Thermosensitive liposomes for triggered release of cytotoxic proteins.

Lysolipid-containing thermosensitive liposomes (LTSL) are clinically-relevant drug nanocarriers which have been used to deliver small molecule cytostatics to tumors in combination with local hyperthermia (42 °C) to trigger local drug release. The objective of this study was to investigate the feasibility of LTSL for encapsulation and triggered release of macromolecular drugs such as plant-derived cytotoxins. As therapeutic protein we used Mistletoe lectin-1 (ML1) - a ribosome-inactivating protein with potent cytotoxic activity in tumor cells. Model macromolecules (dextrans, albumin) and ML1 were encapsulated in small unilamellar LTSL with varying lipid compositions by the thin film hydration method and extrusion. LTSLs showed molecular weight dependent heat-triggered release of the loaded cargo. The most promising composition, ML1 formulated in LTSL composed of 86:10:4 %mol DPPC:MSPC:DSPE-PEG2000, was further studied for bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging showed uptake of released ML1 after mild hyperthermia at 42 °C, subsequently leading to potent cytotoxicity by LTSL-ML1. Our study shows that LTSL in combination with localized hyperthermia hold promise as local tumor delivery strategy for macromolecular cytotoxins.

Development and characterization of an innovative heparin coating to stabilize and protect liposomes against adverse immune reactions.

Liposomes have been recognized as excellent drug delivery systems, but when they come in direct contact with different blood components they may trigger an immediate activation of the innate immune system. The aim of the present study was to produce long-circulating, blood-compatible liposomes by developing a construct of liposomes covered by a novel unique heparin complex (CHC; 70 heparin molecules per complex) to avoid recognition by the innate immune system. Unilamellar, cationic liposomes were produced by hand extrusion through a 100-nm polycarbonate membrane. Coating of liposomes with the macromolecular CHC was accomplished by electrostatic interactions. Dynamic light scattering as well as QCM-D measurements were used to verify the electrostatic deposition of the negatively charged CHC to cationic liposomes. The CHC-coated liposomes did not aggregate when in contact with lepirudin anti-coagulated plasma. Unlike previous attempts to coat liposomes with heparin, this technique produced freely moveable heparin strands sticking out from the liposome surface, which exposed AT binding sites reflecting the anticoagulant potentials of the liposomes. In experiments using lepirudin-anticoagulated plasma, CHC-coated liposomes, in contrast to non-coated control liposomes, did not activate the complement system, as evidenced by low C3a and sC5b-9 generation and reduced leakage from the liposomes. In conclusion, we show that liposomes can be successfully coated with the biopolymer CHC, resulting in biocompatible and stable liposomes that have significant application potential.

PKCbeta is essential for the development of chronic lymphocytic leukemia in the TCL1 transgenic mouse model: validation of PKCbeta as a therapeutic target in chronic lymphocytic leukemia.

The development and the propagation of chronic lymphocytic leukemia (CLL) has been linked to signaling via the B-cell receptor (BCR). Protein kinase C beta (PKCbeta) is an essential signaling element of the BCR and was recently shown to be overexpressed in human CLL. We used the TCL1 transgenic mouse model to directly target PKCbeta in the development of murine CLL. TCL1 overexpression did restore the CD5(+) B-cell population that is absent in PKCbeta-deficient mice. However, PKCbeta-deleted TCL1 transgenic mice did not develop a CLL disease, suggesting a role of PKCbeta in the establishment of the malignant clone. Moreover, targeting of PKCbeta with the specific inhibitor enzastaurin led to killing of human CLL samples in vitro. We thus propose that PKCbeta may be a relevant target for the treatment of CLL.

Arsenic trioxide induces apoptosis preferentially in B-CLL cells of patients with unfavourable prognostic factors including del17p13.

In the last decade, arsenic trioxide (As2O3) has been used very successfully to treat acute promyelocytic leukaemia (APL). Much less is known about the effectiveness of As2O3 in other neoplastic disorders. In this paper, we report that after 18 h in vitro treatment with 4 microM As2O3, 75+/-18% of B cell chronic lymphocytic leukaemia (B-CLL) cells (n=52) underwent apoptosis. It is important to note that B-CLL cells harboring a deletion of chromosome 17p13, which predisposes to fludarabine resistance and has been identified as an important negative predictor of clinical outcome, were more susceptible to As2O3 toxicity than cells lacking this aberration. Furthermore, unfavourable risk profiles such as unmutated IgVH status, high CD38 expression and prior treatment were associated with significantly higher sensitivity of B-CLL cells to As2O3. As2O3 also preferentially killed B-CLL cells compared to B cells from healthy age-matched controls. Molecular analysis revealed that basal superoxide dismutase activity was positively correlated with the pro-apoptotic activity of As2O3 pointing to a role of reactive oxygen species in cell death induction. The high activity of As2O3 in B-CLL cells from high-risk patients makes it a promising drug for high-risk and/or fludarabine-refractory B-CLL patients.

