorescence. When normal human epithelial cells derived from esophageal or foreskin epithelium were examined, the antibody showed significant reactivity with subconfluent growing cells. After the normal cells were allowed to become quiescent, the reactivity with this antibody decreased. All of the localization seen by fluorescence was in a distribution consistent with the previously demonstrated location of p21 scr on the inner aspect of the plasma membrane. Electron microscope localization showed labeling for this antigen on the inner surface of the plasma membrane in both transformed mouse cells and in the human tumor cell lines MCF-7 and HTB-2 (RT4). These results suggest that the amounts of p21-like proteins detectable in human epithelial tumor cells do not necessarily reflect their malignant potential, but may be related to their epithelial nature. The loss of detectable localization at quiescence suggests that p21 levels decrease when these epithelial cells stop growing, and raises the possibility that an analog of p21 may be used by these human epithelial cells to regulate cell growth.     

The Function of Vacuolar ATPase (V-ATPase) a Subunit Isoforms in Invasiveness of MCF10a and MCF10CA1a Human Breast Cancer Cells

The vacuolar H⁺ ATPases (V-ATPases) are ATP-driven proton pumps that transport protons across both intracellular and plasma membranes. Previous studies have implicated V-ATPases in the invasiveness of various cancer cell lines. In this study, we evaluated the role of V-ATPases in the invasiveness of two closely matched human breast cancer lines. MCF10a cells are a non-invasive, immortalized breast epithelial cell line, and MCF10CA1a cells are a highly invasive, H-Ras-transformed derivative of MCF10a cells selected for their metastatic potential. Using an in vitro Matrigel assay, MCF10CA1a cells showed a much higher invasion than the parental MCF10a cells. Moreover, this increased invasion was completely sensitive to the specific V-ATPase inhibitor concanamycin. MCF10CA1a cells expressed much higher levels of both a1 and a3 subunit isoforms relative to the parental line. Isoforms of subunit a are responsible for subcellular localization of V-ATPases, with a3 and a4 targeting V-ATPases to the plasma membrane of specialized cells. Knockdown of either a3 alone or a3 and a4 together using isoform-specific siRNAs inhibited invasion by MCF10CA1a cells. Importantly, overexpression of a3 but not the other a subunit isoforms greatly increased the invasiveness of the parental MCF10a cells. Similarly, overexpression of a3 significantly increased expression of V-ATPases at the plasma membrane. These studies suggest that breast tumor cells employ particular a subunit isoforms to target V-ATPases to the plasma membrane, where they function in tumor cell invasion.     

Uptake of photofrin II,a photosensitizer used in photodynamic therapy,by tumour cells in vitro

Photosensitizing dyes are used in fluorescence diagnostics and photodynamic therapy (PDT). These usually hematoporphyrin derivatives (HpD) accumulate preferentially within neoplastic tissues. HpD is a mixture of ether and ester linked porphyrins. Its partially purified form is known under the commercial name of photofrin II (PII). PII emission spectra were studied in a hydrophilic (PBS) and a lipophilic (PC liposomes) environment. Red shift was observed in their emission maxima from 615 nm in buffer solution to 635 nm in lipid. Identical red shift was obtained when the intracellular fluorescence of two cancer cell lines, MCF 7 and Jurkat, incubated with PII was investigated. Thus, intramembrane localization of PII may be suggested. As determined from the total fluorescence intensity, the uptake of PII was only slightly higher for Jurkat than for MCF 7 cells. Nevertheless the kinetics of the uptake was found to be different for both cell lines.     

Differential Effect of Curcumin on the Nanomechanics of Normal and Cancerous Mammalian Epithelial Cells

