Stanisław Szala

Electrotransfer of gene encoding endostatin into normal and neoplastic mouse tissues: Inhibition of primary tumor growth and metastatic spread

Electroporation-mediated gene transfer relies upon direct delivery of plasmids into cells permeabilized by electric fields, a method more efficient than transfer using nonviral vectors, although neither approaches the transfer efficiency of viral vectors. Here we studied electrotransfer of a gene encoding an angiogenesis inhibitor (endostatin) into primary tumors and muscle tissues, which would serve as a site of synthesis and secretion into the bloodstream of a therapeutic antimetastatic protein with systemic effects. Optimum electroporation conditions (voltage, number and duration of impulses, separation of caliper electrodes) were first established to maximize expression of a reporter gene transferred into murine Renca kidney carcinoma, B16(F10) melanoma, or skeletal muscle tissues. In neoplastic tissues, electrotransfer of plasmid DNA was far more efficient than electroporation with lipoplexes, but no differences between naked DNA and lipoplexes were found in case of electroporated muscles. We then studied the electrotransfer of plasmid DNA carrying the endostatin gene into pre-established experimental Renca tumors. A significant inhibition of tumor growth was observed in animals electroporated with this construct. Electrotransfer of the endostatin gene into muscle tissues resulted in reduced numbers of experimental B16(F10) metastases in the lungs. This study clearly shows that electroporation may be used to efficiently transfer antiangiogenic genes into both normal and neoplastic tissues.

Intra‐tumoral administration of naked plasmid DNA encoding mouse endostatin inhibits renal carcinoma growth

Endostatin is a C‐terminal fragment of collagen XVIII and has potent anti‐angiogenic and anti‐tumor activity. Mouse endostatin‐coding sequences were obtained using PCR and linked to the signal sequence of influenzavirus hemagglutinin. The signal‐sequence endostatin fragment was subcloned into plasmid vectors under the transcriptional control of cytomegalovirus promoter. Murine renal carcinoma (Renca) cells transfected with endostatin‐coding plasmid are shown to secrete full‐length endostatin. Endostatin‐secreting Renca cells demonstrate slower growth in vivo compared to empty vector–transfected cells, but their in vitro growth is unaffected. Anti‐angiogenic activity of secreted endostatin was confirmed in a Matrigel angiogenesis assay in vivo. We report growth inhibition of Renca tumors resulting from intra‐tumoral delivery of plasmid vector encoding secretable endostatin. Elevated local concentrations of endostatin resulted from multiple intra‐tumoral injections of endotoxin‐purified plasmid DNA. Local endostatin levels were high enough to obtain growth arrest of Renca tumors.

The Role of Glycyrrhizin, an Inhibitor of HMGB1 Protein, in Anticancer Therapy

Certain anticancer drugs, such as the peptide CAMEL (aa sequence KWKLFKKIGAULKVL) induce necrotic type of cell death. During this process, a protein termed high mobility group box 1 (HMGB1) is released from cell nucleus into cytoplasm and then into extracellular milieu. Outside of cells, it becomes a proinflammatory cytokine. Its effects range from stimulation of cancer as well as endothelial cell proliferation, to activation of angiogenesis, cell motility and induction of inflammatory conditions. Release of HMGB1 cytokine during the course of anticancer therapy has negative effects upon the therapy itself, since it leads to tumor relapse. We assumed that the inhibition of HMGB1 activity may be conducive towards better therapeutic results in case of drugs inducing necrotic cell death. In this context we studied glycyrrhizin (GR), a triterpenoid saponin glycoside of glycyrrhizic acid and a well-known inhibitor of HMGB1. We have shown that GR inhibits proliferation and migration of cells stimulated by HMGB1 cytokine, as well as HMGB1-induced formation of blood vessels and reduces inflammatory condition (lowering tumor necrosis factor α levels). GR-mediated inhibition of HMGB1 activity (CAMEL-induced release) impedes, in turn, tumor regrowth in mice. As expected, inhibited tumor regrowth is linked to diminished tumor levels of the released HMGB1 and reduced inflammatory condition. To conclude, the use of GR significantly improved anticancer effectiveness of the CAMEL peptide.

Introduction of murine Il-4 gene into B16(F10) melanoma tumors by direct gene transfer with DNA-liposome complexes

Il-4 is a highly pleiotropic cytokine which induces cytotoxic activity when present at the tumor site. Death of tumor cells probably depends on the appearance of an inflammatory infiltrate composed of eosinophils and macrophages. Regression of established tumor masses has been readily observed upon direct intratumor implantation of cells which constitutively produce high amounts of Il-4. We now report a similar potent anti-tumor effect achieved via direct gene transfer, i.e. by injecting Il-4 DNA complexed with cationic liposomes into B16(F10) melanoma tumor in vivo.

A new cholesterol derivative suitable for transfecting certain type of cells in the presence of 10% serum

We developed a new cationic lipid suitable for use as a DNA carrier in the presence of 10% sera. The novel compound (abbreviated as Arg-Chol) contains cholesterol and a dipeptide consisting of glycine and sterically protected arginine. The efficiency of reporter gene transfection using liposomes based on this new reagent was compared with that of liposomes made with other cationic derivatives of cholesterol. Lipoplexes formulated with the newly synthesized lipid mediate in vitro transfection of B16(F10) murine melanoma cells in the presence of 10% sera more efficiently than in other cell lines and compared with other cholesterol derivatives studied.

