Ori Braitbard

Cytotoxic salan-titanium(IV) complexes: high activity toward a range of sensitive and drug-resistant cell lines, and mechanistic insights.

The cytotoxicities of highly efficient salan–TiIV complexes toward a range of cell lines, including drug‐resistant cells, are reported along with preliminary mechanistic insights. Five salan–TiIV complexes were investigated toward eight different human and murine cancer‐derived cell lines, including colon, ovarian, lung, cervical, pancreatic, leukemic, skin, and breast. The salan complexes are more active toward the cells analyzed than cisplatin and the known titanium compound (bzac)2Ti(OiPr)2, and no cell line resistant to the salan complexes was identified. Moreover, the salan–TiIV complexes are highly active toward both cisplatin‐sensitive (A2780) and cisplatin‐resistant (A2780CisR) human ovarian cancer cell lines. Similarly, the salan complexes are cytotoxic toward multi‐drug‐resistant (ABCB1‐expressing) mouse lymphoma cell lines HU‐1 and HU‐2. Importantly, minimal or no activity was observed toward primary murine cells (bone marrow, heart, liver, kidney, spleen, and lung), supporting selectivity for cancer cells. Additionally, the salan complexes maintain high cytotoxicity for up to 24 h following exposure to cell culture medium, whereas reference complexes (bzac)2Ti(OiPr)2 and Cp2TiCl2 rapidly lose much of their activity upon exposure to medium, within ∼1 h. The upregulation of p53 followed by cell‐cycle arrest in G1 phase is likely one mechanism of action of the salan complexes. Taken together, the results indicate that these compounds are selectively toxic to cancer cells and are able to circumvent two independent mechanisms of drug resistance, thus expanding the scope of their potential medicinal utility.

Anti‐proliferative Activity of Nano‐Formulated Phenolato Titanium(IV) Complexes Against Cancer Cells

Nanoparticles of titanium(IV) complexes of phenolato ligands were formed and evaluated for cytotoxicity toward human HT‐29 colon cancer, murine T‐25 lymphoma, and murine HU‐2 multidrug‐resistant (MDR) cells. The nano‐formulation, besides increasing the complexes′ shelf lives, is particularly efficient in overcoming limitations in solubility and cell‐penetration, thus enhancing biological accessibility; large complexes that were inactive when measured in a non‐formulated form showed marked activity when nano‐formulated. For active and accessible small complexes, the effect of the formulation was negligible. Most complexes showed similar activity toward MDR cells and their drug‐sensitive analogues, further increasing their therapeutic potential. An exception is a particularly hydrophobic complex, which is presumably more accessible to interaction with the membrane ABCB1 (MDR1) transporter active in the multidrug resistance of HU‐2 cells. The most efficient compound is a mononuclear complex of a single hexadentate ligand, combining particularly high activity and hydrolytic stability with accessibility aided by the nano‐formulation.

Highly Effective and Hydrolytically Stable Vanadium(V) Amino Phenolato Antitumor Agents

Vanadium(V) oxo complexes with no labile ligands, including six octahedral complexes with pentadentate diaminotris(phenolato) ligands and one pentacoordinate complex with a tetradentate aminotris(phenolato) ligand, were synthesized in high yields. All octahedral complexes demonstrated high hydrolytic stability with no signs of decomposition after days in the presence of water, whereas the pentacoordinate complex decomposed within minutes to release the free ligand, demonstrating the marked impact of coordination number and geometry on the complex electrophilicity. All complexes showed marked cytotoxicity toward human colon HT-29 and ovarian OVCAR-3 cells. In particular, the octahedral complexes exhibited especially high activity, higher than that of cisplatin by up to 200-fold. Selected complexes demonstrated similarly high activity also toward the A2780 and the A2780cis cisplatin-resistant line. High cytotoxicity was also recorded after prolonged incubation in a DMSO solution at 4 and 37 °C temperatures and in biological medium. In vivo studies pointed to high efficacy in reducing tumor size, where no clinical signs of toxicity were detected in the treated mice. These results overall indicate high potential of the tested compounds as antitumor agents.

Specific Design of Titanium(IV) Phenolato Chelates Yields Stable and Accessible, Effective and Selective Anticancer Agents

Octahedral titanium(IV) complexes of phenolato hexadentate ligands were developed and showed very high stability for days in water solutions. In vitro cytotoxicity studies showed that, whereas tetrakis(phenolato) systems are generally of low activity presumably due to inaccessibility, smaller bis(phenolato)bis(alkoxo) complexes feature high anticancer activity and accessibility even without formulations, also toward a cisplatin-resistant cell line. An all-aliphatic control complex was unstable and inactive. A leading phenolato complex also revealed: 1) high durability in fully aqueous solutions; accordingly, negligible loss of activity after preincubation for three days in medium or in serum; 2) maximal cellular accumulation and induction of apoptosis following 24-48 h of administration; 3) reduced impact on noncancerous fibroblast cells; 4) in vivo efficacy toward lymphoma cells in murine model; 5) high activity in NCI-60 panel, with average GI50 of 4.6±2 μm. This newly developed family of Ti(IV) complexes is thus of great potential for anticancer therapy.

The signal peptide of mouse mammary tumor virus-env: a phosphoprotein tumor modulator.