Though the pharmacological activity of curcumin inhibiting the proliferation of certain cancer cells in culture was demonstrated, its effect on early-stage modifications induced in cell mechanics influencing hereby cell growth and cell adhesion are still questionable. We investigate the morphology and the elastic properties of live cultured, non-malignant human mammalian epithelial cells (HMEC) and cancerous breast epithelial cells (MCF7) by atomic force microscopy. We describe the different behavior of the two similar cell lines under curcumin treatment and we use fluorescence microscopy to identify the microtubules as the cytoskeleton structures responding to curcumin. The first changes in the HMEC cell morphology are observed after already 2 h incubation with curcumin. A 6-h long treatment leaves the MCF7 cells morphology non-affected, but the microtubules of HMEC cells disassemble and form a ring-like organization circumscribing the nuclear area. The observed morphological changes were correlated to modifications in cell’s mechanics via elasticity force mapping measurements. Curcumin treatment modified elasticity of the HMEC cells increasing the cell’s average Young’s modulus two- to threefold, especially in the cytoplasmic area. Contrariwise, a slight decrease in the Young’s modulus was noticed for the MCF7 cells, as they become softer due to the action of curcumin. Chemotherapeutic drugs exert their effect via the perturbation of the dynamic instability of the microtubule, hence the cell-specific perturbation induced by curcumin can help in future understanding of drug induced events on the cell behavior.     

Expression of the protein phosphatase 1 inhibitor KEPI is downregulated in breast cancer cell lines and tissues and involved in the regulation of the tumor suppressor EGR1 via the MEK-ERK pathway

KEPI is a protein kinase C-potentiated inhibitory protein for type 1 Ser/Thr protein phosphatases. We found no or reduced expression of KEPI in breast cancer cell lines, breast tumors and metastases in comparison to normal breast cell lines and tissues, respectively. KEPI protein expression and ubiquitous localization was detected with a newly generated antibody. Ectopic KEPI expression in MCF7 breast cancer cells induced differential expression of 95 genes, including the up-regulation of the tumor suppressors EGR1 (early growth response 1) and PTEN (phosphatase and tensin homolog), which is regulated by EGR1. We further show that the up-regulation of EGR1 in MCF7/KEPI cells is mediated by MEK-ERK signaling. The inhibition of this pathway by the MEK inhibitor UO126 led to a strong decrease in EGR1 expression in MCF7/KEPI cells. These results reveal a novel role for KEPI in the regulation of the tumor suppressor gene EGR1 via activation of the MEK-ERK MAPK pathway.     

Curcumin induces apoptosis in human gastric carcinoma AGS cells and colon carcinoma HT-29 cells through mitochondrial dysfunction and endoplasmic reticulum stress

In the present study, we investigate the effect of curcumin, a major active component isolated from rhizomes of Curcuma longa, on the cytotoxicity of three human carcinoma cell lines (AGS, HT-29 and MGC803) in gastrointestinal tract and a normal gastric epithelial cell line GES-1, and the mechanism of curcumin-induced apoptosis. The results indicated that curcumin inhibited the gastrointestinal carcinoma cell growth in a dose-dependent manner and cytotoxicity was more towards the gastric carcinoma cell AGS and colon carcinoma cell HT-29 compared to normal gastric cell GES-1, and increased externalization of phosphatidylserine residue was observed by Annexin V/PI staining in the two cell lines. Treatment of AGS and HT-29 cells with curcumin enhanced the cleavage of procaspase-3, -7, -8 and -9. Meanwhile, curcumin induced endoplasmic reticulum (ER) stress and mitochondrial dysfunction as evidenced by up-regulation of CCAAT/enhancer binding protein homologous protein (CHOP), phosphorylation of JNK and down-regulation of SERCA2ATPase, release of cytochrome c, decrease of Bcl-2 and reduction of mitochondrial membrane potential in both AGS and HT-29 cells. Overexpression of bax, total JNK, phospho-FADD and total FADD were also observed in curcumin-treated HT-29 cells. Moreover, curcumin decreased cytosolic and ER Ca²⁺, but increased mitochondrial Ca²⁺ in the two cell lines. 2-Aminoethoxydiphenyl borate, an antagonist of inositol 1, 4, 5-triphosphate receptor, partly blocked curcumin-induced cytosolic Ca²⁺ decrease in AGS and HT-29 cells. Additionally, carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial Ca²⁺ uptake, reversed curcumin-triggered AGS and HT-29 cells growth inhibition. siRNA to CHOP markedly reduced curcumin-induced apoptosis. These results suggest that curcumin can impact on ER stress and mitochondria functional pathways in AGS and HT-29 cells, death receptor pathway was also involved in curcumin-treated HT-29 cells, thus identifying specific well-defined molecular mechanisms that may be targeted by therapeutic strategies.     