Can inhibition of angiogenesis and stimulation of immune response be combined into a more effective antitumor therapy

Cancer initiation and progression is strongly influenced by the tumor microenvironment consisting of various types of host cells (inflammatory cells, vascular cells and fibroblasts), extracellular matrix and non-matrix molecules. Host cells play a defining role in two major processes crucial for tumor growth: angiogenesis and escape from immune surveillance. The interdependence of these processes resemble the principles of Yin and Yang, as the stimulation of tumor angiogenesis inhibits effective immune responses, while angiogenesis inhibition may have the opposite effect. These considerations may be useful in developing anticancer strategies based on the potentially synergistic combinations of antiangiogenic and immunostimulatory drugs.

Combined Tumor Cell-Based Vaccination and Interleukin-12 Gene Therapy Polarizes the Tumor Microenvironment in Mice

Tumor progression depends on tumor milieu, which influences neovasculature formation and immunosuppression. Combining immunotherapy with antiangiogenic/antivascular therapy might be an effective therapeutic approach. The aim of our study was to elaborate an anticancer therapeutic strategy based on the induction of immune response which leads to polarization of tumor milieu. To achieve this, we developed a tumor cell-based vaccine. CAMEL peptide was used as a B16-F10 cell death-inducing agent. The lysates were used as a vaccine to immunize mice bearing B16-F10 melanoma tumors. To further improve the therapeutic effect of the vaccine, we combined it with interleukin (IL)-12 gene therapy. IL-12, a cytokine with antiangiogenic properties, activates nonspecific and specific immune responses. We observed that combined therapy is significantly more effective (as compared with monotherapies) in inhibiting tumor growth. Furthermore, the tested combination polarizes the tumor microenvironment, which results in a switch from a proangiogenic/immunosuppressive to an antiangiogenic/immunostimulatory one. The switch manifests itself as a decreased number of tumor blood vessels, increased levels of tumor-infiltrating CD4+, CD8+ and NK cells, as well as lower level of suppressor lymphocytes (Treg). Our results suggest that polarizing tumor milieu by such combined therapy does inhibit tumor growth and seems to be a promising therapeutic strategy.

Evolving models of tumor origin and progression

History of cancer disease models clearly illustrates the evolving nature of these concepts. Since such models undergo continual revisions and additions as a result of underlying medical research, they also tend to reorganize knowledge and allow perceiving previously unseen relationships. Growth of medical thought has been influenced for many centuries by an ancient Hippocratic concept of disease seen as a disturbance in bodily “humors.” True mechanisms of cell and tissue injury started to be elucidated only with the advent of postmortem pathological findings. Concerning cancer, when first disease-producing bacteria were identified in the nineteenth century, also neoplasms were treated as infectious diseases. Foreign organisms were thought to be present inside tumors. However, this hypothesis could not be confirmed by microscopic or histochemical studies. The latter suggested, instead, that tumors were rather formed by abnormal cells. Cancer was then started to be regarded as a disease of cells. This interpretation was radically altered by later developments in genetics which suggested that neoplasms can be treated as genetic diseases as pathologic cellular lesions are caused by mutations in specific genes. More recent models have compared carcinogenesis to evolutionary processes. Due to genetic instability, successive mutations, appearing in cells, lead to selection of cancer cells which feature specific phenotypic traits. The newest data indicate that there may be also a link between cancer and mutated stem cells. The review discusses main concepts of tumor origin forwarded since the beginnings of the nineteenth century.

Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice

Aims The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. Methods and Results MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. Conclusion In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.

Nowotworowe naczynia krwionośne

Growth of tumors usually depends on the development of the tumor’s own vasculature. Small avascular tumors (1–2 mm3) cannot continue growth provided an equilibrium between pro-angiogenic and anti-angiogenic factors is maintained within the tumor environment. Angiogenesis is not the only factor responsible for tumor blood vessels forming, as vasculogenic mimicry plays an equally substantial role in this process. Vessel-like structures formed during this process are made up from cancer cells, macrophages and mast cells. Certain neoplasms are capable of growing without developing their own vasculature; instead they secure growth via normal blood vessels of the host. Slowed-down blood flow through an abnormally built tumor vascular network is the main reason for cancer cells’ underoxygenation (hypoxia). Defective blood vessels, with hypoxia resulting, play a major role in tumor progression. Underoxygenation induces formation of novel vessels and these new defective vessels are in turn the principal reason for hypoxia. The latter increases cancer cells’ malignancy and invasiveness. A particular process, called transdifferentiation, takes place in tumor vasculature when hypoxia is present and involves neoplastic cells transforming into endothelial cells. Since growth of a tumor is dependent on its own blood supply, inhibition of such vascular network growth and/or damage to this network should exert a strong impact on tumor growth. Long-term administration of anti-angiogenic drugs, however, encounters unexpected problems. Anti-angiogenic drug resistance, together with paradoxical stimulation of invasiveness and metastasis by these drugs, has lately become a dominant issue in anticancer therapy.