Adhesion molecules and chemokines: the navigation system for circulating tumor (stem) cells to metastasize in an organ-specific manner.

To date, cancer is still the second most prevalent cause of death after cardiovascular diseases in the industrialized word, whereby the primary cause of cancer is not attributed to primary tumor formation, but rather to the growth of metastases at distant organ sites. For several years it was considered that the well-known phenomenon of organ-specific spreading of tumor cells is mostly a mechanical process either directed passively due to size constraints (mechanical trapping theory) or due to a fertile environment provided by the organ in which tumor cells can proliferate (seed and soil hypothesis). Both mechanisms strongly depend on the adhesive properties of tumor cells either to endothelial cells and/or cancer cells, which are facilitated by a variety of cell adhesion molecules including carbohydrates and integrins. Within the past years it became evident that the organ-specific metastatic spreading of tumor cells does not only rely on heterotypic and homotypic adhesive interactions, but also on the interplay of chemokines and their appropriate receptors. Moreover, the identification of cancer stem cells in various tumor tissues has opened new questions. Cancer stem cells possess self-renewal, differentiation, and tumor-initiating capacities. Thus these cells are ideal candidates to be the seed of a secondary tumor. In the present review we will give a brief overview about the complex process of organ-specific metastasis formation depending on the interplay of adhesion molecules, chemokines, and the putative role of cancer stem cells in metastasis formation.

Visualization of tumor cell extravasation.

In cancer the blood-borne spread of tumor cells leads to the formation of secondary tumors at distant loci, whereby the extravasation of tumor cells is a prerequisite step during hematogenous metastasis. In regard to the fate of endothelial cells located at the site of tumor cell infiltration, tumor cell-endothelial interactions were analyzed using an in vitro real-time model. This model shows the complete sequence of the transmigration process and gave new insights into the complex and dynamic cell-cell and cell-matrix interactions which occur during tumor cell transmigration across the endothelial barrier. An in vitro real-time apoptosis assay permits the distinction between apoptotic cell death from necrotic cell death. This model indicates that transmigration of tumor cell clusters derived from the invasive human bladder carcinoma cell line T24 irreversibly damages the endothelial cells by inducing apoptosis at the site of tumor cell infiltration. It is postulated here that apoptosis induction facilitates the removal of detached endothelial cells, thereby forestalling a local inflammatory response which might be detrimental to extravasating tumor cells.

Role of the beta1-integrin subunit in the adhesion, extravasation and migration of T24 human bladder carcinoma cells.

The abilities of tumor cells to extravasate from the blood vessel system and to migrate through the connective tissue are prerequisites in metastasis formation. Both processes are chiefly mediated by integrins, which mediate both cell-cell and cell-matrix interactions. We investigated the role of integrin subunits in the adhesion, extravasation and migration of the highly invasive human bladder carcinoma cell line T24. Here we show that inhibition of the beta(1)-integrin subunit using the specific beta(1)-integrin blocking antibody 4B4 significantly reduces the adhesion to HUVEC and transmigratory activity of T24 cells. The blockade of the beta(1)-integrin subunit also resulted in a significantly reduced locomotory activity of T24 cells. A detailed cell migration analysis on a single cell level revealed that blockade of the beta(1)-integrin subunit leads to an altered migration pattern of single cells but does not influence migration per se. Migration parameters such as time active, velocity and distance migrated were significantly reduced as compared to untreated control cells. Our observations strongly suggest a central role for the beta(1)-integrin subunit in forming the cell-cell and cell-matrix bonds necessary for adhesion, extravasation and migration.

3D-extravasation model -- selection of highly motile and metastatic cancer cells.

Extravasation has been described as a rate-limiting step in the process of hematogeneous metastasis formation. Thereby, transendothelial migration of tumor cells consists of a complex series of events involving multiple cell-cell and cell-matrix interactions. 3D-extravasation assays are valuable tools for the identification of genes, which are the key players at switchboards of the intracellular signaling pathways. In consequence, the combination of 3D-modeling and whole genome expression analysis lead to unravel molecular parameters which descripe distinct clinical phenotypes of cancer and therefore, work as prognosticators, predictors of therapy and new therapy targets.