Cytoskeleton alterations induced by Geodia corticostylifera depsipeptides in breast cancer cells

Crude extracts of the marine sponge Geodia corticostylifera from Brazilian Coast have previously shown antibacterial, antifungal, cytotoxic, haemolytic and neurotoxic activities. The present work describes the isolation of the cyclic peptides geodiamolides A, B, H and I (1-4) from G. corticostylifera and their anti-proliferative effects against sea urchin eggs and human breast cancer cell lineages. Its structure-activity relationship is discussed as well. In an initial series of experiments these peptides inhibited the first cleavage of sea urchin eggs (Lytechinus variegatus). Duplication of nuclei without complete egg cell division indicated the mechanism of action might be related to microfilament disruption. Further studies showed that the geodiamolides have anti-proliferative activity against human breast cancer cell lines (T47D and MCF7). Using fluorescence techniques and confocal microscopy, we found evidence that the geodiamolides A, B, H and I act by disorganizing actin filaments of T47D and MCF7 cancer cells, in a way similar to other depsipeptides (such as jaspamide 5 and dolastatins), keeping the normal microtubule organization. Normal cells lines (primary culture human fibroblasts and BRL3A rat liver epithelial cells) were not affected by the treatment as tumor cells were, thus indicating the biomedical potential of these compounds.     

Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2.

Neu differentiation factor (NDF)-induced signaling involves the activation of members of the ErbB family of receptor tyrosine kinases. Although ectopic expression of recombinant ErbB receptors has yielded valuable insight into their signaling properties, the biological function and in vivo interplay of these receptors are still poorly understood. We addressed this issue by studying NDF signaling in various human cell lines expressing moderate levels of all known ErbB receptors. NDF-induced phosphorylation of ErbB-2 and ErbB-3 was found in the breast epithelial cell line MCF10A, the breast tumor cell lines T47D and MCF7, and the ovarian tumor cell line OVCAR3. Despite similar expression levels, NDF-induced phosphorylation of ErbB-4 was cell specific and only detected in T47D and OVCAR3 cells. Blocking cell surface expression of ErbB-2 by intracellular expression of a single-chain antibody revealed that in these two cell lines, ErbB-2 significantly enhanced phosphorylation of ErbB-4. Efficient NDF-induced phosphorylation of ErbB-3 was strictly ErbB-2 dependent in the breast tumor cell lines T47D and MCF7, while it was largely ErbB-2 independent in MCF10A and OVCAR3 cells. Consequently, NDF-stimulated intracellular signaling and induction of a biological response displayed a cell-specific requirement for ErbB-2. Thus, while ErbB-2 cooperates with NDF receptors in the breast tumor cell lines, ErbB-2 independent mechanisms seem to prevail in other cellular contexts.     

Curcumin induces concentration-dependent alterations in mitochondrial function through ROS in C2C12 mouse myoblasts

Curcumin exhibits antioxidant properties in normal cells where the uptake is low, unlike in tumor cells where uptake is high and curcumin increases reactive oxygen species (ROS) production and cell death. Mitochondria are the main source and primary target of cellular ROS. We hypothesized that curcumin would regulate cellular redox status and mitochondrial function, depending on cell sensitivity and/or curcumin concentration in normal cells. We examined the differences between low and high concentrations of curcumin, with specific attention focused on ROS levels, mitochondrial function, and cell viability in mouse C2C12 myoblast under normal and simulated conditions of diabetes. Cells incubated with high concentrations of curcumin (10–50 μM) resulted in decreased cell viability and sustained robust increases in ROS levels. Mechanistic studies showed that increased ROS levels in cells incubated with 20 μM curcumin induced opening of mitochondrial permeability transition pores and subsequent release of cytochrome c, activation of caspases 9 and 3/7, and apoptotic cell death. Low concentrations of curcumin (1–5 μM) did not affect cell viability, but induced a mild increase in ROS levels, which peaked at 2 hr after the treatment. Incubation with 5 μM curcumin also induced ROS-dependent increases in mitochondrial mass and membrane potential. Finally, pretreatment with 5 μM curcumin prevented high glucose-induced oxidative cell injury. Our study suggests that mitochondria respond differentially depending on curcumin concentration-dependent induction of ROS. The end result is either cell protection or death. Curcumin may be an effective therapeutic target for diabetes and other mitochondrial diseases when used in low concentrations.     

