sq-23377 and Neoplasms

The natural polyether ionophore antibiotics might be important chemotherapeutic agents for the treatment of cancer. In this article, the pharmacology and anticancer activity of the polyether ionophores undergoing pre-clinical evaluation are reviewed. Most of polyether ionophores have shown potent activity against the proliferation of various cancer cells, including those that display multidrug resistance (MDR) and cancer stem cells (CSC). The mechanism underlying the anticancer activity of ionophore agents can be related to their ability to form complexes with metal cations and transport them across cellular and subcellular membranes. Increasing evidence shows that the anticancer activity of polyether ionophores may be a consequence of the induction of apoptosis leading to apoptotic cell death, arresting cell cycle progression, induction of the cell oxidative stress, loss of mitochondrial membrane potential, reversion of MDR, synergistic anticancer effect with other anticancer drugs, etc. Continued investigation of the mechanisms of action and development of new polyether ionophores and their derivatives may provide more effective therapeutic drugs for cancer treatments.

Topics: Antineoplastic Agents; Apoptosis; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Humans; Ionophores; Neoplasms; Polyesters

Adoptive immunotherapy (AIT) of cancer with T lymphocytes may be limited by the need to activate tumor antigen-sensitized cells in vitro. In murine models, we have shown that AIT with tumor-sensitized T cells that have been pharmacologically activated with bryostatin 1 and ionomycin plus interleukin-2 can induce tumor regression. A Phase I clinical trial was carried out to assess the feasibility and toxicity associated with using tumor- or vaccine-draining lymph node cells, activated pharmacologically and expanded in culture with low-dose interleukin-2 and infused intravenously, followed by IL-2 infusion. Nine patients were entered into the trial, and six were treated as planned. Average expansion of cell numbers over 13 to 27 days in culture was 118-fold. No patient's cells reached the target cell number (2.5 x 10(10)). Infusion of these cells did not result in any unexpected toxicities. The toxicities observed were related to IL-2 infusion, and conformed to the expected range of side-effects. Based on these Phase I results, additional trials, with tumor antigen vaccine-sensitized DLN and technical modifications of the culture technique, are planned.

Topics: Adult; Bryostatins; Cells, Cultured; Female; Humans; Immunotherapy, Adoptive; Interleukin-2; Ionomycin; Lactones; Lymph Nodes; Lymphocyte Activation; Macrolides; Male; Middle Aged; Neoplasms; Pilot Projects; T-Lymphocytes

Proteomic analysis of ionomycin-treated and untreated mammary epithelial MCF10A cells elucidated differences in Ku80 cleavage. Ku80, a subunit of the Ku protein complex, is an initiator of the non-homologous, end-joining (NHEJ), double-strand breaks (DSBs) repair pathway. The nuclear Ku80 was cleaved in a calcium concentration-dependent manner by m-calpain but not by m-calpain. The cleavage of nuclear Ku80 at its α/β domain was validated by Western blotting analysis using flag-tagged expression vectors of truncated versions of Ku80 and a flag antibody and was confirmed in m-calpain knock-down cells and in vitro cell-free evaluation with recombinant proteins of calpains, Ku70, and Ku80. In addition, the cleaved Ku80 still formed a Ku heterodimer and promoted DNA DSB repair activity. Taken together, these findings indicate that translocated m-calpain enhances the NHEJ pathway through the cleavage of Ku80. Based on the present study, m-calpain in DNA repair pathways might be a novel anticancer drug target, or its mechanism might be a possible route for resistance acquisition of DNA damage-inducing chemotherapeutics.

