monensin and Breast-Neoplasms

Topics: Adenylyl Cyclases; Animals; Breast Neoplasms; Calcitonin; Cells, Cultured; Cyclic AMP; Humans; Kidney; Monensin; Osteoclasts; Rats; Receptors, Calcitonin; Receptors, Cell Surface; Second Messenger Systems; Tumor Cells, Cultured

Breast cancer is a malignant tumor that occurs in the epithelial tissue of the breast gland, the morbidity, and mortality of which continue to increase. Therefore, it is crucial to find new drugs to treat breast cancer. Monensin is a carrier antibiotic that has been reported to inhibit the growth of cancer cells; however, its impacts on breast cancer cells have not been reported. In this article, the cell survival rate was measured by CCK-8. Colony formation assay was utilized to detect the level of cell proliferation. Transwell was used to measure the ability of cell invasion, and wound healing was used to measure the ability of cell migration. RT-qPCR and western blot were, respectively, used to detect the expression of related genes and proteins. The level of apoptosis was detected by flow cytometry. Cell transfection technique was used for overexpressing UBA2. We found that Monensin inhibited the proliferation and migration of breast cancer cells and inhibited the expression of MMP-2 and MMP-9. In addition, Monensin promoted the apoptosis accompanied by the increase of Bax, caspase3, caspase7, and caspase9 and the decreased of bcl-2 of breast cancer cells. Monensin was also found to inhibit UBA2 expression in breast cancer cells. Subsequently, after overexpression of UBA2, the impacts of Monensin on proliferation, migration, and apoptosis of breast cancer cells was inhibited. In conclusion, Monensin can inhibit the proliferation and migration and activate apoptosis of breast cancer cells via downregulating the expression of UBA2.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line; Cell Movement; Cell Proliferation; Down-Regulation; Female; Humans; Monensin; Ubiquitin-Activating Enzymes; Wound Healing

Previously, we showed that monensin, Na+ ionophore, potently inhibited the growth of acute myelogenous leukemia and lymphoma cells. Here, we demonstrate that monensin inhibited the proliferation of solid tumor cells with IC50 of about 2.5 micro M. Monensin induced a G1 or a G2-M phase arrest in these cells. When we examined the effects of this drug on SNU-C1 cells, monensin decreased the levels of CDK2, CDK4, CDK6, cyclin D1 and cyclin A proteins. While p27 was increased by monensin, p21 was not. In addition, monensin markedly enhanced the binding of p27 with CDK2, CDK4 and CDK6. Furthermore, the activities of CDK2-, CDK4- and CDK6-associated kinase were reduced in association with hypophosphorylation of Rb protein. Monensin also induced apoptosis in solid tumor cells. Apoptotic process of SNU-C1 cells was associated with the changes of Bax, caspase-3 and mitochondria transmembrane potential (deltapsim). Taken together, these results demonstrated for the first time that monensin inhibited the growth of solid tumor cells, especially SNU-C1 cells, via cell cycle arrest and apoptosis.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspases; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Drug Screening Assays, Antitumor; Female; HeLa Cells; Humans; Inhibitory Concentration 50; Intracellular Membranes; Ionophores; Membrane Potentials; Mitochondria; Monensin; Neoplasm Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma Protein; Sodium; Tumor Cells, Cultured; Tumor Suppressor Proteins; Uterine Cervical Neoplasms

Despite intensive efforts to understand cell signaling from endosomes, there is no direct evidence demonstrating that endosomal signaling is sufficient to activate signal transduction pathways or that endosomal signaling can produce biological responses. The lack of breakthrough is due in part to the inability to generate endosomal signals in isolation from plasma membrane signals. In this Protocol, we describe a system in which epidermal growth factor (EGF) receptor (EGFR) is specifically activated when it is endocytosed into endosomes. We treated cells with EGF in the presence of AG1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks recycling of EGFR. This treatment led to the internalization of nonactivated EGF-EGFR complex into endosomes. The endosome-associated EGFR was then activated by removing AG1478 and monensin. During this procedure, we did not observe any detectable surface EGFR phosphorylation. We also achieved specific activation of endosome-associated EGFR without using monensin. Specific activation of endosome-associated EGFR provides a unique tool to study endosomal signaling of EGFR. This method may also be applied to other receptor tyrosine kinases to study whether they, too, can signal from endosomes.

Topics: Animals; Breast Neoplasms; Cell Line; Dogs; Endosomes; ErbB Receptors; Fluorescent Antibody Technique, Indirect; Humans; Kidney; Membrane Proteins; Mice; Monensin; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Quinazolines; Signal Transduction; Tumor Cells, Cultured; Tyrphostins

Tamoxifen has been reported to have numerous physiological effects that are independent of the estrogen receptor, including sensitization of resistant tumor cells to many chemotherapeutic agents. Drug-resistant cells sequester weak base chemotherapeutics in acidic organelles away from their sites of action in the cytosol and nucleus. This work reports that tamoxifen causes redistribution of weak base chemotherapeutics from acidic organelles to the nucleus in drug-resistant cells. Agents that disrupt organelle acidification (e.g., monensin, bafilomycin A1) cause a similar redistribution. Measurement of cellular pH in several cell lines reveals that tamoxifen inhibits acidification of endosomes and lysosomes without affecting cytoplasmic pH. Similar to monensin, tamoxifen decreased the rate of vesicular transport though the recycling and secretory pathways. Organellar acidification is required for many cellular functions, and its disruption could account for many of the side effects of tamoxifen.