Heterogeneous breast tumoroids: An in vitro assay for investigating cellular heterogeneity and drug delivery

Breast tumors are typically heterogeneous and contain diverse subpopulations of tumor cells with differing phenotypic properties. Planar cultures of cancer cell lines are not viable models of investigation of cell-cell and cell-matrix interactions during tumor development. This article presents an in vitro coculture-based 3-dimensional heterogeneous breast tumor model that can be used in drug resistance and drug delivery investigations. Breast cancer cell lines of different phenotypes (MDAMB231, MCF7, and ZR751) were cocultured in a rotating wall vessel bioreactor to form a large number of heterogeneous tumoroids in a single cell culture experiment. Cells in the rotating vessels were labeled with Cell Tracker fluorescent probes to allow for time course fluorescence microscopy to monitor cell aggregation. Histological sections of tumoroids were stained with hematoxylin and eosin, progesterone receptor, E-cadherin (E-cad), and proliferation marker ki67. In vitro tumoroids developed in this study recapture important features of the temporal-spatial organization of solid tumors, including the presence of necrotic areas at the center and higher levels of cell division at the tumor periphery. E-cad-positive MCF7 cells form larger tumoroids than E-cad-negative MDAMB231 cells. In heterogeneous tumors, the irregular surface roughness was mainly due to the presence of MDAMB231 cells, whereas MCF7 cells formed smooth surfaces. Moreover, when heterogeneous tumoroids were placed onto collagen gels, highly invasive MDAMB231 cell-rich surface regions produced extensions into the matrix, whereas poorly invasive MCF7 cells did not. The fact that one can form a large number of 1-mm tumoroids in 1 coculture attests to the potential use of this system at high-throughput investigations of cancer drug development and drug delivery into the tumor.     

Raft-dependent endocytosis of autocrine motility factor is phosphatidylinositol 3-kinase-dependent in breast carcinoma cells

Autocrine motility factor (AMF) is internalized via a receptor-mediated, dynamin-dependent, cholesterol-sensitive raft pathway to the smooth endoplasmic reticulum that is negatively regulated by caveolin-1. Expression of AMF and its receptor (AMFR) is associated with tumor progression and malignancy; however, the extent to which the raft-dependent uptake of AMF is tumor cell-specific has yet to be addressed. By Western blot and cell surface fluorescence-activated cell sorter (FACS) analysis, AMFR expression is increased in tumorigenic MCF7 and metastatic MDA-231 and MDA-435 breast cancer cell lines relative to dysplastic MCF10A mammary epithelial cells. AMF uptake, determined by FACS measurement of protease-insensitive internalized fluorescein-conjugated AMF, was increased in MCF7 and MDA-435 cells relative to MCF-10A and caveolin-1-expressing MDA-231 cells. Uptake of fluorescein-conjugated AMF was dynamin-dependent, methyl-beta-cyclodextrin- and genistein-sensitive, reduced upon overexpression of caveolin-1 in MDA-435 cells, and increased upon short hairpin RNA reduction of caveolin-1 in MDA-231 cells. Tissue microarray analysis of invasive primary human breast carcinomas showed that AMFR expression had no impact on survival but did correlate significantly with expression of phospho-Akt. Phospho-Akt expression was increased in AMF-internalizing MCF7 and MDA-435 breast carcinoma cells. AMF uptake in these cells was reduced by phosphatidylinositol 3-kinase inhibition but not by regulators of macropinocytosis such as amiloride, phorbol ester, or actin cytoskeleton disruption by cytochalasin D. The raft-dependent endocytosis of AMF therefore follows a distinct phosphatidylinositol 3-kinase-dependent pathway that is up-regulated in more aggressive tumor cells.     