Mouse mammary tumor virus (MMTV) is associated primarily with mammary carcinomas and lymphomas. The signal peptide of the MMTV envelope precursor is uniquely targeted to nucleoli of cells that harbor the virus, where it can function as a nuclear export factor for intron-containing transcripts. Antibodies to this signal peptide, which we refer to as p14, were previously shown to label nucleoli in a subset of human breast cancers. To look for additional cellular functions of p14, different mutants were ectopically expressed in the MCF-7 human breast cancer cell line. This approach identified motifs responsible for its nucleolar targeting, nucleocytoplasmic shuttling, target protein (B23, nucleophosmin) binding, and phosphorylation at serine 18 and 65 both in situ and in vitro. To test the role of these phosphorylation sites, we carried out in vivo tumorigenesis studies in severe combined immunodeficient mice. The findings show that the p14-Ser65Ala mutation is associated with impaired tumorigenicity, whereas the p14-Ser18Ala mutation is associated with enhanced tumorigenicity. Microarray analysis suggests that phosphorylation at serine 18 or at serine 65 is associated with transcriptional regulation of the L5 nucleolar ribosomal protein (a p14 target) and the Erb-B signal transduction pathway. Taken together, these results show that the phosphorylation status of p14 determines whether it functions as a pro-oncogenic or antioncogenic modulator. Mol Cancer Res; 10(8); 1077–86. ©2012 AACR.

Highly Stable Tetra-Phenolato Titanium(IV) Agent Formulated into Nanoparticles Demonstrates Anti-Tumoral Activity and Selectivity

Titanium(IV) complexes exhibit high potential as anti-tumor agents, particularly due to their low intrinsic toxicity and cytotoxicity toward cisplatin resistant cells. Nevertheless, Ti(IV) complexes generally undergo rapid hydrolysis that previously hampered their utilization as anticancer drugs. We recently overcame this difficulty by developing a highly stable Ti(IV) complex that is based on tetra-phenolato, hexadentate ligand, formulated into organic nanoparticles. Herein we investigated the activity of this complex in vitro and in vivo. Although inactive when tested directly due to poor solubility, when formulated, this complex displayed (a) high cytotoxicity toward cisplatin resistant human ovarian cells, A2780-cp, with resistance factor of 1.1; (b) additive behavior in combination with cisplatin toward ovarian and colon cancer cells; (c) selectivity toward cancer cells as implied by its mild activity toward non-cancerous, fibroblast lung cells, MRC-5; (d) high stability and durability as manifested by the ability to maintain cytotoxicity, even following one week of incubation in 100% aquatic medium solution; and (e) in vivo efficacy toward solid tumors of human colon cancer cells, HT-29, in nude mice without any clinical signs of toxicity. These features support the formulated phenolato Ti(IV) complex being an effective and selective anti-tumoral agent.

Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

BackgroundWe describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i) products of the proteolytic (e.g. tryptic) digestion of the protein to be identified and (ii) unique to the target protein, as far as one can know from the published sequences.ResultsIn this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems.ConclusionThe data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plate

A new immunization and treatment strategy for mouse mammary tumor virus (MMTV) associated cancers.

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers.

Reversing ABCB1-mediated multi-drug resistance from within cells using translocating immune conjugates

Multi-drug resistance (MDR) is still a major cause of the eventual failure of chemotherapy in cancer treatment. Different approaches have been taken to render these cells drug sensitive. Here, we attempted sensitizing drug-resistant cells from within, using a translocating immune conjugate approach. To that effect, a monoclonal antibody, C219, directed against the intracellular ATP-binding site of the membrane-anchored MDR transporter ABCB1 [P-glycoprotein (P-gp), MDR1], was conjugated to human immunodeficiency virus [HIV(37–72)Tat] translocator peptide through a disulfide bridge. Fluorescence-labelled IgG-Tat conjugates accumulated in drug resistant Chinese hamster ovary (CHO) cells within less than 20 min. Preincubation with C219-S-S-(37–72)Tat conjugate augmented calcein accumulation in drug-resistant CHO and mouse lymphoma cells, indicating reduction in ABCB1 transporter activity. A thioether conjugate C219-S-(37–72)Tat was ineffective, as were disulfide and thioether conjugates of an irrelevant antibody. Furthermore, in the presence of C219-S-S-(37–72)Tat, drug resistant cells were sensitized to colchicine and doxorubicin. Taken together, these findings demonstrate, as proof of principle, a novel approach for the reversal of MDR from within cells, by delivery of translocating immune conjugates as sensitizing agents towards chemotherapy.

An ELISA-based procedure for assaying proteins in digests of human leukocytes and cell lines, using specifically selected peptides and appropriate antibodies.

BackgroundWe describe the application of an ELISA-based assay (the Peptidomatrix) that can be used to simultaneously identify and quantitate a number of proteins in biological samples. The biological sample (blood component, biopsy, culture or other) is first lysed to release all the proteins, without any additional separation. The denatured proteins in the sample are then digested in bulk with the desired proteolytic enzyme(s). The peptides in the digest are then assayed by appropriate antibodies, using a competition ELISA protocol.ResultsAs an example of its use, the present paper applies the Peptidomatrix to the assay of four membrane proteins MDR1 (P-glycoprotein or ABCB1), MRP1 (ABCC1), BCRP/MXR (ABCG2) and the alpha subunit of the Na, K_ATPase (ATP1A1), present in a number of cell lines and in human lymphocytes. We show that we can detect and quantitate these proteins, using a series of peptide-antibody pairs, and that we can differentiate between cell lines or cell preparations that express the target proteins and those that do not.ConclusionWe have devised a simple, ELISA-based proteomics assay that enables the quantitation of designated proteins in a cell or tissue sample, and that can be used in any laboratory, with minimal specialized equipment.