Topics: Antineoplastic Agents; Calcium; Calcium Ionophores; Calpain; Cell Line; Cell-Free System; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Drug Resistance, Neoplasm; Epithelial Cells; Gene Knockdown Techniques; Humans; Ionomycin; Ku Autoantigen; Molecular Targeted Therapy; Neoplasms; Protein Binding; Protein Domains; Protein Multimerization; Protein Transport; Proteolysis; Proteomics; Recombinant Proteins; RNA Interference; RNA, Small Interfering

We recently reported that an N-terminally truncated retinoid X receptor-α (tRXRα) produced in cancer cells acts to promote cancer cell growth and survival through AKT activation. However, how RXRα is cleaved and how the cleavage is regulated in cancer cells remain undefined. In this study, we demonstrated that calpain II could cleave RXRα protein in vitro, generating two truncated RXRα products. The cleavage sites in RXRα were mapped by Edman N-terminal sequencing to Gly(90)↓Ser(91) and Lys(118)↓Val(119). Transfection of the resulting cleavage product RXRα/90, but not RXRα/118, resulted in activation of AKT in cancer cells, similar to the effect of tRXRα. In support of the role of calpain II in cancer cells, transfection of calpain II expression vector or its activation by ionomycin enhanced the production of tRXRα, whereas treatment of cells with calpain inhibitors reduced the levels of tRXRα. Co-immunoprecipitation assays also showed an interaction between calpain II and RXRα. In studying the regulation of tRXRα production, we observed that treatment of cells with lithium chloride or knockdown of glycogen synthase kinase-3β (GSK-3β) significantly increased the production of tRXRα. Conversely, overexpression of GSK-3β reduced tRXRα expression. Furthermore, we found that the inhibitory effect of GSK-3β on tRXRα production was due to its suppression of calpain II expression. Taken together, our data demonstrate that GSK-3β plays an important role in regulating tRXRα production by calpain II in cancer cells, providing new insights into the development of new strategies and agents for the prevention and treatment of tRXRα-related cancers.

Topics: Amino Acid Sequence; Animals; Calcium Ionophores; Calpain; Cell Line, Tumor; Down-Regulation; Enzyme Activation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Hep G2 Cells; Humans; Ionomycin; Lithium Chloride; MCF-7 Cells; Mice; Neoplasms; Proto-Oncogene Proteins c-akt; Retinoid X Receptor alpha; RNA Interference; RNA, Small Interfering; Sequence Analysis, Protein; Signal Transduction

Human VRK2 (vaccinia-related kinase 2), a kinase that emerged late in evolution, affects different signaling pathways, and some carcinomas express high levels of VRK2. Invasion by cancer cells has been associated with NFAT1 (nuclear factor of activated T cells) activation and expression of the COX-2 (cyclooxygenase 2) gene. We hypothesized that VRK proteins might play a regulatory role in NFAT1 activation in tumor cells. We demonstrate that VRK2 directly interacts and phosphorylates NFAT1 in Ser-32 within its N-terminal transactivation domain. VRK2 increases NFAT1-dependent transcription by phosphorylation, and this effect is only detected following cell phorbol 12-myristate 13-acetate and ionomycin stimulation and calcineurin activation. This NFAT1 hyperactivation by VRK2 increases COX-2 gene expression through the proximal NFAT1 binding site in the COX-2 gene promoter. Furthermore, VRK2A down-regulation by RNA interference reduces COX-2 expression at transcriptional and protein levels. Therefore, VRK2 down-regulation reduces cell invasion by tumor cells, such as MDA-MB-231 and MDA-MB-435, upon stimulation with phorbol 12-myristate 13-acetate plus ionomycin. These findings identify the first reported target and function of human VRK2 as an active kinase playing a role in regulation of cancer cell invasion through the NFAT pathway and COX-2 expression.

Topics: Animals; Binding Sites; Cell Line, Tumor; Cyclooxygenase 2; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Intracellular Signaling Peptides and Proteins; Ionomycin; Isoenzymes; Mice; Models, Biological; Muscle Proteins; Neoplasm Invasiveness; Neoplasms; NFATC Transcription Factors; Phosphorylation; Phosphoserine; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Response Elements; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic

Nonsteroidal anti-inflammatory drugs (NSAID) have shown chemopreventive effects in both preclinical and clinical studies; however, the precise molecular mechanism governing this response remains unclear. We used DNA microarray techniques to search for genes whose expression is induced by the NSAID indomethacin in human gastric carcinoma (AGS) cells. Among identified genes, we focused on those related to tight junction function (claudin-4, claudin-1, and occludin), particularly claudin-4. Induction of claudin-4 by indomethacin was confirmed at both mRNA and protein levels. NSAIDs, other than indomethacin (diclofenac and celecoxib), also induced claudin-4. All of the tested NSAIDs increased the intracellular Ca2+ concentration. Other drugs that increased the intracellular Ca2+ concentration (thapsigargin and ionomycin) also induced claudin-4. Furthermore, an intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] inhibited the indomethacin-dependent induction of claudin-4. These results strongly suggest that induction of claudin-4 by indomethacin is mediated through an increase in the intracellular Ca2+ concentration. Overexpression of claudin-4 in AGS cells did not affect cell growth or the induction of apoptosis by indomethacin. On the other hand, addition of indomethacin or overexpression of claudin-4 inhibited cell migration. Colony formation in soft agar was also inhibited. Suppression of claudin-4 expression by small interfering RNA restored the migration activity of AGS cells in the presence of indomethacin. Based on these results, we consider that the induction of claudin-4 and other tight junction-related genes by NSAIDs may be involved in the chemopreventive effect of NSAIDs through the suppression of anchorage-independent growth and cell migration.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Biomarkers, Tumor; Calcium; Cell Movement; Chemoprevention; Claudin-4; Colony-Forming Units Assay; Enzyme Inhibitors; Gene Expression Profiling; Humans; Indomethacin; Ionomycin; Ionophores; Membrane Proteins; Neoplasms; Oligonucleotide Array Sequence Analysis; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Tight Junctions; Tumor Cells, Cultured

The major histocompatibility complex nonrestricted cytotoxic leukemic T cell line T acute lymphoblastic leukemia (TALL)-104 is being pursued as a therapeutic agent for cancer. However, the receptors and effector mechanisms responsible for its broad tumoricidal function remain undefined. Here, we examined the roles played by natural cytotoxicity receptors (NCR), killer cell immunoglobulin-like receptors, cytolytic granule components, and tumor necrosis factor (TNF) family members in tumor recognition and lysis by TALL-104 cells. The perforin-granzyme pathway, TNF-related apoptosis-inducing ligand (TRAIL), and Fas were each involved in the lysis of particular tumor targets by TALL-104. Furthermore, phorbol 12-myristate 13-acetate/ionomycin treatment induced surface expression of Fas-L and TRAIL. In addition, supernatants from CD3-stimulated TALL-104 cultures exhibited antiproliferative activity, which was blocked 50-90% by anti-TNF-alpha monoclonal antibody (mAb). Although negative for the NCR natural killer (NK)p44, this cell line was found to express NKp46. An anti-NKp46 antibody strongly blocked TALL-104-mediated lysis of certain targets and directly induced cytokine production, granule release, and redirected lysis responses. Anti-NKG2D and anti-2B4 also stimulated redirected cytotoxicity by TALL-104. By contrast, anti-NKG2A mAb did not stain the cells or inhibit killing responses. Alternatively, KIR3DL2 was detected on TALL-104, and expression of its reported ligand, human leukocyte antigen (HLA)-A, on target cells provided protection from cytotoxicity. Thus, NKp46, NKG2D, and 2B4 are activating receptors, and KIR3DL2 is an inhibitory receptor on TALL-104. The data demonstrate the ability of TALL-104 cells to recognize a wide variety of tumors with NK cell receptors and kill them with a broad arsenal of cytolytic effector mechanisms, including cytolytic granules and TNF family ligands.