Quantitative fluorescence cytometric analysis of Bcl-2 levels in tumor cells exhibiting a wide range of inherent Bcl-2 protein expression: correlation with Western blot analysis

BACKGROUND: A protocol to measure a wide range of Bcl-2 protein expression using quantitative fluorescence cytometry (QFCM) in different cell types was developed for use with flow cytometry. Bcl-2 measurements obtained by flow cytometry were correlated with Western blot Bcl-2 measurements to confirm specificity of the Bcl-2-FITC staining. This protocol was applied to measure absolute levels of Bcl-2 protein in different tumor cell lines including Bcl-2-transfected breast carcinoma cell lines and in peripheral blood lymphocytes (PBL). METHODS: HL-60, K562, DOHH2, Jurkat, MDA435/LCC6, MCF7 cell lines, and PBL derived from normal donors were fixed, permeabilized, stained with anti-Bcl-2-FITC antibody and evaluated by QFCM. In parallel, the same cells were evaluated for Bcl-2 protein expression by Western blot analysis. Mitochondrial localization of anti-Bcl-2-FITC antibody inside cells was confirmed using fluorescence imaging microscopy. RESULTS: Bcl-2 expression in different cell types could be accurately quantified based on antibody-binding capacity (ABC) ranging from 12.6 x 10(3) antibody-binding sites in HL-60 cells to 1.64 x 10(6) antibody-binding sites in a Bcl-2-transfected MDA435/LCC6 clone. The data from flow cytometry analysis correlated well with Western analysis (R(2) = 0.78). Bcl-2-FITC staining colocalized with dyes specific for mitochondria. CONCLUSIONS: The Bcl-2 staining protocol described here was shown to be specific, sensitive, and it was able to provide higher resolution as well as more reproducible quantitation of Bcl-2 protein content in cells when compared with Western blot methods. Quantitation of Bcl-2 content in cells by QFCM may be useful for monitoring Bcl-2 expression in cells undergoing various treatments in vitro and in vivo.     

Curcumin analogue 1,5-bis(4-hydroxy-3-((4-methylpiperazin-1-yl)methyl)phenyl)penta-1,4-dien-3-one mediates growth arrest and apoptosis by targeting the PI3K/AKT/mTOR and PKC-theta signaling pathways in human breast carcinoma cells

Recent developments in the literature have demonstrated that curcumin exhibit antioxidant properties supporting its anti-inflammatory, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Despite the valuable findings of curcumin against different cancer cells, the clinical use of curcumin in cancer treatment is limited due to its extremely low aqueous solubility and instability, which lead to poor in vivo bioavailability and limited therapeutic effects. We therefore focused in the present study to evaluate the anti-tumor potential of curcumin analogues on the human breast carcinoma cell lines MDA-MB-231 and MCF-7, as well as their effects on non-tumorigenic normal breast epithelial cells (MCF-10). The IC₅₀ values of curcumin analogue J1 in these cancer cell lines were determined to be 5 ng/ml and 10 ng/ml, in MDA-MB-231 and MCF-7 cells respectively. Interestingly, at these concentrations, the J1 did not affect the viability of non-tumorigenic normal breast epithelial cells MCF-10. Furthermore, we found that J1 strongly induced growth arrest of these cancer cells by modulating the mitochondrial membrane potentials without significant effect on normal MCF-10 cells using JC-1 staining and flow cytometry analysis. Using annexin-V/PI double staining assay followed by flow cytometry analysis, we found that J1 robustly enhanced the induction of apoptosis by increasing the activity of caspases in MDA-MB-231 and MCF-7 cancer cells. In addition, treatment of breast cancer cells with J1 revealed that, in contrast to the expression of cyclin B1, this curcumin analogue vigorously decreased the expression of cyclin A, CDK2 and cyclin E and subsequently sensitized tumor cells to cell cycle arrest. Most importantly, the phosphorylation of AKT, mTOR and PKC-theta in J1-treated cancer cells was markedly decreased and hence affecting the survival of these cancer cells. Most interestingly, J1-treated cancer cells exhibited a significant inhibition in the activation of RhoA followed by reduction in actin polymerization and cytoskeletal rearrangement in response to CXCL12. Our data reveal the therapeutic potential of the curcumin analogue J1 and the underlying mechanisms to fight breast cancer cells.     

Apoptotic behaviour of hepatic and extra-hepatic tumor cell lines differs after Fas stimulation.