Topics: Anti-Bacterial Agents; Biological Transport; Boron Compounds; Breast Neoplasms; Cytoplasm; Doxorubicin; Drug Resistance, Neoplasm; Endosomes; Female; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Lysosomes; Macrolides; Monensin; Neuroblastoma; Receptors, Estrogen; Tamoxifen; Transferrin; Tumor Cells, Cultured

Multidrug resistance (MDR) is a significant problem in the treatment of cancer. Chemotherapeutic drugs distribute through the cyto- and nucleoplasm of drug-sensitive cells but are excluded from the nucleus in drug-resistant cells, concentrating in cytoplasmic organelles. Weak base chemotherapeutic drugs (e.g., anthracyclines and vinca alkaloids) should concentrate in acidic organelles. This report presents a quantification of the pH for identified compartments of the MCF-7 human breast tumor cell line and demonstrates that (a) the chemotherapeutic Adriamycin concentrates in acidified organelles of drug-resistant but not drug-sensitive cells; (b) the lysosomes and recycling endosomes are not acidified in drug-sensitive cells; (c) the cytosol of drug-sensitive cells is 0.4 pH units more acidic than the cytosol of resistant cells; and (d) disrupting the acidification of the organelles of resistant cells with monensin, bafilomycin A1, or concanamycin A is sufficient to change the Adriamycin distribution to that found in drug-sensitive cells, rendering the cell vulnerable once again to chemotherapy. These results suggest that acidification of organelles is causally related to drug resistance and is consistent with the hypothesis that sequestration of drugs in acidic organelles and subsequent extrusion from the cell through the secretory pathways contribute to chemotherapeutic resistance.

Topics: Anti-Bacterial Agents; Antibiotics, Antineoplastic; Antineoplastic Agents; Breast Neoplasms; Cell Compartmentation; Doxorubicin; Drug Resistance, Multiple; Female; Humans; Hydrogen-Ion Concentration; Ionophores; Macrolides; Monensin; Tumor Cells, Cultured

Mucinous glycoproteins are present on the surfaces of tumor cells. Knowledge of which parts of the mucin molecule are accessible targets for cells of the immune system is important in the development of successful therapeutic approaches. One breast (ZR-75-1), two colon (Colo 205 and SW1116), and three pancreas (Capan-2, HPAF and SW1990) cancer cell lines were examined. The reactivities of antibodies HMFG-2, specific for the tripeptide (DTR) in the 20 amino acid tandem repeat of MUC1, and SM-3 (PDTRP) were greatly enhanced by pre-treating cells with an inhibitor of O-glycosylation, benzyl-alpha-N-acetylgalactosamide. However, desialylation of cell surfaces with neuraminidase or pre-treatment with an inhibitor of carbohydrate processing, monensin, also greatly enhanced the reactivities of HMFG-2, SM-3 and HMFG-1 (PDTR). Thus, sialic acids on termini of neighboring oligosaccharides significantly limit access to the peptide region recognized by antibodies HMFG-1/2 and SM-3.

Topics: Amino Acid Sequence; Antibodies; Breast Neoplasms; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Colonic Neoplasms; Epitopes; Glycosylation; Humans; Immunoassay; Membrane Glycoproteins; Molecular Sequence Data; Monensin; Mucin-1; Mucins; Neoplasm Proteins; Neuraminidase; Pancreatic Neoplasms; Sialic Acids; Tumor Cells, Cultured

Recombinant ricin A chain was chemically linked to monoclonal antibodies directed toward human breast cancer cells, a human T-cell differentiation antigen, and mouse transferrin receptor. Three types of immunotoxins were prepared; in two of them the antibody was linked to recombinant ricin A chain by a disulfide bond and in the third, a nonreducible thioether bond was used. Immunotoxins containing a nonreducible linkage may have some advantage over conjugates containing a reducible linkage because of improved stability in vivo. Conjugation of recombinant ricin A chain through either the endogenous thiol group or through a derivatized amino group produced immunotoxins with comparable cytotoxicity. The thioether conjugate was 1000-fold less cytotoxic to target tumor cells than the respective disulfide-linked immunotoxin. However, addition of monensin, a monocarboxylic ionophore, greatly enhanced the cytotoxicity of the thioether-linked immunotoxin. Monensin increased the immunotoxin activity better than other lysosomotropic reagents that were tested. The increase in activity of recombinant ricin A chain-containing immunotoxins mediated by monensin argues against a role for contaminating ricin B chain in potentiation.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Cell Line; Humans; Immunization, Passive; Immunotoxins; Lysosomes; Monensin; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Recombinant Proteins; Ricin