Topics: Adoptive Transfer; Antibodies, Monoclonal; Antigens, CD; Apoptosis Regulatory Proteins; Carcinogens; CD3 Complex; Cell Degranulation; Cytotoxicity, Immunologic; Fas Ligand Protein; Histocompatibility Antigens Class I; Humans; Immediate-Early Proteins; Ionomycin; Ionophores; Jurkat Cells; Killer Cells, Natural; Lymphocyte Activation; Membrane Glycoproteins; Monomeric GTP-Binding Proteins; Natural Cytotoxicity Triggering Receptor 1; Neoplasms; NK Cell Lectin-Like Receptor Subfamily C; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Immunologic; Receptors, KIR; Receptors, KIR3DL2; Receptors, Natural Killer Cell; Signal Transduction; Signaling Lymphocytic Activation Molecule Family; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; U937 Cells

The knowledge of tumor-associated T-cell epitopes is important for the understanding of tumor biology and the development of cancer vaccines. We describe here a biochemical approach for the identification of tumor-associated T-cell epitopes. Peptides are extracted from immunoaffinity isolated MHC class I molecules of tumor cells and separated by HPLC. The HPLC fractions are then tested for biological activity of the peptides which are then sequenced by mass spectrometry. The tumor association of the identified T-cell epitopes is confirmed using synthetic analogs and T-cells of cancer patients.

Topics: Algorithms; Antibodies, Monoclonal; Antigens, CD; Antigens, Neoplasm; Brefeldin A; Chromatography, Affinity; Chromatography, High Pressure Liquid; Databases, Protein; Epitope Mapping; Epitopes, T-Lymphocyte; Flow Cytometry; Histocompatibility Antigens Class I; Humans; Interferon-gamma; Ionomycin; Leukocytes, Mononuclear; Lymphocyte Activation; Neoplasms; Peptide Library; Peptides; Sequence Analysis, Protein; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Alterations in gene expression may represent an underlying cause of undesired side-effects mediated by the immunosuppressant cyclosporin A (CsA). We employed the method of differential display PCR to identify new genes whose expression is modulated by CsA. Human peripheral blood mononuclear cells (PBMCs), or subpopulations thereof, were simultaneously stimulated with the phorbol ester 4beta-phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. We identify the gene encoding the DNA repair enzyme DNA polymerase beta (Pol beta) as a novel CsA-sensitive transcription unit. Our data show that transcription of pol beta mRNA is induced by Ca2+ and that CsA significantly inhibits PMA/ionomycin- and ionomycin-mediated upregulation of both pol beta mRNA and Pol beta protein. The CsA-mediated inhibition of pol beta upregulation is maintained for at least 21 h after gene activation and is exerted via the phosphatase calcineurin. FK506, another immunosuppressant that targets calcineurin, also inhibits pol beta upregulation, while rapamycin competes with FK506 action. This work identifies Ca2+ as an inducer of pol beta gene activity in primary blood cells. The demonstrated CsA sensitivity of this process suggests a novel molecular mechanism that may contribute to the increased tumor incidence in patients receiving CsA treatment.

Topics: Animals; Base Sequence; Calcineurin; Calcium; Cyclosporine; DNA Polymerase beta; DNA Primers; DNA Repair; Humans; Immunosuppressive Agents; In Vitro Techniques; Ionomycin; Ionophores; Leukocytes, Mononuclear; Neoplasms; RNA, Messenger; Tacrolimus; Tetradecanoylphorbol Acetate; Up-Regulation

Human tumour-infiltrating lymphocytes (TIL) were prepared by enzyme digestion from a series of different tumours and were purified on a fluorescence-activated cell sorter (FACS II) according to their CD4+ and CD8+ phenotype. CD4+ and CD8+ TIL were stimulated separately in a low density microculture system with phytohaemagglutinin (PHA) or with ionomycin plus phorbol-12, 13-dibutyrate (PDBu). The PHA-induced proliferation of TIL was highly decreased when compared with control peripheral blood lymphocytes. A decreased proliferation of TIL was also observed when cells were stimulated with ionomycin plus PDBu, a combination which is thought to circumvent early events associated with lymphocyte activation. Some TIL were also plated in limiting dilution where they showed decreased frequencies of proliferating T cell precursors. The data suggest that one component of the inhibition of TIL must be acting 'downstream' of the early events of lymphocyte activation.

Topics: CD4-Positive T-Lymphocytes; Cell Separation; Flow Cytometry; Humans; Ionomycin; Lymphocyte Activation; Neoplasms; Phorbol 12,13-Dibutyrate; T-Lymphocytes, Cytotoxic