Fas-induced apoptosis is one form of programmed cell death responsible for hepatocyte demise. However, the role of this cell surface receptor in the death of tumoral hepatic cells is still being debated. It has been shown that some hepatoma cell lines may escape apoptosis because of abnormal Fas localization correlated with non-functionality of the Fas protein or dysfunctionality in the Fas pathway cascade. The aim of this study was to investigate the behaviour of four hepatoma cell lines, HepG2, Hep3B, SKHep1 and Chang-Liver and two extrahepatic cell lines, MCF7, a mammary tumoral cell line and OVCAR-3, an ovarian tumoral cell line, when they were treated with an agonistic anti-Fas antibody alone, with interferon gamma (IFNgamma), an up-regulator of Fas protein expression, alone or with a combination of both agents. We first performed immunofluorescence and flow cytometry to confirm that Fas was present on the cell surface of each cell line in the normal state. Apoptosis was then investigated after induction with the various treatments, by DAPI staining, agarose gel DNA electrophoresis and PARP cleavage. Caspase 8 and 3 expression, as well as two anti-apoptotic proteins Bcl-2 and HSP70, and one proapoptotic protein Bax were also investigated by immunoblot allowing identification of several apoptotic pathways based on the behaviour of the different studied proteins. HepG2 and OVCAR-3 cells were sensitive to the anti-Fas antibody alone. Hep3B was resistant to Fas-induced apoptosis but sensitive to IFNgamma-induced apoptosis. MCF7 was resistant to anti-Fas antibody and IFNgamma Chang-Liver and SKHep1 were sensitive to IFNgamma and anti-Fas antibody but at different degrees. Chang-Liver used the Fas and IFNgamma pathways, while SKHep1 involved mostly the Fas pathway. These results show that each tumor cell line is characterized by different apoptotic behaviour in relation to Fas and/or IFNgamma-induced apoptosis. In addition, despite the high level of Bcl-2 and HSP70 proteins in the tumoral cells investigated here, they were not fully protected against apoptosis, except for MCF7. This emphasizes the necessity to analyse the different proteins responsible for apoptosis to adapt anti-tumoral therapeutics.     

Global enhancement of nuclear localization-dependent nuclear transport in transformed cells

Fundamental to eukaryotic cell function, nucleocytoplasmic transport can be regulated at many levels, including through modulation of the importin/exportin (Imp/Exp) nuclear transport machinery itself. Although Imps/Exps are overexpressed in a number of transformed cell lines and patient tumor tissues, the efficiency of nucleocytoplasmic transport in transformed cell types compared with nontransformed cells has not been investigated. Here we use quantitative live cell imaging of 3 isogenic nontransformed/transformed cell pairs to show that nuclear accumulation of nuclear localization signal (NLS)-containing proteins, but not their NLS-mutated derivatives, is increased up to 7-fold in MCF10CA1h human epithelial breast carcinoma cells and in simian virus 40 (SV40)-transformed fibroblasts of human and monkey origin, compared with their nontransformed counterparts. The basis for this appears to be a significantly faster rate of nuclear import in transformed cell types, as revealed by analysis using fluorescence recovery after photobleaching for the human MCF10A/MCF10CA1h cell pair. Nuclear accumulation of NLS/nuclear export signal-containing (shuttling) proteins was also enhanced in transformed cell types, experiments using the nuclear export inhibitor leptomycin B demonstrating that efficient Exp-1-mediated nuclear export was not impaired in transformed compared with nontransformed cells. Enhanced nuclear import and export efficiencies were found to correlate with 2- to 4-fold higher expression of specific Imps/Exps in transformed cells, as indicated by quantitative Western blot analysis, with ectopic expression of Imps able to enhance NLS nuclear accumulation levels up to 5-fold in nontransformed MCF10A cells. The findings indicate that transformed cells possess altered nuclear transport properties, most likely due to the overexpression of Imps/Exps. The findings have important implications for the development of tumor-specific drug nanocarriers in anticancer therapy.     

