Ryszard Smolarczyk

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.

Active heat shock transcription factor 1 supports migration of the melanoma cells via vinculin down-regulation

Heat shock transcription factor 1 (HSF1), the major regulator of stress response, is frequently activated in cancer and has an apparent role in malignant transformation. Here we analyzed the influence of the over-expression of a constitutively active transcriptionally-competent HSF1 mutant form on phenotypes of mouse and human melanoma cells. We observed that the expression of active HSF1 supported anchorage-independent growth in vitro, and metastatic spread in the animal model in vivo, although the proliferation rate of cancer cells was not affected. Furthermore, active HSF1 enhanced cell motility, reduced the adherence of cells to a fibronectin-coated surface, and affected the actin cytoskeleton. We found that although the expression of active HSF1 did not affect levels of epithelial-to-mesenchymal transition markers, it caused transcriptional down-regulation of vinculin, protein involved in cell motility, and adherence. Functional HSF1-binding sites were found in mouse and human Vcl/VCL genes, indicating a direct role of HSF1 in the regulation of this gene. An apparent association between HSF1-induced down-regulation of vinculin, increased motility, and a reduced adherence of cells suggests a possible mechanism of HSF1-mediated enhancement of the metastatic potential of cancer cells.

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.

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.

Oxidation of carbidopa by tyrosinase and its effect on murine melanoma

Oxidation of the anti-Parkinsonian agent carbidopa by tyrosinase was investigated. The products of this reaction were identified as 3-(3,4-dihydroxyphenyl)-2-methylpropanoic acid and 6,7-dihydroxy-3-methylcinnoline. These results demonstrate that after oxidation of the catechol moiety to an o-quinone either a redox exchange with the hydrazine group or a cyclization reaction occur. The cyclization product underwent additional oxidation reactions leading to aromatization. The cyclization reaction is undesired in the case of hydrazine-containing anti-melanoma prodrugs and will have to be taken into account in designing such compounds. Carbidopa was tested against B16(F10) melanoma cells in culture and showed cytotoxicity significantly higher than either of its oxidation products and l-dopa. This effect, however, was not specific to this cell line.

M1-like macrophages change tumor blood vessels and microenvironment in murine melanoma

Tumor-associated macrophages (TAMs) play a significant role in at least two key processes underlying neoplastic progression: angiogenesis and immune surveillance. TAMs phenotypic changes play important role in tumor vessel abnormalization/ normalization. M2-like TAMs stimulate immunosuppression and formation of defective tumor blood vessels leading to tumor progression. In contrast M1-like TAMs trigger immune response and normalize irregular tumor vascular network which should sensitize cancer cells to chemo- and radiotherapy and lead to tumor growth regression. Here, we demonstrated that combination of endoglin-based DNA vaccine with interleukin 12 repolarizes TAMs from tumor growth-promoting M2-like phenotype to tumor growth-inhibiting M1-like phenotype. Combined therapy enhances tumor infiltration by CD4+, CD8+ lymphocytes and NK cells. Depletion of TAMs as well as CD8+ lymphocytes and NK cells, but not CD4+ lymphocytes, reduces the effect of combined therapy. Furthermore, combined therapy improves tumor vessel maturation, perfusion and reduces hypoxia. It caused that suboptimal doses of doxorubicin reduced the growth of tumors in mice treated with combined therapy. To summarize, combination of antiangiogenic drug and immunostimulatory agent repolarizes TAMs phenotype from M2-like (pro-tumor) into M1-like (anti-tumor) which affects the structure of tumor blood vessels, improves the effect of chemotherapy and leads to tumor growth regression.

Antitumor activity of opiorphin, sialorphin and their conjugates with a peptide klaklakklaklak

This is the study on the effect of opiorphin, sialorphin and their analogs on antitumor activity. We demonstrated that conjugation of opiorphin and sialorphin with a proapoptotic, antimicrobial peptide klak (klaklakklaklak) led to compounds (opio‐klak and sialo‐klak) that were cytotoxic against cancer cells (LN18, PC3, A549, HCT116 and B10‐F16) in the MTT test. The conjugated analogs were designed to increase the effectiveness of the peptide. The opio‐klak derivative was the most effective in the in vitro assays and led to a decrease in viability of cancer cells over time as compared with that of untreated controls. In contrast, treatment with either the untargeted klak peptide or opiorphin as a negative control led to a negligible loss in viability. Antitumor effect of the opio‐klak was also observed in vivo in murine melanoma tumor‐bearing mice. Cessation of peptide administration resulted in tumor regrowth. Our results are seemingly valuable for the development of opiorphin analogs with potential clinical applications. Copyright

Polarization of Tumor Milieu: Therapeutic Implications

During neoplastic progression, cancer cells recruit inflammatory cells (monocytes, neutrophils, mast, and dendritic cells, etc.), which become “educated” under the influence of factors released by cancer cells (mainly cytokines). As a consequence, the former lose their ability to present antigens. Instead, they become cells involved in remodeling of extracellular matrix and stimulate the formation of blood vessels (angiogenesis). Proangiogenic factors released by inflammatory cells act as immunosuppressants and the tumor milieu becomes proangiogenic and immunosuppressive. Latest studies have demonstrated the possibility of reverting such proangiogenic/immunosuppressive microenvironment which inhibits tumor growth. Reverted tumor microenvironment becomes anti-angiogenic and immunostimulatory. Reversal of tumor microenvironment is especially feasible with combinations of anti-angiogenic and immunomodulatory factors. For instance, combinations of VEGF, VEGFR2, or TGF-β activity inhibitors with immunostimulants such as anticancer vaccines, CpG sequences, or IL-12 were effective in inhibiting growth of experimental tumors. In our hands, a DNA vaccine directed against endoglin (CD105), a tumor vascular endothelial cell-surface protein, when combined with IL-12, led to a ca. 30 % cure rate in mice bearing experimental melanoma tumors. It appears that attempts to therapeutically revert tumor microenvironment might merit further consideration.