In the perspective of therapeutic approaches the monoclonal antibody, MBrl, with a quite restricted spectrum of reactivity for human breast carcinoma, was coupled to restrictocin (Res), a ribosome inactivating protein produced by Aspergillus restrictus. In a cell-free system this toxin was found to have an activity comparable to that of other plant toxins, but its in vitro toxicity was shown to be low on different cell lines. Three batches of MBr1-Res conjugate were prepared and their specificity, efficiency, and maximum level of cytotoxicity were analyzed on the cell line MCF-7 expressing the relevant antigen, on several irrelevant tumor cell lines, and on normal cells. Conjugates were from 600 to 1500 times more efficient than the uncoupled derivatized Res towards MCF-7 cells and were completely ineffective on the other target cells. The antigen-driven cytotoxicity was confirmed by the nontoxicity of an irrelevant conjugate on MCF-7 cells. The cytotoxic efficiency of MBr1-Res was low when compared to the binding level of MBr1 at the same concentration and a portion of treated cells (from 10% to 30%) survived the treatment. The heterogeneity of expression of the relevant antigen, together with its only partial internalization, could account for these limitations. The lysosomotropic agent ammonium chloride and the carboxylic ionophore monensin were tested as potentiating agents but in both cases the cytotoxicity remained unmodified. A neutralization assay performed on a xenogenic model indicated that the MBr1-Res conjugate was capable of reducing the tumor take. These data indicate the possibility of using the Res to prepare a reproducible and highly selective breast cancer conjugate. However, there are still a number of problems which must first be solved before we can consider its clinical application.

Topics: Allergens; Ammonium Chloride; Animals; Antibodies, Monoclonal; Antigens, Plant; Antineoplastic Agents; Binding Sites, Antibody; Breast Neoplasms; Cytotoxins; Drug Screening Assays, Antitumor; Drug Synergism; Fungal Proteins; Humans; Immunotoxins; Mice; Monensin; Peptides; Protein Synthesis Inhibitors; Ribonucleases; Tumor Cells, Cultured

Monensin is a carboxylic ionophore which dissipates proton gradients across cell membranes. Monensin is known to potentiate the cytotoxic activity of immunotoxins (antibody-toxin conjugates) directed against several human tumor-associated antigens. We have investigated the effect of monensin on an immunotoxin cytotoxic to the human breast cancer cell line MCF-7. This immunotoxin is composed of an antibody directed against a human breast cancer membrane antigen, and ricin A chain, which has been produced by recombinant DNA techniques. In a 16-hour cytotoxicity assay, monensin reduced 34-fold the median inhibitory dose, from 1.4 X 10(-8)M (without monensin) to 4.1 X 10(-10)M (with monensin). In timed cytotoxicity assays, 50% of control protein synthesis was reached in immunotoxin treated cells 8-fold faster in the presence of monensin (0.5 hours) than in its absence (4 hours). Monensin produced no enhancement of immunotoxin effect on a control cell line, nor on a control immunotoxin on MCF-7 cells, demonstrating specificity of monensin effect. In addition, specific immunotoxin alone or with monensin produced no toxicity on MCF-7 cells maintained at 23 degrees C. These results suggest that both binding and internalization of immunotoxin are necessary for the monensin effect. Monensin was a potent enhancer of immunotoxin effect on human breast cancer cells. This effect occurs without the presence of ricin B chain in the conjugate.

Topics: Antigens, Neoplasm; Breast Neoplasms; Cell Survival; Dose-Response Relationship, Drug; Female; Humans; Immunotoxins; Leucine; Monensin; Ricin; Temperature; Tumor Cells, Cultured

A 52K glycoprotein is secreted by human breast cancer cells in culture after estrogen stimulation. Using monoclonal antibodies, we have quantitated and characterized the corresponding proteins of the cell compartment. Using pulse-chase experiments, we have shown that about 40% of the 52K protein is secreted, the majority being successively processed into a 48K and a 34K protein. This last protein is very stable. The processing is inhibited by lysosomotropic agents and leupeptin, suggesting that it occurs in acidic vesicles, such as lysosomes or endosomes. Estradiol increased the intracellular level of immunoreactive 52K related proteins by 4-fold. Its effect is, however, more obvious in the medium, since there is a constitutive level in the cell. The stimulatory effects of estradiol on [3H]mannose and [35S]methionine incorporation into these proteins were similar and the endoglycosydase H sensitivity of the proteins was not altered, suggesting that estradiol did not modulate the glycosylation step. Antiestrogens did not stimulate synthesis and glycosylation of the 52K related proteins. Estradiol also increased the stability of the 52K precursor as well as that of total proteins. We conclude that the secreted 52K protein is the precursor of two cellular proteins of 48K and 34K. Estradiol stimulates both the intracellular accumulation of these proteins and the secretion of the precursor.

Topics: Ammonium Chloride; Antibodies, Monoclonal; Breast Neoplasms; Cell Compartmentation; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation; Glycosylation; Humans; Leupeptins; Molecular Weight; Monensin; Neoplasm Proteins; Protein Processing, Post-Translational