Pharmacokinetics of (111)In-labeled triplex-forming oligonucleotide targeting human N-myc gene

The radiolabeled triplex-forming oligonucleotide (TFO) demonstrated the potential for sequence-specific DNA binding and destruction. In this study, by selecting the polypurine-polypyrimidine stretch (2950-2978) in the human N-myc gene as a target, the (111)In-labeled TFO targeting human N-myc gene (N-mycTFO(111)In) was tested for its cellular uptake and nuclear localization in vitro and in vivo. This is because the deregulated N-myc expression is strongly implicated in the pathogenesis of several important human malignancies, including breast carcinoma and neuroblastoma. N-mycTFO(111)In was bound selectively to the N-myc sequence in vitro. The total cellular uptake of TFO after the incubation of various normal and cancer cells with TFO for 24 h was 20-54.8% of the injected dose (%ID), and the nuclear localization was 6.59-30.0%ID, depending on cell lines. The highest cellular uptake was found in the human neuroblastoma SK-N-DZ (54.8%ID), human mammary ductal carcinoma T47-D (54%ID), human acute T cell leukemia Jurkat (54%ID), and multidrug-resistant human breast adenocarcinoma MCF7/TH (49.5%ID). The lowest was in the human normal mammary epithelium MCF10A (20.0%ID). The highest nuclear localization was found in MCF7/TH (30%ID) and SK-N-DZ (28.7%ID). The lowest was in MCF11A (6.59%ID). We next injected TFO into human mammary tumor-xenografted Balb/c nude mice. Tumor targeting of TFO in vivo reached its maximum peak 5 h after the intravenous injection in three types of tumor models. They are 21.0 +/- 3.23%ID per gram of tissue (%ID/g) for MCF7/TH, 7.77 +/- 2.11%ID/g for MCF7, and 4.53 +/- 1.20%ID/g for MCF10A. The TFO blood level decreased from 8.00 +/- 0.90%ID/g 15 min after the injection, to 1.30 +/- 0.30%ID/g after 19 h. The kidney TFO level increased rapidly from 5.93 +/- 0.94%ID/g after 15 min, to 25.1 +/- 5.60%ID/g after 19 h. A high TFO level (19.7-24.5%ID/g) in the liver was maintained until 19 h after the injection. Therefore, we suggest that the (111)In-labeled N-myc-targeting TFO, a promising modality for nanoexplosive gene therapy, could effectively target the nucleus of the multidrug-resistant breast carcinoma MCF7/TH in vitro and in vivo. It has approximately 130 min of half-life of blood TFO.     

Hugl1 and Hugl2 in Mammary Epithelial Cells: Polarity,Proliferation, and Differentiation

Loss of epithelial polarity is described as a hallmark of epithelial cancer. To determine the role of Hugl1 and Hugl2 expression in the breast, we investigated their localization in human mammary duct tissue and the effects of expression modulation in normal and cancer cell lines on polarity, proliferation and differentiation. Expression of Hugl1 and Hugl2 was silenced in both MCF10A cells and Human Mammary Epithelial Cells and cell lines were grown in 2-D on plastic and in 3-D in Matrigel to form acini. Cells in monolayer were compared for proliferative and phenotypic changes while acini were examined for differences in size, ability to form a hollow lumen, nuclear size and shape, and localization of key domain-specific proteins as a measure of polarity. We detected overlapping but distinct localization of Hugl1 and Hugl2 in the human mammary gland, with Hugl1 expressed in both luminal and myoepithelium and Hugl2 largely restricted to myoepithelium. On a plastic surface, loss of Hugl1 or Hugl2 in normal epithelium induced a mesenchymal phenotype, and these cells formed large cellular masses when grown in Matrigel. In addition, loss of Hugl1 or Hugl2 expression in MCF10A cells resulted in increased proliferation on Matrigel, while gain of Hugl1 expression in tumor cells suppressed proliferation. Loss of polarity was also observed with knockdown of either Hugl1 or Hugl2, with cells growing in Matrigel appearing as a multilayered epithelium, with randomly oriented Golgi and multiple enlarged nuclei. Furthermore, Hugl1 knock down resulted in a loss of membrane identity and the development of cellular asymmetries in Human Mammary Epithelial Cells. Overall, these data demonstrate an essential role for both Hugl1 and Hugl2 in the maintenance of breast epithelial polarity and differentiated cell morphology, as well as growth control.