Vladislav V. Glinsky

Modified citrus pectin anti-metastatic properties: one bullet, multiple targets

In this minireview, we examine the ability of modified citrus pectin (MCP), a complex water soluble indigestible polysaccharide obtained from the peel and pulp of citrus fruits and modified by means of high pH and temperature treatment, to affect numerous rate-limiting steps in cancer metastasis. The anti-adhesive properties of MCP as well as its potential for increasing apoptotic responses of tumor cells to chemotherapy by inhibiting galectin-3 anti-apoptotic function are discussed in the light of a potential use of this carbohydrate-based substance in the treatment of multiple human malignancies.

Apoptosis and metastasis: increased apoptosis resistance of metastatic cancer cells is associated with the profound deficiency of apoptosis execution mechanisms.

Programmed cell death, particularly adhesion-dependent regulation of cell survival and apoptosis, is recognized as one of the main homeostatic mechanisms designed to control cell positioning, eliminate misplaced cells and block metastatic dissemination. Recently we reported that highly metastatic cancer cells exhibit a higher resistance to the programmed cell death compared to their poorly metastatic counterparts (Cancer Lett., 101, 43-51, 1996). However, the molecular and genetic basis for the association of aggressive metastatic phenotype with resistance toward apoptosis remains to be elucidated. Here we extended our investigation on apoptosis and metastasis using a panel of nine murine and human cancer cell lines with different metastatic potential. We examined the relationship of the metastatic ability and the sensitivity to apoptosis as well as determined the status of two major apoptosis execution mechanisms (induction of nuclear Ca2+-dependent endonucleases and activation of ICE-like proteases) in cancer cells with distinct metastatic potential and different sensitivity to apoptosis. We found that high metastatic potential is strictly associated with the increased resistance to apoptosis, diminished level of nuclear Ca2+-dependent endonucleases, and significantly reduced activity of CPP32/Yama death protease. We concluded that high resistance to apoptosis of metastatic cancer cells is associated with and may depend upon the profound deficiency of major apoptosis execution mechanisms.

Resistance to apoptosis in human cells conferred by telomerase function and telomere stability

Cell senescence and programmed cell death (apoptosis) are two fundamental biological mechanisms that regulate proliferative capacity, survival potential, aging, and death of cells. Here we report several independent lines of experimental evidence that support the hypothesis that telomerase function and telomere length perform important roles in cell survival during apoptosis. First, with serum starvation and matrix‐independent survival experiments, we found that young normal diploid cells were more resistant to apoptosis than their older counterparts. In addition, normal cells with stable telomere lengths caused by ectopic expression of telomerase maintained an increased resistance to serum starvation– and matrix‐deprivation–induced programmed cell death compared with aged normal cells without telomerase. Second, we found that telomerase‐positive immortalized SW39 cells had a higher survival ability and resistance to apoptosis than their telomerase‐negative immortalized counterparts, SW13 and SW26. Third, we showed that telomerase‐positive cells with experimentally elongated telomeres (GTR‐IDH4 and GTR‐DU145) acquired increased survival ability and higher resistance to apoptosis than the parental cell lines with shorter telomeres (IDH4 and DU145). Higher resistance to apoptosis of these cells was associated with a deficiency in two major apoptosis execution pathways: induction of nuclear calcium‐dependent endonucleases and activation of the interleukin‐1β‐converting enzyme–family of proteases (caspases). Taken together, these results provide the first direct experimental evidence supporting the hypothesis that telomerase activity and maintenance of telomere stability are associated with increased cellular resistance to apoptosis. Mol. Carcinog. 25:241–248, 1999.

Apoptosis and metastasis: a superior resistance of metastatic cancer cells to programmed cell death

We studied the response to different external signals leading to apoptosis of several poorly and highly metastatic cell lines employing a murine B16 melanoma experimental metastasis model. We found that highly metastatic cells exhibit a superior survival ability and resistance to apoptosis compared to poorly metastatic cells which would give the former an obvious selective growth advantage during tumor progression. Our results indicate that there is a genetic link between aggressive metastatic phenotype and resistance to apoptosis.

Identification and Characterization of Peptides That Bind Human ErbB-2 Selected from a Bacteriophage Display Library

The ErbB-2 receptor, a member of the tyrosine kinase type 1 family of receptors, has been implicated in many human malignancies. The overexpression of ErbB-2 in cancer cells as well as its extracellular accessibility makes it an attractive target for the development of tumor-specific agents. In this study, random peptide bacteriophage display technology was employed to identify peptides that bound the extracellular domain of human ErbB-2. The peptide KCCYSL, most frequently occurring in the affinity-selected phage population, was chemically synthesized and characterized for its binding activities to ErbB-2. The synthetic peptide exhibited high specificity for ErbB-2 and an equilibrium dissociation constant of 30 μM. Peptide binding to ErbB-2 positive human breast and prostate carcinoma cells was visualized in direct cell binding assays. In conclusion, the peptide KCCYSL has the potential to be developed into a cancer imaging or therapeutic agent targeting malignant cells overexpressing the ErbB-2 receptor.

Inhibition of colony formation in agarose of metastatic human breast carcinoma and melanoma cells by synthetic glycoamine analogs

We studied the influence of 10 synthetic glycoamine analogs on colony formation in 0.3 and 0.9% agarose by metastatic human breast carcinoma (MDA-MB-435) and melanoma (TXM-13) cells. Nine synthetic analogs significantly inhibited the colony formation in 0.9% agarose of MDA-MB-435 human breast carcinoma cells; five compounds caused a 73–83% reduction of colony formation. Seven synthetic glycoamines caused a significant inhibition of colony formation in 0.9% agarose by TXM-13 melanoma cells with the inhibitory effect ranging from 71 to 87%. The 50% inhibition (I50) doses and relative activity rank of the compounds were similar for both breast carcinoma and melanoma cell lines. The murine B16 melanoma cell aggregation assay was employed to elucidate the potential mechanism(s) of the inhibitory activity of synthetic glycoamines. The relative activity ranks of the compounds based on the independently determined I50 doses for both cell aggregation and clonogenic growth assays were very similar for the four most active synthetic analogs and clearly indicated the importance of hydrophobic amino acid in mediating the bioactivity of synthetic glycoamines. In both experimental systems (clonogenic growth in agarose and cell aggregation assay) the leading compound was N-(1-deoxy-d-fructos-1-y1)-d-leucine (Fru-d-Leu) and the least active analog was N-(1-deoxy-d-fructos-1-yl)-glycine (Fru-Gly). These results show that synthetic glycoamines may act by competing for specific carbohydrate-lectin interactions, particularly those involving β-galactoside-specific lectins expressed on metastatic cells.

Glycobioinformatics: Current strategies and tools for data mining in MS‐based glycoproteomics

Glycobioinformatics is a rapidly developing field providing a vital support for MS‐based glycoproteomics research. Recent advances in MS greatly increased technological capabilities for high throughput glycopeptide analysis. However, interpreting MS output, in terms of identifying glycan structures, attachment sites and glycosylation linkages still presents multiple challenges. Here, we discuss current strategies used in MS‐based glycoproteomics and bioinformatics tools available for MS‐based glycopeptide and glycan analysis. We also provide a brief overview of recent efforts in glycobioinformatics such as the new initiative UniCarbKB directed toward developing more comprehensive and unified glycobioinformatics platforms. With regards to glycobioinformatics tools and applications, we do not express our personal preferences or biases, but rather focus on providing a concise description of main features and functionalities of each application with the goal of assisting readers in making their own choices and identifying and locating glycobioinformatics tools most suitable for achieving their experimental objectives.

Continuous real time ex vivo epifluorescent video microscopy for the study of metastatic cancer cell interactions with microvascular endothelium

Recent studies suggest that only endothelium-attached malignant cells are capable of giving rise to hematogenous cancer metastases. Moreover, tumor cell adhesion to microvascular endothelium could be crucial in metastasis predilection to specific organs or tissues. However, the existing in vitro and in vivo techniques do not provide for sufficient delineation of distinct stages of a dynamic multi-step intravascular adhesion process. Here we report the development of an experimental system allowing for prolonged continuous ex vivo real-time observation of malignant cell adhesive interactions with perfused microvessels of a target organ in the context of its original tissue. Specifically, the vasculature of excised dura mater perfused with prostate cancer cells is described. An advantage of this technique is that selected fluorescently labeled tumor cells can be followed along identified vascular trees across the entire tissue specimen. The techniques provide for superior microvessel visualization and allow for uninterrupted monitoring and video recording of subsequent adhesion events such as rolling, docking (initial reversible adhesion), locking (irreversible adhesion), and flattening of metastatic cancer cells within perfused microvasculature on a single cell level. The results of our experiments demonstrate that intravascular adhesion of cancer cells differs dramatically from such of the leukocytes. Within dura microvessels perfused at physiological rate, non-interacting, floating, tumor cells move at velocities averaging 7.2×103 μm/s. Some tumor cells, similarly to leukocytes, exhibit rolling-like motion patterns prior to engaging into more stable adhesive interactions. In contrast, other neoplastic cells became stably adhered without rolling showing a rapid reduction in velocity from 2×103 to 0 μm/s within fractions of a second. The experimental system described herein, while developed originally for studying prostate cancer cell interactions with porcine dura mater microvasculature, offers great flexibility in adhesion experiments design and is easily adapted for use with a variety of other tissues including human.

Combinatorial evolution of high-affinity peptides that bind to the Thomsen-Friedenreich carcinoma antigen.

Thomsen-Friedenreich (TF) antigen occurs on approximately 90% of human carcinomas, is likely involved in carcinoma cell homotypic aggregation, and has clinical value as a prognostic indicator and marker of metastasized cells. Previously, we isolated anti-TF antigen peptides from bacteriophage display libraries. These bound to TF antigen on carcinoma cells but were of low affinity and solubility. We hypothesized that peptide amino acid sequence changes would result in increased affinity and solubility, which would translate into improved carcinoma cell binding and increased inhibition of aggregation. The new peptides were more soluble and exhibited up to fivefold increase in affinity (Kd ≅ 60 nM). They bound cultured human breast and prostate carcinoma cells at low concentrations, whereas the earlier peptides did not. Moreover, the new peptides were potent inhibitors of homotypic aggregation. The maturated peptides will have expanded applications in basic studies of the TF antigen and particular utility as clinical carcinoma-targeting agents.

Intravascular cell-to-cell adhesive interactions and bone metastasis

Metastatic cancer spread to bones, causing intractable pain, pathological fractures, spinal cord compression, and ultimately death, represents massive clinical problem. Intravascular cell-to-cell heterotypic (between cancer and other types of cells) and homotypic (between cancer cells) adhesive interactions, leading to the establishment of metastatic deposits in bone marrow vasculature, represent important rate-limiting steps in bone metastasis. In this review, we discuss molecular and cellular mechanisms underpinning metastasis-associated intravascular cell-to-cell adhesive interactions, their role in a multi-step metastatic cascade, and a potential for therapeutic targeting of early metastasis-associated adhesive events.